A method of forming biopolymer fibres

Abstract

The invention provides a method of forming biopolymer fibres, the method comprising; (i) providing a first and a second aqueous solution, wherein one of the first or the second aqueous solution comprises at least one water-soluble polycationic biopolymer and the other first or second aqueous solution comprises at least one water-soluble polyanionic biopolymer; and (ii) adding the first aqueous solution to the second aqueous solution to form a complexation mixture, wherein the complexation mixture comprises polyelectrolyte biopolymer complexes formed by complexation of the polycationic and polyanionic biopolymers of the first and second aqueous solutions; and (iii) effecting laminar fluid flow of the complexation mixture, which applies shear stress to the polyelectrolyte complexes to form biopolymer fibres.

Description

[0001] A Method of Forming Biopolymer Fibres

[0002] Technical Field of the Invention

[0003] The present invention relates to a method of forming biopolymer fibres. The invention further relates to a method of forming fibrous hydrogels.

[0004] Background to the Invention

[0005] Hydrogels are three-dimensional polymer networks with great water absorbance ability. Hydrogels formed from biopolymers (such as polysaccharides, polypeptides and nucleic acids) have varied characteristics and have a wide range of applications. One common application is in the field of biomedicine, for example, in drug delivery, tissue engineering and cell expansion.

[0006] Hydrogels have been commonly used in tissue engineering applications because of their ability to closely mimic the extracellular matrix (ECM) which in turn promotes tissue repair and regeneration. The high water content of a hydrogel can provide an ideal environment for cell survival and its structure can provide support for the cells in the engineered tissues. There are a range of biopolymer materials that have been shown to be suitable for tissue engineering applications, for example, collagen, gelatine, elastin, alginate, cellulose, glycosaminoglycans and DNA. Biopolymer-based hydrogels allow for the treatment and repair of tissues without the need for major invasive medical procedures. The hydrogel pore size is an important factor in tissue engineering applications, with cell-sized pores shown to increase levels of cell invasion. Furthermore, cell- sized pores improve wound healing outcomes as hydrogels comprising cell- sized pores have faster ingrowth of vasculature which subsequently reduces scarring after wound closure. More recently, injectable hydrogels have been shown to have great potential for applications such as tissue engineering. Shear-thinning hydrogels allow for cell delivery via injection and can conform to the shape of the target site, allowing them to fill irregular spaces, for example, a wound site. There are several methods for generating porous hydrogels that have the shear thinning properties required for injectability. For example, methods involving microfluidics to create microspheres (M. Musiime et al., Cells, 2021, 10, 662), methods of forming fragmented bulk hydrogels via extrusion (M. Musiime et. al, Cells, 2021, 10, 662) and methods of forming hydrogel micro-strands when extruded through pores (X. Li et al., Sci. Transl. Med., 2019, 11). However, the hydrogel pores obtained through such known methods are generally larger than those found in the extracellular matrix (in the order of tens of microns in diameter). Hydrogels with larger pores are less effective for tissue engineering applications as larger pores mean that cells cannot assemble collagen properly.

[0007] The majority of skin extracellular matrix is made of collogen. Collogen typically comprises 600-800 nm wide fibrils having a basket weave morphology with interconnected pores. Fibrous hydrogels have been formed using synthetic materials that mimic the structure of collagen. Known methods for producing such fibrous hydrogels involve a first step of forming individual fibres via electrospinning and non-scalable fragmenting methods, followed by cross-linking the fibres to form a hydrogel (M. A. Johns et al., ACS Omega, 2018, 3, 937; J. G. Martins et al., Carbohydr. Polym., 2018, 197, 47). These known methods are not scalable and / or cannot be translated into clinical applications due to the large number of discrete operations that need to be validated, the low throughput and the high cost of fabrication machinery. US7323540B2 discloses the formation of individual polymer microrods (such as polystyrene) through a method of fluid templating. This method does not disclose the formation of biopolymer-based fibres and hydrogels.

[0008] It is therefore an aim of the present invention to provide a method of forming a fibrous biopolymer-based hydrogel that is scalable and can be translated for use in clinical applications.

[0009] It is a further aim of the present invention to provide a method of forming biopolymer fibres that are shear-thinning and capable of being injected and / or sprayed to a target site.

[0010] It is a further aim of the present invention to provide biopolymer fibres that once injected and / or sprayed to a target site, can gel and form a fibrous hydrogel. The present invention also aims to provide a hydrogel that can improve cell regeneration and growth.

[0011] It is also an aim of embodiments of the invention to overcome or mitigate at least one problem of the prior art, whether disclosed herein or not.

[0012] Summary of the Invention

[0013] According to a first aspect of the invention there is provided a method of forming biopolymer fibres, the method comprising;

[0014] (i) providing a first and a second aqueous solution, wherein one of the first or the second aqueous solutions comprises at least one water-soluble polycationic biopolymer and the other first or second aqueous solution comprises at least one water-soluble poly anionic biopolymer; and (ii) adding the first aqueous solution to the second aqueous solution to form a complexation mixture, wherein the complexation mixture comprises polyelectrolyte biopolymer complexes formed by complexation of the polycationic and poly anionic biopolymers of the first and second aqueous solutions; and

[0015] (iii) effecting laminar fluid flow of the complexation mixture, which applies shear stress to the poly electrolyte complexes to form biopolymer fibres.

[0016] The term “water-soluble” is used herein to describe biopolymers that are completely soluble in water, as well as those that are partially or substantially water- soluble.

[0017] The complexation mixture may comprise a mixture of different polyelectrolyte biopolymer complexes, for example, different ratios of polycationic polymer and polyanionic polymer.

[0018] In some embodiments, the first and / or second aqueous solution comprises one or more miscible organic solvents. The one or more organic solvents may be added to the first and / or second solution to improve the solubility of the polyanionic and / or polycationic biopolymers. Alternatively, or additionally, the complexation mixture may comprise one or more miscible organic solvents.

[0019] In some embodiments, the solution comprising the or each polycationic biopolymer has a pH of no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, or no more than 3. Preferably, the or each polycationic biopolymer has a pH of between 3-8, or more preferably, a pH of between 4-7. In some embodiment, the solution comprising the or each polyanionic biopolymer has a pH of no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, or no more than 2. Preferably, the or each polyanionic biopolymer has a pH of between 2-7, or more preferably, a pH of between 3-6.

[0020] In some embodiments, the solution comprising the or each polycationic biopolymer has a zeta potential of at least +20 mV. In some embodiments, the solution comprising the polyanionic biopolymer has a zeta potential of no more than -20 mV.

[0021] In some embodiments, the first and / or second solution further comprises one or more additives. Alternatively, or additionally, the complexation mixture may further comprise one or more additives. The or each additive may be independently selected from the group consisting of: a therapeutic agent (e.g. chlorhexidine), an emulsifier, a stabilizer, collagen, a decellularised extracellular matrix, a surfactant, a cross-linking agent, a lubricant, a thickening agent, a leachable and any combination of the aforementioned. In alternative embodiments, the one or more additives may be added in a separate step of the method.

