Injectable Compositions

Abstract

Injectable compositions are disclosed. Such compositions may be formed from cellulosic fibers and a deep eutectic solvent. Also disclosed are methods of preparing the injectable compositions, the methods including (i) contacting cellulosic fibers with a deep eutectic solvent to provide deep eutectic solvent-treated cellulosic fibers, (ii) washing the deep eutectic solvent-treated cellulosic fibers, and (iii) homogenizing or mechanically micronizing the washed deep eutectic solvent-treated cellulosic fibers to provide the injectable composition. Also disclosed are uses of and methods related to the compositions.

Description

[Technical field]

[0001] The present invention relates inter alia to injectable compositions formed from or including cellulosic fibers, methods of making same and uses of said compositions. [Background technology]

[0002] Where a prior art publication is referred to herein, it will be expressly understood that this reference is not an admission that this publication forms part of the common general knowledge in the field in Australia or any other country. Although the following description is particularly directed to dermal fillers, the subject matter of this application is not limited to this use.

[0003] Dermal fillers (also known as soft tissue fillers) are used to add fullness and smoothness to areas of the skin, especially the face. This can be particularly desirable when volume has been lost, especially due to aging. Many visible signs of aging can be attributed to volume loss. Thinning of the skin due to aging is commonly seen on the cheeks, lips, and around the mouth. Thus, dermal fillers are frequently used to replace lost volume in areas including the lips, nasolabial folds, wrinkles around the mouth, and cheeks.

[0004] Dermal fillers are typically injected directly into the area to be treated, often after application of a topical anesthetic cream. The effects of dermal fillers are immediate and can last from 2 to 18 months, depending on the type of product, the area being treated, and individual patient factors such as metabolism and whether the patient has had previous treatments.

[0005] One of the most common types of dermal fillers is hyaluronic acid, which is estimated to account for up to 80% of the dermal filler market. Hyaluronic acid has a good safety profile because it is found naturally in the body, and the results are temporary because the body gradually and naturally absorbs hyaluronic acid after a period of time. To achieve the desired viscosity and persistence, hyaluronic acid products are synthetically crosslinked, and the degree of crosslinking can result in different dermal filler products based on anatomical requirements. However, such crosslinked products can result in more viscous gels, which can result in more difficult injectability and greater patient discomfort, limiting their use at a certain threshold. Furthermore, such crosslinked products can form discrete particles, which can still be felt in tissue even if such particles are hundreds of microns in diameter. Therefore, there is a need to develop alternative dermal fillers.

[0006] Hyaluronic acid is also found naturally in tissues throughout the body. In particular, hyaluronic acid functions as a lubricant in joints, and therapeutic hyaluronic acid is used to improve joint function, for example, due to arthritis. In particular, hyaluronic acid is used to treat knee osteoarthritis by intra-articular injection. Summary of the Invention [Means for solving the problem]

[0007] In view of the above, the present invention relates in one embodiment to an injectable composition that can be useful as a dermal filler.In another embodiment, the present invention relates to an injectable composition that can be used instead of or together with hyaluronic acid products.In a further embodiment, the present invention can at least partially overcome at least one of the above-mentioned shortcomings or provide consumers with a useful or commercial alternative.

[0008] In a first aspect, the present invention provides an injectable composition formed from cellulosic fibers and a deep eutectic solvent. In one embodiment, the composition is a homogenized composition. In one embodiment, the composition is a mechanically micronized composition, such as a milled or sonicated composition. In another embodiment, the injectable composition is formed from cellulose nanofibers and a deep eutectic solvent.

[0009] In a second aspect, the present invention provides an injectable composition comprising deep eutectic solvent processed cellulosic fibers. In one embodiment, the deep eutectic solvent processed cellulosic fibers are deep eutectic solvent processed cellulose nanofibers.

[0010] In a third aspect, the present invention provides an injectable composition comprising cellulose nanofibers. The cellulose nanofibers may be treated or composited with a deep eutectic solvent (DES). In one embodiment, the cellulose nanofibers may be DES-treated cellulose nanofibers.

[0011] The first to third aspects of the present invention may be characterized as follows.

[0012] As used herein, terms such as "deep eutectic solvent treated cellulosic fibers" or "deep eutectic solvent treated cellulose nanofibers" or "DES treated cellulose nanofibers" do not imply that the composition comprises a deep eutectic solvent. Rather, the terms mean that the cellulosic fibers or cellulose nanofibers have been treated or composited with a deep eutectic solvent.

[0013] The term "deep eutectic solvent" will be known to those skilled in the art. For example, a review of deep eutectic solvents is provided in Smith, EL et al. (2014) Chemical Reviews, 114, 11060-11082. Deep eutectic solvents (or DES) are systems formed from eutectic mixtures of Lewis or Bronsted acids and bases. Deep eutectic solvents may include a variety of anionic and / or cationic species. DESs may be Type I, Type II, Type III, or Type IV DESs.

[0014] DES may be formed from a Lewis acid and a Lewis base. The Lewis base may include a nitrogen atom, an amide group, a urea group, a carbamate group, or an ammonium group (such as a quaternary ammonium salt). The Lewis base may be urea or acetamide, particularly urea. The Lewis acid may include an ammonium, phosphonium or sulfonium cation, an amine, an amide, a carboxylic acid, or a polyol, particularly an ammonium cation. The Lewis acid may be sulfamic acid. In one embodiment, the Lewis acid is sulfamic acid and the Lewis base is urea. In one embodiment, the Lewis acid is choline chloride. In one embodiment, the Lewis acid is choline chloride and the Lewis base is urea.

[0015] The Lewis acid and Lewis base can be present in the DES in any suitable molar ratio. In one embodiment, the molar ratio of Lewis acid:Lewis base is 1:5 to 1:1, 1:4 to 1:2, or about 1:3. The molar ratio of Lewis acid:Lewis base can be 1:5, 1:4, 1:3, 1:2, or 1:1, particularly 1:4, 1:3, or 1:2.

[0016] Deep eutectic solvents may modify cellulosic fibers (or cellulose nanofibers). For example, deep eutectic solvents may sulfate cellulosic fibers. Without wishing to be bound by theory, deep eutectic solvents may modify cellulosic fibers or nanofibers by ionic or covalent bonding, complexation, or dissolution of components of the cellulosic fibers that are then removed with the deep eutectic solvent.

[0017] In one embodiment, the composition is substantially free of deep eutectic solvents, in particular free of deep eutectic solvents. The composition may be substantially free of residual deep eutectic solvents and their components. The term "substantially free" or "substantially free" means that the amount of deep eutectic solvents (or their components) in the composition is below the toxic level of deep eutectic solvents (or their components). For example, the composition may contain less than 2 wt.% deep eutectic solvents (or their components), in particular less than 1.5 wt.%, 1 wt.%, 0.9 wt.%, 0.8 wt.%, 0.7 wt.%, 0.6 wt.%, 0.5 wt.%, 0.4 wt.%, 0.3 wt.%, 0.2 wt.%, 0.1 wt.% or 0.05 wt.% deep eutectic solvents (or their components).

[0018] When a composition is formed (or a cellulosic fiber or nanofiber is treated) with a deep eutectic solvent that includes sulfamic acid, the amount of sulfamic acid in the composition is less than 150 mg / L, 50 mg / L, 25 mg / L, 10 mg / L, 5 mg / L, 1 mg / L, 0.5 mg / L, 0.3 mg / L, 0.1 mg / L, 0.08 mg / L, 0.05 mg / L, 0.03 mg / L, or 0.01 mg / L.

[0019] When a composition is formed (or cellulosic fibers or nanofibers are treated) with a deep eutectic solvent that includes urea, the amount of urea in the composition is less than 20 mg / L, 10 mg / L, 5 mg / L, 2.5 mg / L, 1 mg / L, 0.8 mg / L, 0.5 mg / L, 0.3 mg / L, 0.1 mg / L, 0.08 mg / L, 0.05 mg / L, 0.03 mg / L, or 0.01 mg / L.

[0020] Advantageously, the inventors have found that the compositions of the first to third aspects have advantageous rheological properties, mechanical integrity, and from the experiments carried out, appear to be safe to use, biocompatible, and slowly degraded or absorbed by the body over time. The cellulosic fibers (or nanofibers) may also be obtained from safe and abundant plant sources. The compositions may also be injectable through a 30 or 31 gauge needle (as is the current practice for hyaluronic acid compositions). Finally, if the injection of the composition is a mistake, enzymes may be used to rapidly degrade the cellulose nanofibers in situ.

