Polysaccharide formulation

Abstract

A formulation comprises polysaccharide aggregates suspended in an aqueous medium and a salt. The only polysaccharides present in the formulation are the polysaccharides which form the aggregates. The formulation may find use in the treatment or prevention of fibrosis or scarring. The formulation can be prepared by dissolving a polysaccharide in an aqueous medium at a temperature of at least 50 °C to form a solution, cooling the solution, and adding a salt to the cooled solution while stirring.

Description

POLYSACCHARIDE FORMULATIONField of the inventionThe present invention relates to a formulation comprising polysaccharide aggregates suspended in an aqueous medium, and a salt. Also provided are uses of the formulation, in particular in the treatment of fibrosis, and methods of preparing the formulation.Background to the inventionFibrotic diseases account for 45% of all deaths in the developed world, and progressive scarring conditions, such as epidermolysis bullosa, result in a significant reduction in quality of life. Excessive scarring, for example following burn injury, can cause both physical impairment and psychosocial harm. Scarring in the eye can lead to blindness, and fibrosis in other organs, including the lungs, liver and kidneys, can be fatal.Because scarring is an innate part of wound healing and comprises a multistage series of overlapping phases with an ever-changing chemical, biological, and material environment, it is difficult to pinpoint a specific factor as being responsible for pathological fibrosis. A cytokine that is often implicated in aberrant scarring is transforming growth factor beta 1 (TGFpi). Therefore, TGFpi can be considered a target for the prevention of fibrosis. However, systemic TGFpi inhibition causes serious cardiac valve issues, systemic autoimmunity, and early death. Previous attempts to target TGFpi have largely been unsuccessful. These attempts include the use of TGFpi antibodies, peptides, and receptor decoys. Additionally, non-selective small molecule treatments have been attempted, however, these have been to be more prone to side effects. It would therefore be of particular benefit to effectively inhibit TGFpi without side effects.The current invention has been devised with these issues in mind.Summary of the inventionAccording to a first aspect of the invention, there is provided a formulation comprising: polysaccharide aggregates suspended in an aqueous medium; and a salt, wherein the only polysaccharide(s) present in the formulation is(are) the polysaccharide(s) which forms the aggregates.It will be understood that the formulation does not contain any polysaccharides other than the polysaccharide, or mixture of polysaccharides, from which the aggregates are formed. Thus, in the formulation of the invention, the therapeutic agent (i.e. the polysaccharide) also serves as the delivery vehicle. By providing a self-delivering therapeutic, which may contain only a single, regulatory-approved component, clinical translation is facilitated.In some embodiments, the formulation consists of: polysaccharide aggregates suspended in an aqueous medium; and a salt.In some embodiments, the polysaccharide is an anionic polysaccharide.In some embodiments, the anionic polysaccharide is selected from carrageenan, gellan, dextran sulphate, alginate, pectin, xanthan gum, gum Arabic, carboxymethyl cellulose, heparin, heparan sulphate, chondroitin sulphate, hyaluronic acid, keratan sulphate, dermatan sulphate, derivatives thereof, and mixtures thereof (i.e. mixtures of two or more of any of the disclosed polysaccharides, or their derivatives).In some embodiments, the anionic polysaccharide is selected from carrageenan, gellan, dextran sulphate, alginate, derivatives thereof, and mixtures thereof.In some embodiments, the formulation comprises a single polysaccharide (i.e. the polysaccharide which forms the aggregates). The polysaccharide may be any of those disclosed herein.In some embodiments, the anionic polysaccharide is carrageenan. The carrageenan may be any suitable form of carrageenan, such as iota, lambda or kappa carrageenan. In some embodiments, the carrageenan is iota carrageenan.Polysaccharides are advantageous because they are far more stable than biologies, have proven low toxicity and have been used in the food industry for decades. For example, iota carrageenan is a low-cost, regulatory-approved polysaccharide.Surprisingly, it has been found that carrageenan (e.g. iota carrageenan) and other polysaccharides, in particular anionic polysaccharides, can prevent collagen fibril formation, a key step in scar formation. The formulation of the invention may therefore be effective in the treatment and prevention of fibrosis.In some embodiments, the anionic polysaccharide is present in the formulation at a concentration of at least 0.0001 %w / v, at least 0.000125 %w / v, at least 0.00025 %w / v, at least 0.0005 %w / v, at least 0.00075 %w / v, at least 0.001 %w / v, at least 0.0025 %w / v, at least 0.005 %w / v, at least 0.0075 %w / v, at least 0.01 %w / v, at least 0.025 %w / v, at least 0.5 %w / v, at least 0.7 %w / v, or at least 1 .0 %w / v.In some embodiments, the anionic polysaccharide is present in the formulation at a concentration of from 0.01 to 2.0 %w / v, from 0.025 to 1 .5 %w / v, from 0.05 to 1 .2 %w / v, or from 0.75 to 1.0 %w / v.In some embodiments, the anionic polysaccharide is carrageenan (e.g. iota carrageenan), which is present in the formulation at a concentration of from 0.025 %w / v to 0.75 %w / v.In some embodiments, the anionic polysaccharide is gellan. The gellan may be present in the formulation at a concentration of greater than 0.9 %w / v, e.g. at least 1.0 %w / v, at least 1 .5 %w / v or at least 2.0 %w / v.In some embodiments, the aggregates (e.g. microparticles) have a largest dimension which is less than about 100 pm, less than about 50 pm, less than about 30 pm or less than about 15 pm. In some embodiments, the aggregates have a largest dimension which is from about 0.2 pm to about 50 pm, from about 0.5 pm to about 20 pm, or from about 1 pm to about 10 pm.In some embodiments, the salt comprises divalent cations. The divalent cations may be calcium ions, magnesium ions, zinc ions, ferrous iron ions, strontium ions or a mixture thereof.In some embodiments, the salt comprises monovalent cations. The monovalent cations may be lithium ions, sodium ions, potassium ions, copper ions, silver ions, gold ions, or a mixture thereof. In some embodiments, the monovalent cations are sodium ions.In some embodiments, the salt comprises multivalent cations. The multivalent cations may be ferric iron ions, cobalt ions, manganese ions, aluminium ions, nickel ions, tin ions, or a mixture thereof.In some embodiments, the divalent cations are calcium