[0022] In some embodiments, the at least one poly anionic and / or polycationic biopolymer is capable of cross-linking in the presence of a cross-linking agent. The cross-linking of the or each polyanionic and / or polycationic biopolymer may result in gelation.

[0023] The molecular weight of the or each polycationic and / or polyanionic biopolymer may be selected to enable sufficient entanglement of the biopolymers during complexation.

[0024] The or each polyanionic biopolymer may have a mean average molecular weight of at least 1 kDa, at least 10 kDa, at least 20 kDa, at least 30 kDa, at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, at least 110 kDa, at least 120 kDa, at least 130 kDa, at least 140 kDa, at least 150 kDa, at least 160 kDa, at least 170 kDa, at least 180 kDa, at least 190 kDa, at least 200 kDa, at least 210 kDa, at least 220 kDa, at least 230 kDa, at least 240 kDa, at least 250 kDa, at least 260 kDa, at least 270 kDa, at least 280 kDa, at least 290 kDa, or at least 300 kDa. Preferably, the or each polyanionic biopolymer may have a mean average molecular weight of between 20-250 kDa.

[0025] The or each polycationic biopolymer may have a mean average molecular weight of at least 100 kDa, at least 200 kDa, at least 300 kDa, at least 400 kDa, at least 500 kDa, at least 600 kDa, at least 700 kDa, at least 800 kDa, at least 900 kDa, at least 1000 kDa, at least 1100 kDa, at least 1200 kDa, at least 1300 kDa, at least 1400 kDa, or at least 1500 kDa. Preferably, the or each polycationic biopolymer may have a mean average molecular weight of between 800 kDa - 1600 kDa, or more preferably, around 1250 kDa.

[0026] The or each polycationic biopolymer may have an average hydrodynamic radius of at least 100 nm, at least 200 nm, at least 300 nm, at least 400 nm, at least 500 nm, at least 600 nm, at least 700 nm, at least 800 nm, at least 900 nm, at least 1000 nm, at least 1200 nm, at least 1400 nm, at least 1600 nm, at least 1800 nm, at least 2000 nm, at least 2200 nm, at least 2400 nm, or at least 2600 nm. Preferably, the or each polycationic biopolymer has a hydrodynamic radius of around 300-700 nm.

[0027] The or each polyanionic biopolymer may be independently selected from the group consisting of: pectin, methylcellulose, gum arabic, hyaluronic acid, Xanthan, gellan, alginate, carrageenan (kappa, iota or lambda), and any combination thereof. The or each polycationic complex may be independently selected from the group consisting of: chitosan, poly-L-lysine and derivatives thereof (e.g. e-poly-L-lysine), proteins, nucleic acids, negatively-charged synthetic polymers and any combination thereof.

[0028] In some embodiments, the first aqueous solution comprises at least one water- soluble polyanionic biopolymer and the second solution comprises at least one polycationic biopolymer. Adding the or each polyanionic biopolymer to the or each polycationic biopolymer is preferred as this method has been shown to produce longer and thinner fibres.

[0029] Preferably, the first aqueous solution may comprise only one water-soluble polyanionic biopolymer; more preferably, the first aqueous solution may comprise pectin. Preferably, the second aqueous solution may comprise only one water-soluble polycationic biopolymer; more preferably, the second aqueous solution may comprise chitosan.

[0030] In preferred embodiments, the first aqueous solution comprises pectin in water and the second aqueous solution comprises chitosan in 1% (v / v) acetic acid solution. Preferably, the second aqueous solution comprises high molecular weight chitosan.

[0031] The first aqueous solution may be added to the second aqueous solution by spraying, injecting, pipetting, dripping, or pouring the first aqueous solution into the second aqueous solution. The aforementioned addition methods may be performed through a needle, conduit, tube, or pipette, for example. The addition method may be performed manually or using a pump (such as a peristaltic pump). The first aqueous solution may be added to the second aqueous solution at a set or variable flow rate or fluid velocity. In some embodiments, the first aqueous solution is added to the second aqueous solution at a flow rate of at least 5 mL / min, at least 10 mL / min, at least 15 mL / min, at least 20 mL / min, at least 25 mL / min, at least 30 mL / min, at least 40 mL / min, or at least 50 mL / min. In preferred embodiments, the first aqueous solution is added to the second solution at a flow rate of around 10 mL / min.

[0032] The first aqueous solution may be added to the second aqueous solution at a fluid velocity of at least 0.50 m / s, 0.55 m / s, 0.60 m / s, 0.65 m / s, 0.70 m / s, 0.75 m / s, 0.80 m / s, 0.85 m / s, 0.90 m / s, 0.95 m / s, 1.0 m / s, 1.05 m / s, 1.10 m / s, 1.15 m / s, 1.20 m / s, 1.25 m / s, 1.30 m / s, 1.35 m / s, 1.40 m / s, 1.45 m / s, 1.50 m / s, 1.55 m / s, 1.60 m / s, 1.65 m / s, 1.70 m / s,

[0033] 1.75 m / s, 1.80 m / s, 1.85 m / s, 1.90 m / s, 1.95 m / s, 2.00 m / s, 2.05 m / s, 2.10 m / s, 2.15 m / s,

[0034] 2.20 m / s, 2.25 m / s, 2.30 m / s, 2.35 m / s, 2.40 m / s, 2.45 m / s, 2.50 m / s, 2.55 m / s, 2.60 m / s,

[0035] 2.65 m / s, 2.70 m / s, 2.75 m / s, 2.80 m / s, 2.85 m / s, 2.90 m / s, 2.95 m / s, 3.00 m / s, 3.05 m / s,

[0036] 3.10 m / s, 3.15 m / s, 3.20 m / s, 3.25 m / s, 3.30 m / s, 3.35 m / s, 3.40 m / s, 3.45 m / s, 3.50 m / s,

[0037] 3.55 m / s, 3.60 m / s, 3.65 m / s, 3.70 m / s, 3.75 m / s, 3.80 m / s, 3.85 m / s, 3.90 m / s, 3.95 m / s,

[0038] 4.00 m / s, 4.05 m / s, 4.10 m / s, 4.15 m / s, 4.20 m / s, 4.25 m / s, 4.30 m / s, 4.35 m / s, 4.40 m / s,

[0039] 4.45 m / s or at least 4.50 m / s. The first aqueous solution may be added to the second aqueous solution at a fluid velocity of between 0.60-4.00 m / s, or preferably between 1.00-2.00 m / s.