[0021] The composition may be a medical or cosmetic injectable composition. The composition may be suitable for injection into a human or animal for medical treatment purposes, e.g. to treat, prevent or ameliorate a condition, disorder or disease. The composition may be suitable for injection into a human or animal for cosmetic treatment purposes, e.g. as a dermal filler. In one embodiment, the composition comprises a therapeutic agent. In another embodiment, the composition does not comprise a therapeutic agent. In one embodiment, the composition is pharmacologically inactive.

[0022] The cellulosic fibers (or nanofibers) may be bleached cellulosic fibers (or nanofibers). Suitable bleaching agents will be known to those skilled in the art. In one embodiment, the bleaching agent may be a chlorite or hypochlorite, in particular a chlorite. The bleaching agent may be present in an acidic solution, such as an acidic solution of a chlorite. The bleaching agent may be sodium chlorite. The cellulosic fibers (or nanofibers) may be delignified. The cellulosic fibers (or nanofibers) may be bleached and delignified cellulosic fibers (or nanofibers). In some embodiments, the cellulosic fibers (or nanofibers) are bleached and / or delignified fibers of one or more plants as described in paragraphs 0024-0027.

[0023] In alternatives of the first to third aspects, the cellulosic fibres (or nanofibres) may be replaced with a cellulosic material (or nanocellulosic material). The cellulosic material (or nanocellulosic material) may comprise (or may be) a cellulosic fibre (or nanofibre). The cellulosic material may comprise (or may be) a cellulosic particle (or nanoparticle). Thus, in an alternative aspect, the invention may relate to an injectable composition formed from a cellulosic material (or nanocellulosic material) and a deep eutectic solvent. In an alternative aspect, the invention may relate to an injectable composition comprising a deep eutectic solvent treated cellulosic material. In a further alternative aspect, the invention may relate to an injectable composition comprising a cellulosic material.

[0024] The cellulosic fibres (or cellulose nanofibres, or cellulosic material, or nanocellulosic material) may be derived from a plant of the subtribe Triodiinae. The plant of the subtribe Triodiinae may be a plant of the genera Triodia, Monodia or Symplectrodia, in particular a plant of the genus Triodia. The cellulosic fibres (or cellulose nanofibres, or cellulosic material, or nanocellulosic material) may be derived from a drought tolerant or dry grass species.

[0025] Cellulosic fibres (or cellulose nanofibres, or cellulosic materials, or nanocellulose materials) may be derived from the Australian spinifex grass. Spinifex (also known as "porcupine" and "hummock" grass) is a long-established common name for three genera including Triodia, Monodia and Symplectrodia (not to be confused with the grass genus Spinifex, which is restricted to coastal dune systems in Australia). Hummock grassland communities in arid Australia are dominated by spinifex species of the "Triodia" genus. About 70 species of Triodia have been described, which are long-lived and deep-rooted, with root growth penetrating tens of metres underground. Of this species, the most abundant are two soft species called T. pungens and T. sindjii, and two hard species called T. basedowii and T. longiceps. T. pungens has been found to have an approximate composition of cellulose (37%), hemicellulose (36%), lignin (25%) and ash (4%) in unwashed form, such that the hemicellulose content constitutes 37% of the lignocellulose content.

[0026] In one embodiment, the cellulosic fibers (or cellulose nanofibers, or cellulosic material, or nanocellulosic material) are derived from grass species with C4 leaf anatomy. Exemplary grasses with C4 leaf anatomy include the Australian spinifex grass described in the previous paragraph. Other examples of grasses with C4 leaf anatomy that may be used to form cellulose nanofibers include Digitalia sanguinalis (L.) scopoli, Panicum coloratum L. var. Macaricarrhenius goossens, Brachiara brizantha (Hoechst ExA. Rich) Staff, D. violascens Link, P. dichotomiflorum michokeus, B. decimense schiense, and others. staff, Echinochloa cruz-galli P. Vove, P. miliasium L., B. fumidicola (Rendl) Schweich, Paspalum gisticum L., B. muthica (Forsk) Staff, Setaria glauca (L.) P., Cynodon dactylon (L.) Persoon, Panicum maximum Jacques, S. viridis (L.) P. Vove, Eleuthine coracana (L.) Ger toner, Urochlore texana (Buckley) Webster, Sorghum sandanense Staff, E. indica (L.) Gartner, Stogiopogon cottrifer (Sambu) Hackel, Eragrostis syrianensis (Allioni) Vignolo-lutani, Chloris gayana kuns, Eragrostis curvula, Leptochloa dubia, Mahlenbergia liguti, E. ferruginia (Sambu) P. Vobe, Sporobolus indicus R. Br. var. Purpureo-safsus (Owi) T. Koyama, Andropogon galardii, Leptochloa chinensis (L.) Nice, Miscanthus grasses (elephant grass), Salsola plants including Russian thistle, rice straw, wheat straw, corn stover, and Zoysia tenuifolia Wild.

[0027] Because Triodia grass grows under dry conditions, the inventors believe that other arid zone grasses that grow in Australia and other parts of the world may also be used in the present invention. The most drought tolerant grass genera in Australia (although they do require water for the first year or two) include Anigozanthos, Austrodanthonia, Austrostipa, Barocyon pallens, Baumea juncia, Ukiagara, Chiapiripedium, Carex bikenoviana, Carex goudichoudiana, Carex apressa, C. terreticoulis, Coutis, Centrolepis, Oryza sativa, Corysandra, Conostylis, Cyperus serrata, Cyperus serrata, Desmocladus flexuosa, Scutellaria baicalensis, Dichaete serrata, and Scutellaria baicalensis. These include Latunae, Casexa, Eurycorda complanata, Evandora aristata, Ficcinia nodosa, Ghania, Button grass, Haemathria uncinata, Hipporeana, Imperata japonica, Johnsonia, Joycea parido, Rush grass, Kingia australis, Lepidosperma, Ampera, Leptocarpus, Lomandra, Meeboldina, Mesomelaena, Neurachne alpecroidea, Nothodanthonia, Patersonia, Poa, Spinifex, Semdo triandra, Tremulina tremula, Sibana, Triodia and Xanthoea. Dry zone grasses that grow in other parts of the world in which the invention can also be used include Aristida pallens (wiregrass), Andropogon (big bluestem), Bouteloa enopoda (black grama), Chloris loxburghiana (horsetail grass), Megalucaya (red grass), Panicum trifoliata (switchgrass), Pennisetum ciliaris (buffel grass), Schizacrium scoparium (schizachyrium), Sorghum nutans (Indian grass), and Esparto (needle grass).

[0028] Along with cellulose and lignin, hemicellulose is believed to be a major component of the various materials that make up cellulosic fibers (or cellulose nanofibers). Without wishing to be bound by theory, it is believed that hemicellulose is likely distributed throughout the cellulosic fiber (or nanofiber) both on the surface of the fiber and between the primary cellulose fibrils (or nanofibrils) when the cellulosic fiber (or nanofiber) is composed of bundles of primary (elementary) cellulose fibrils (or nanofibrils). Cellulose is a more rigid crystalline material, while hemicellulose is amorphous and therefore has weaker mechanical properties. When hemicellulose is distributed throughout the structure of the cellulosic fiber (or nanofiber), without wishing to be bound by theory, the inventors believe that hemicellulose acts to increase the flexibility of the nanocellulose, possibly acting as a plasticizer or reinforcing agent between the cellulosic fibers, allowing the individual cellulosic fibers to creep and stretch against each other. This may reduce the stiffness of the cellulosic fibres (or nanofibers) but potentially increase their fracture toughness (indeed, when the inventors homogenise base treated spinifex, which is an example of a suitable source of cellulosic fibres (or nanofibers) for use in the present invention, and compare this to other softwood or hardwood pulps, they find that spinifex can be exposed to much higher mechanical energies without fibril fracture, which appears to indicate a much higher toughness of the cellulose / nanocellulose).

