Methods for producing water-resistant chitosan adhesive and uses thereof

Abstract

A method for preparing an adhesive, said method comprising - providing catechol substituted chitosan in an aqueous solution with a pH in the range of pH 3-5, - admixing said aqueous solution with a salt to obtain a polymer-rich phase and a polymer-poor phase; and - isolating the polymer-rich phase to obtain said adhesive. Described is also an adhesive obtainable from said method and uses thereof.

Description

[0001] METHODS FOR PRODUCING WATER-RESISTANT CHITOSAN ADHESIVE AND USES THEREOF

[0002] FIELD

[0003] The field of the invention is provision of adhesives based on biomolecules.

[0004] BACKGROUND

[0005] It is a growing desire to provide 100% biodegradable products for chemicals used in different industries. Biopolymers have received considerable attention in recent years given the environmental concern related to the incorrect disposal of materials based on synthetic plastics.

[0006] This is also true in the field of adhesives, where renewable materials from biopolymers to monomers derived from renewable resources are increasingly investigated.

[0007] The sustainability of biomolecules-based (bio-based) products is, however, not a sufficient argument for their commercialisation. For example, the bio-based adhesives should perform at least comparably to commercial counterparts, which is a challenging benchmark.

[0008] In this context, poor water-resistance is a critical shortcoming of bio-based adhesives that makes them unfavorable for outdoor applications. Water penetration within the bio-based adhesives produces swelling, plasticization, and leaching of the adhesive joint, all of which can cause a cohesive failure. Aside from cohesive weakening, the adhesive-substrate interfacial adhesion is also diminished in wet conditions.

[0009] The development of materials with novel functionalities such as underwater adhesion and self-healing has been greatly inspired by mussels. A mechanism which is linked to the mussel adhesive's chemistry pertains to the physical properties of the mussel adhesive, suggesting that mussel adhesive proteins, in particular mfp-3, in the coacervate form can easily spread onto underwater surfaces without being dispersed into the water.

[0010] Various strategies have been employed in the design of mussel-inspired materials. However, a common requirement among the suggested strategies is the inclusion of at least two isolated components that possess both catechol and amine functionalities and can undergo complexation / coacervation between them. SUMMARY

[0011] The problem underlying the invention is the fact that bio-based adhesives, in general, cannot perform as well as the synthetic ones. A major issue with bio-based adhesives is their poor water-resistance. This means the adhesive, when exposed to humidity or liquid water, becomes significantly weaker and often falls apart. Thus, bio-based adhesives in general are not suitable for wet applications, such as underwater or tissue adhesion.

[0012] The problem underlying the invention is to provide new compositions based on the biomolecules, that can be used as a single component or multi component adhesive with improved properties, wherein the adhesive mainly consists of self-complexed catechol substituted chitosan (Chi-C).

[0013] The problem of the invention has been solved by the subject-matter as defined in the independent claims.

[0014] The invention provides a bio-based adhesive obtainable by preparing a catechol substituted chitosan, with a catechol content of 20%-60%, and admixing to said Chi-C in an aqueous solution with a pH in the range of pH 3-5, an amount of salt, that induces the formation of two phases (phase separation) , namely a polymer-rich (water-poor phase) phase and a polymer-poor phase (water-rich phase) and isolating the polymer-rich phase. The obtained polymer-rich phase contains a self-complexed material of Chi-C with strong cohesive interactions and cross-linking.

[0015] Accordingly, the present invention provides a method for preparing an adhesive, said method comprising providing catechol substituted chitosan in an aqueous solution with a pH in the range of pH 3-5, admixing said aqueous solution with a salt to obtain a polymer-rich phase and a polymer-poor phase and isolating the polymer-rich phase to obtain said adhesive.

[0016] Preferably the salt, preferably NaCI, is present in an amount in the range of 0.18-2M.

[0017] In embodiments, the catechol content in the catechol substituted chitosan (Chi-C) is in the range of 15-60 % (wt.%), and the chitosan has a degree of deacetylation of chitosan in the range of 70-99%, preferably in the range of 75-85%. In embodiments of said method the salt is NaCI and the pH of the aqueous solution is about pH 3 and; the catechol content in the catechol substituted chitosan is in the range of 15-22%, preferably about 20%, and the salt content is in the range of 1.15-3 M, preferably in the range of 1 .2-2M; the catechol content in the catechol substituted chitosan is in the range of 23-30%, preferably about 25%, and the salt content is in the range of 0.85-1.95 M, preferably in the range of 1.0-1.8 M; the catechol content in the catechol substituted chitosan is in the range of 30-40%, preferably about 34%, and the salt content is in the range of 0.5-1.0M, preferably in the range of 0.6-0.9M; the catechol content in the catechol substituted chitosan is in the range of 41-50%, preferably about 43%, and the salt content is in the range of 0.35-0.55M, preferably in the range of 0.4-0.5M; or the catechol content in the catechol substituted chitosan is in the range of 50-60%, preferably about 54%, and the salt content is in the range of 0.2-0.4M, preferably about 0.3M.

[0018] Alternatively, the salt is NaCI and the pH of the aqueous solution is about pH 4.5 and; the catechol content in the catechol substituted chitosan is in the range of 15-22%, preferably about 20%, and the salt content is in the range of 0.9-1.5 M, preferably about 1 M; the catechol content in the catechol substituted chitosan is in the range of 23-30%, preferably about 25%, and the salt content is in the range of 0.5-1.5 M, preferably in the range of 0.6-1 .25 M; the catechol content in the catechol substituted chitosan is in the range of 30-40%, preferably about 34%, and the salt content is in the range of 0.3-1.0M, preferably in the range of 0.5-0.7M; the catechol content in the catechol substituted chitosan is in the range of 41-50%, preferably about 43%, and the salt content is in the range of 0.2-0.6M, preferably in the range of 0.3-0.5M; the catechol content in the catechol substituted chitosan is in the range of 50-60%, preferably about 54%, and the salt content is in the range of 0.2-0.4M, preferably in the range of 0.2-0.3M. Said isolation step may comprise a step of centrifugation, filtration, sedimentation, decantation, or flocculation or any combination thereof. Alternatively, to centrifuging the mixture it is possible to vacuum filter the mixture to obtain the polymer-rich phase. As mentioned above, other methods may also be used such as sedimentation, decantation or flocculation or any combination thereof.

[0019] Additionally, said method may further comprise a step of curing the isolated adhesive, such as enzymatic curing, such as e.g., enzymatic curing using a mixture of Horseradish peroxidase (HRP) and an oxidative agent, preferably H2O2. Preferably, the Chi-C:HRP weight ratio is in the range of 150:1-10.000:1 , preferably 1875:1 or 1500:1 , and the Chi- C:H2C>2 weight ratio is in the range of 50:1 to 6500:1 , preferably 625:1.