[0040] The shear stress applied to the polyelectrolyte complexes ensures that biopolymer fibres of the desired length and diameter are formed. The shear stress may be altered by varying the flow rate or the fluid velocity of the complexation mixture, or by varying the rate of stirring the complexation mixture. Additionally, the viscosity of the complexation mixture may be varied to alter the shear stress. Alternatively, the viscosity of the first and / or second aqueous solution may be varied to alter the viscosity of the complexation mixture.

[0041] The first and / or second aqueous solution may have a viscosity of between 0.002- 0.014 Pa / s.

[0042] The concentration of the or each biopolymer in the first and / or second aqueous solution may be around 75 mg / mL.

[0043] In some embodiments, increasing the viscosity of the first and / or second aqueous solutions and / or the complexation mixture results in the formation of biopolymer fibres with a crimped structure. In some embodiments, the viscosity of the first and / or second aqueous solution is increased to a viscosity of between 0.011-0.020 Pa / s in order to form crimped fibres. Crimped nanofibers have the advantage of promoting vascularisation in vascular grafts that mimic the mechanical properties of native vasculature.

[0044] The complexation mixture may be in a flowing (recirculating) stream. The flow rate or the fluid velocity of the complexation mixture may be a suitable flow rate or fluid velocity for the flow to be laminar.

[0045] The complexation mixture may be continuously stirred using a stirring device, such as a magnetic stirrer bar and magnetic stirrer plate for example. The rate of stirring may be a suitable flow rate for the flow to be laminar.

[0046] The flow rate or the fluid velocity of the complexation mixture may be variable. In some embodiments, fluid velocity of the third solution is at least 0.05 m / s, at least 0.10 m / s, at least 0.15 m / s, at least 0.20 m / s, at least 0.25 m / s, at least 0.30 m / s, at least 0.35 m / s, at least 0.40 m / s, at least 0.45 m / s, at least 0.50 m / s, at least 0.55 m / s, at least 0.60 m / s, at least 0.65 m / s, at least 0.70 m / s, at least 0.75 m / s, at least 0.80 m / s, at least 0.85 m / s, at least 0.90 m / s, at least 0.95 m / s, or at least 1.00 m / s. The fluid velocity of the complexation mixture may be no more than 0.95 m / s, no more than 0.90 m / s, no more than 0.85 m / s, no more than 0.80 m / s, no more than 0.75 m / s, no more than 0.70 m / s, no more than 0.65 m / s, no more than 0.60 m / s, no more than 0.55 m / s, no more than 0.5 m / s, no more than 0.45 m / s, no more than 0.40 m / s, no more than 0.35 m / s, no more than 0.30 m / s, no more than 0.25 m / s, no more than 0.20 m / s, no more than 0.15 m / s, or no more than 0.10 m / s. Preferably, the fluid velocity of the complexation mixture may be between 0.05-1.00 m / s, or more preferably between 0.05-0.50 m / s.

[0047] In embodiments where the complexation mixture is continuously stirred by a stirring device, the complexation mixture stirring device operates at no more than 300 rpm, no more than 400 rpm, no more than 500 rpm, no more than 600 rpm, no more than 700 rpm, no more than 800 rpm, no more than 900 rpm, no more than 1000 rpm, no more than 1100 rpm, no more than 1200 rpm, no more than 1300 rpm, no more than 1400 rpm, no more than 1500 rpm, or no more than 2000 rpm. Preferably, the stirring device may operate at between 300-1000 rpm, or more preferably, 500-800 rpm. Most preferably, the stirring device may operate at 600-700 rpm.

[0048] The flowing or stirred complexation mixture may have a Reynolds number of no more than 2000, no more than 1800, no more than 1600, no more than 1400, no more than 1200, no more than 1000, no more than 800, no more than 600, or no more than 400. The flowing or stirred complexation mixture may have a Reynolds number of at least 200, at least 400, at least 600, at least 800, at least 1000, at least 1200, at least 1400, at least 1600, or at least 1800. Preferably, the flowing or stirred complexation mixture may have a Reynolds number of between 200-2000, or more preferably, between 450-1800. In some embodiments, the second aqueous solution is also subjected to laminar fluid flow, and wherein the fluid flow is as described for the complexation mixture.

[0049] A set constant flow rate / fluid velocity and / or rate of stirring of the complexation mixture ensures that that the biopolymer fibres formed are substantially uniform.

[0050] In a specific embodiment, a first aqueous solution is sprayed through a needle into a second aqueous solution that is constantly stirred using a stirring device and wherein the first aqueous solution is sprayed through a one-inch long 23 G needle at a flow rate of 10 mL / min (fluid velocity of around 1.24 m / s) and the stirring device operates between 600-700 rpm.

[0051] In an alternative specific embodiment, a first solution is injected through a needle into a recirculating flowing stream of a second aqueous solution and wherein the first aqueous solution is injected through a one-inch long 23G needle at a flow rate of 10 mL / min (fluid velocity of around 1.24 m / s) and the second aqueous solution is continuously recirculated though 8 mm tubing at a flow rate of between 200-800 mL / min (fluid velocity of between 0.03-0.13 m / s).

[0052] In some embodiments, the composition and size of the biopolymer fibres formed by the method of the first aspect of the invention are substantially uniform.

[0053] The biopolymer fibres formed by the method may be nanofibers.

[0054] The biopolymer fibres may have a mean average length of no more than 3000 pm, 2500 pm, 2400 pm, 2300 pm, 2200 pm, 2100 pm, 2000 pm, 1900 pm, 1800 pm, 1700 pm, 1600 pm, 1500 pm, 1400 pm, 1300 pm, 1200 pm, 1100 pm, 1000 pm, 900 pm, 800 pm, 700 pm, 600 pm, 500 pm, 400 pm, 300 pm, 200 pm, 100 pm, or no more than 50 pm. The biopolymer fibres may have a mean average length of at least 20 pm, 50 pm, 100 pm, 200 pm, 300 pm, 400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, 1000 pm, 1100 pm, 1200 pm, 1300 pm, 1400 pm, 1500 pm, 1600 pm, 1700 pm, 1800 pm, 1900 pm, 2000 pm, 2100 pm, 2200 pm, 2300 pm, 2400 pm, or at least 2500 pm. The biopolymer fibres may have a mean average length in the range of 50-3000 pm, 50- 2500 pm, 50-2400 pm, 50-2300 pm, 50-2200 pm, 50-2100 pm, 50-2000 pnp.50-1900 pm, 50-1800 pm, 50-1700 pm, 50-1600 pm, 50-1500 pm, 50-1400 pm, 50-1300 pm, 50- 1200 pm, 50-1100 pm, 50-1000 pm, 50-900 pm, 50-800 pm, 50-700 pm, 50-600 pm, 50-500 pm, 50-400 pm, 50-300 pm, or 50-200 pm. The biopolymer fibres may have a mean average length in the range of 2400-2500 pm, 2300-2500 pm, 2200-2500 pm, 2100-2500 pm, 2000-2500 pm, 1900-2500 pm, 1800-2500 pm, 1700-2500 pm, 1600- 2500 pm, 1500-2500 pm, 1400-2500 pm, 1300-2500 pm, 1200-2500 pm, 1100-2500 pm, 1000-2500 pm, 900-2500 pm, 800-2500 pm, 700-2500 pm, 600-2500 pm, 500-2500 pm, 400-2500 pm, 300-2500 pm, 300-2500 pm, or 200-2500 pm. Preferably, biopolymer fibres may have a mean average length in the range of 50-2500 pm, or more preferably, 100-2500 pm.