ions. In some embodiments, the salt is CaCh.The salt may be dissolved in the aqueous medium. The aqueous medium may be water,In some embodiments, the salt (or the divalent cations) is present in the formulation at a concentration of from 0.001 M to 2.0 M, from 0.05 to 1.5 M, from 0.075 to 1.0 M, or from 0.01 M to 0.5 M.In some embodiments, the salt or divalent cations (e.g. calcium ions) are present at a concentration of from 0.05 M to 0.4 M.In some embodiments, the polysaccharide is carrageenan (e.g. iota carrageenan) and the divalent cation is a calcium ion. In some embodiments, the polysaccharide is carrageenan and the salt is calcium chloride.Suitable formulations may comprise carrageenan (e.g. iota carrageenan) and a salt comprising a divalent cation (e.g. Ca2+) in the amounts described in Table 1 :Table 1In some embodiments, the formulation comprises a further therapeutic agent. The formulation of the invention may therefore, in addition to providing a therapeutic benefit, may also be used as a delivery vehicle for one or more further therapeutic agents.The further therapeutic agent may be an antimicrobial (e.g. an antibiotic, antifungal, antiviral and / or antiparasitic), an anti-inflammatory agent (e.g. a non-steroidal anti-inflammatory agent), an analgesic, a chemotherapeutic agent, an anti-tumorigenic agent, a hormone, an antihistamine, an expectorant, a coagulant or anti-coagulant, a vitamin, a nutrient, a nutraceutical or any combination thereof.In some embodiments, the formulation is shear thinning. In some embodiments, the formulation is not shear thinning.According to a second aspect of the invention there is provided a container comprising the formulation of any preceding claim. The container may be of any suitable form, such as a bottle, tube, cannister, or carton. The container may comprise a dispensing means for dispensing the formulation, e.g. a dropper, pump or spray cap. In some embodiments, the container is a spray bottle.According to a third aspect of the invention there is provided the formulation of the first aspect for use in the treatment or prevention of fibrosis or scarring, or in the treatment of a wound.The fibrosis, scarring or wound may be of any part of the body, such as the skin, the eyes or an internal organ (e.g. the lungs, heart, liver, kidneys), or the oral, nasal, oesophageal, laryngeal, intestinal, anal or genital mucosa. Thus, in some embodiments, the fibrosis, scarring or wound is of the epithelium.In some embodiments, the formulation of the invention is for use in the treatment or prevent of fibrosis, scarring or a wound of the oral mucosa.It may be that the fibrosis or scarring is or was caused by an injury, a burn, surgery ora disease or condition. As used herein, “injury” will be understood as including damage to the body caused by any means, including but not limited to physical injury, radiation, chemical damage or injury caused by a biological agent or organism (e.g. a bite or infection).The formulation of the invention may be used as a liquid wound dressing. The wound may be a cut, a scratch, a burn, an abrasion, an incision, a tear or a puncture.In some embodiments, the fibrosis or scarring is caused by a disease or condition selected from mucous membrane pemphigoid, epidermolysis bullosa, oral submucous fibrosis, keloids, intestinal fibrosis, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, or ulcerative jejunoileitis.In some embodiments, the fibrosis or scarring is caused by a disease or condition selected from systemic sclerosis, cardiovascular fibrosis (e.g. atherosclerosis, arterial stiffness), renal fibrosis, hepatic fibrosis (e.g. alcoholic liver fibrosis, non-alcoholic liver fibrosis), pulmonary fibrosis (e.g. idiopathic pulmonary fibrosis, cystic fibrosis), fibrothorax, glial scarring, arthrofibrosis, Peyronie’s disease, Dupuytren’s disease, infection (e.g. bacterial or viral).Thus, the formulation of the invention is for use in the treatment or prevention of fibrosis or scarring in a subject suffering from any of the diseases or conditions disclosed herein.In some embodiments, the fibrosis or scarring is caused by a disease or condition selected from mucous membrane pemphigoid, epidermolysis bullosa, oral submucous fibrosis, or keloids.In some embodiments, the fibrosis or scarring is caused by epidermolysis bullosa. Thus, in some embodiments the formulation of the invention is for use in the treatment or prevention of fibrosis or scarring in a subject suffering from epidermolysis bullosa.In a further aspect, the invention provides a method of treating or preventing fibrosis or scarring, the method comprising administering the formulation described herein to a subject in need thereof.In yet a further aspect, the invention provides a method of treating a wound, the method comprising administering the formulation described herein to a subject in need thereof.In another aspect, the invention provides a method of inhibiting collagen fibrillogenesis and / or inhibiting myofibroblast differentiation, the method comprising administering the formulation described herein to a subject in need thereof.The formulation may be administered by any suitable means, such as topically or injection. In some embodiments, the formulation is administered topically, e.g. by spraying, coating or spreading, via the application of drops, or by dipping or immersing the affected area into the formulation. In some embodiments, the formulation is administered by spraying. For the treatment of internal tissues, the formulation may be administered by injection or during surgery.The use of the formulation may comprise administering the formulation topically to the skin or eyes, or to the oral, nasal, oesophageal, laryngeal, intestinal, anal or genital mucosa.In a further aspect, the invention provides the use of a polysaccharide, or a formulation as described herein, in the prevention or treatment of scarring, or in the treatment of a wound.In another aspect, the invention provides the use of a polysaccharide, or a formulation as described herein, as a moisturiser.The polysaccharide may be any polysaccharide as described herein. In some embodiments, the polysaccharide is in the form of an aggregate. The aggregates may have any of the properties described herein. The aggregates may be suspended, e.g. in an aqueous medium.According to a further aspect of the invention, there is provided a method of preparing a formulation, the method comprising:(i) dissolving a polysaccharide in an aqueous medium, optionally at a temperature of at least 50 °C, to form a solution;(ii) optionally cooling the solution; and(iii) adding a salt to the solution while stirring.In some embodiments the polysaccharide is an anionic polysaccharide. The anionic polysaccharide may be any of those described herein. For example, the