[0020] Moreover, the present invention relates to an adhesive obtainable by providing catechol substituted chitosan in an aqueous solution with a pH in the range of pH 3-5, admixing said aqueous solution with a salt to obtain a polymer-rich phase and a polymer-poor phase; and isolating the polymer-rich phase to obtain said adhesive. In embodiments, such adhesive is characterized by an uncured underwater adhesive strength measured using a Universal Testing System of at least 100 kPa, when said adhesive is evenly applied underwater to aluminum substrates. In further embodiments, such an adhesive is characterized by an underwater adhesive strength measured using a Universal Testing System of at least 300 kPa, when said adhesive is evenly applied underwater to aluminum substrates and enzymatically cured underwater for 10-30 min. In further embodiments, such an adhesive is characterized by a tensile adhesion strength measured using a Universal Testing System of at least 200 kPa when said adhesive is evenly applied to aluminum substrates. In further embodiments, such an adhesive is characterized by a tensile adhesion strength measured using a Universal Testing System of at least 500 kPa when said adhesive is evenly applied to aluminum substrates, and enzymatically cured for 10-30 min.

[0021] In additional aspects that invention also relates to the use of said adhesive as a surgical adhesive.

[0022] In further aspects the invention relates to a gel consisting of catechol substituted chitosan, wherein catechol substituted chitosan is present in the gel in a total concentration of from 1 to 25 wt% such as from 5 to 20 wt%, from 10 to 15 wt%, such as about 15 wt%. In additional aspects the invention relates to a kit comprising; a tissue sealant comprising a catechol substituted chitosan with a catechol content in the range of 15-60 %, A curing agent, and optionally, Instructions for use.

[0023] BRIEF DESCRIPTION OF FIGURES

[0024] FIGURE 1. Phase separation of Chi-C solution at pH 3.0 by mixing NaCI solution with a Chi- C solution.

[0025] FIGURE 2. Verification of catechol conjugation to chitosan, (a) UV-Vis spectra of Chi-C solutions at 0.1 mg / mL and (b) UV-Vis standard curve using dopamine solutions.

[0026] FIGURE 3. Phase separation of Chi-C solution at pH 3.0 effected on catechol content and ionic strength. Black indicates the formation of a high amount of precipitate collected following centrifugation, where the precipitate can be described as an elastic tough material with less fluid properties and limited spread ability. Dark grey indicates a relative high amount of precipitate collected after centrifugation, where the precipitate can be described as a soft hydrogel which can be applied onto a substrate or injected using a syringe. Midgrey indicates a relatively small amount of precipitate collected by centrifugation. Light grey indicates conditions where no phase separation was observed.

[0027] FIGURE 4. Phase separation of Chi-C solutions for different catechol / salt combinations at pH 3.

[0028] FIGURE 5. Adhesion strength underwater as a function of ionic strength and catechol content. Grey bars indicate an injectable glue-like precipitate. Black bars exhibit a solid- / rubbery-like precipitate. The data plotted as the average ± standard deviations (n = 5).

[0029] FIGURE 6. Mechanical properties investigation of Chi-C precipitate with and without HRP:H2O2. The rheological properties evaluated as a function of curing time a) in the beginning and b) curing after 15 minutes. Solid / dashed lines represent storage / loss modulus, respectively. FIGURE 7. Visual investigation showing compression and resilience behaviors, (top row) precipitated Chi-C phase without HRP:H2O2 added (bottom row) precipitated Chi-C phase with HRP:H2C>2 added.

[0030] FIGURE 8. Adhesion strength under water of cured and uncured adhesive.

[0031] FIGURE 9. Adhesion strength under water of the optimized two-component Chi-C adhesive vs. time.

[0032] FIGURE 10. Tissue adhesion test on pig skin, using uncured Chi-C, HRP:H2O2 cured Chi-C against commercially available BSA / glutaraldehyde tissue glue (BIOGLUE®) and N-butyl cyanoacrylate based glue (3M Vetbond Tissue glue).

[0033] FIGURE 11 . Phase diagram of Chi-C solutions at pH 5. Black indicates the formation of a high amount of precipitate collected following centrifugation, where the precipitate can be described as an elastic tough material with less fluid properties and limited spread ability. Dark grey indicates a relative high amount of precipitate collected after centrifugation, where the precipitate can be described as a soft hydrogel which can be applied onto a substrate or injected using a syringe. Mid-grey indicates a relatively small amount of precipitate collected by centrifugation. Light grey indicates conditions where no phase separation was observed.

[0034] DETAILED DESCRIPTION

[0035] Adhesives are man-made products that are used in almost any industry. Despite their favourable functionality, most commercial adhesives are petroleum-based (synthetic polymers) and contain harmful volatile substances e.g., formaldehyde. To reduce the health and environmental footprints of these adhesives, research is dedicated to developing novel adhesives that are made from natural, bio-based materials, such as proteins, polysaccharides, and plant phenolics. The issue is that bio-based adhesives, in general, cannot perform as well as the synthetic ones. A major issue with bio-based adhesives is their poor water-resistance. This means the adhesive, when exposed to humidity or liquids such as water or blood, becomes significantly weaker and often falls apart. Thus, bio-based adhesives in general are not suitable for outdoor (exterior) or surgical applications. This shortcoming significantly hampers the widespread use of bio-based adhesive. The present invention addresses this problem and relates to a method of obtaining a fully bio-based adhesive that can bind to wet surfaces and is water resistant, with commerciallevel water-resistance.

[0036] Mussel adhesive proteins, particularly mfp-3, undergo self-complexation without the need for another complexing agent. This occurs due to the electrostatic screening in sea water where the relatively high ionic strength shields long-range electrostatic interactions between individual proteins and allows short-range inter- and intra-chain interactions in the form of cation-pi and pi-pi interactions.

[0037] Drawing inspiration from this phenomenon, the inventors herein suggest that other molecules with amine and catechol functionalities could be designed to undergo phase separation / self-complexation resulting in a condense precipitated phase.

[0038] Accordingly, the adhesive is a single component adhesive. The adhesive consists of a polysaccharide component (chitosan e.g., derived from crustaceans such as, e.g., shrimp shell, or mushrooms) which is deacetylated and subsequently conjugated with catechol groups (e.g., Dihydroxybenzoic acid (DHBA). Under optimal conditions (e.g., pH and ionic strength) and at specific degrees of catechol conjugation the Chi-C undergo self- complexation / precipitation. This means that by mixing aqueous solutions of Chi-C under specific ionic and pH conditions it is possible to obtain a precipitated material, that can be isolated and used as an adhesive.

[0039] Thus, in a first aspect the present invention relates to a method for preparing an adhesive, said method comprising providing catechol substituted chitosan in an aqueous solution with a pH in the range of pH 3-5, admixing said aqueous solution with a salt to obtain a polymer-rich phase and a polymer-poor phase; and isolating the polymer-rich phase to obtain said adhesive.

[0040] Method for preparing catechol modified chitosan adhesive

[0041] The present invention relates to a method for preparing a single component catechol modified chitosan adhesive which mimics mussel adhesive in its formation of a hydrogel like material which is suitable for application by smearing or injection. As it had previously been demonstrated that amine and catechol functionalities could also undergo self-complexation, when short-ranged attractive interactions and long-ranged electrostatic repulsion is balanced by addition of salt, the inventors envisioned that the amine-rich structure of deacetylated chitosan combined with the substitution on some of the amine groups with a catechol group, the short-ranged attractive interactions and long-ranged electrostatic repulsion could be balanced by salt such that the Chi-C self- complexes / precipitates, which results in two phases, which can be described as a polymer rich phase containing Chi-C in condensed state and a polymer poor phase, containing less mostly soluble / non-condensed Chi-C.