[0029] In some embodiments of the present invention, the hemicellulose content of the cellulosic fibres (from which the composition of the first aspect is formed) is at least 20% or 30% by weight of the lignocellulosic component of the cellulosic fibres from which the composition of the first aspect is formed. Preferably, the hemicellulose content is 20-55% w / w, 30-55% w / w, 30-50% w / w, 36-48% w / w, 40-48% w / w or 42-47% w / w or any intermediate range within the above ranges including 20-30% w / w, 30-40% w / w or 40-50% w / w. These high hemicellulose contents may be achieved by any means, including, but not limited to, using plant materials that are naturally high in hemicellulose and using subsequent processing to produce cellulosic fibers (or nanofibers) that retain a high hemicellulose content, or alternatively, using a cellulosic material (or nanocellulosic material) that has a lower hemicellulose content and mixing it with a separately produced hemicellulose material to obtain a mixture that provides cellulose (or nanocellulose) with a high hemicellulose content.

[0030] In some embodiments of the second and third aspects of the invention, the hemicellulose content of the deep eutectic solvent treated cellulosic fibers (or cellulose nanofibers) is less than 20% by weight of the lignocellulosic component of the cellulosic fibers. In one embodiment, the hemicellulose content of the deep eutectic solvent treated cellulosic fibers (or cellulose nanofibers) is less than 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% by weight of the lignocellulosic component of the cellulosic fibers, in particular less than 10%, 6%, 5%, or 4% by weight. In one embodiment, the cellulose content of the deep eutectic solvent treated cellulosic fibers (or cellulose nanofibers) is greater than 50% by weight of the lignocellulosic component of the cellulosic fibers. In one embodiment, the cellulose content of the deep eutectic solvent treated cellulosic fibers (or cellulose nanofibers) is less than 80%, 75%, 70%, 65%, 60% or 55% by weight of the lignocellulosic component of the cellulosic fibers, in particular less than 65% or 55%. In one embodiment, the lignin content of the deep eutectic solvent treated cellulosic fibers (or cellulose nanofibers) is less than 20% by weight of the lignocellulosic component of the cellulosic fibers. In one embodiment, the lignin content of the deep eutectic solvent treated cellulosic fibers (or cellulose nanofibers) is less than 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% by weight of the lignocellulosic component of the cellulosic fibers, in particular less than 5% or 3%. Deep eutectic solvent treatment can reduce the amount of lignocellulose, cellulose, hemicellulose and / or lignin in cellulosic fibers.

[0031] In some embodiments, the cellulosic fibers (or cellulosic materials) are derived from plant material having a hemicellulose content of 20% or 30% or more (w / w), in other embodiments, the hemicellulose content of the plant material is 20-55% w / w, 30-55% w / w, 30-50% w / w, 36-48% w / w, 40-48% w / w, or 42-47% w / w, or any intermediate range within the above ranges, including 20-30% w / w, 30-40% w / w, or 40-50% w / w.

[0032] The cellulose nanofibers may be of any suitable dimensions. As used herein, the term "nanofiber" means that the length of the fiber is typically less than 1000 nm. The fiber is longer than the width. In one embodiment, the composition may include cellulose nanofibers. In one embodiment, the length of the nanofiber is about 50 nm to 1,500 nm, about 50 nm to 1,400 nm, about 100 nm to 1,400 nm, about 200 nm to 1,300 nm, about 300 nm to 1,200 nm, about 400 nm to 1,100 nm, or about 500 nm to 1,000 nm. In one embodiment, the width or diameter of the cellulose nanofiber is 0.5 nm to 20 nm, 1 nm to 15 nm, 1 nm to 10 nm, 1 nm to 6 nm, 2 nm to 5 nm, or about 3 nm. Without wishing to be bound by theory, the inventors believe that the cellulose nanofibers may be long and thin, which advantageously helps to make the composition strong while still being very flexible. The cellulose nanofibers may be substantially cylindrical. The length to width ratio (aspect ratio) of the cellulose nanofibers may be from 150:1 to 500:1, particularly from 150:1 to 400:1. Spinifex grass has the highest cellulosic fiber length to width ratio of any biomass reported. It will be understood that the fiber length and aspect ratio values ​​of any given sample of cellulose nanofibers will comprise a distribution of values, and the quoted values ​​approximately represent the average of the values ​​of the different fibers in the sample.

[0033] The cellulosic fibres (or nanofibers) may carry an electric charge. The cellulosic fibres may carry a positive or negative charge, in particular a negative charge.

[0034] In one embodiment, the cellulosic fibers (or nanofibers) are crosslinked. The cellulosic fibers (or nanofibers) may be crosslinked by a crosslinking agent. Suitable crosslinking agents may include compounds that include moieties containing epoxides, alkenes, aldehydes, imides, amines, carboxylic acids, and acrylamides. Crosslinking agents may include compounds that include at least two moieties selected from the group consisting of epoxides, alkenes, aldehydes, imides, amines, carboxylic acids, ureas, and acrylamides. In one embodiment, the crosslinking agent may be an amino acid. The crosslinking agent may be selected from the group consisting of 1,4-butanediol diglycidyl ether, divinyl sulfone, 1,2,7,8-diepoxyoctane, hexamethylene diamine, glycine, epichlorohydrin, urea, and methylene bisacrylamide. The crosslinking agent may be 1,4-butanediol diglycidyl ether, divinyl sulfone, 1,2,7,8-diepoxyoctane, glycine, or urea. In one embodiment, the cellulose nanofibers are not crosslinked or treated with a crosslinking agent.

[0035] The composition may be homogenized or mechanically micronized. The composition may be homogenized at a pressure of at least 400 bar, or at least 500 bar, 600 bar, 700 bar, 800 bar, 900 bar, or 1,000 bar. The composition may be homogenized at a pressure of about 1,100 bar. The composition may be mechanically micronized, for example, by grinding or sonicating the composition. The composition may be homogenized multiple times, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0036] The cellulosic fibers (or cellulose nanofibers, or DES cellulosic fibers, or DES cellulose nanofibers) may be present in the composition in any suitable concentration. In one embodiment, the composition may comprise less than 10% w / v of the cellulosic fibers (or cellulose nanofibers, or DES cellulosic fibers or DES cellulose nanofibers), in particular less than 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% w / v of the cellulosic fibers (or cellulose nanofibers, or DES cellulosic fibers or DES cellulose nanofibers). The composition may comprise between 0.01% and 5% w / v of the cellulosic fibers (or cellulose nanofibers, or DES cellulosic fibers or DES cellulose nanofibers), or between 0.05% and 4% w / v, between 0.1% and 2% w / v, between 0.1% and 1.5% w / v, or between 0.1% and 1% w / v of the cellulosic fibers (or cellulose nanofibers, or DES cellulosic fibers or DES cellulose nanofibers). The composition may comprise 0.2%, 0.4%, 0.6%, 0.8%, 0.88% or 1.0% w / v cellulosic fibers (or cellulose nanofibers, or DES cellulosic fibers, or DES cellulose nanofibers).

[0037] The composition may include any suitable solvent for injection. In one embodiment, the solvent is an aqueous solvent. The solvent may be saline, particularly sterile saline. The solvent may be a buffered aqueous solution. The solvent may be phosphate buffered saline (PBS). The buffered aqueous solution may be one that maintains the composition at near physiological pH or at least within the range of about pH 6.0 to 9.0.

[0038] The composition may have any pH suitable for injection, hi one embodiment, the pH of the composition is 4-9, particularly 5-8, or 5.4-7.

[0039] The composition may be in any form suitable for injection. The composition may be in the form of a liquid or gel, particularly in the form of a gel. The composition may have any suitable viscosity. In one embodiment, the complex viscosity of the composition is 10-10,000 Pa·s at 1 rad / s and 25°C, 50-5,000 Pa·s at 1 rad / s and 25°C, or 100-1,000 Pa·s at 1 rad / s and 25°C. The loss modulus of the composition may be 10-1,000 Pa at 1 rad / s and 25°C, 10-500 Pa at 1 rad / s and 25°C, or 10-100 Pa at 1 rad / s and 25°C.