anionic polysaccharide may be selected from carrageenan, gellan, dextran sulphate, alginate, pectin, xanthan gum, gum Arabic, carboxymethyl cellulose, heparin, heparan sulphate, chondroitin sulphate, hyaluronic acid, keratan sulphate, dermatan sulphate, derivatives thereof, and mixtures thereof.In some embodiments, the anionic polysaccharide is selected from carrageenan, gellan, dextran sulphate, alginate, derivatives thereof, and mixtures thereof.In some embodiments, the method comprises dissolving a single polysaccharide in the aqueous medium. The polysaccharide may be any of those disclosed herein.In some embodiments, the anionic polysaccharide is carrageenan. The carrageenan may be any suitable form of carrageenan, such as iota, lambda or kappa carrageenan. In some embodiments, the carrageenan is iota carrageenan.In some embodiments, the polysaccharide is dissolved in the aqueous medium at a temperature of at least 50 °C, at least 60 °C or at least 70°C, e.g. 80 °C. The aqueous medium may be heated to the required temperature prior to addition of the polysaccharide.In some embodiments, the salt comprises divalent cations. The divalent cations may be calcium ions, magnesium ions, zinc ions, ferrous iron ions, strontium ions, or a mixture thereof. In some embodiments, the divalent cations are calcium ions. In some embodiments, the salt is CaCh.In some embodiments, the salt comprises monovalent cations. The monovalent cations may be lithium ions, sodium ions, potassium ions, copper ions, silver ions, gold ions, or a mixture thereof. In some embodiments, the monovalent cations are sodium ions.In some embodiments, the salt comprises multivalent cations. The multivalent cations may be ferric iron ions, cobalt ions, manganese ions, aluminium ions, nickel ions, tin ions, or a mixture thereof.The aqueous medium may be water, preferably deionised water.In some embodiments, the solution is cooled prior to the addition of the salt. In some embodiments, the solution is cooled to below 50 °C, below 40 °C, below 30 °C or below 25 °C, prior to the addition of the salt. In some embodiments, the solution is cooled to room temperature (e.g. about 20 °C), prior to the addition of the salt. The solution may be actively cooled (e.g. in a fridge, or using a cooling jacket), or it may be allowed to cool passively.Surprisingly, it has been found that the addition of a salt (e.g. CaCh) when the solution is cold causes a change in the microstructure of the formulation, resulting in the formation of aggregates. Without being bound by theory, it is believed that the formation of small aggregates, or microparticles, improves the spray coverage of the formulation.In a further aspect, the invention provides a formulation obtainable by the method described herein.A formulation of the invention, such as a formulation prepared according to the methods described herein, may have one or more of the following properties: i. a viscosity of less than 50 Pa s, less than 40 Pa s, less than 30 Pa s, less than 20 Pa s, less than 10 Pa s or less than 5 Pa s, at a shear stress of 0.1 Pa, at 25 °C; ii. a viscosity of 1 Pa s at a shear stress of 1 Pa, at 25 °C; iii. a light absorbance which is at least 1 .0, at least 1 .5 or at least 2.0 times greater than that of a reference (e.g. water); iv. an elastic modulus (G’) of less than 1 Pa when subjected to oscillatory strain of 0.5% at a frequency of 0.1 Hz; v. a viscous modulus (G”) of less than 1 Pa when subjected to oscillatory strain of 0.5% at a frequency of 0.1 Hz;vi. a coefficient of friction of less than 2.5, less than 2, less than 1.5, or less than 1.0, at a rotational velocity of 0.1 mm s-1 .Advantageously, the formulation of the polysaccharide as a suspension of aggregates provides good lubrication, which can reduce the shear forces that initiate progressive scarring which occurs, for example, in diseases such as epidermolysis bullosa. The suspension also allows spraying for even coverage on hard-to-reach anatomical sites, like the oral mucosa.The microstructure of the formulation (i.e. the presence of polysaccharide aggregates) may be determined through light microscopy and / or by measuring the turbidity of the formulation, using the methods described herein. A significant increase in turbidity is indicative of the formation of aggregates. The turbidity of the formulation can be assessed by measuring its light absorbance.As will be appreciated by the skilled person, the light absorbance of a sample will depend on the path length (i.e. amount of sample), which is arbitrary, and on the absorbance of the container in which the sample is contained during measurement. Thus, the light absorbance of the formulation may be more accurately defined relative to a reference sample (e.g. water), which is measured under the same conditions, including the same path length and the same type of container.The viscosity, elastic modulus, viscous modulus and coefficient of friction of a formulation can be determined using the methods described herein.In some embodiments, the viscosity, elastic modulus, viscous modulus and / or coefficient of friction is determined using a Kinexus Ultra+ device. In some embodiments, the viscosity, elastic modulus, viscous modulus and / or coefficient of friction is determined using a 0.5% w / v solution, using a tribometer attachment with a 1 mm gap.Not only does the present invention provide a novel therapy for fibrosis treatment and prevention, but the inventors have also demonstrated the potential of self-delivering immunomodulatory polysaccharides as a safe, cost-effective, stable platform, which can more easily be translated into a clinical benefit.Brief description of figuresEmbodiments of the invention will now be described by way of example with reference to figures in which:Figure 1 shows the chemical structure of A) low acyl gellan, B) iota carrageenan, C) dextran sulphate, D) alginate, and E) methyl cellulose;Figure 2 shows the change in 405 nm absorbance over time of mixtures of collagen, PBS and A) water, B) gellan (G), C) iota carrageenan (iC), D) dextran sulphate (DS), E) alginate (Alg), and F) methylcellulose (MC). An increase in relative absorbance indicates collagen fibril formation;Figure 3 shows the time taken to reach 95% of the plateau value for polysaccharide concentrations that did not completely inhibit fibrillogenesis; gellan (G), iota carrageenan (iC), dextran sulphate (DS), alginate (Alg), methylcellulose (MC);Figure 4 shows the fold-change in A) COL1 , B) aSMA, C) SMAD3 and D) SMAD4 mRNA levels, as measured by RT-qPCR, in fibroblasts exposed for 48 hours to: normal culture conditions (NC), media doped with 10 ng mL-1 TGFpi (PC), and media with TGFpi plus gellan (G), iota carrageenan (iC), dextran sulphate (DS), alginate (Alg), and methyl cellulose (MC). Statistical differences