[0042] The present invention thus relates to a method for preparing an adhesive, said method comprises providing catechol substituted chitosan in an aqueous solution with a pH in the range of pH 1 .0-5.5, preferably in the range of pH 2-5, more preferred 3-5, such as pH 3.0, pH 3.1 , pH 3.2, pH 3.3, pH 3.4, pH 3.5, pH 3.6, pH 3.7, pH 3.8, pH 3.9, pH 4.0, pH 4.1 , pH 4.2, pH 4.3, pH 4.4, pH 4.5, pH 4.6, pH 4.7., pH 4.8, pH 4.9, or such as pH 5.0. As is shown in example 1 and 2, the pH is essential for both the production of catechol substituted chitosan, since chitosan is not easily dissolved at a pH above pH 5, which hinders the catechol substitution. Additionally, as is shown in example 2, self-complexation / phase separation by the addition of salt is more apparent at pH 3 than at pH 5.

[0043] Catechol substituted chitosan (Chi-C) is generally produced as described in example 1 , by reductive amination, where a carboxylic acid modified catechol e.g., DHBA may be conjugated to the deacetylated chitosan backbone, to varying degree, depending on the amount of deacetylation of the chitosan and the amount of added catechol in the substitution. The catechol content in the catechol substituted chitosan may e.g., be in the range of 1-65 % (wt.%%). As is shown in example 2, the amount of substituted catechol impacts the Chi-C phase separation, where there is a general inverse correlation between the amount of catechol substitution and the amount of salt required to induce Chi-C phase separation, where a larger amount of substituted catechol resulted in a lower amount of salt required to induce the phase separation. It is preferred that the catechol content in the catechol substituted chitosan is in the range of 1-60 % (wt. %), such as in the range of 15- 60%, such as in the range of 20-55 %, or such as about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or such as about 60%. Substitution of the chitosan polymers is as such not limited to catechol substitution. Chitosan may also be conjugated with alternative isomeric phenolic acids, such as e.g., hydroxybenzoic acids such as e.g., monohydroxybenzoic acids, dihydroxybenzoic acids, or trihydroxybenzoic acids, or hydroxycinnamic acids such as e.g., caffeic acid, ferulic acid, p- coumaric acid, sinapic acid and cinnamic acid. In the examples of the present disclosure, dihydroxybenzoic acid DHBA is used, but suitable alternatives will be evident to the skilled person. Examples of monohydroxybenzoic acids are e.g., selected from the group consisting of salicylic acid, p-hydroxybenzoic acid and m-hydroxybenzoic acid. Examples of dihydroxybenzoic acids may e.g., be selected from the group consisting of gentisic acid, protocatechuic acid, Catechol, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid and 2,6-dihydroxybenzoic acid. Examples of trihydroxybenzoic acids may e.g., be selected from the group consisting of gallic acid (3,4,5-trihydroxybenzoic acid), phloroglucinolcarboxylic acid (3,5,6-trihydroxybenzoic acid), 2,3,4-trihydroxybenzoic acid and 2,4,6-trihydroxybenzoic acid.

[0044] Chitosan is a linear polysaccharide composed of randomly distributed p-(1 — >4)-linked D- glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). It is made by treating the chitin from shrimp and other crustaceans with an alkaline solution, such as sodium hydroxide.

[0045] Chitosan may be obtained from chitin by chemical or enzymatic methods. In chemical methods, both acidic and alkaline extraction can be used, but the alkaline method is used more frequently.

[0046] Chitosan is produced commercially by deacetylation of chitin, which is the structural element in the exoskeleton of crustaceans (such as crabs and shrimp) and cell walls of fungi. The degree of deacetylation (%DD) can be determined by NMR spectroscopy, and the %DD in commercial chitosan ranges from 60 to 100%. For commercially produced chitosan, the viscosity range (1 wt. % in 1% acetic acid at 25 °C) is generally 5 - 5000 cps, corresponding to an average MW of 20,000-2,000,000 Daltons.

[0047] A common method for obtaining chitosan is the deacetylation of chitin using sodium hydroxide in excess as a reagent and water as a solvent. The reaction follows first-order kinetics though it occurs in two steps; the activation energy barrier for the first stage is estimated at 48.8 kJ-mol-1at 25-120 °C and is higher than the barrier to the second stage. The amino group in chitosan has a pKavalue of ~6.5, which leads to some protonation in neutral solution, increasing with increased acidity (decreased pH) and the %DD-value. This makes chitosan water-soluble and a bio-adhesive which readily binds to negatively charged surfaces such as mucosal membranes. The free amine groups on chitosan chains can make crosslinked polymeric networks with dicarboxylic acids to improve chitosan's mechanical properties. Some chitosan grades may have been obtained by chemical modifications so that they are more soluble in neutral pH.

[0048] Chitosan suitable for use in an adhesive according to the invention has a degree of deacetylation corresponding to the degree of deacetylation of commercially available chitosan (60 to 99%). Herein, it is preferred that the chitosan used to produce the catechol substituted chitosan (Chi-C) has a has a degree of deacetylation of chitosan in the range of 70-99%, preferably 75-85%, or such as above 75%.

[0049] Chitosan suitable for use in an adhesive according to the invention has a molecular weight corresponding to the molecular weight of commercially available chitosan (5,000-2,000,000 Daltons). Generally, in the context of the present invention low molecular weight chitosan is 50,000-190,000 Da, medium molecular weight chitosan is 190,000-310,000 Da and high molecular weight chitosan is about 310,000 to >375,000 Da. Typically, the molecular weight of a chitosan suitable for use in an adhesive of the invention is in a range from 50,000 to 750,000 Daltons such as from 50.000-190.000 Daltons, from 190.000-310.000 Daltons, from 310.000-375.000, such as about 50kDa, 55kDa, 60kDa, 65kDa, 70kDa, 75kDa, 80kDa, 85kDa, 90kDa, 95kDa, 100kDa, 110kDa, 120kDa, 130kDa, 140kDa, 150kDa, 160kDa, 170kDa, 180kDa, 190kDa, 200kDa, 210kDa, 220kDa, 230kDa, 240kDa, 250kDa, 260kDa, 270kDa, 280kDa, 290kDa, 300kDa, 310kDa, 320kDa, 330kDa, 340kDa, 350kDa, 360kDa, 370kDa or such as about 375kDa.

[0050] Chitosan suitable for use in an adhesive according to the invention has a viscosity corresponding to the viscosity of commercially available chitosan (5 - 5000 cps, 1 wt.% in 1% acetic acid at 25 °C). The viscosity is in a range of from 50 to 300 cps, such as 50 cps, 60 cps, 70 cps, 80 cps, 90 cps, 100 cps, 110 cps, 120 cps, 130 cps, 140 cps, 150 cps, 160 cps, 170 cps, 180 cps, 190 cps, 200 cps, 210 cps, 220 cps, 230 cps, 240 cps, 250 cps, 260 cps, 270 cps, 280 cps, 290 cps, 300 cps, or such as from 75 to 200 cps, or such as from 75 to 150 cps, or such as about 100cps Chitosan is soluble in dilute organic acid solutions but is insoluble in high concentrations of hydrogen ions and is precipitated as a gel-like compound. Chitosan is positively charged by amine groups, making it suitable for binding to negatively charged molecules. However, it has disadvantages such as low mechanical strength and low-temperature response rate; it must be combined with other gelling agents to improve its properties.