[0055] The length of the biopolymer fibres may depend on the polycationic and / or polyanionic biopolymers used.

[0056] The biopolymer fibres may have a mean average diameter of no more than 1600 nm, no more than 1400 nm, no more than 1200 nm, no more than 1000 nm, no more than 800 nm, no more than 600 nm, no more than 400 nm, no more than 200 nm, or no more than 100 nm. The biopolymer fibres may have a mean average diameter of at least 50 nm, at least 100 nm, at least 200 nm, at least 400 nm, at least 600 nm, at least 800 nm, at least 1000 nm, at least 1200 nm, or at least 1400 nm. The biopolymer fibres may have a mean average diameter of between 100-1000 nm, more preferably, between 200-800 nm, or most preferably, around 500 nm.

[0057] The biopolymer fibres may have a mean averaged aspect ratio of at least 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:1100, 1:1200, 1:1300, 1:1400, 1:1500, 1:1600, 1:1700, 1:1800, 1:1900, 1:2000, 1:2100, 1:2200, 1:2300, 1:2400, 1:2500, 1:2600, 1:2700, 1:2800, 1:2900, 1:3000, 1:3100, 1:3200, 1:3300, 1:3400, 1:3500, 1:3600, 1:3700, 1:3800, 1:3900, 1:4000, 1:4100, 1:4200, 1:4300, 1:4400, or at least 1:4500. In some embodiments, biopolymer fibres with a higher aspect ratio are preferred. Biopolymer fibres with a higher aspect ratio provide for the formation of higher viscosity materials at rest which, for example, improves properties for coatings as the material is more likely to stay on a surface (i.e. less likely to drip off).

[0058] In some embodiments, the method comprises a further method step (step (iv)) comprising; separating the biopolymer fibres formed in step (iii) from the complexation mixture. The further method step (step (iv)) may also comprise packing the biopolymer fibres. The separation and / or the fibre-packing step (step (iv)) may follow step (iii), the separation and / or the fibre-packing step (step (iv)) may immediately follow step (iii). The separation and / or fibre-packing step (step (iv)) may form a packed hydrogel precursor. In some embodiments, the packed hydrogel precursor is loose-packed having larger pore sizes. In other embodiments, the packed hydrogel precursor is tight-packed having smaller pore sizes. The preference the formation of a loose-packed hydrogel precursor or a tight-packed hydrogel precursor depends on the application. Both the loose-packed hydrogel precursor and the tight-packed hydrogel precursor formed by the method of the first aspect of the invention are shear-thinning. In some embodiments, the packed hydrogel precursor is shear- thinning. The hydrogel precursor may be capable of being injected and / or sprayed to a target site. In some embodiments, the hydrogel precursor may be capable of being injected and / or sprayed through a needle, pipe, or conduit having an internal diameter of at least 100 pm, at least 110 pm, at least 120 pm, at least 130 pm, at least 140 pm, at least 150 pm, at least 160 pm or at least 170 pm, at least 180 pm, at least 190 pm, or at least 200 pm. Preferably, the hydrogel precursor may be capable of being injected and / or sprayed through a needle, pipe, or conduit having an internal diameter of at least 150 pm. The hydrogel precursor may be capable of being injected through a 32G needle, a 31G needle, a 30G needle, a 29G needle, or a 28G needle. In some embodiments, the hydrogel precursor is injected and / or sprayed whilst maintaining pore size.

[0059] The fibre-packing step may comprise centrifugation. Centrifugation may occur at a speed of at least 500G, 600G, 700G, 800G, 900G, 1000G, 2000G, 3000G, 4000G, 5000G, 6000G, 7000G, 8000G, 9000G, 10,000G, ll,000G, 12,000G, 13,000G, 14,000G, 15,000G, 16,000G, 17,000G, 18,000G, 19,000G, 20,000G, 25,000G, or atleast 30,000G. Centrifugation may occur at a suitable speed to form a loose-packed fibrous material, this may be achieved using a centrifugation speed of between 700-7000G. Centrifugation may occur at a suitable speed to form tight-packed fibrous material, this may be achieved using a centrifugation speed of around 17000G. The complexation mixture may be centrifuged for less than 30 minutes, less than 20 minutes, less than 15 minutes, less than 10 minutes, or less than 5 minutes. Preferably, the complexation mixture is centrifuged for around 10 minutes. In some embodiments, the fibre-packing step may comprise one or more steps of centrifugation. Each centrifugation step may be performed at a different speed and / or for a different period of time. In preferred embodiments wherein loose- packed materials are formed, the fibre-packing step may comprise three centrifugation steps, the first at 1000G for 10 minutes, the second at 1000G for 10 minutes, and the third at 6000G for 10 minutes. Each step of centrifugation may involve the removal of the supernatant and the resuspension of the pellet prior to the following centrifugation step. The resuspended pellet may be subject to the same centrifugation steps as described above.

[0060] In other embodiments, the separation step comprises a tangential flow filtration (TFF) method, or an analogous method thereof.

[0061] In some embodiments, the hydrogel precursor has a mean average pore size of at least 0.1 pm, at least 1 pm, at least 5 pm, at least 10 pm, at least 15 pm, at least 20 pm, at least 25 pm, at least 30 pm, at least 35 pm, at least 40 pm, or at least 45 pm. The hydrogel precursor may have a mean average pore size of no more than 50 pm, no more than 45 pm, no more than 40 pm, no more than 35 pm, no more than 30 pm, no more than 25 pm, no more than 20 pm, no more than 15 pm, no more than 10 pm, no more than 5 pm, or no more than 1 pm. Preferably, the hydrogel precursor has a mean average pore size of between 0.1-50 pm, or more preferably, between 1-25 pm.

[0062] In some embodiments, the method further comprises a further method step (step (v)) comprising; adding one or more cross-linking agent(s) to the hydrogel precursor. The one or more cross-linking agent may result in gelation of the hydrogel precursor (comprising the packed biopolymer fibres) to form a fibrous hydrogel.

[0063] The cross-linking step may follow the fibre-packing step (step (iv)).