calculated with respect to PC for each gene;Figure 5 shows A) Quantification of aSMA from immunocytochemistry images. B) Quantification of collagen laydown after 48 hours of culture by picrosirius red staining. C) Quantification of aSMA from immunocytochemistry images taken after 5 days of normal culture conditions (NC1), 5 days in media doped with 10 ng mL-1 TGFpi (PC), 2 days in TGFpi then 3 days in normal media (NC2), or media doped with gellan (G), iota carrageenan (iC), dextran sulphate (DS), alginate (Alg), and methyl cellulose (MC). Statistical differences calculated with respect to PC for each condition;Figure 6 shows A) Quantification of aSMA from immunocytochemistry images, where the polysaccharides were mixed with the TGFpi containing media immediately prior to application for 48 hours. Statistical differences calculated with respect to PC for each condition. B) Particle size distribution of iota carrageenan (iC), TGFpi (TGFpi) and their mixture (Mix), measured in cell culture media by DLS;Figure 7 shows frequency sweeps of A) 0.5% iota carrageenan solution, 0.5% iota carrageenan solution mixed while hot with B) sodium (NaCI-H) and C) calcium (CaCI2-H), then cooled under shear, and 0.5% iota carrageenan solution mixed following cooling with D) sodium (NaCI-C) and E) calcium (CaCI2-C). F) Shear stress ramps of these formulations;Figure 8 shows microscopy images of A) 0.5% iota carrageenan solution, 0.5% iota carrageenan solution mixed while hot with B) sodium (NaCI-H) and C) calcium (CaCI2-H), then cooled under shear, and 0.5% iota carrageenan solution mixed following cooling with D) sodium (NaCI) and E) calcium (CaCI2-C). F) Turbidity of each formulation, measured through its light absorbance;Figure 9 shows spray coverage of formulations of A) 0.005% and B) 0.5% iota carrageenan. C) Friction curves for various formulations. D) Comparison of the coefficient of friction at 0.1 mm s-1 and 10 mm s-1 ;Figure 10 shows a statistically significant increase in the spray area between 0.1 and 0.75 %w / v carrageenan at a calcium concentration of 0.1 M; andFigure 11 shows a statistically significant increase in spray area between 0.05 and 0.4 M calcium at 0.25% carrageenan, 0.05 and 0.3 M calcium for 0.5% carrageenan, and 0.05 and 0.15 M calcium for 0.75% carrageenan.Example 1IntroductionIn this study, a range of common polysaccharides (shown in Figure 1) are investigated fortheir ability to prevent fibrosis at multiple length scales, including inhibiting collagen fibrillogenesis and TGFpi -induced myofibroblast transdifferentiation. The most promising was then investigated to create a novel self-delivering, microstructured formulation, which can be sprayed to give even coverage over hard-to-reach locations such as the oral mucosa, and provide lubrication to minimise the shear trauma that ultimately gives rise to oral scarring in EB.Materials and methodsMaterialsAll water used was MilliQ type 1 deionised water unless otherwise stated. Collagen (type 1 , rat tail) was purchased from Corning. Gellan (low acyl) was purchased from Kelcogel. lota carrageenan (commercial grade), dextran sulphate (sodium salt from Leuconostoc spp, for molecular biology), alginate (BioReagent grade), methyl cellulose, bovine serum albumin (BSA), Sirius red, picric acid solution, acetic acid, sodium hydroxide, sodium chloride and calcium chloride dihydrate were purchased from Sigma. TGFpi (human recombinant) was purchased from ProteinTech. Phosphate buffered saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM), foetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco.Collagen FibrillogenesisAll reagents were refrigerated at 4 °C before and throughout this protocol, to prevent fibrillogenesis occurring prior to incubation. Collagen was diluted from its initial concentration (3.71 mg mL-1) to 0.8 mg mL-1with deionised water. Polymer solutions were made bydissolving in deionised water at 0.5 %w / v, and concentrations of 0.005, 0.0005 and 0.00005 %w / v were made by serial dilution. 150 pL of 1x PBS was placed to each well of a 96 well plate, and 75 pL of polymer solution was added, and mixed thoroughly pipette mixing. Then, 75 pL of 0.8 mg mL’1collagen solution was added to each well, and was mixed thoroughly by pipette mixing, taking care to avoid the formation of bubbles. The plate was then placed in a plate reader (Spark, Tecan), pre-heated to 30 °C, and the 405 nm absorbance was read over 6 hours. The data was normalised by dividing through by the initial absorbance value, then analysed by fitting an exponential plateau plus dead time equation:Where A(t) (-) is the normalised 405 nm absorbance as a function of time, Amax (-) is the plateau value at infinite time, A0 (-) is the initial value (set to 1 as data is normalised), k (min-1) is a rate constant, t is the experiment time (min) and td is the dead time (min). This model was then used to find the time at which the absorbance reached 95% of the plateau value, by rearranging Eq. X:Cell CultureHuman dermal fibroblasts were purchased from Sigma. Adherent 2D cultures were maintained in high glucose DMEM supplemented with 10% FBS, 2 mM L-glutamine and 100 II mL-1penicillin-0.1 mg ml’1streptomycin under 5% CO2 at 37°C. For experiments this medium with 1 % FBS was used to minimise the effects of unknown cytokines in the serum. When used, TGFpi was added directly to the media. To introduce polysaccharides, autoclaved solutions were made up a concentration on 0.1 %w / v to supplement the DMEM at a 1 in 20 dilution, to achieve a final concentration of 0.005%.Gene ExpressionTo ensure sufficient RNA harvest, cells were seeded in 6-well plates at a density of 150,000 cells per well and allowed to settle for 72 hours in DMEM with 10% FBS. Media was then changed to 1% FBS for 24 hours to allow the cells to acclimatise. Experimental media, which was prepared 24 hours in advance, was then applied for 48 hours: negative control (NC) was a replacement of 1% FBS DMEM; positive control (PC) was 1 % FBS DMEM plus 10 ng mL’1TGFpi ; the other experimental medias were 1% FBS DMEM plus 10 ng mL’1TGFpi and 0.005% gellan (G), iota carrageenan (iC), dextran sulphate (DS), alginate (Alg), or methyl cellulose (MC).Cells were then lysed and RNA extracted using an RNEasy mini kit (Qiagen), the purity checked by ensuring the 260 / 280 absorbance ratio was >2, before converting to cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems), following the manufacturer’s instructions. Gene expression analysis was performed on each sample for COL1 , aSMA, SMAD3, SMAD4, and GAPDH (housekeeper), using a SYBR green (Applied Biosystems) based RT-qPCR, with QuantiTect primer assays (Qiagen) and an AriaMx Real- Time PCR system (Agilent Technologies). qRT-PCR data were analysed using the Delta Delta Ct method as described previously