[0051] In the method for obtaining an adhesive, the Chi-C may be dissolved in water and then the pH may be adjusted to a pH in the range of 3.0 to 5.0. Because chitosan in general is very difficult to dissolve at higher pH values, such as above pH 5.0, the common approach is to first dissolve it in 1-2% acetic acid, and after dissolution, pH is adjusted to 3.0-5.0. Partial precipitation of chitosan generally occurs, but with time this can re-dissolved. Depending on the type of chitosan, one may be able to avoid the dissolution-in-acetic acid step, and add chitosan to water, and adjust the solution to pH to 3.0-.5.0 afterwards or immediately dissolve chitosan in the solution / buffer with pH of 3.0-5.0. Furthermore, it is possible to use chitosan derivatives such as chitosan hydrochloride, carboxymethyl chitosan, chitosan acetate, etc. which are much easier to dissolve in water. For example, when chitosan hydrochloride is used it dissolves immediately in water and reduces the pH value of the solution, so there is no need for initial dissolution in acetic acid.

[0052] Alternatively, the chitosan may be dissolved in an aqueous solution having a pH in the range of 3.0-5.0. The aqueous solution may be a suitable buffer solution such as an acetic acid / acetate buffer solution.

[0053] Isolation

[0054] The self-complexation / phase separation is obtained by addition of a salt to the Chi-C. The addition of salt may be done e.g., by admixing to the Chi-C solution a salt to obtain a polymer-rich phase and a polymer-poor phase.

[0055] A "polymer-rich phase" as referred to herein refers to a distinct portion within a heterogeneous system, typically composed of a mixture of substances such as e.g., a saline solution of catechol substituted chitosan, in which polymers, such as the catechol substituted chitosan are present in higher concentrations compared to an adjacent "polymer-poor phase”. The polymer-rich phase is generally recognised as the phase which may be isolated in the bottom of a solution following isolation by e.g., centrifugation or sedimentation, as is shown in figures 1 and 12.

[0056] Following formation of the polymer-rich phase and a polymer-poor phase, the polymer rich phase may be isolated from the polymer poor phase. Said isolation step may comprise a step of centrifugation, filtration, sedimentation, decantation, or flocculation any or combination thereof. Additional suitable methods that allow for isolation of the polymer-rich phase will be evident to the skilled person.

[0057] The phase separated Chi-C which are isolated as the polymer-rich phase may comprise the following properties: it is a gel (contains water, not a dry solid material),

[0058] It has a white-milky color, It is opaque.

[0059] Salt and catechol content

[0060] The physical properties of the isolated Chi-C were found to be highly dependent on the catechol content and the salt content (as shown in Figure 3 and 10). Discrete values of both salt and catechol resulted in injectable glue-like materials which optimal for the applications as an adhesive, which e.g., needs to be applied to surfaces. As shown in Figure 3 and 10, increasing the amount of salt in samples which otherwise produced the glue-like materials (for example an increase in salt content from 0.9M to 1.0 and above at a catechol content of 34%) resulted in a rubber-like material which was harder and more granular, which was less favored for the application as injectable glue-like adhesive, but may be useful in other applications. Generally, the harder rubber like materials also showed a lesser adhesive strength, as is also shown in Figure 5.

[0061] As is shown in example 2 and figure 3, 4, 5 and 11 the salt content, pH and catechol content greatly influence the properties of the Chi-C material isolated once phase separated / precipitated. In embodiments, the amount of salt admixed to the Chi-C solution results in a salt content in the range of 0.18-2M. Preferably, the salt content is in the range of 1.15-3 M, 0.85-1.95 M, 0.5-1 .0M, 0.35-0.55M or in the range of 0.2-0.4M, more preferably in the range of 1 .2-2M, 1 .0-1 .8 M, 0.6-0.9M, 0.4-0.5M, or in the range of 0.25-0.35M, for a catechol content of about 20%, 25%, 34%, 43%, or 54%, respectively.

[0062] In embodiments, the catechol content in the catechol substituted chitosan is in the range of

[0063] 10-60%, such as 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%,

[0064] 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%,

[0065] 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%,

[0066] 55%, 56%, 57%, 58%, 59%, or such as 60%. In embodiments, the catechol content in the catechol substituted chitosan is in the range of 15-22%, preferably about 20%.

[0067] In embodiments, the catechol content in the catechol substituted chitosan is in the range of23-30%, preferably about 25%.

[0068] In embodiments, the catechol content in the catechol substituted chitosan is in the range of30-40%, preferably about 34%.

[0069] In embodiments, the catechol content in the catechol substituted chitosan is in the range of 41-50%, preferably about 43%.

[0070] In embodiments, the catechol content in the catechol substituted chitosan is in the range of range of 50-60%, preferably about 54%.

[0071] Specifically, salt ranges which are preferred to specific catechol contents at a pH of the solution of about pH 3 are e.g.;

[0072] For a catechol content in the catechol substituted chitosan in the range of 15-22%, preferably about 20%, the salt content is in the range of 1.15-3 M, preferably in the range of 1.2-2M; or

[0073] For a catechol content in the catechol substituted chitosan in the range of 23-30%, preferably about 25%, the salt content is in the range of 0.85-1.95 M, preferably in the range of 1 .0-1 .8 M; or

[0074] For a catechol content in the catechol substituted chitosan in the range of 30-40%, preferably about 34%, the salt content is in the range of 0.5-1 .0M, preferably in the range of 0.6-0.9M; or

[0075] For a catechol content in the catechol substituted chitosan in the range of 41-50%, preferably about 43%, the salt content is in the range of 0.35-0.55M, preferably in the range of 0.4-0.5M; or

[0076] For a catechol content in the catechol substituted chitosan in the range of 50-60%, preferably about 54%, the salt content is in the range of 0.2-0.4M, preferably about 0.3M.

[0077] Specifically, salt ranges which are preferred to specific catechol contents at a pH of the solution of about pH 5 are e.g.;

[0078] For a catechol content in the catechol substituted chitosan in the range of 15-22%, preferably about 20%, the salt content is in the range of 0.9-1 .5 M, preferably about 1M; or For a catechol content in the catechol substituted chitosan in the range of 23-30%, preferably about 25%, the salt content is in the range of 0.5-1 .5 M, preferably in the range of 0.6-1.25 M; or

[0079] For a catechol content in the catechol substituted chitosan in the range of 30-40%, preferably about 34%, the salt content is in the range of 0.3-1 .0M, preferably in the range of 0.5-0.7M; or

[0080] For a catechol content in the catechol substituted chitosan in the range of 41-50%, preferably about 43%, the salt content is in the range of 0.2-0.6M, preferably in the range of 0.3-0.5M; or

[0081] For a catechol content in the catechol substituted chitosan in the range of 50-60%, preferably about 54%, the salt content is in the range of 0.2-0.4M, preferably in the range of 0.2-0.3M.

[0082] In embodiments, the salt is NaCI and the pH of the aqueous solution is about pH 3 and; the catechol content in the catechol substituted chitosan is 20%, and the salt content is in the range of 0.1 -3M; or the catechol content in the catechol substituted chitosan is 25%, and the salt content is in the range of 0.2-2M; or the catechol content in the catechol substituted chitosan is 34%, and the salt content is in the range of 0.1-1.5M; or the catechol content in the catechol substituted chitosan is 43%, and the salt content is in in the range of 0.1-1.5M; or the catechol content in the catechol substituted chitosan is 54%, and the salt content is in the range of 0.1-1 M.