[0040] The composition may have any suitable storage modulus (G'). In one embodiment, the storage modulus of the composition is 10 to 10,000 Pa at a frequency of 0.1 to 1000 rad / s (or 0.1, 1, 10, or 100 rad / s), 50 to 5,000 Pa at a frequency of 0.1 to 1000 rad / s (or 0.1, 1, 10, or 100 rad / s), or 100 to 1,000 Pa at a frequency of 0.1 to 1000 rad / s (or 0.1, 1, 10, or 100 rad / s). In one embodiment, the composition has a storage modulus of 10 to 10,000 Pa at a frequency of 0.1 to 1,000 rad / s (or 0.1, 1, 10 or 100 rad / s) at 25°C, 50 to 5,000 Pa at a frequency of 0.1 to 1,000 rad / s (or 0.1, 1, 10 or 100 rad / s) at 25°C, or 100 to 1,000 Pa at a frequency of 0.1 to 1,000 rad / s (or 0.1, 1, 10 or 100 rad / s) at 25°C.

[0041] In one embodiment, the composition may include an additional agent. The additional agent may be a rheology modifier. The rheology modifier may be, for example, hyaluronic acid or cross-linked hyaluronic acid. The composition may also include an active agent. For example, in the case of arthritis treatment, the composition may include a steroid. Alternatively, the composition may include platelets (or platelet-rich plasma). This may be advantageous for platelet-rich plasma therapy.

[0042] The composition may be formulated in unit dose form.For example, the composition may be present in ampoules, pre-filled syringes, small injectables or multi-dose containers.Such compositions may contain preservatives.

[0043] In a fourth aspect, the present invention relates to a method for preparing an injectable composition, said method comprising the steps of: (i) contacting a cellulosic fiber with a deep eutectic solvent to provide a deep eutectic solvent treated cellulosic fiber; (ii) washing the cellulosic fibers treated with the deep eutectic solvent; and (iii) homogenizing or mechanically micronizing the washed, deep eutectic solvent-treated cellulosic fibers to provide the injectable composition.

[0044] In one embodiment, the composition comprises cellulose nanofibers. In one embodiment, step (iii) provides an injectable composition comprising cellulose nanofibers. Without wishing to be bound by theory, the inventors believe that homogenization or mechanical micronization of the composition may generate shear forces that disperse the fibers throughout the resulting composition and may fragment or fibrillate the cellulosic fibers to provide the cellulose nanofibers.

[0045] The method of the fourth aspect may produce the composition of the first, second or third aspect.

[0046] In one embodiment of the fourth aspect, the cellulosic fibres used in step (i) are bleached cellulosic fibres. Thus, prior to step (i), the method may comprise the step of bleaching the cellulosic fibres to provide bleached cellulosic fibres. As mentioned above, suitable bleaching agents will be known to those skilled in the art. In one embodiment, the cellulosic fibres may be bleached with a chlorite or hypochlorite, in particular a chlorite or sodium chlorite. The bleaching agent may be a 1% w / v aqueous solution of sodium chlorite. The cellulosic fibres may be bleached with any suitable concentration of bleaching agent at any suitable temperature and any suitable pH. The ratio of cellulosic fibre to bleaching agent may be from 10:1 to 100:1, in particular from 20:1 to 40:1, or about 30:1. The cellulosic fibres may be bleached at 40°C to 90°C, 50°C to 90°C, 60°C to 80°C, or about 70°C. Prior to contacting the bleached cellulosic fibers with the deep eutectic solvent, the bleached cellulosic fibers may be washed before drying. The bleached cellulosic fibers may be washed with water at a temperature greater than 50°C. The bleached cellulosic fibers may be washed until the pH of the pulp is greater than 6.5. The bleached cellulose nanofibers may be dried at 40°C to 90°C, 50°C to 90°C, 60°C to 80°C, or about 70°C.

[0047] The cellulosic fibres may be delignified prior to bleaching. Thus, the cellulosic fibres used in the bleaching step may be delignified cellulosic fibres. In one embodiment, the method may comprise a step of delignifying the cellulosic fibres prior to bleaching. Since bleaching may be optional, in one embodiment, the cellulosic fibres used in step (i) are delignified cellulosic fibres. Thus, prior to step (i), the method may comprise a step of delignifying the cellulosic fibres to provide delignified cellulosic fibres. For the avoidance of doubt, delignifying the cellulosic fibres means that the amount of lignin in the cellulosic fibres is reduced, and after this step the cellulosic fibres may still contain some lignin. The delignification step may be carried out using a base, in particular a hydroxide, more particularly sodium hydroxide. The sodium hydroxide may be 2 or 3% w / v sodium hydroxide. Delignification may be carried out using any suitable concentration of delignification agent (in particular a base) and at any suitable temperature. The ratio of cellulosic fibre to delignification agent may be 5:1 to 50:1, in particular 10:1 to 30:1, or about 20:1. The cellulosic fibre may be bleached at 50°C to 100°C, 70°C to 90°C, or about 80°C. Before contacting the delignified fibre with the bleaching agent, the delignified fibre may be washed. The delignified fibre may be washed multiple times, for example at least two or three times. The delignified fibre may be washed with an aqueous solvent, in particular water. The aqueous solvent may be at any suitable temperature, in particular 30°C to 90°C, 40°C to 80°C, 50°C to 70°C, or about 60°C.

[0048] The cellulosic fibers may be suspended in a solvent prior to delignification. Thus, the cellulosic fibers used in the delignification step may be soaked cellulosic fibers. In one embodiment, the cellulosic fibers may be suspended in a solvent prior to delignification. The solvent may be an aqueous solvent, in particular water, or reverse osmosis water. The aqueous solvent may be at any suitable temperature. For example, the aqueous solvent may be at 20°C to 70°C, 30°C to 70°C, 40°C to 60°C, or about 50°C. The cellulosic fibers may be suspended for any suitable length of time, for example at least 5 hours, at least 10 hours, or at least 12 hours. The delignification agent may be added to the soaked cellulosic fibers without filtration.

[0049] The cellulosic fibers may be milled prior to soaking. In one embodiment, the method may include milling the cellulosic fibers to provide milled cellulosic fibers prior to soaking. The cellulosic fibers may be milled using a cutting mill. The cellulosic fibers may be passed through a mesh, in particular a mesh of less than 5 mm, a mesh of less than 3 mm, or a mesh of 1 mm.

[0050] The cellulosic fibres may be mulched prior to being milled. Thus, the cellulosic fibres used in the milling step may be mulched cellulosic fibres. Thus, the method may comprise the step of mulching the cellulosic fibres prior to milling to provide mulched cellulosic fibres. After mulching, the mulched cellulosic fibres may be washed. The mulched cellulosic fibres may be washed multiple times, for example at least two or three times. The washing may be carried out with an aqueous solvent, in particular water. The aqueous solvent may be at a temperature of 50°C to 100°C, 70°C to 90°C, or about 80°C. The mulched and washed cellulosic fibres may be dried prior to being milled.

[0051] Thus, in one embodiment, the method comprises: (i) delignifying the cellulosic fibers; (ii) optionally bleaching the delignified cellulosic fibers; (iii) contacting the cellulosic fiber of step (i) or step (ii) with a deep eutectic solvent to provide a deep eutectic solvent-treated cellulosic fiber; (iv) washing the cellulosic fibers treated with the deep eutectic solvent; and (v) homogenizing or mechanically micronizing the washed, deep eutectic solvent-treated cellulosic fibers to provide the injectable composition.

[0052] In a further embodiment, the method comprises: (i) mulching cellulosic fibers to provide mulched cellulosic fibers; (ii) grinding the mulched cellulosic fibers to provide ground cellulosic fibers; (iii) providing cellulosic fibers obtained by suspending and soaking the pulverized cellulosic fibers in a solvent; (iv) delignifying the soaked cellulosic fibers to provide delignified cellulosic fibers. (v) optionally bleaching the delignified cellulosic fibers to provide bleached cellulosic fibers; (vi) contacting the cellulosic fiber of step (iv) or step (v) with a deep eutectic solvent to provide a deep eutectic solvent treated cellulosic fiber; (vii) washing the deep eutectic solvent treated cellulosic fibers; and (viii) homogenizing or mechanically micronizing the washed, deep eutectic solvent-treated cellulosic fibers to provide the injectable composition.

[0053] The deep eutectic solvent used in the step of contacting the cellulosic fiber with the deep eutectic solvent may be as described above. This step may include forming a deep eutectic solvent. The deep eutectic solvent may be formed by heating the Lewis acid and Lewis base. The heating may be at a temperature of 50°C to 100°C, 70°C to 90°C, or about 80°C. The molar ratio of the cellulosic fiber to the deep eutectic solvent may be 1:2 to 1:50, 1:2 to 1:40, 1:2 to 1:30, 1:5 to 1:20, or about 1:10. The step of contacting the cellulosic fiber with the deep eutectic solvent may be carried out at any suitable temperature, such as 100°C to 250°C, 100°C to 200°C, 120°C to 180°C, 130°C to 170°C, 140°C to 160°C, or about 150°C. The contacting step may be carried out for any suitable length of time, such as from 10 minutes to 2 hours, particularly about 30 minutes.