[0064] Gelation of the hydrogel precursor occurs rapidly. The one or more cross-linking agent may be independently selected from the group consisting of: UV light irradiation, divalent ions, divalent ions, citric acid, calcium ions, iron ions, zinc ions, triphosphates, and any combination of the aforementioned. In a specific embodiment, the biopolymer fibres comprise pectin and the pectin fibres are cross-linked using calcium ions, for example using calcium chloride.

[0065] The hydrogel precursor may be used to form fibrous hydrogels in a variety of shaped structures for example noodles, spheres, oblate spheroids etc. To do so, the hydrogel precursor may be extruded through a needle into a cross-linking bath containing a cross-linking agent. Different shapes may be created by using different sized / shaped needles, different concentrations of cross-linking agent, and / or different extrusion techniques. The hydrogel precursor maintains its shape after extrusion before contacting the cross-linking bath at which point gelation occurs and the fibrous hydrogel is formed. Spheres may be formed using the following methods; for example, electro-spraying, jet cutter, centrifugal spinner, or two phase separation using PEG / Dextran.

[0066] According to a second aspect of the invention there is provided biopolymer fibres prepared by the method of the first aspect of the invention.

[0067] The biopolymer fibres may be as described for the first aspect of the invention.

[0068] According to a third aspect of the invention there is provided a fibrous hydrogel prepared by the method of the first aspect of the invention.

[0069] The fibrous hydrogel may be as described for the first aspect of the invention.

[0070] The fibrous hydrogel formed from the biopolymer fibres and hydrogel precursor as described above may be used in a variety of applications, some of which are outlined below. In some embodiments, the fibrous hydrogel is used in the treatment of wounds. In such embodiments, the fibrous hydrogel may be formed from the hydrogel precursor which may be shear-thinning and loose-packed. The fibrous hydrogel may be sprayed or injected directly to the wound site. The use of injectable hydrogels allows the hydrogel to fill irregular wound cavities, under hanging areas, and hard to reach areas (e.g. periodontal cavities). Once applied to the target wound site, cross-linking of the hydrogel precursor may occur by exposure with a cross-linking agent. Exposure with a crosslinking agent may be effected via calcium ions present at the wound site. Cross-linking of the hydrogel precursor may result in gelation and the formation of a fibrous hydrogel.

[0071] In some embodiments, the fibrous hydrogel is used as a nasal filter. In such embodiments, the hydrogel precursor may be shear-thinning and loose-packed and may by sprayed into the nasal cavity to form a fibrous hydrogel. The spray formulation may be prepared by freeze-drying the hydrogel precursor, resuspending the hydrogel precursor in ethanol and palmitic acid, and then washing the hydrogel precursor. The hydrogel precursor may then be sprayed nasally, and a fibrous hydrogel may form when the hydrogel precursor contacts a cross-linking agent in the nasal cavity, such as calcium ions.

[0072] In some embodiments, the fibrous hydrogel is used as a microcarrier for culturing meat. Using the fibrous hydrogel as a microcarrier for culturing meat is useful for foodgrade materials that do not require relevant cell types to be detached from the growth surface. Such microcarriers increase cell productivity and viability, as well as reducing costs (as it removes the need for trypsinisation). The fibrous hydrogel may be used as a microcarrier in a hollow fibre bioreactor, a stirred tank bioreactor, or a fluidised bed bioreactor. Alternatively, the fibrous hydrogel may form a Pickering emulsion for use as a microcarrier for cultured meat.

[0073] In some embodiments, the fibrous hydrogel is incorporated as a porogen agent in the manufacture of medical grade silicone. Using the fibrous hydrogel in such application has the advantage of creating interconnected pore structures in the medical grade silicone, allowing for greater ingrowth of cells into medical products, consequently increasing their performance at a lower cost. In such embodiments, the hydrogel precursor may be used to form the fibrous hydrogel. The hydrogel precursor may be shear-thinning and loose-packed or tight-packed. The fibrous hydrogel may be capable of being injected through a needle for the formation of small structures (e.g. noodles, spheres, oblate spheroids). The hydrogel precursor maintains its shape after extrusion and then is crosslinked to form a fibrous hydrogel. Cross-linking of the fibrous hydrogel occurs rapidly by contacting the hydrogel precursor with a cross-linking bath comprising a cross-linking agent (e.g. calcium ions or zinc ions).

[0074] Accordingly, according to another aspect of the invention there is provided use of the fibrous hydrogel of the invention in any of the aforementioned applications.

[0075] Detailed Description of the Invention

[0076] In order that the invention may be more clearly understood embodiments thereof will now be described, by way of example only, with reference to the accompanying drawings, of which:

[0077] Figure 1 illustrates the fabrication of pectin / chitosan fibres, wherein (a) shows a simplified diagram of the experimental set up used in a “stirring” method, according to a first embodiment of a method of the first aspect of the invention, (b) shows a simplified diagram of the experimental set up used in an “injecting into flowing stream” method according to a second embodiment of a method of the first aspect of the invention, (c) shows a confocal micrograph of Rhodamine B labelled pectin / chitosan fibres formed from the stirring method (scale bar 10 pm), and (d) shows a confocal micrograph of Rhodamine B labelled pectin / chitosan fibres formed from the injecting into flowing stream method (scale bar 200 pm).

[0078] Figure 2 is (a) a scatter diagram showing pectin / chitosan fibre lengths formed via the stirring method shown in Figure 1; (b) a scatter diagram showing pectin / chitosan fibre diameters formed via the stirring method shown in Figure 1 ; (c) a graph showing the lengths of pectin / chitosan fibres formed via the injection method shown in Figure 1; (d) is a graph showing the length of gellan / chitosan fibres formed via the injection method shown in Figure 1. Individual fibres were measured using ImageJ.

[0079] Figure 3 shows a confocal micrograph of Rhodamine B labelled pectin / chitosan “crimped” fibres formed according to a third embodiment of a method of the first aspect of the invention with 10% PVP added to chitosan solution (scale bar 100 pm).

[0080] Figure 4 shows (a) a confocal micrograph of Rhodamine B labelled pectin / chitosan packed hydrogel precursor formed by the stirring method shown in Figure 1 followed by centrifugation (prepared at 700 RPM and loose packing (6000G)), and (b) a confocal micrograph of Rhodamine B labelled pectin / chitosan packed hydrogel precursor formed by the stirring method shown in Figure 1 followed by centrifugation (prepared at 700 RPM and tight packing (17000G)).

[0081] Figure 5 shows (a) a graph of a shear stress sweep of pectin / chitosan hydrogel precursors similar to those formed in Figure 4 (prepared at 17000G and 6000G) (N=3), and (b) a graph of a shear stress sweep of pectin / chitosan hydrogel precursors formed by the injecting into flowing stream method shown in Figure 1 followed by centrifugation (N=3).