[0083] In embodiments, the salt is NaCI and the pH of the aqueous solution is about pH 4.5 and; the catechol content in the catechol substituted chitosan is 20%, and the salt content is in the range 0.5-2M; or the catechol content in the catechol substituted chitosan is 25%, and the salt content is in range of 0.5-2M; or the catechol content in the catechol substituted chitosan is 34%, and the salt content is in range of 0.1-1 .5M; or the catechol content in the catechol substituted chitosan is 43%, and the salt content is in the range of 0.1-1.5M; or the catechol content in the catechol substituted chitosan is 54%, and the salt content is in the range of 0.1-1 M. In embodiments, the salt is NaCI and the pH of the aqueous solution is about pH 3 and the catechol content in the catechol substituted chitosan is 20%, and the salt content is in the range of 0.1-3M, such as 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1 M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M or such as 3M, such as in the range of in the range of 1.15-3 M, preferably in the range of 1.2-2M.

[0084] In embodiments, the salt is NaCI and the pH of the aqueous solution is about pH 3 and the catechol content in the catechol substituted chitosan is 25%, and the salt content is in the range of 0.2-2M such as 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1 M, 1.1M, 1.2M, 1 ,3M, 1 ,4M, 1 ,5M, 1 ,6M, 1 ,7M, 1 ,8M, 1 ,9M or such as 2M, such as in the range of in the range of 0.85-1 .95 M, preferably in the range of 1 .0-1 .8 M.

[0085] In embodiments, the salt is NaCI and the pH of the aqueous solution is about pH 3 and the catechol content in the catechol substituted chitosan is 34%, and the salt content is in the range of 0.1-1 ,5M, such as 0.1 M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1 M, 1.1 M, 1 ,2M, 1 ,3M, 1 ,4M or such as 1.5M, such as in the range of 0.5-1.0M, preferably in the range of 0.6-0.9M.

[0086] In embodiments, the salt is NaCI and the pH of the aqueous solution is about pH 3 and the catechol content in the catechol substituted chitosan is 43%, and the salt content is in in the range of 0.1-1M, such as 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, or such as 1 M, or such as in the range of 0.35M-0.55M, preferably in the range of 0.4-0.5M.

[0087] In embodiments, the salt is NaCI and the pH of the aqueous solution is about pH 3 and the catechol content in the catechol substituted chitosan is 54%, and the salt content is in the range of 0.1-1M, such as 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, or such as 1 M, or such as in the range of 0.2-0.4M, preferably about 0.3M.

[0088] In embodiments, the salt is NaCI and the pH of the aqueous solution is about pH 4.5 and the catechol content in the catechol substituted chitosan is 20%, and the salt content is in the range 0.5-2M, such as 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1 M, 1.2M, 1.3M, 1.4M, 1.5M, 1 ,6M, 1 ,7M, 1 ,8M, 1 ,9M or such as 2M, or such as in the range of 0.9-1.5 M, preferably about 1 M. In embodiments, the salt is NaCI and the pH of the aqueous solution is about pH 4.5 and the catechol content in the catechol substituted chitosan is 25%, and the salt content is in range of 0.5-2M, such as 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1 M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1 ,7M, 1 ,8M, 1 ,9M or such as 2M, or such as in the range of 0.5-1.5 M, preferably in the range of 0.6-1.25 M.

[0089] In embodiments, the salt is NaCI and the pH of the aqueous solution is about pH 4.5 and the catechol content in the catechol substituted chitosan is 34%, and the salt content is in range of 0.1-1.5M, such as 0.1 M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1M, 1 ,2M, 1 ,3M, 1 ,4M, or such as 1 .5M, or such as in the range of 0.3-1.0M, preferably in the range of 0.5-0.7M.

[0090] In embodiments, the salt is NaCI and the pH of the aqueous solution is about pH 4.5 and the catechol content in the catechol substituted chitosan is 43%, and the salt content is in the range of 0.1-1 ,5M, such as 0.1 M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1 M, 1.1 M, 1 ,2M, 1 ,3M, 1 ,4M, or such as 1.5M, or such as in the range of 0.2-0.6M, preferably in the range of 0.3-0.5M.

[0091] In embodiments, the salt is NaCI and the pH of the aqueous solution is about pH 4.5 and the catechol content in the catechol substituted chitosan is 54%, and the salt content is in the range of 0.1-1M, such as 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M or such as 1M, or such as in the range of 0.2-0.4M, preferably in the range of 0.2-0.3M.

[0092] In the examples provided herein the salt is NaCI, but other salts are also suitable to induce the Chi-C self-complexation. As such the salt is not limited to monovalent salts, but may also be bivalent, trivalent, or multivalent salts. Examples of alternative salts are e.g., KCI, CaCI2, MgCI2, KC4H5O6, NH4CI, NaHCO3, CaSO4, MgSO4, C2H3NaO2, K2SO4, CaCO3, Na2SO4, K2CO3, NaNO3, MgCO3, Ca3(PO4)2, AICI3, FeCI3, AI2(SO4)3, Fe2(SO4)3. Further suitable alternative salts which are capable of inducing phase separation are well know to the skilled person. In preferred embodiments, the salt is NaCI.

[0093] Phase separation

[0094] It is contemplated that phase separation takes place leading to a so-called “self-complexed” or “precipitated” or “coacervate” polymer-rich phase, alternatively also described in literature as liquid-liquid phase separation (LLPS), or a general phase separation of a molecular complex, so the aqueous medium containing Chi-C becomes opaque / turbid, and a precipitation of Chi-C coacervate / complex takes place. In the present context, the terms “complex”, “cross-linked product”, “precipitate” and “coacervate” is used for the product obtained when introducing a sufficient amount of salt, e.g., NaCI to the Chi-C containing solution in an aqueous medium having a pH of from pH 3-5.

[0095] In general, precipitation is considered the process of solute molecules coming out of a solution to form solid particles due to a change in conditions like e.g., pH or concentration. Phase separation, generally, involves the separation of a solution into two liquid phases resulting from specific molecular interactions, resulting in a phase rich in solutes i.e. , in the present case a polymer-rich phase, comprising most of the Chi-C. Generally, precipitation forms solid particles, while coacervation creates liquid droplets. Precipitation generally occurs when solubility limits are exceeded, while coacervation arises from phase separation driven by molecular interactions.

[0096] Accordingly, the phase separation occurring upon addition of salt to Chi-C may be investigated to evaluate if the produced complex has a precipitation or a coacervated nature.

[0097] Formation of a coacervate may be investigated in a number of ways known to the skilled person such as for example by visual observation, light microscopy, behavioral assessments (such as e.g., dilution of the polymer-rich phase) and / or rheological measurements.

[0098] In example, microscopic inspection using a light microscope, such as e.g., using a light microscope at e.g., 20x-1000x magnification, of the mixture can be used to determine the nature of the produced product, where coacervation generally results in the formation of dynamic oil-like droplets, while precipitation generally leads for formation of solid particles, wherein both states are generally visible when performing e.g., light microscopy.