[0052] with the negative control samples used as the calibrator and GAPDH as the endogenous control gene. Relative quantification values are presented as fold changes in gene expression relative to the control group, which was normalised to one.Protein ExpressionCells were seeded in 8-well chamber slides at a density of 5,000 cells per well and allowed to settle for 72 hours in DM EM with 10% FBS. Media was replaced with 1% FBS for 24 hours to allow the cells to acclimatise. For 2 day experiments (data presented in Figure 5A,B&C), the experimental media was prepared 24 hours in advance, then applied for 48 hours; experimental media are described above. For 2-day experiments assessing the inhibition mechanism (data presented in Figure 6A), the same procedure was followed, but the experimental media was prepared immediately prior to application. For 5-day experiments (data presented in Figure 5D), group NC1 was treated with 1% FBS DMEM, and all other experimental groups were treated with 1 % FBS DMEM plus 10 ng mL'1TGFpi , for 48 hours. The media was then changed again: groups NC1 and NC2 had 1 % FBS DMEM, group PC had 1 % FBS DMEM plus 10 ng mL-1TGFpi , and the remaining groups had 1% FBS DMEM plus 0.005% of their respective polymer. These conditions were retained for 72 hours, with a change of media at 48 hours.For immunocytochemical analysis, cells were washed with PBS, fixed with 10% formalin at ambient temperature for 10 minutes, then washed again with PBS. Cells were blocked with 0.1 % triton X-100, 3% BSA in PBS, for 30 mins. Antibodies were made up in 0.5% Tween 20, 3% BSA in PBS. Primary antibodies for collagen 1 (polyclonal rabbit, Abeam) and aSMA (monoclonal mouse, Sigma) were made up 1 in 200, and added to each well for 1 hour. Secondary antibodies (594 goat anti-rabbit and 488 goat anti-mouse, Alexa Fluor, Invitrogen) were made up 1 in 500 and, following PBS washing, added up each well for 1 hour, protecting from light. Finally, each well was washed with PBS, DAPI was added for 10 mins to stain nuclei, and well were washed again before the chamber was removed and a cover slip applied to the slides. Cells were imaged using an Evos M5000 fluorescent microscope equipped with a dark box, ensuring the same camera settings for every image. Fluorescence intensityquantification was carried out in Imaged, thresholding to remove all areas not containing cells, then assessing the average intensity of the remaining area.For picrosirius red staining, cells were washed with PBS, then fixed in methanol overnight at -20 °C. Wells were washed with PBS again, then stained with picrosirius red (0.1 % Sirius red in saturated picric acid) at ambient temperature for 1 hour. Wells were then washed with 0.1 % acetic acid 5 times to remove non-specifically bound stain. The collagen-bound stain was then eluted with 0.1 M NaOH, and the absorbance of the eluent was read at 540 nm in a plate reader (Spark, Tecan).DLSTo replicate the conditions in the cell experiments as far as possible, DMEM (without phenol red indicator as this may interfere with measurement) was used as the dispersant medium. TGFpi was dispersed at a concentration of 0.0005 g L'1(500 ng mL'1) to achieve a measurable concentration, lota carrageenan was prepared as in the cell experiments above and added to the DMEM with and without TGFpi . Samples were made 24 hours before measurement, to replicate conditions in the cell experiments. Particle size was measured by dynamic light scattering (DLS), using a Zetasizer (Nano ZS, Malvern). lota Carrageenan FormulationStock iota carrageenan solution was made by dissolving at a concentration 0.555 %w / v in deionised water at 80 °C, which was diluted 100-fold for the stock dilute solution. Sodium chloride and calcium chloride solutions were made at a concentration of 5 M in volumetric flasks. To prepare formulations, 45 mL of stock solution was stirred at 1200 rpm, and 5 mL of water or salt solution was added (resulting in final concentrations of 0.5 or 0.005% iota carrageenan, and 0.5 M salt, where added). For ‘hot mixes’, denoted ‘H’, the salt was added while the iota carrageenan solution was still at 80 °C, and was then stirred for 30 minutes until cool. For ‘cold mixes’, denoted ‘C’, the iota carrageenan was allowed to cool to room temperature, then the salt solution was added under stirring.Light MicroscopyA drop of each formulation was placed on a microscope slide, covered with a glass cover slip, and imaged using a light microscope (Evos XL Core).Turbidity1 mL of formulation was syringed into a 48 well plate, and the absorbance at 350 nm was measured using a plate reader (Spark, Tecan).RheologyRheological characterisation was carried out on a Kinexus Ultra+ (Netsche). For dilute samples (0.005%), a double gap geometry was used to maximise contact area, and for concentrated samples (0.5%), a parallel plate geometry with a 1 mm gap. Stress ramps were carried out from 0.01 to 100 Pa, over 10 minutes, at 25 °C.SprayabilityFormulations were mixed with 0.001% rhodamine 6G for visualisation, then loaded into a standard hand-pump spray bottle. The pump was primed, then sprayed vertically downwards onto A5 paper from a distance of 5 cm. Once dry, the paper was imaged using a fluorescent scanner (iBright 1500, Invitrogen), and images were loaded in Imaged, cropped, thresholded, and particle analysis carried out to assess the percentage area of the cropped square, which was then converted to a real surface area.TribologyLubrication was assessed using a 3-ball tribological attachment for the rheometer, with silicone elastomer (Sylgard 184, Dow) used as the lower surface. Formulation was added so as to cover the lower surface. A normal force of 1 N was employed, and tests were carried out at 25 °C. The upper geometry was accelerated from 0.1 to 10 mm s’1.ResultsInhibition of Collagen FibrillogenesisFibrosis is the excessive laydown of ECM, primarily collagen type 1. The ability to delay or entirely prevent collagen fibril formation has been explored as a strategy to inhibit scar formation at the ECM level. The effect of several natural polysaccharides on collagen fibrillogenesis was thus tested in vitro. In the absence of any inhibitors, collagen formed fibrils at increased temperatures, forming a turbid gel over time (Figure 2A). However, some natural polysaccharides were able to slow, or entirely prevent, this process. Specifically, at concentrations above 0.00125%, gellan (Figure 2B), iota carrageenan (Figure 2C), dextran sulphate (Figure 2D), and alginate (Figure 2E), which are all anionic, appeared to completely inhibit fibrillogenesis. Interestingly methyl cellulose, an uncharged polymer, had no significant effect on collagen fibrillogenesis at any tested concentration (Figure 2F, p>0.05).The effect of the polymer concentrations that did not entirely prevent fibrillogenesis were analysed by comparing the time taken to get to 95% of the plateau, to that of the water onlycontrol (38 min) (Fig. 3). Despite being able to inhibit gelation at high concentration, gellan did not significantly slow fibrillogenesis below 0.000125%. At 0.000125%, iota carrageenan (123 min, p<0.0001) and dextran sulphate (119 min, p<0.0001 at 0.000125%, 59 min, p<0.01 at 0.0000125%) significantly retarded collagen fibrillogenesis, as did alginate (79 min, p<0.0001 at 0.000125%, 68 min, p<0.0001 at 0.0000125%). This shows that, at low concentrations, iota carrageenan and dextran sulphate are the most effective, suggesting that in addition to negative charge, the possession of sulphate groups specifically may be important to prevent fibrillogenesis. This is interesting as proteoglycans and glycosaminoglycans, molecules implicated in directing collagen fibrillogenesis in vivo, are often heavily sulphated.Prevention of Myofibroblast TransdifferentiationMyofibroblasts, the cells responsible for rapid ECM production and contraction in fibrosis, are transdifferentiated from fibroblasts primarily through TGFpi signalling. The ability to prevent fibroblasts from transdifferentiating into myofibroblasts should thus limit the overaccumulation of collagen associated with scarring. The ability of the polysaccharides to prevent fibrosis was tested in an in vitro model of scarring, where exposure to TGFpi induced myofibroblast transdifferentiation.A key feature of myofibroblasts is their increased collagen production compared to fibroblasts. At the genetic level, exposure to TGFpi increased transcription of collagen 1 (COL1) twofold (Figure 4A, negative control (NC) vs positive control (PC), p<0.01), and it was found that iota carrageenan (p<0.0001), alginate (p<0.01) and methyl cellulose (p<0.01) significantly downregulated COL1 , with iota carrageenan having the greatest effect.Myofibroblasts are characterised primarily by increasing aSMA expression; this form