[0082] Figure 6 shows (a) a graph of an amplitude sweep of a pectin / chitosan CaCh crosslinked hydrogels formed from hydrogel precursors similar to those shown in Figure 5 (N=3), and (b) a bar graph of a comparison of values for G’ of the pectin / chitosan CaCh cross-linked hydrogels at different concentrations of CaCh (N=3).

[0083] Figure 7 illustrates (a) the thresholding coverage of pectin / chitosan cross-linked hydrogel sprayed on a 5 mm x 5 mm section of paper, (b) an image of droplets of pectin / chitosan cross-linked hydrogel deposited on 5% porcine gelatin, and (c) an image of inverted droplets of pectin / chitosan crosslinked hydrogel deposited on 5% porcine gelatin showing adherence to the surface.

[0084] Figure 8 shows (a) a light microscope image of pectin / chitosan macrofibres formed according to a fourth embodiment of a method of the first aspect of the invention (scale bar 300 pm), (b) a graph of the quantification of the pectin / chitosan macrofibre diameter (N=3) for two different concentrations of cross-linking solutions (200 mM CaCh and 400 mM CaCh), (c) a light microscope image of a freeze-dried fibrous pectin / chitosan hydrogel (scale bar 450 pm), and (d) a photograph of palmitic acid-modified pectin / chitosan hydrogel.

[0085] Figure 9 shows (a) a confocal image of human dermal fibroblasts cultured in pectin / chitosan hydrogels cross-linked with CaCh (prepared at 6000G) and stained with CellTracker® Red, (b) a confocal image of live human dermal fibroblasts cultured in pectin / chitosan hydrogels cross-linked with CaCh (prepared at 6000G) and stained with Calcein AM, and (c) a bar chart showing the quantitative analysis of cell circularity at day 1 and day 5 post seeding. Individual dots represent individual cells measured in microscopy images.

[0086] Figure 10 shows (a) a confocal image of fibrous pectin / chitosan hydrogels crosslinked with CaCh (scale bar 200 m), (b) a confocal image of a Pickering emulsion of pectin / chitosan nanofibers formed according to a fifth embodiment of a method of the first aspect of the invention (scale bar 300 pm), and (c) a confocal image of a Pickering emulsion of pectin / chitosan nanofibers added to PBS and suspended in water (scale bar 400 pm).

[0087] Figure 11 illustrates the formation of polyelectrolyte complexes of chitosan (0.75 mg / mL chitosan) with different food-grade materials according to embodiments of the method of the invention; (a) 25 mg / mL iota carrageenan, (b) 150 mg / mL pectin, (c) 50 mg / mL pectin, (d) 50 mg / mL kappa carrageenan, (e) 100 mg / mL kappa carrageenan, and (f) 100 mg / mL pectin.

[0088] Figure 12 shows a graph of the oscillatory rheology analysis of iota carrageenan / chitosan fibres formed according to a fifth embodiment of the method of the invention.

[0089] Figure 13 illustrates a light microscopy image of gellan / chitosan fibres formed according to a sixth embodiment of the method of the invention.

[0090] Examples 1-5 described below focus on fibres and hydrogels composed of pectin and chitosan. However, the same or similar methods may be applied for the formation of alternative poly electrolyte biopolymer fibres, such as iota carrageenan / chitosan fibres as shown in Example 6, or gellan / chitosan fibres as shown in Example 7.

[0091] Example 1 - Fabrication of pectin / chitosan nanofibers

[0092] Pectin solution was added to chitosan solution via two different addition approaches, the first via spraying into a stirred solution (Figure 1A), and the second via injecting into a flowing stream (Figure IB). The method of formation of the pectin / chitosan fibres using the two approaches is described in more detail below.

[0093] Approach 1 - Spraying into a stirred solution:

[0094] A solution of 0.75 mg / mL chitosan was prepared by dissolving medium molecular weight chitosan (1,250,00 Mw, Batch number 920IYF, GP8956 deacetylation 97%) in 1% (v / v) acetic acid for 30 minutes. A 0.75 mg / mL solution of low-methoxy (27-32%) citrus peel derived Pectin (Glentham Biosciences GC7250) was prepared by dissolving in MilliQ water for 30 minutes.

[0095] The pectin solution was then sprayed (using a peristaltic pump) at a flow rate of 10 mL / min through a one-inch long 23 G needle in the top of a Duran bottle into 100 mL of the chitosan solution contained within the Duran bottle. The chitosan solution was continuously stirred at 700 RPM, until 75 mL of the pectin solution had been added. Figure 1C illustrates the pectin / chitosan fibres formed via this method (labelled with rhodamine-B isothiocyanate fluorescent dye).

[0096] Approach 2 - Injecting into a flowing stream:

[0097] A solution of 200 mL of 0.75 mg / mL chitosan (prepared as described in Approach 1) was recirculated at a flow rate of either 200 mL / min or 800 mL / min (using a peristaltic pump) through 8 mm tubing. 150 mL of 0.75 mg / mL pectin was injected into the tubing at a flow rate of 10 mL / min. Figure ID illustrates the pectin / chitosan fibres formed via this method (labelled with rhodamine-B isothiocyanate fluorescent dye).

[0098] The lengths of the pectin / chitosan fibres produced using the stirring method above are shown in Figure 2A. Figure 2B shows the diameter of the pectin / chitosan fibres produced using the stirring method above.

[0099] Fibre length can be modified by varying the stirring speed or the flow rate of the chitosan solution. Figure 2C shows how changing the flow rate when using the injecting into a flowing stream method can affect the length of the biopolymer fibres formed.

[0100] Increasing the flow rate (up to a threshold value) can increase the fibre length. Example 2 - Fabrication ofpectin / chitosan “ crimped” fibres

[0101] Increasing the viscosity of the chitosan solution adds crimps to the pectin / chitosan fibres, likely through the compressional bulking of the fibres as they are gelated. To form crimped fibres as shown in Figure 3, 5% polyvinylpyrrolidone (40 kDa) was dissolved in the 0.75 mg / mL chitosan solution in 1% (v / v) acetic acid. 100 mL of this solution was placed in a 500 mL Duran bottle and stirred at 700 RPM while 20 mL of pectin solution was added in a similar approach to that described in Approach 1 above.

[0102] Example 3 - Fabrication and characterisation of pectin / chitosan hydrogel precursor

[0103] To create a hydrogel precursor (i.e. packed fibres) from the pectin / chitosan fibres formed in Example 1, the pectin / chitosan fibres in a dilute solution of chitosan (i.e. the mixture resulting from Example 1) were centrifuged at 1000G for 10 minutes. Any unreacted chitosan in the supernatant was removed and the pellet was resuspended in 50 mL of deionized water, which was then centrifuged again for 10 minutes at 1000G. The supernatant was removed and final pellet was centrifuged at 6000G for loose-packed hydrogel precursor (Figure 4A) or 17000G for tight-packed hydrogel precursor (Figure 4B) for 10 minutes and the supernatant discarded to leave the corresponding hydrogel precursor.