[0054] The step of washing the deep eutectic solvent treated cellulosic fiber may be performed any suitable number of times. In one embodiment, the deep eutectic solvent treated cellulosic fiber is washed at least 2, 3, 4, 5, 6, 7 or 8 times. The washing may be performed with an aqueous solvent, particularly water, more particularly purified water. The washing may be performed at any suitable temperature with an aqueous solvent. For example, the solvent may be from freezing point (0°C) to boiling (100°C). In one embodiment, the washing is performed with a solvent at at least two different temperatures. The washing may be performed with a first solvent at 50°C to 100°C, particularly 70°C to 100°C, 80°C to 100°C, or 90°C to 100°C. The washing may be performed with a second solvent at 0°C to 50°C, 0°C to 30°C, 0°C to 20°C, or 0°C to 10°C. The washing may be performed with the first solvent at least once, twice, three or four times. Washing may be performed at least 1, 2, 3 or 4 times with the second solvent.

[0055] The washing step may include exchanging the solvent of the deep eutectic solvent treated cellulosic fibers. The solvent may be exchanged with saline (particularly sterile saline), or an aqueous buffer solution (such as phosphate buffered saline (PBS)).

[0056] The method may include diluting the washed deep eutectic solvent treated cellulosic fiber (or solvent exchanged fiber) to a desired concentration. The desired concentration may be less than 10% w / v of deep eutectic solvent treated cellulosic fiber, particularly less than 9% w / v, 8% w / v, 7% w / v, 6% w / v, 5% w / v, 4% w / v, 3% w / v or 2% w / v of deep eutectic solvent treated cellulosic fiber. The desired concentration may be 0.01%-5% w / v of deep eutectic solvent treated cellulosic fiber, or 0.05%-4% w / v, 0.1%-2% w / v, 0.1%-1.5% w / v, 0.1%-1% w / v of deep eutectic solvent treated cellulosic fiber. The desired concentration may be 0.2% w / v, 0.4% w / v, 0.6% w / v, 0.8% w / v, 0.88% w / v or 1.0% w / v of deep eutectic solvent treated cellulosic fiber.

[0057] In one embodiment, the deep eutectic solvent treated cellulosic fibers may be washed with an acid prior to homogenization or mechanical micronization. The acid wash may be a mild acid wash. The acid may be, for example, sulfuric acid or phosphoric acid.

[0058] The step of homogenizing the washed deep eutectic solvent treated cellulosic fibers may be at any suitable pressure, but in particular at high pressure. The pressure may be at least 400 bar, or at least 500 bar, 600 bar, 700 bar, 800 bar, 900 bar or 1,000 bar. The pressure may be about 1,100 bar. The step of mechanically micronizing the washed deep eutectic solvent treated cellulosic fibers may be or include grinding or ultrasonication of the washed deep eutectic solvent treated cellulosic fibers.

[0059] Features of the fourth aspect of the invention may be as described for the first, second or third aspects.

[0060] In a fifth aspect, the present invention provides an injectable composition prepared by the method of the fourth aspect.

[0061] In a sixth aspect, the present invention relates to the use of a composition according to the first, second, third or fifth aspect of the invention as a dermal filler, or for treating, preventing or ameliorating the symptoms of arthritis, or in platelet rich plasma therapy. In one embodiment, the arthritis is osteoarthritis.

[0062] As used herein, the terms "treatment" (or "treating") and "prevention" (or "preventing") should be considered in their broadest context. For example, the term "treatment" does not necessarily mean that a patient is treated until complete recovery. The term "treatment" includes amelioration of symptoms of a disease, disorder, or condition, or reducing the severity of a disease, disorder, or condition. Similarly, "prevention" does not necessarily mean that a subject will never contract a disease, disorder, or condition. "Prevention" may be considered as reducing the likelihood of development of a disease, disorder, or condition, or preventing or otherwise reducing the risk of developing a disease, disorder, or condition.

[0063] As used herein, the term "subject" or "individual" or "patient" may refer to any subject for which therapy is desired, particularly a vertebrate subject, and even more particularly a mammalian subject. Suitable vertebrates include, but are not limited to, primates, birds, livestock animals (e.g., sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs) and captive wild animals (e.g., foxes, deer, jingos). A preferred subject is a human.

[0064] In a seventh aspect, the present invention relates to a method of treating, preventing or ameliorating symptoms of arthritis in a subject, comprising injecting a composition of the first, second, third or fifth aspect of the invention into the subject. The arthritis may be osteoarthritis. In one embodiment, the composition is injected into a joint of the subject.

[0065] In an eighth aspect, the present invention provides the use of cellulosic fibers (or nanofibers) in the manufacture of a composition of the first, second, third or fifth aspect administered by injection for treating, preventing or ameliorating symptoms of arthritis in a subject. The arthritis may be osteoarthritis. In one embodiment, the composition is formulated for injection into a joint of the subject.

[0066] In a ninth aspect, the present invention provides a composition of the first, second, third or fifth aspect of the present invention for use in treating, preventing or ameliorating symptoms of arthritis in a subject, the composition being administered by injection. The arthritis may be osteoarthritis. In one embodiment, the composition is for injection into a joint of a subject.

[0067] In a tenth aspect, the present invention relates to a method of increasing volume in or under the skin of a subject, comprising injecting a composition of the first, second, third or fifth aspect of the invention into or under the skin of the subject.

[0068] The composition may be injected in any suitable manner. For example, the composition may be injected subcutaneously, intradermally or intramuscularly, particularly subcutaneously or intradermally. The composition may be injected into the face or chest of a subject. The composition may be injected to increase the volume of the lips, chest or cheeks of a subject. The method may provide cosmetic surgery to a subject. The method may improve the appearance of nasolabial folds or wrinkles around the mouth. The method may improve the appearance of wrinkles on the skin of a subject.

[0069] In an eleventh aspect, the present invention relates to the use of a composition according to the first, second, third or fifth aspect of the invention for increasing volume in or under the skin of a subject. Features of the eleventh aspect of the invention may be as described in the tenth aspect of the invention.

[0070] In a twelfth aspect, the present invention relates to a method of promoting healing in a subject comprising administering a composition of the first, second, third or fifth aspect of the invention in a platelet-rich plasma therapy.

[0071] In a thirteenth aspect, the present invention relates to the use of cellulosic fibres (or nanofibres) in the manufacture of a composition according to the first, second, third or fifth aspect of the invention for platelet-rich plasma therapy to treat injury in a subject.

[0072] In the sixth to thirteenth aspects of the present invention, the composition may be administered in an effective amount. As used herein, "effective amount" refers to administration of a composition in an amount sufficient to at least partially achieve a desired response or achieve a desired effect. It is expected that the "effective amount" will fall within a broad range that can be determined through routine trials. Determination of dosage, etc. is within the skill of the physician or veterinarian responsible for the care of the patient.

[0073] Embodiments of the sixth, twelfth and thirteenth aspects of the invention relate to platelet-rich plasma therapy. In these embodiments, the composition administered will comprise platelets, in particular platelet-rich plasma. The platelet-rich plasma is administered by injection. Such therapy may be useful in wound healing and in the treatment of injuries, particularly to tendons and / or ligaments.

[0074] The features of the sixth to thirteenth aspects of the present invention may be as described for the first to fifth aspects of the present invention.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0076] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more combinations.

[0077] In this specification and claims, the term "comprising," as well as its derivatives, including "comprises" and "comprise," includes each of the stated elements but does not exclude the inclusion of one or more additional elements.