of actin gives them their contractile ability, and indeed it was demonstrated that TGFpi induced a 73- fold increase in aSMA gene transcription (Figure 4B, NC vs PC, p<0.0001). This was significantly reduced to only a twofold increase by iota carrageenan (p<0.0001), but was not significantly affected by the other polymers.Binding of TGFp to its type II serine / threonine receptor, TGFPR2, activates the type I receptor TGFPR1 , also termed activin receptor-like kinase 5 (ALK5), to intracellularly phosphorylate receptor-regulated SMAD proteins (R-SMADs). Activated R-SMADs, such as SMAD3, form complexes with co-factor SMAD4 and these complexes translocate to the nucleus where they regulate gene transcription. This process is key to TGFpi induced signalling pathway of myofibroblast transdifferentiation. Interestingly, exposure to TGFpi actually downregulated SMAD3 gene transcription (Figure 4C, NC vs PC, p<0.01), a finding that has been notedpreviously, lota carrageenan, alone of the polymers tested, significantly dampened SMAD3 gene transcription further (p<0.001). This may be beneficial in itself; it has been shown that mice lacking SMAD3 show accelerated wound healing. SMAD 4 gene transcription was also downregulated by iota carrageenan (Figure 4D, p<0.01), despite TGFpi itself not having a significant effect.In addition to downregulating transcription of key genes, it is important to show that this translates to the protein level, particularly for collagen 1 and aSMA. Exposure to TGFpi for 48 hours induced extensive myofibroblast transdifferentiation, which could be visualised through immunocytochemistry by their high aSMA expression. Quantification of immunocytochemistry images showed a significant increase in aSMA protein expression on exposure to TGFpi (Figure 5A, NC vs PC, p<0.0001), however this was significantly reduced by iota carrageenan (p<0.0001) and alginate (p<0.01). The change in collagen 1 protein expression could not be detected through immunocytochemistry quantification after 48 hours (NC vs PC, p>0.05, data not shown), however a difference could be seen through quantitative picrosirius red staining (Figure 5B). This technique showed collagen production was increased by TGFpi signalling (NC vs PC, p<0.01), but was significantly reduced by iota carrageenan (p<0.01). While not statistically significant, alginate also appeared to slightly dampen collagen production.The ability of the polysaccharides not only to prevent, but to reverse the scarring response was examined by exposing the fibroblasts to TGFpi for 48 hours (other than NC1), then changing to media without TGFpi (other than PC) with the polysaccharides. Again, exposure to TGFpi significantly increased aSMA expression (Figure 5C, NC1 vs PC, p<0.0001). Interestingly, exposure to TGFpi for 48 hours then to normal, TGFpi-free media for 72 hours produced the same response as exposure to TGFpi for the full 5 days (NC2 vs PC, p>0.05). This may suggest that initiation of myofibroblast transdifferentiation occurs quickly, and extended TGFpi exposure has no effect, or that the myofibroblasts transdifferentiated by 48 hours then produce TGFpi themselves to propagate the process. The latter is supported by the greater absolute values of fluorescence intensity in the 5-day experiment compared to the 2-days for all groups exposed to TGFpi (Figure 5A vs Figure 5C). Further, autocrine production of TGFpi has been observed previously in myofibroblasts, including those from hypertrophic scars. Again, iota carrageenan (p<0.01) and alginate (p<0.05) were able to reduce this response.In the previous experiments (Figure 5), experimental media (containing polysaccharides and TGFpi) was created 24 hours in advance of application to cells. To examine how thepolysaccharides might be interacting with the system, the experimental media was made immediately prior to application and, interestingly, none of the polymers appeared to have a significant therapeutic effect (Figure 6A). This led to the hypothesis that the therapeutic effect was caused by interaction between the polysaccharides and TGFpi , rather than between the polysaccharides and receptors on the cell. Complexation between iota carrageenan and TGFpi was studied directly by measuring the colloidal size of the polysaccharide and protein separately, and combined as in the cell system (Figure 6B). lota carrageenan alone displayed 2 peaks, perhaps indicating the presence of unaggregated (at around 9 nm) and aggregated (at around 106 nm) polymers. TGFpi , meanwhile, displayed a single peak at 164 nm. The mixed system displayed both peaks for the iota carrageenan, as well a third peak at 5560 nm. This suggests the formation of large aggregates from the combination of iota carrageenan and TGFpi . lota carrageenan forming a complex with TGFpi would explain how it inhibits myofibroblast transdifferentiation at the molecular level. This may be by covering the active site on the protein that couples with the receptor on the cell, or sterically preventing binding between the protein and its due to the size of the complex. While iota carrageenan was most effective of the polysaccharides investigated, this mechanism is non-specific, and may be extended to other polysaccharides, such as alginate, that also showed some efficacy. As the mechanism of binding is most likely electrostatic, between the positively charged protein and negatively charged polysaccharide, this shows why methyl cellulose, an uncharged polymer, had little effect in this study. This mechanism may also explain why iota carrageenan and alginate appear able to impede transdifferentiation once started (Figure 5C), as it can sequester the TGFpi produced by the already transdifferentiated myofibroblasts, breaking the positive feedback loop and preventing further transdifferentiation. lota Carrageenan Formulation lota carrageenan appears to be highly effective at preventing fibrosis, both by inhibiting collagen fibril formation, and preventing myofibroblast transdifferentiation. Dilute solutions (0.005%) have material properties similar to water, yielding very low retention on the oral mucosa, while concentrated solutions (0.5%) spray poorly. This has been overcome previously by blending with another polysaccharide to disrupt the bulk network.Carrageenans undergo gelation by transitioning from random coil structures when hot to double helices when cooled, which then aggregate to form a bulk network. As polyanions, this process is influenced by the presence of cations. It was thus investigated whether cations,added under shear, could be used to create a particulate microstructure to facilitate improved spraying.Without the addition of cations, 0.5% iota carrageenan solution was elastically dominated at all frequencies tested (G’>G”, Figure 7A). This shows that the polymers have formed a bulk network capable of storing energy, and indicates bulk double helix aggregation. Interestingly, the addition of high concentrations (0.5 M) of either NaCI (Figure 7B) or CaCh (Figure 7C) to the solution prior to cooling under shear (NaCI-H and CaCh-H, respectively), did not appear to alter this network structure, as the oscillatory rheological profiles appear similar. This also seems to be the case for NaCI added under shear to the iota carrageenan solution when cool (NaCI-C, Figure 7D). Only CaCh, added to the cold solution under shear (CaCh-C), appreciably altered the rheological profile, lowering both the elastic and viscous moduli (Figure 7E). This decreased stiffness suggests interruption of the bulk network structure, and indeed the phase angle is appreciably higher (data not shown) for CaCh-C, suggesting less energy can be stored in the system.A similar trend was seen in the shear stress profile, lota carrageenan solution was found to be shear thinning, with a high viscosity (100 Pa s) at low shear stress, which dropped almost four orders of magnitude at high shear stress (Figure 7F). The addition of sodium, either when hot (NaCI-H) or cold (NaCI-C), did not appreciably change this profile, and the addition of calcium when hot (CaCh-H) appeared only to slightly reduce the stress required for shear thinning, but did not change the upper or lower viscosity thresholds. However, when added cold (CaCh-C), calcium slightly reduced the low stress viscosity, but notably reduced the onset of shear thinning to a much lower stress value. This is likely because, rather than breaking the double helix aggregates apart, which requires large forces, the shear thinning in this system represents the disentanglement of individual particles, which requires notably less stress.The microstructure of each formulation was assessed through light microscopy. In the absence of added cations (Figure 8A), NaCI-H (Figure 8B), CaCh-H (Figure 8C) or NaCI-C (Figure 8D), no appreciable microstructure