[0104] To create hydrogel precursor (i.e. packed fibres) from the crimped pectin / chitosan fibres formed in Example 2, the crimped pectin / chitosan fibres in a dilute solution of chitosan (i.e. the mixture resulting from Example 2) were centrifuged at 2000G for 30 minutes (due to the increased viscosity of the chitosan solution). The supernatant was removed and the pellet resuspended in 50 mL of double distilled water, which was then again centrifuged at 1000G for 10 minutes. Finally, the supernatant was removed, and the final pellet was centrifuged at 17000G for tight-packed hydrogel precursor for 10 minutes and the supernatant discarded to leave the fibrous hydrogel material.

[0105] The mechanical properties of the shear-thinning hydrogel precursors were measured using dynamic shear rheometry. A shear rate sweep between 10-4-102s1was performed on three independently prepared pectin / chitosan hydrogel precursors (i.e. packed fibres), each prepared via the stirring method at 700 RPM (and packing of 17000G and 6000G), and via the injection method. Figures 5 A and 5B confirm the shear- thinning properties of the hydrogel precursors by showing a decrease in viscosity as shear is applied. This indicates that the packed pectin / chitosan fibres can slide past each other under strain. In addition, repeated stress ramps do not change the relationship between shear stress and recovery. This demonstrates that the pectin / chitosan shear-thinning hydrogel precursors exhibit self-healing properties after application of strain.

[0106] Example 4 - Gelation of pectin / chitosan shear-thinning hydrogel precursors

[0107] The addition of 200 mM CaCh to the pectin / chitosan shear-thinning hydrogel precursor of Example 3 (non-crimped fibres, loose-packed) results in gelation, as shown in Figure 6 with a crossover in G7G’ ’ . This is a result of cross-linking of the pectin. At 1 Hz, 0.1% shear stress, indicative of the forces the cells impart on their environment, the average values of G’ and G’ ’ are 364 Pa and 475 Pa respectively. These properties fall in the range of biomaterials that have been shown to promote outgrowth of endothelial cells and dermal cell spreading without leading to aberrant scarring and collagen deposition in mouse models. They also show a high loss tangent indicating they are viscoelastic. This may indicate a higher degree of cell spreading in vitro.

[0108] Example 5 - Applications of pectin / chitosan hydrogels i. Pectin / chitosan hydrogels for use as a spray in biomedical applications:

[0109] Hydrogel sprays have been previously used in the treatment of wounds, as a mucoadhesive delivery agent and as a nasal spray. Here it is shown that the pectin / chitosan hydrogel precursors have good spray coverage.

[0110] Pectin / chitosan hydrogel precursors (as described in Example 3, loose-packed at 1000G) were dyed with 0.1% (w / v) activated charcoal and 100 pL sprayed at a 45° angle onto paper. The sprayed paper was then scanned to show thresholding coverage (48%) of a 5 mm by 5 mm (Figure 7A).

[0111] 20 pL pipette drops of pectin / chitosan hydrogel precursors (as described in Example 3, loosed-packed at 1000G) were added (by pipetting) to 5% (w / v) porcine gelatin to simulate the surface of wounded dermis. Visually, the contact angle was lower for the pectin / chitosan hydrogels than the contact angle typically seen for water (Figure 7B). When the gelatin was inverted, the pectin / chitosan hydrogels still adhered, suggesting adherence to the dermis (Figure 7C). ii. Pectin / chitosan macrofibres for the formation of a woven mat for use in wound dressings:

[0112] Earger diameter strands have a wide range of applications, for example, they can be woven into mats to form a wound dressing. Pectin / chitosan fibres can be used to create larger 100-600 |im diameter strands (Figure 8A). This was done through extruding pectin / chitosan hydrogel precursors in a 200 mM CaCh bath. The strand structure from the shape of the needle was maintained, whilst also maintaining pore size. Increasing the concentration of CaCh as a crosslinking agent decreases the width of the strands, as shown in Figure 8B. This is due to an increase in cross-linking speed so the strands have less time to relax after injection. Figure 8C shows that the fibres can be freeze-dried to create mats that retain their fibrillar structure. Further, the addition of palmitic acid renders the fibrous hydrogel hydrophilic, as shown in Figure 8D. iii. Pectin / chitosan hydrogels for cell growth and cell spreading of human dermal fibroblasts:

[0113] To assess the effect of pectin / chitosan hydrogels on human cells, adult dermal fibroblasts were suspended at 500,000 cells per mL of loose-packed pectin / chitosan hydrogel in low attachment 24- well plates with 300,000 cells per well. DMEM / F12 with 10% FBS was used to cross-link the pectin / chitosan hydrogel precursor, which formed an irregular shape on the bottom of the well plate.

[0114] The samples were imaged one day after seeding and five days post-seeding. Figure 9 A shows evidence of cell spreading after five days, which was confirmed by Otsu thresholding (Figure 9B and 9C), with an increase in cell spreading after by the end of the imaging period. Cell spreading co-occurs with collagen deposition and growth in in vitro systems. iv. Pectin / chitosan hydrogels as microcarriers for cultured meat: For cultured meat applications, the fibrous hydrogels can be used as a microcarrier for use in the expansion of animal stem cells.

[0115] The pectin / chitosan hydrogels of the present invention may be modified into many different formats, for example, pectin / chitosan microcarrier fibres (Figure 10A) for use in a hollow fibre bioreactor, a stirred tank bioreactor, or a fluidised bed bioreactor. The pectin / chitosan microcarrier fibres shown in Figure 10A are formed using Approach 1 described above and form a loose-packed hydrogel precursor (fibres centrifuged at 6000G). The pectin / chitosan hydrogel precursor was loaded into a 27G needle mounted in a falcon tube with the tip 10 mm away from the surface of a 5 mL solution of 200 mM CaCh. The tube was centrifuged at 90G for 50 minutes so that the fibrous material was ejected from the mounted needle in separate droplets of 100-400 pm in diameter and then cross-linked once contacting the CaCh solution in the tube. This allows cell encapsulation / cell growth on a surface and the microcarriers can be suspended in a stirred tank (bioreactor) for cell expansion.

[0116] In addition to a polymer format, the pectin / chitosan hydrogels can be modified to form a Pickering emulsion (Figure 10B and 10C, showing confocal and optical micrographs of the Pickering emulsions after 4 hours, respectively). The Pickering emulsions shown in Figures 10B and 10C were formed using Approach 1 above. The solution of the pectin chitosan fibres were not subjected to a separated or fibre-packing step, and instead was added to sunflower oil (0.8 mL sunflower oil, 0.1 mL pectin / chitosan fibres, 0.1 mL DMEM). This mixture was vortexed to create an emulsion and imaged after 4 hours. The proposed method for the stabilisation of the oil / water interface is due to the pectin / chitosan fibre absorption on the surface, the pectin / chitosan fibres are positively charged (dimensions approx. 500 nm by 200 pm). Potential applications include the use for cell expansion on the surface, and or stabilisation of sauces and emulsions used in the food and drink industry. Emulsions have the potential in the scale up of cells, due to the ability to separate cells and microcarriers. The emulsions are stable in a small-scale bioreactor. Emulsions have the potential to allow for higher cell density than when using conventional microcarriers.