[0099] In the present invention, a salt concentration that induces the formation of a polymer-rich phase and a polymer-poor phase is to be understood as a salt concentration at which the solution changes from a clear solution to an opaque solution containing at least two phases e.g., a polymer-rich and a polymer poor phase, and wherein the polymer-rich phase may be isolated as an injectable glue-like material. Such transition may be investigated in a plethora of ways known to the skilled person, such as by visual inspection, UV-vis absorbance and / or centrifugation and inspection of the pelleted fraction, as also disclosed in the examples presented herein.

[0100] The concentration of Chi-C in the mixture obtained is up to 80 mg / mL such as from 1 mg / mL to 75 mg / mL, from 2 mg / mL to 70 mg / mL, from 3 mg / mL to 60 mg / ml, from 3 mg / mL to 50 mg / mL, from 4 mg / mL to 40 mg / mL, from 4 mg / mL to 30 mg / mL, from 5 mg / mL to 20 mg / mL from 5 mg / mL to 15 mg / mL, from 6 mg / mL to 10 mg / mL or about 5 mg / mL, 6 mg / mL, 7mg / ml, 8 mg / mL, 9 mg / mL or 10 mg / mL, or such as up to 50 mg / mL before centrifugation and isolation of the polymer-rich fraction.

[0101] An adhesive

[0102] The present invention also relates to an adhesive comprising catechol substituted chitosan (Chi-C), wherein the adhesive has a glue-like structure, which is suitable for smearing or injection. Such adhesive comprises a chitosan with a deacetylation level of 75%-85%, and an amount of substituted catechol of about 20% to about 54%. Such an adhesive is obtainable via the methods described herein. The invention also relates to a gel consisting of catechol substituted chitosan, wherein catechol substituted chitosan is present in the gel in a total concentration of from 1 to 25 wt% such as from 5 to 20 wt%, from 10 to 15 wt%, such as about 15 wt%.

[0103] The adhesive is suitable for underwater applications and tissue adhesion, as well as adhesion of dry materials / substrates.

[0104] Substrates may e.g., be aluminum, poly(methyl methacrylate) (Acrylic), poly(carbonate) (PC), steel, plywood, and / or tissue. The shear strength also depend on the combination of the materials to be bound together, e.g., aluminum binding to poly(methyl methacrylate), aluminum binding to poly(carbonate) (PC), aluminum binding to steel, aluminum binding to plywood, plywood binding to plywood, tissue, such as skin, muscle or bone, binding to suitable substrates, or tissue, binding to tissue, or any alternative combination of the aforementioned. Further suitable substrates and combinations thereof to be adhered will be evident to the skilled person.

[0105] The surface of the substrate(s) to be joined also greatly influences the measured shear strength(s). Substrates such as e.g., plastics which are generally surface treated (using adhesion primers, plasma, etc.) to improve the adhesion strength, which are joined by an adhesive according to the present invention, are expected to have a higher shear strength, such as a lap shear strength, compared to a substrate with a low surface energy / non- reactive nature.

[0106] On the other hand, substrates with a low surface energy / non-reactive nature e.g., poly(methyl methacrylate) (Acryl), joined by an adhesive according to the present invention are to be expected to have a lesser shear strength, compared to substrates where the surface is treated to improve the adhesion strength.

[0107] In addition, the adhesive as disclosed herein is exceptionally suitable for underwater adhesion. Generally, adhesion to substrates underwater or in wet environments presents significant challenges due to the inherent properties of water. Water molecules create a barrier that prevents traditional adhesives from effectively bonding surfaces. First, water interferes with traditional adhesives ability to establish direct contact with the surfaces, inhibiting the formation of strong molecular bonds. Second, many traditional adhesives rely on a drying or curing process, which is hindered by water's presence, as it slows down evaporation and / or various chemical reactions. Additionally, water can seep into the adhesive, causing it to weaken or degrade over time.

[0108] As is shown in the examples the adhesives as disclosed herein are especially suited to underwater adhesion, both before and after curing, as is evident from examples 3 and 7.

[0109] Accordingly, the adhesive of the present invention may be characterized by an uncured underwater adhesive strength measured using a Universal Testing System of at least 100 kPa, when said adhesive is evenly applied underwater to aluminum substrates.

[0110] The adhesive of the present invention may further be characterized by an underwater adhesive strength measured using a Universal Testing System of at least 300 kPa, when said adhesive is evenly applied underwater to aluminum substrates and enzymatically cured, as described herein, underwater for 10-30 min.

[0111] The adhesive of the present invention may further be characterized by a tensile adhesion strength measured using a Universal Testing System of at least 200 kPa when said adhesive is evenly applied to aluminum substrates.

[0112] The adhesive of the present invention may further be characterized by a tensile adhesion strength measured using a Universal Testing System of at least 500 kPa when said adhesive is evenly applied to aluminum substrates, and enzymatically cured for 10-30 min. The adhesive of the present invention may further be characterized by a tensile adhesion strength measured using a Universal Testing System of at least 15 kPa when said adhesive is evenly applied to skin and aluminum substrates. The adhesive of the present invention may further be characterized by a tensile adhesion strength measured using a Universal Testing System of at least 25 kPa when said adhesive is evenly applied to skin and aluminum substrates, and enzymatically cured for 10-30 min.

[0113] Accordingly, the adhesive presented herein is also suited for use as a surgical adhesive. The reason for this is the injectability of the adhesive, where it is shown that the glue may be prepared in a way that either renders it as an injectable gel or as a rubbery material, which is less suited for surgical applications.

[0114] The adhesive presented herein may this be used as a surgical adhesive, either as a cured or uncured adhesive. The use as a surgical adhesive / sealant often requires the curing to be completed within seconds to minutes.

[0115] In example 4 the curing of the adhesive using different Chi-C:HRP and Chi-C:H2O2 weight ratios was tested to illustrate the potential possible tailoring of the curing properties of the adhesive. Firstly, the Chi-C:HRP ratio was fixed to 1500:1 and a number of different Chi- C:H2C>2 weight ratios was tested (From 50:1 to 6250:1 ). The variation of the Chi-C:H2O2 weight ratios showed that the gelling time is tuneable by the amount of H2O2, from a gelation time from approx. 1 minute to about 60 minutes. Furthermore, the Chi-C:H2O2 ratio of 625:1 provides a reasonable curing time for tissue adhesion testing.

[0116] From the initial test a Chi-C:H2O2 ratio of 625:1 was selected as baseline and the Chi-C:HRP was varied from 150:1 to 9375:1 . This showed that the gelling time is also tuneable by changing the Chi-C:HRP ratio as well.

[0117] Horseradish Peroxidase (HRP) is an enzyme isolated from horseradish roots, which catalyses the oxidation of substrates by hydrogen peroxide, and is widely used due to its high turnover rate and stability.