was visible. However, for CaCh-C, a distinct microstructure was visible that appeared as small aggregates on the order of 1 - 10 pm in size (Figure 8E). At the macroscale, this microstructure was observable by a significant increase in turbidity (Figure 8F, p<0.001). This is indicative of the formation of large aggregates (observed in Figure 8E), a phenomenon observed in gellan, which also gels by formation and aggregation of double helices, at high calcium concentrations. In iota carrageenan, increasing the calcium concentration above a critical value has been shown to increase the local heterogeneity and lead to phase separation. Further, at comparable concentrations of bothcalcium and polymer used in this study, iota carrageenan has been shown to precipitate. Here, by constantly mixing during this process, the precipitate has been blended in to yield the observed particulate microstructure. The high ionic forces that cause precipitation may prevent the polymers from rearranging, as they would in a ‘weak’ gel conformation, from the formed microstructure. However, this is also why the microstructured formulation has slightly lower viscosity and moduli, because the precipitated polymer is less able to structure the surrounding water.It is interesting that CaCh causes this drastic change in microstructure, and thus material properties, only when added once the iota carrageenan is cold. It is well know that iota carrageenan has a higher affinity for divalent cations, such as calcium, than monovalent ones, such as sodium, and that divalent cations result in stronger, if still technically ‘weak’, gels. It has been suggested that monovalent cations elicit intramolecular interactions only, increasing attraction within double helices rather than between them. This may explain why NaCI formulations did not produce a notable microstructure, as the interhelical aggregation was not affected. Conversely, divalent cations are able to bridge neighbouring double helix structures, promoting such interhelical aggregation. However, it has been suggested that intramolecular bridges may form preferentially to intermolecular ones. It is thus hypothesised that, when added while the polymers are in a random coil formation in hot solution, calcium is bound to form intramolecular bridges during double helix formation on cooling, and is therefore less available for interhelix aggregation. Conversely, when added after double helix formation, the added calcium is available to form interhelical bridges, and aggregate the microstructure to such an extent that phase separation is induced. This would explain the marked differences between the formulations containing the same components, in the same concentrations, where only the formulation method differs.Formulation Sprayability and LubricationSpray delivery is a convenient way to provide an even layer of material to difficult-to-reach anatomical locations, such as the nasal and oral mucosa. By forming a microstructure and discretising the polymers into small particles, such formulations may spray more readily than a bulk network. In dilute systems, where the polymers do not overlap and thus act as discrete particles, spray area was high (Figure 9A). Interestingly, even in this system the addition of cations appeared to slightly increase spray area, though not significantly. This may be due to charge screening allowing increased intramolecular interaction, effectively decreasing particle size. In the concentrated system, iota carrageenan solution sprayed poorly, as previously noted, because the bulk polymer network cannot be disrupted sufficiently during the short spraying time. Interestingly, even the formulations that did not alter the oscillatory or shearrheology, nor induce a visible microstructure, significantly increased the spray area (Figure 9B); approximately a 4-fold increase for NaCI-H (p<0.01), a 2-fold increase for CaCh-H (p<0.05), and a 5-fold increase for NaCI-C (p<0.001). It has been noted previously that, despite altering the polymer conformation, iota carrageenan is Theologically insensitive to cations. This provides further evidence that that sprayability is not necessarily related to rheological properties in structured fluids. However, with an 18-fold increase in spray area (p<0.01), the CaCh-C formulation had the same spray coverage as the dilute solutions. This shows that creating discrete microparticles, rather than the continuous entangled network seen in concentrated polymer solutions, is paramount for sprayability.A final consideration is prevention of the initial stimulus that initiates the fibrotic cascade. In epidermolysis bullosa, for example, minimal shear trauma can lead to progressive blistering, ulceration and subsequent scarring, including on the internal mucosa. If shear trauma can be minimised by lubricating the oral mucosa, this may allow normal eating and oral hygiene, without leading to scarring. Lubrication of the formulations was thus investigated. The dilute system was a poor lubricant, displaying a reduction in friction only at high speeds, while both concentrated systems reduced the coefficient of friction at much lower speeds (Figure 9C). Interestingly, the microstructured system was significantly more lubricating than the iota carrageenan alone in solution at low speeds (Figure 9D, p<0.01). At high speeds the lubrication ability of both concentrated systems was similar, though both significantly reduced friction compared to the dilute system (p<0.05).ConclusionsAll anionic polysaccharides tested, but not neutrally charged methyl cellulose, were able to prevent or inhibit collagen fibri llogenesis, depending on concentration. In an in vitro model of fibrosis, many of the anionic polysaccharides had some effect, but iota carrageenan alone significantly downregulated COL1, aSMA, SMAD3 and SMAD4 gene transcription, as well as COL1 and aSMA protein expression. This is likely due to iota carrageenan forming a complex with TGFpi, preventing it from binding to cell receptors to initiate myofibroblast transdifferentiation, lota carrageenan can be microstructured by adding calcium, but only when cold following double helix formation. This microstructured formulation can be sprayed for high surface coverage, and provides a lubricating effect. By preventing myofibroblast transdifferentiation at the molecular / cell level, inhibiting collagen laydown at the ECM level, and lubricating to limit shear-induced trauma at the macro level, iota carrageenan could present a promising new therapy to prevent the persistent oral scarring seen in epidermolysis bullosa.Example 2Materials and methods lota carrageenan solutions were made by dissolving in deionised water at 80 °C, at a concentration 11.1% higher than required in the final formulation, e.g. a concentration of 0.278 %w / v, 0.555 %w / v and 0.833 %w / v, for final formulation concentrations of 0.25 %w / v, 0.5 %w / v, and 0.75 %w / v, respectively. Calcium chloride was made at a concentration of 0.5 M in a volumetric flask, and diluted to produce the required concentration - tenfold that of the final concentration, e.g. 1 M for a final formulation concentration of 0.1 M. To prepare formulations, 45 mL of carrageenan solution was stirred at 1200 rpm, and 5 mL of water or salt solution was added, to produce the required final concentrations.Formulations were mixed with 0.001% rhodamine 6G for visualisation, then loaded into a standard hand-pump spray bottle. The pump was primed, then sprayed vertically downwards onto A5 paper from a distance of 5 cm. Once dry, the paper was imaged using a fluorescent scanner (iBright 1500, Invitrogen), and images were loaded in Imaged, cropped, thresholded, and particle analysis carried out to assess the percentage area of the cropped square, which was then converted to a real surface area.ResultsIt was found that adding calcium to iota carrageenan in a specific way can vastly improve its sprayability. Both the concentration of carrageenan, the polymer, and the concentration of calcium, the salt, have an effect, and both have a minimum and maximum in which the effect occurs. A composition range in which this occurs was identified. Figure 10 shows the difference in spray area in the presence and absence of 0.1 M calcium, for different concentrations of iota carrageenan (iC). A statistically significant increase in the spray area was observed between 0.1 and 0.75 %w / v carrageenan, inclusive.With reference to Figure 11, the calcium concentration was then varied for a few different concentrations of carrageenan. A statistically significant increase in spray area was observed between 0.05 and 0.4 M calcium at 0.25% carrageenan (Fig. 11a), between 0.05 and 0.3 M calcium for 0.5% carrageenan (Fig. 11b), and between 0.05 and 0.15 M calcium for 0.75% carrageenan (Fig. 11c).