[0117] These microcarriers can be used to encapsulate cells, which will then deposit collagen and act as a feeder layer to expand other cell types. As they are created from food safe materials they do not have to be separated from cells and can be incorporated into the final product adding to the texture of the meat product.

[0118] Example 6 - Fabrication of iota carrageenan / chitosan fibres

[0119] Iota carrageenan / chitosan fibres were formed using the method of the present invention as described in Example 1, as shown in Figure 11. Similarly to the pectin / chitosan fibres described above, the iota carrageenan / chitosan fibres can be packed (via centrifugation steps described in Example 3) and also show shear-thinning properties, as shown in Figure 12.

[0120] Example 7 - Fabrication of gellan / chitosan fibres

[0121] Gellan / chitosan fibres were formed using the method of the present invention as described in Example 1, Approach 2, as shown in Figure 13. Similarly to the pectin / chitosan fibres described above, the gellan / chitosan fibres can be packed (via similar centrifugation steps described in Example 3) and also show shear-thinning properties. Figure 2D shows that gellan / chitosan fibres formed have longer lengths than the pectin / chitosan fibres formed (but have similar diameters).

[0122] The above embodiments are described by way of example only. Many variations are possible without departing from the scope of the invention.

Claims

CLAIMS1. A method of forming biopolymer fibres, the method comprising;(i) providing a first and a second aqueous solution, wherein one of the first or the second aqueous solution comprises at least one water- soluble polycationic biopolymer and the other first or second aqueous solution comprises at least one water-soluble polyanionic biopolymer; and(ii) adding the first aqueous solution to the second aqueous solution to form a complexation mixture, wherein the complexation mixture comprises poly electrolyte biopolymer complexes formed by complexation of the polycationic and polyanionic biopolymers of the first and second aqueous solutions; and(iii) effecting laminar fluid flow of the complexation mixture, which applies shear stress to the poly electrolyte complexes to form biopolymer fibres.

2. The method according to claim 1 , wherein the first aqueous solution comprises at least one water-soluble polyanionic biopolymer and the second aqueous solution comprises at least one water-soluble polycationic biopolymer.

3. The method according to claim 1 or claim 2, wherein the aqueous solution comprising the polycationic biopolymer has a zeta potential of at least +20 mV and the aqueous solution comprising the polyanionic biopolymer has a zeta potential of no more than -20 mV.

4. The method according to any preceding claim, wherein the or each polycationic biopolymer has an average molecular weight of between 800- 1600 kDa, or more preferably around 1250 kDa.

5. The method according to any preceding claim, wherein the or each polyanionic biopolymer has an average molecular weight of between 20-250 kDa.

6. The method according to any preceding claim, wherein the or each polycationic biopolymer has a hydrodynamic radius of between 300-700 nm.

7. The method according to any preceding claim, wherein the or each polyanionic biopolymer is independently selected from the group consisting of; pectin, methylcellulose, gum arabic, hyaluronic acid, Xanthan, gellan, alginate, carrageenan (kappa, iota or lambda), and any combination thereof.

8. The method according to any preceding claim, wherein the or each polycationic biopolymer is independently selected from the group consisting of: chitosan, poly-L-lysine and derivatives thereof (e.g. e-poly-L-lysine), proteins, nucleic acids, negatively-charged synthetic polymers and any combination thereof.

9. The method according to any preceding claim, wherein the first aqueous solution comprises pectin dissolved in water and wherein the second aqueous solution comprises chitosan dissolved in 1% (v / v) acetic acid solution.

10. The method according to any preceding claim, wherein the Reynolds number of the complexation mixture is between 450-1800.

11. The method according to any preceding claim, wherein the viscosity of the first and / or second solution is between 0.002-0.014 Pa / s.

12. The method according to any preceding claim, wherein the first aqueous solution is added into a flowing stream of the second aqueous solution, and optionally wherein the fluid velocity of the complexation mixture is between 0.05-1.00 m / s, or more preferably between 0.05-0.50 m / s.

13. A method according to any preceding claim, wherein the first aqueous solution is added into a stirred solution of the second aqueous solution, and optionally wherein the second aqueous solution is stirred at a stirring rate of between 300-1000 rpm, more preferably between 500-800 rpm, or most preferably between 600-700 rpm.

14. The method according to any preceding claim, wherein the biopolymer fibres are nanofibers.

15. The method according to any preceding claim, wherein the biopolymer fibres prepared by the method have an average length of between 50-250 pm, or more preferably between 100-2500 pm.

16. The method according to any preceding claim, wherein the biopolymer fibres prepared by the method have an average diameter of between 200-800 nm, or more preferably around 500 nm.

17. Biopolymer fibres prepared by the method of any one of claims 1 to 16.

18. The method according to any one of claims 1 to 16, further comprising the step;(iv) separating the biopolymer fibres from the complexation mixture and any unreacted reagents, and packing the biopolymer fibres to form a packed hydrogel precursor.

19. The method according to claim 18, wherein fibre-packing step (iv) comprises one or more rounds of centrifugation.

20. The method according to claim 18 or claim 19, wherein the method comprises packing the biopolymer fibres to form a packed hydrogel precursor with an average pore size of between 0.1-50 pm, or more preferably between 1-25 pm.

21. The method according to any one of claims 18 to 20, wherein the packed hydrogel precursor prepared by the method is shear-thinning.

22. The method according to any one of claims 18 to 21, further comprising the step:(v) adding one or more cross-linking agent(s) to the packed hydrogel precursor to form a fibrous hydrogel.

23. The method as claimed in claim 22, wherein the or each crosslinking agent comprises an agent independently selected from the group consisting of: UV light irradiation, divalent ions, trivalent ions, citric acid, calcium ions, iron ions, zinc ions, triphosphates, and any combination of the aforementioned.

24. A fibrous hydrogel prepared according to the method of claims 22 or 23.

25. The fibrous hydrogel as claimed in claim 24, for use in the treatment of wounds.

26. The fibrous hydrogel as claimed in claim 25, wherein the hydrogel precursor is sprayed or injected to a target wound site and the fibrous hydrogel forms on application in use.

27. The use of the fibrous hydrogel as claimed in claim 24, as a nasal filter; and optionally, wherein the hydrogel precursor is sprayed and applied nasally and the fibrous hydrogel forms on application in use.

28. The use of the fibrous hydrogel as claimed in claim 24, as a microcarrier for cultured meat.

Images