[0118] Accordingly, in embodiments, the curing of the adhesive is done using a Chi-C:HRP weight ratio of about 1500:1 and a Chi-C:H2O2 weight ratio in the range of 1250:1 to 6250:1 , or a Chi-C:H2O2 weight ratio of about 625:1 and a Chi-C:H2O2 weight ratio in the range of 150:1 to 9375:1 . As is shown in the examples the specific ratio of Chi-C:H2O2 and Chi-C:HRP can be tailored to the specific need, depending on the required curing time. In embodiments, the Chi-C:H2O2 weight ratio is about 1500:1 and the Chi-C:H2O2 weight ratio is about 625:1. In embodiments, the curing time if the adhesive is tailored to a curing time in the range of 1 s to 2 hrs, such as in the range of 1s-1 min, such as in the range of 1min-10 min, such as in the range of 10min-30min, such as in the range of 30min-60min, or such as 60min-120min. In example 5 it was shown that curing of Chi-C with HRP:H2O2 increases the storage modulus (G’), where the cured Chi-C showed at least two times higher G’ values (Figure 6B), indicating that the curing of Chi-C with HRP:H2O2 increases Chi-C crosslinking and significantly improve the mechanical strength of the hydrogels.

[0119] Curing

[0120] The isolated adhesive may be used in absence or presence of curing depending on the intended use.

[0121] The presence of catechol and amine groups in the Chi-C material allows for a vast range of different curing strategies. Catechol oxidation can be achieved through various methods, including exposure to air, metal ions, heat, UV radiation, and enzymatic catalysis. For instance, it has been shown that UV radiation can be used to oxidize catechol and create cross-linkable curable materials. Examples of Chi-C curing methods are e.g., use of metal ions, enzymatic catalysis, use of strong oxidants such as periodate, and heat oxidation. In addition, reagents with amine reactivity, such as e.g., aldehydes, epoxides may also be used to cure the adhesive through amine cross-linking. Further examples of amine reactive cross linker will be evident to the skilled person.

[0122] Herein, curing generally refers to the controlled hardening of the obtained adhesive through methods like UV-radiation, heat, or by addition of additives. Curing ensures structural integrity and durability of the adhesive. Thus, curing is a process that extends the usefulness and quality of the adhesive disclosed herein. The curing can be done at various conditions. For instance, by thermal curing, enzymatic curing or chemical curing, or any combination thereof. In embodiments, the adhesive provided herein can be used as a cured wet hydrogel adhesive. In other embodiments, the adhesive provided herein can be a dried cross-linked adhesive, e.g., cured by thermal curing.

[0123] Furthermore, catechol groups in the catechol substituted chitosan may also be converted into quinones by UV radiation, heat, and / or oxidation such as e.g., by raising pH, addition of sodium periodate, hydrogen peroxide, or similar oxidizing agents.

[0124] For surgical applications, enzymatic curing may be used to cure the adhesive disclosed herein. Enzymatic curing is a process where an enzyme, such as Horseradish Peroxidase (HRP) is added to aid catechol oxidation, which results in the formation of covalent bonds between the Chi-C polymers, thereby crosslinking the polymers, and potentially also crosslinking the polymers to the substrate. In addition, oxidation may also be used for curing. Several oxidative agents may be used, such as Hydrogen peroxide (H2O2), sodium periodate (NalOzi), silver nitrate (AgNOs), and iron(lll) chloride (FeCh), preferably H2O2 is used as the oxidative agent. Preferably a combination of enzymatic curing and chemical curing e.g., by oxidation, is used to cure the Chi-C adhesive.

[0125] As is shown in examples 7 and 8, the curing of the Chi-C adhesive using HRP:H2C>2 in different ratios relative to the amount of Chi-C greatly influences the properties and adhesive strength of the adhesive (Figure 8, 9 and 10).

[0126] Accordingly, in embodiments, the method for producing the adhesive further comprises a step of curing the isolated adhesive. In embodiments, the curing is conducted via. thermal curing. In other embodiments, the curing is conducted via. enzymatically assisted curing. Preferably, the curing includes enzymatic curing using a mixture of HRP and an oxidative agent, preferably H2O2.

[0127] As is shown in example 4, a two-component adhesive may be prepared by providing an adhesive as disclosed herein and a curing agent, wherein mixing of the adhesive and the curing agent induces the curing of the adhesive.

[0128] Accordingly, the invention also relates to a two-component adhesive comprising the phase separated isolated polymer rich fraction as disclosed herein and secondly one or more curing agents, preferably an oxidation agent, more preferably H2O2 and HRP.

[0129] Preferably, the Chi-C:HRP weight ratio is in the range of 150:1-10.000:1 , such as 150:1 , 375:1 , 937.5:1 , 1875:1 , 3750:1 , or 9375:1 , preferably 1875:1 or 1500:1. Additionally, it is preferred that the Chi-C:H2O2 weight ratio is in the range of 50:1 to 6500:1 , such as in the range of 50:1 , 500:1 , 625:1 , 1250:1 , 3000:1 , 5000:1 or such as 6250:1 , preferably the Chi- C:H2C>2 weight ratio is 625:1 . For some applications a Chi-C:HRP weight ratio of 1500:1 and a Chi-C:H2O2 weight ratio may be preferred. In addition, the curing time, i.e., the time it takes for the hardening of the adhesive may be in the range of 1 s to 5 hrs, such as in the range of 30s to 90 minutes, depending on the type and amount of curing agent used. In example, the curing time may be 0.5-1 hr when a Chi- C:HRP weight ratio of 1500:1 and a Chi-C:H2O2 weight ratio of between 50:1 and 500:1 is used. In examples, the curing time may be 10-15 minutes when a Chi-C:HRP weight ratio of 1500:1 and a Chi-C:H2O2 weight ratio of between 625:1 is used. In further examples, the curing time may be 1-5 minutes, when a Chi-C:HRP weight ratio of 1 :1500 and a Chi-C:H2O2 weight ratio of between 1250:1 and 6250:1 is used. In further examples, the curing time may be 30s to 30 minutes when a Chi-C:H2O2 weight ratio of 625:1 and a Chi-C:HRP weight ratio of between 150:1 and 9375:1 is used. In further examples, the curing time may be about 30 minutes when a Chi-C:H2O2 weight ratio of 6250:1 and a Chi-C:HRP weight ratio of between 150:1 and 9375:1 is used.

[0130] It will be evident to the skilled person that curing of the adhesive disclosed herein may be conducted in a plethora of ways, not limited to the curing methods disclosed herein, but will largely depend on the application of the adhesive.

[0131] A kit

[0132] The present invention also relates to a kit comprising a tissue sealant, comprising a catechol substituted chitosan as disclosed herein. As such the kit optimally comprise a glue-like formulation of the Chi-C as described herein, with mechanical properties which makes it suitable for injection via a syringe.