Claims

Claims1. A formulation comprising: polysaccharide aggregates suspended in an aqueous medium; and a salt, wherein the only polysaccharide(s) present in the formulation is(are) the polysaccharide(s) which form(s) the aggregates.

2. The formulation of claim 1 , wherein the polysaccharide is an anionic polysaccharide.

3. The formulation of claim 2, wherein the anionic polysaccharide is selected from carrageenan, gellan, dextran sulphate, alginate, pectin, xanthan gum, gum Arabic, carboxymethyl cellulose, heparin, heparan sulphate, chondroitin sulphate, hyaluronic acid, keratan sulphate, dermatan sulphate and mixtures thereof.

4. The formulation of claim 3, wherein the anionic polysaccharide is carrageenan, optionally iota carrageenan.

5. The formulation of any one of claims 1 to 3, wherein the anionic polysaccharide is present at a concentration of at least 0.000125 %w / v, optionally at least 0.005 %w / v, further optionally at least 0.5 %w / v.

6. The formulation of any preceding claim, wherein the anionic polysaccharide is iota carrageenan which is present at a concentration of from 0.1 to 1.0 %w / v, optionally a concentration of from 0.25 to 0.75 %w / v.

7. The formulation of any preceding claim, wherein a largest dimension of the aggregates is less than 100 pm, optionally from about 1 pm to about 10 pm.

8. The formulation of any preceding claim, wherein the salt comprises divalent cations, optionally wherein the divalent cations are calcium ions.

9. The formulation of any preceding claim, wherein the salt is present at a concentration of from 0.01 M to 0.5 M.

10. The formulation of claim 8, wherein the calcium ions are present at a concentration of from 0.05 M to 0.4 M.

11. The formulation of any preceding claim, wherein the formulation is not a shear thinning fluid gel.

12. The formulation of any one of claims 1 to 11 for use in the treatment or prevention of fibrosis or scarring, optionally wherein the fibrosis or scarring is of the epithelium.

13. The formulation for use of claim 12, wherein the fibrosis or scarring is fibrosis or scarring of the skin, the eyes or an internal organ (e.g. the lungs, heart, liver, kidneys), or the oral, nasal, oesophageal, laryngeal, intestinal, anal or genital mucosa.

14. The formulation for use of claim 12 or claim 13, wherein the fibrosis or scarring is caused by an injury, a burn, surgery or a disease or condition.

15. The formulation for use of any one of claims 12 to 14, wherein the fibrosis or scarring is caused by a disease or condition selected from mucous membrane pemphigoid, epidermolysis bullosa, oral submucous fibrosis, keloid, intestinal fibrosis, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, ulcerative jejunoileitis, systemic sclerosis, cardiovascular fibrosis (e.g. atherosclerosis, arterial stiffness), renal fibrosis, hepatic fibrosis (e.g. alcoholic liver fibrosis, non-alcoholic liver fibrosis), pulmonary fibrosis (e.g. idiopathic pulmonary fibrosis, cystic fibrosis), fibrothorax, glial scarring, arthrofibrosis, Peyronie’s disease, Dupuytren’s disease, infection (e.g. bacterial or viral).

16. The formulation for use of any one of claims 12 to 15, wherein said use comprises administering the formulation topically or by injection.

17. A method of preparing a formulation, the method comprising:(i) dissolving a polysaccharide in an aqueous medium at a temperature of at least 50 °C to form a solution, optionally wherein the polysaccharide is an anionic polysaccharide;(ii) cooling the solution; and(iii) adding a salt to the cooled solution while stirring.

18. A formulation obtainable by the method of claim 17.

19. The formulation of any preceding one of claims 1 to 16 or 18, wherein the formulation has: i. a viscosity of less than 50 Pa s at a shear stress of 0.1 Pa, at 25 °C; ii. a viscosity of 1 Pa s at a shear stress of 1 Pa, at 25 °C; iii. a light absorbance which is at least 1 .5 greater than that of water;iv. a light absorbance of at least 0.4 at 350 nm; v. an elastic modulus (G’) of less than 1 Pa when subjected to oscillatory strain of 0.5% at a frequency of 0.1 Hz; vi. a viscous modulus (G”) of less than 1 Pa when subjected to oscillatory strain of 0.5% at a frequency of 0.1 Hz; vii. a coefficient of friction of less than 2 at a rotational velocity of 0.1 mm s’1; and / or viii. a coefficient of friction of less than 1.5 at a rotational velocity of 0.1 mm s’1.

20. A container comprising the formulation of any one of claims 1 to 16, 18 or 19, optionally wherein the container is a spray bottle.

Images