[0078] Any of the features described herein may be combined in any combination with any one or more of the other features described herein within the scope of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0079] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0080] [Figure 1] 1 illustrates a process for producing a composition according to the present invention. [Figure 2A] 1 is a graph showing the rheology of a composition according to the invention where a gel was prepared in sterile saline; FIG. 2 is a graph showing how the complex modulus varies with frequency for the samples (the lower graph is a black and white version of the upper graph); [Figure 2B] 1 is a graph showing the rheology of a composition according to the invention where a gel was prepared in sterile saline, and FIG. 2 is a graph showing how the complex viscosity varies with frequency for the samples (the lower graph is a black and white version of the upper graph). [Figure 3A] FIG. 1 is a graph showing the rheology of a composition according to the invention, where the gel was prepared in phosphate buffered saline (PBS), and how the complex modulus varies with frequency for the samples (the lower graph is a black and white version of the upper graph), comparing this rheology to the published rheology of commercially available dermal fillers. [Figure 3B] Figure 1 shows the rheology of a composition according to the invention, where the gel was prepared in phosphate buffered saline (PBS). Figure 2 shows how the complex viscosity varies with frequency for the samples (the lower graph is a black and white version of the upper graph). This rheology is compared to the published rheology of commercially available dermal fillers. [Figure 4A] FIG. 2 is a graph showing the rheology of a composition according to the present invention compared to a Restylane™ commercial dermal filler sample, and how the storage and loss moduli vary with frequency for the samples (lower graph is a black and white version of the upper graph). [Figure 4B] FIG. 2 is a graph showing the rheology of a composition according to the present invention compared to a Restylane™ commercial dermal filler sample, and how the complex viscosity varies with frequency for the samples (lower graph is a black and white version of the upper graph). [Figure 5A] FIG. 1 is a graph showing the rheology of a composition according to the present invention compared to a sample of Juvederm™, a commercially available dermal filler; and FIG. 2 is a graph showing how the storage and loss moduli vary with frequency for the samples (the lower graph is a black and white version of the upper graph). [Figure 5B] 1 is a graph showing the rheology of a composition according to the present invention compared to a sample of Juvederm™, a commercially available dermal filler, and how the complex viscosity varies with frequency for the samples (lower graph is a black and white version of the upper graph). [Figure 6A]1 is a graph relating to the injectability of compositions according to the invention;FIG. 2 is a graph showing the force required to displace various compositions of the invention (lower graph is a black and white version of the upper graph); [Figure 6B] Graph relating to the injectability of compositions according to the present invention. Graph showing the glide force of the compositions using 30 and 31 gauge needles (lower graph is a black and white version of upper graph). [Figure 7] FIG. 1 is a graph (full and zoomed) showing the force required to inject a composition according to the present invention (0.88% w / v DES CNF) compared to a commercially available dermal filler sample (lower graph is a black and white version of upper graph). [Figure 8] 1 is a graph showing how the rheology of a gel according to the invention (0.8% w / v DES CNF) changes over time when stored at 4° C. for up to 90 days (lower graph is a black and white version of upper graph). [Figure 9] FIG. 1 is a graph showing how the rheology of a composition according to the invention (0.8% w / v DES CNF) changes over time when stored at 23° C. for up to 90 days (lower graph is a black and white version of upper graph). [Figure 10] FIG. 1 is a graph showing how the rheology of a composition according to the invention (0.8% w / v DES CNF) changes over time when stored at 55° C. for up to 90 days (lower graph is a black and white version of upper graph). [Figure 11] FIG. 1 is a graph showing how the rheology of a composition according to the invention (0.8% w / v DES CNF) changes over time when stored at 37° C. in 5% CO2 for up to 90 days (lower graph is a black and white version of upper graph). [Figure 12A] FIG. 1 is a graph showing how the rheology (at 0.1 Hz) of a composition according to the invention changes over a 60 day period when stored at 55° C. in comparison to a commercial dermal filler sample. Storage modulus is shown (lower graph is a black and white version of upper graph). [Figure 12B]FIG. 2 is a graph showing how the rheology (at 0.1 Hz) of a composition according to the invention changes over a 60 day period when stored at 55° C. in comparison to a commercial dermal filler sample. The loss modulus over this period is shown (lower graph is a black and white version of the upper graph). [Figure 13] FIG. 13 is a transmission electron microscope image of a freeze-dried sample of homogenized deep eutectic solvent processed cellulose nanofibers. EXAMPLES

[0081] Preferred features, embodiments and variations of the present invention can be identified from the following examples which provide sufficient information for one skilled in the art to practice the invention and should not be construed as limiting the scope of the foregoing summary of the invention in any way.

[0082] Example 1: Preparation of cellulose nanofibers Spinifex grass (Triodia pungens) was collected from Camweale, Queensland, Australia.

[0083] The grass was pre-sorted and the leaves were selected and cut from the woody stems. This pre-sorted material is called the "tips". The tips were then mulched, washed six times with water at 80°C for 1 h, air-dried and ground to a fine powder using a Retsch cutting mill with a 1 mm mesh.

[0084] The crushed washed grass was soaked overnight in reverse osmosis (RO) water at 50°C (water to grass ratio of 20:1) and then treated with 3% (w / v) sodium hydroxide at 80°C for 2 hours. The resulting pulp was washed three times with hot water (60°C) to remove dissolved materials. This step delignifies the cellulose nanofibers.

[0085] The alkaline pulp was then bleached twice with 1% (w / v) aqueous sodium chlorite at 70°C for 1 hour at pH 4 with a solvent to grass ratio of 30:1 (with the addition of glacial acetic acid). After this, the mixture was poured into a sieve and boiling deionized water was slowly poured over the pulp in the sieve and the pulp was turned over to wash evenly. This was followed by pouring water at 55°C over the pulp in the sieve. The pulp was then transferred to a beaker, water was added and the bleaching process was repeated as described. After washing the bleached pulp as described above, the pulp was further washed with hot water (55°C) and boiling deionized water. Once it was determined that the pH of the pulp was greater than 6.5, the pulp was removed from the sieve and dried in a convection oven at 70°C for 18 hours.

[0086] The components of the deep eutectic solvent (DES) treatment (1:3 molar ratio of sulfamic acid:urea) were mixed together on an oil bath at 80°C until a clear solution was obtained (approximately 2 hours). Dried bleached cellulose pulp (1:10 molar ratio of cellulose:sulfamic acid) was immersed in the DES. The temperature of the oil bath was then increased to 150°C and the reaction was allowed to proceed for 30 minutes. The reaction was stopped by adding excess water, followed by extensive centrifugation and washing (4 cycles with boiling MilliQ™ purified water and 4 cycles with cold MilliQ™ purified water). Small aliquots of supernatant were collected from the centrifuged gel batches after each washing step for (a) visual observation (color, turbidity) and (b) HPLC-MS analysis (urea) and LC-MS (sulfamic acid) if required to detect residual levels of urea, sulfamic acid, and any other potential by-products (such as glucose and its glucose derivatives of sulfamic acid and urea, and sulfamic acid derivatives of urea and N-substituted ureas, although these were expected to be highly unlikely at a sulfamic acid:urea ratio of 1:3).

[0087] The washed DES-treated cellulosic fibers were centrifuged and diluted with cold phosphate buffered saline (PBS) (although sterile saline could be used instead) and the cellulose was washed with four cycles of washing and centrifugation. Small aliquots of the supernatant were collected from the centrifuged gel batch after each washing step for analysis by (a) visual observation (color, turbidity) and (b) to detect residual levels of urea and sulfamic acid as described above.

[0088] The washed cellulose in PBS was diluted to a concentration of 1 wt% with MilliQ™ purified water or 2 wt% with PBS (or sterile saline) and then passed through a high-pressure homogenizer (GEA, PandaPlus2000) several times: once at 400 bar, once at 700 bar, and three times at 1,100 bar.

[0089] Residual levels of sulfamic acid in the wash supernatants were assessed by LC-MS and residual levels of urea were assessed by HPLC-MS (Invitrogen urea assay) as outlined above. The results are provided in Tables 1 and 2. For Table 2, the urea concentration in the PBS control was below the lower limit of detection. Furthermore, urea of ​​approximately 7-20 mg / dL is considered normal in human blood.

[0090] [Table 1]

[0091] [Table 2]

[0092] From a single batch of DES cellulose nanofiber (CNF) gel prepared above, a series of dilutions were prepared for rheology and injectability studies. Gels were prepared with DES CNF at concentrations including 0.88%, 0.8%, 0.7%, 0.6%, and 0.4% (w / v).

[0093] DES CNF gels were also prepared in saline solution rather than PBS. The procedure for preparing these gels was the same as outlined above, except that the final centrifugation and washing was performed in saline solution (0.9 wt% NaCl) (four cycles in cold saline). Small aliquots of supernatant were collected from the centrifuged gel batches after each washing step for analysis, as described above, to detect (a) visual observation (color, turbidity) and (b) residual levels of urea and sulfamic acid. Additionally, DES cellulose was diluted in saline to a concentration of 1.5 wt% and then passed through a high-pressure homogenizer (GEA, PandaPlus2000) several times, once at 400 bar, once at 700 bar, and three times at 1,100 bar. Gels were prepared with DES CNF at concentrations including 1.1%, 1%, 0.9%, 0.8% and 0.7% (w / v). A transmission electron microscope image of homogenized deep eutectic solvent-treated cellulose nanofibers in MilliQ™ water is provided in FIG.

[0094] After completing washing with hot MilliQ™ water (4 times) and cold MilliQ™ water (4 times), the dry mass of CNF in the prepared gel was measured by a Mettler Toledo moisture content analyzer (HX204 moisture analyzer). To calculate the amount of salt after solvent exchange with PBS buffer, the dry mass of CNF + PBS salt was also measured by a Mettler Toledo moisture content analyzer.

[0095] The ratio of CNF to salt (e.g., from PBS) was determined by thermogravimetric analysis (TGA) of the dried gels, which also allowed accurate normalization of the gel formulations in terms of the true and exact spinifex CNF content (w / v%) for subsequent rheological and injectability measurements.

[0096] The above steps were carried out with high purity reagents where applicable.

[0097] A very similar process is shown in Figure 1, where the milled and washed cellulose nanofibers are shown at 2, which are delignified at 4. The nanofibers are bleached at 6 and then dried at 8, before being contacted with a deep eutectic solvent at 10. The cellulose nanofibers complexed with the deep eutectic solvent are washed at 12 and then homogenized at 14 to provide an injectable composition 16. The washing step 12 may include a solvent exchange step 13.

[0098] Example 2: Quality Control Assessment For residual metal contaminants, samples were submitted for inductively coupled plasma-optical emission spectroscopy (ICP-OES) after each step of the treatment, and the results are provided in Table 3 below.

[0099] [Table 3]

[0100] X-ray photoelectron spectroscopy (XPS) surface analysis of the pulp before and after DES treatment showed that N and S were present only after DES treatment, indicating DES sulfation.

[0101] Example 3: Rheology of DES gels and commercial dermal fillers The gels prepared in the aforementioned experiments were tested for their rheological properties and the results were compared with published results for the hyaluronic acid (HA) dermal fillers Restylane™, Restylane™ LIPP, Restylane™ SubQ and Juvederm™ 24HV (Falcone, SJ and R.A Berg, Crosslinked hyaluronic acid dermal fillers: A comparison of rheological properties. Journal of Biomedical Materials Research Part A, 2008.87A(1):p.264-271). The results are shown in Figures 2 and 3. In Figures 2 and 3, BL DES stands for the gel prepared according to Example 1. BL DES gel is the gel prepared in sterile saline (Figure 2). BL DES PBS gel is the gel prepared in PBS (Figure 3).

[0102] Figures 4 and 5 show the rheology of the gels prepared according to Example 1 compared to actual commercially available dermal filler samples. The commercially available dermal filler samples include Figures 4A and 4B - Restylane™ and Restylane™ LYPS (gels containing 20 mg / mL hyaluronic acid and lidocaine), Figures 5A and 5B - Juvederm™ Volift (gels containing 17.5 mg / mL hyaluronic acid and lidocaine) and Juvederm™ Ultra XC (gels containing 24 mg / mL hyaluronic acid and lidocaine). Comparing Figures 4 and 5, the CNF of the gel in Figure 1 is between 6 and 10 mg / mL.

[0103] In Figures 2-5, dynamic rheological measurements were performed on a TA Instruments rheometer (AR1500) with a cone and plate geometry (40 mm diameter). All measurements were performed at 25 °C. Dynamic measurements were performed over a frequency range of 0.1-100 Hz. Experiments were performed in triplicate.

[0104] Example 4: Injectability of DES gel and commercial dermal fillers Measurements of the injection force required to push the gel through a 30G needle were performed in compression mode on an Instron mechanical testing machine with a 500N load cell. A 1mL syringe filled with 1mL of gel formulation (no air bubbles) and fitted with a 30G or 31G needle was placed face down in the holder. The plunger end of the needle was placed in contact with the load cell assembly. Tests were performed at a crosshead speed of 1mm / s, which is representative of manual syringe delivery to a patient. The load force required to displace the plunger was measured as a function of displacement. The following parameters were determined: -Initial slippage force / plunger loosening force: the force required to initiate plunger movement, - Maximum force: the maximum force measured before the plunger completes its stroke; - Dynamic sliding force: the force required to maintain the plunger motion and expel the contents of the syringe.

[0105] The force values ​​were normalized by the cross-sectional area of ​​the cylindrical plunger. Experiments were performed in triplicate.

[0106] The results are shown in Figures 6A and 6B. Figure 6A shows the force required to displace the various gels of Example 1. Figure 6B shows the sliding force of the DES gel (in saline) using 30 and 31 gauge needles. As shown in Figure 6B, the force required for the 30 and 31 gauge needles is very similar.

[0107] Compared to Restylane™, Restylane™ LYPS, Juvederm™ Ultra XC and Juvederm™ Volift, the force required to inject the 0.88% w / v DES CNF gel of Example 1 is significantly less. This is shown in Figure 7. Restylane contains particles of cross-linked hyaluronic acid of approximately 250 microns. The gel of Example 1 contains rods approximately 500-1,000 nm long by approximately 3 nm wide.

[0108] Example 5: Cell proliferation / cytotoxicity assay Gels washed with MilliQ™ purified water (no PBS) were prepared and supplied in sterile containers at two starting concentrations (0.8% and 0.4% (w / v)). For cytotoxicity assays, fibroblast (3T3) cell lines were seeded in 96-well plates at 2.5-5 × 103 cells / well in DMEM. CNF gels were diluted as follows and sterilized by microwave treatment (5 seconds at 60% power until just before boiling):

[0109] The final gel concentration was 0.5% (final volume 3.2 mL) containing: -0.8% gel solution -2.0mL - 10x Dulbecco's Modified Eagle Medium (DMEM) - 0.32 mL - Fetal calf serum (FCS) - 0.32 mL -Distilled water (DW) - 1.2mL

[0110] Final gel concentration 0.1% (final volume 4.0 mL) -0.4% gel solution 1.0mL -10 times DMEM 0.4 mL -FCS 0.4mL -DW 2.2mL

[0111] Plate 1 - Cell proliferation was measured by MTT assay. 0% (medium only), 0.1% and 0.5% gel were added to the wells and incubated for 24 or 48 hours. 0.01 mL of MTT reagent (Sigma Aldrich) was added for 8 hours, followed by SDS reagent overnight at 37°C (5% CO2). The absorbance of the formazan product was read on a Tecan plate reader (at 570 nm, according to the manufacturer's instructions).

[0112] Plate 2-3T3 cells at approximately 5 × 10 3Cells / well were seeded into black clear bottom 96-well plates. Cellulose gel (or media control) was added to the wells. Media was removed, washed once with Optimem media without phenol red, and PI / Hoechst33342 dye 1 / 1000 in Optimem was added for 1 hour before reading fluorescence on a Tecan plate reader.

[0113] Plate 3-CNF gel (0.5%, 0.1%) was added to wells of a 96-well plate (0.1 mL / well) and left overnight. 3T3 cells were seeded at this point (approximately 5 × 10 cells in DMEM + 10% FCS) onto the CNF gel and onto empty well tissue culture plastic. 3 cells / well) and incubated for 5 days before being examined under a light microscope at two magnifications (×10, ×20).

[0114] Tests showed that the cellulose gel of Example 1 did not kill the cells and the cells did not grow on the gel. The cells did not grow in the presence of cellulose gel as well as in its absence, but the cells that grew in the presence of cellulose gel were viable.

[0115] Example 6: Gel aging of DES gel and commercial dermal fillers Test conditions were set up to evaluate the in vitro stability of both MilliQ™ purified water and PBS gel preparations. 20 mL glass vials were filled with approximately 15 mL of DES CNF gel sample and the vials were sealed with their caps. For each DES CNF sample, seven glass vials were prepared for each temperature condition (one glass vial per time point and two additional glass vials were used for visual inspection). Samples were stored for the required period either in a refrigerator at 4° C., on a laboratory bench at 23° C., or in an oven set at 55° C. Shelf life studies were conducted with (a) DES CNF 0.8% (w / v) in MilliQ™ purified water and (b) DES CNF 1% (w / v) in PBS, with samples taken at 0, 7, 14, 30, 60, and 90 days at 4° C. (i.e., refrigerated storage period), 23° C. (i.e., room temperature storage period), and 55° C. (i.e., accelerated storage period). Testing at 50°C for 7.5 weeks or 60°C for 3.7 weeks corresponds to approximately 1 year for plastics (General Aging Theory and Simplified Protocol for Accelerated Aging of Medical Devices, Karl J. Hemmerich, July 1, 1998, Testing). Rheological measurements are provided in Figures 8-10. Figure 8 provides aging results at 4°C, Figure 9 at 23°C, and Figure 10 at 55°C. Figures 8-10 are for DES CNF 0.8% (w / v) in MilliQ™ purified water. Similar results were obtained for DES CNF 1% (w / v) in PBS. In Figures 8-10, D0 is day 0, D7 is day 7, D14 is day 14, etc.

[0116] Further, shelf-life studies were conducted with (a) DES CNF 0.8% (w / v) in MilliQ™ purified water and (b) DES CNF 1% (w / v) in PBS at 37°C (i.e., biological stability) under 5% CO2 atmosphere, with samples taken on days 0, 7, 14, 30, 60, and 90. In this experiment, 20 mL glass vials were filled with approximately 15 mL of DES CNF gel sample and each cap was loosely closed (i.e., quarter turn). For each DES CNF sample, seven glass vials were prepared for each temperature condition (one glass vial per time point and two additional glass vials were used for visual inspection). The samples were then stored in an incubator at 37°C under 5% CO2 atmosphere for the required period. The water dish at the bottom of the incubator was checked weekly and water was replenished as necessary. Figure 11 provides the aging results of DES CNF 0.8% (w / v) in MilliQ™ purified water. Similar results were obtained for DES CNF 1% (w / v) in PBS.

[0117] During the course of these experiments, it was ensured that the samples did not dry out (i.e., samples were replenished with liquid as necessary).

[0118] The results were compared to those of commercially available dermal fillers including Restylane™, Restylane™ LYPS, Juvederm™ Volift, and Juvederm™ Ultra XC. The results are presented in Figures 12A and 12B (aging at 55°C, G' or G" at 0.1 Hz).

[0119] The injectability of DES CNF samples was also evaluated for (a) DES CNF 0.8% (w / v) in MilliQ™ purified water and (b) DES CNF 1% (w / v) in PBS. The sliding force (N) was evaluated for samples stored at 4°C, 23°C, 37°C, under 5% CO2, and 55°C for 0, 7, 14, 30, 60, and 90 days. For DES CNF 0.8% (w / v), the sliding force on day 0 was about 4.7 N and increased to about 5.7 N to 6.7 N over 90 days. For DES CNF 1% (w / v) in PBS, the sliding force on day 0 was about 1.6 N and increased to about 2.5 N to 3.2 N over 90 days.

[0120] Example 7: Reversibility Test Cellulase from T. reesei (Sigma, ≥700 units / g) was dissolved in PBS at various concentrations (0.1%, 0.5%, 1% and 2% by weight) and added to DES CNF 1% (w / v) incubated at 37° C. Visual observations were performed over the incubation period.

[0121] Cellulose activity was assessed by evaluating the presence of free carbonyl groups (C=O) (reducing sugars) at different incubation times using the DNS (dinitrosalicylic acid) assay (Miller (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31, 3 p. 426-428). Rheology was performed on the enzymatically degraded DES gels at different incubation times. The results are provided in Table 4 below. As the time or cellulase concentration increases, the rate at which cellulose degrades increases.

[0122] [Table 4]

[0123] Similarly, the storage modulus (G') and loss modulus (G") of the samples decreased upon exposure to cellulase and were affected by the cellulase concentration. For example, after the addition of 1 mL of 2% cellulase for 1 h at 37 °C, G' decreased by almost 10-fold.

[0124] Example 8: Animal studies C57BI / 6 mice were subcutaneously injected with DES CNF gel (1.0% DES CNF gel in saline), 0.1 mL per injection, at 4 different dorsal sites per mouse (left and right flanks, left and right shoulders, control (saline) administered at left shoulder and flank, sample administered at right shoulder and flank). Mice received either 1.0% DES CNF gel in saline as sample or commercial hyaluronic acid (HA) (Restylane™) as sample. The study also included control mice that did not receive any injections. Mice were monitored daily for signs of irritation or swelling. At weekly intervals, mice were euthanized at 7, 14, 21, 28 and 56 days after gel implantation (n=2 or 3 per time point). The implanted gels and surrounding tissues were removed for histological analysis to determine the cellular response to the gel. Prior to injection, the cellulose was UV irradiated for 50 minutes to sterilize the composition.

[0125] After mice were sacrificed, tissue samples were fixed with 4% paraformaldehyde (PFA) and stained with hematoxylin and eosin (H&E). When necessary, tissues were stored in 70% ethanol until processing. 6 μm tissue sections were taken for each sample.

[0126] It was observed that the "softer" cellulose gel (or DES CNF gel) produced a longer bolus after 7 days than the HA control (which was rounder).

[0127] After 14 days, the HA boluses lost their "round" shape and appeared to be less able to withstand histological processing (presumably because the HA degraded over time), however, after 14 days the cellulose treatments were largely intact and still able to withstand histological processing.

[0128] After 56 days, the volume of the bolus of the cellulose sample was estimated to be approximately 50% of its original volume on day 0. The bolus of the HA sample also degraded.

[0129] Whole blood was collected from each mouse at necropsy to determine whether there was an increase in circulating white blood cells. No differences in white blood cell counts were found between treatment groups on days 7 and 14. There was no sign of a systemic inflammatory response, and any inflammation was most likely localized to the injection site.

[0130] In accordance with the statute, the invention has been described in language more or less specific to structural or systematic features. It is to be understood that the invention is not limited to the specific features shown or described, since the means described herein include preferred forms of carrying out the invention. The invention is therefore claimed in any of its forms or modifications within the proper scope of the appended claims as appropriately interpreted by those skilled in the art.

Claims

1. 1. A method for preparing an injectable composition, said method comprising: (i) contacting cellulosic fibers with a deep eutectic solvent to provide deep eutectic solvent-treated cellulosic fibers; (ii) washing the deep eutectic solvent-treated cellulosic fibers; and (iii) homogenizing or mechanically micronizing the washed, deep eutectic solvent-treated cellulosic fibers to provide the injectable composition. A method comprising:

2. The method described in claim 1, wherein the injectable composition is for cosmetic use.

3. 3. The method of claim 2, wherein step (i) of the method is preceded by a step of delignifying the cellulosic fibers and optionally bleaching the delignified cellulosic fibers.

4. 4. The method of claim 2 or claim 3, wherein the step of contacting the cellulose nanofibers with a deep eutectic solvent is carried out at a temperature of 120°C to 180°C and at a molar ratio of cellulose nanofibers to deep eutectic solvent of 1:2 to 1:

30.

5. The deep eutectic solvent is formed from a Lewis acid and a Lewis base, the Lewis acid comprises an ammonium, phosphonium, or sulfonium cation, an amine, an amide, a carboxylic acid, or a polyol; The method of any one of claims 2 to 4, wherein the Lewis base comprises a nitrogen atom or comprises an amide group, a urea group, a carbamate group, or an ammonium group.

6. 6. The method of claim 5, wherein the deep eutectic solvent is urea sulfamate.

7. The method according to any one of claims 2 to 6, wherein the cellulosic fibres are derived from plants of the subfamily Triodinae.

8. A cosmetic injectable composition prepared by the method of any one of claims 1 to 7.

9. 10. Use of the injectable composition of claim 8 as a dermal filler.

10. The injectable composition of claim 8 for platelet-rich plasma therapy, further comprising platelets.

11. 10. A cosmetic method for increasing volume in or under the skin of a subject, comprising injecting the injectable composition of claim 8 into or under the skin of the subject.

12. The method of claim 11 , wherein the method is for increasing lip, breast, or cheek volume of the subject.

Images