[0133] For instance, phase separated Chi-C, such as Chi-C with 25% catechol conjugation (Chi- C25%) may be mixed with HRP or H2O2 in separate vials. In some embodiments, the weight ratio of Chi-C25%:HRP is 1500: 1 and the weight ratio of Chi-C25%:H2O2 is 50:1. In some embodiments, the weight ratio of Chi-C25% / HRP is 1500: 1 and the weight ratio of Chi- C25%:H2C>2 is 500:1. In some embodiments, the weight ratio of Chi-C25%:HRP is 1500: 1 and the weight ratio of Chi-C25%:H2O2 is 625:1. In some embodiments, the weight ratio of Chi-C25%:HRP is 1500: 1 and the weight ratio of Chi-C25%:H2O2 is 1250:1. In some embodiments, the weight ratio of Chi-C25%:HRP is 1500: 1 and the weight ratio of Chi- C25%:H2C>2 is 3000:1. In some embodiments, the weight ratio of Chi-C25%:HRP is 1500: 1 and the weight ratio of Chi-C25%:H2O2 is 5000:1. In some embodiments, the weight ratio of Chi-C25%:HRP is 1500: 1 and the weight ratio of Chi-C25%:H2O2 is 6250:1. In some embodiments, the weight ratio of Chi-C25%:HRP is 150:1 and the weight ratio of Chi- C25%:H2C>2 is 625:1 . In some embodiments, the weight ratio of Chi-C25%:HRP is 375:1 and the weight ratio of Chi-C25%:H2O2 is 625:1 . In some embodiments, the weight ratio of Chi- C25%:HRP is 937.5:1 and the weight ratio of Chi-C25%:H2O2 is 625:1. In some embodiments, the weight ratio of Chi-C25%:HRP is 1875:1 and the weight ratio of Chi- C25%:H2O2 is 625:1 . In some embodiments, the weight ratio of Chi-C25%:HRP is 3750:1 and the weight ratio of Chi-C25%:H2O2 is 625:1 . In some embodiments, the weight ratio of Chi-C25%:HRP is 9375:1 and the weight ratio of Chi-C25%:H2O2 is 625:1 . In some embodiments, the weight ratio of Chi-C25%:HRP is 150:1 and the weight ratio of Chi- C25%:H2O2 is 6250:1. In some embodiments, the weight ratio of Chi-C25%:HRP is 375:1 and the weight ratio of Chi-C25%:H2O2 is 6250:1. In some embodiments, the weight ratio of Chi-C25%:HRP is 937.5:1 and the weight ratio of Chi-C25%:H2O2 is 6250:1 . In some embodiments, the weight ratio of Chi-C25%:HRP is 1875:1 and the weight ratio of Chi- C25%:H2O2 is 6250:1. In some embodiments, the weight ratio of Chi-C25%:HRP is 3750:1 and the weight ratio of Chi-C25%:H2O2 is 6250:1. In some embodiments, the weight ratio of Chi-C25%:HRP is 9375:1 and the weight ratio of Chi-C25%:H2O2 is 6250:1. Preferably, the vials are mixed in equal volume and / or weight. For example, the curable adhesive may be prepared by mixing 0.5 g of phase separated Chi-C25% with 100 pL of 0.1 wt% HRP in a first vial and mixing 0.5 g of phase separated Chi-C25% with 100 pL of 0.8 wt% H2O2 in a second vial. The two vials may then be mixed to cure the Chi-C.

[0134] In embodiments, the present invention relates to a kit comprising; a tissue sealant comprising a catechol substituted chitosan with a catechol content in the range of 15-60 % according to the present disclosure, A curing agent, and optionally, Instructions for use.

[0135] In further embodiments, the present invention relates to a kit comprising; a tissue sealant comprising a catechol substituted chitosan with a catechol content in the range of 15-60 %, a curing agent, preferably H2O2, an enzymatic curing catalysator, preferably HRP, and optionally, Instructions for use.

[0136] In embodiments, the tissue sealant, curing agent and / or enzymatic curing catalysator is provided in individual vials, as a powders or as a gel. Preferably, the tissue sealant is provided as an injectable gel, and the curing agent and enzymatic curing catalysator are provided as liquid solutions. In further embodiments, the present invention relates to a kit comprising; a tissue sealant comprising a catechol substituted chitosan with a catechol content in the range of 15-60 %, - a water-miscible epoxide / aldehyde compound, and optionally

[0137] Instructions for use.

[0138] GENERAL

[0139] It should be understood that any feature and / or aspect discussed above in connections with the compounds according to the invention apply by analogy to the methods described herein.

[0140] The following figures and examples are provided below to illustrate the present invention. They are intended to be illustrative and are not to be construed as limiting in any way.

[0141] EXAMPLES

[0142] Materials

[0143] Chitosan (Chi, 75-85% deacetylated with low molecular weight, e.g., product no. 448869 from SigmaAldrich), sodium cyanoborohydride (NaCNBHs, > 95%, for synthesis), 3,4- dihydroxybenzaldehyde (DHBA, > 97%), horseradish peroxidase (HRP, lyophilized powder, ~ 150 U / mg), hydrogen peroxide (H2O2, 30 wt% in water), and sodium chloride (NaCI) were purchased from Sigma-Aldrich and used without further purification. All solutions were prepared using ultrapure water (Arium® Pro) with a resistivity of 18.2 MO cm. pH adjustment was conducted using a HCI solution.

[0144] Example 1 - Preparation of catechol conjugated chitosan

[0145] The aim of the current example is to demonstrate one way of preparing the catechol substituted chitosan of the present disclosure.

[0146] Methods

[0147] Chi-C with different catechol contents (10%, 20%, 25%, 34% 43%, and 54%) were synthesized as described below.

[0148] Table 1 Summary of the conditions employed for achieving various degrees of catechol conjugations.

[0149] Mass of Reaction Reducing pH Values

[0150] Target Reducing

[0151] DHBA dd d i i

[0152] Chi-C10% 0.16 1

[0153] In a volumetric flask, 2 g of chitosan was dissolved in 40 mL of water containing 0.8 mL of glacial acetic acid. The mixture was stirred overnight, and the pH was then adjusted to 4.5 using 1 M HCI. The resulting solution was clear and pale yellow in color. DHBA was dissolved in a water / methanol mixture (2:1 volume ratio, pH adjusted to 4.5) in various concentrations (see Table 2). The pH of the mixture was kept constant at 4.5 during the synthesis process.

[0154] Table 2 Regent compositions of Chi-C synthesis and catechol content determinations

[0155] Next, a reductive amination reaction was conducted. 2.0 g solid NaCNBHs was dissolved in water with a pH of 4.5 and added dropwise. After 4 hours, the pH was adjusted to 6.5 by adding 2 M NaOH dropwise under vigorous stirring to obtain precipitated Chi-C. The excess water was removed, and 50 ml 1 wt. % HCI solution was added to the reaction flask. The mixture was stirred to re-dissolve Chi-C. The solution was then dialyzed against HCI solution (pH 3-3.5) for 48 hours (in the beginning, four exchanges every 2h) using a regenerated cellulose dialysis membrane (MWCO: 3.5 KDa). The solution was lastly dialyzed against water for 4 hr, followed by freeze-drying for two days. Around 1.8 g dried Chi-C product was obtained and stored in a desiccator at a temperature of -20°C, covered by aluminum foil for further use. Reactions mixtures and solutions were preserved from light during the entire synthesis and dialysis process.

[0156] Results

[0157] To confirm / quantify the catechol substitution, Chi-C products were dissolved in acetate buffer (pH 3) at a concentration of 1 wt%. The solutions were then diluted to 0.1 mg / mL for UV-Vis testing. The absorbance was recorded over a range of 200 to 800 nm using a NanoDrop 2000 / 2000c Spectrophotometer (Thermo Fisher Scientific; Wilmington, USA). The absorbance peak at 280 nm (Figure 2(a)), confirms catechol grafting.

[0158] The degree of catechol conjugation was determined using a standard calibration curve (Figure 2(b)). The absorbance intensity at 280 nm was measured for dopamine (DA) solutions of varying concentrations (0.002, 0.01 , 0.02, 0.03, and 0.04 mg / mL). Example 2 - Chi-C phase separation

[0159] The aim of the current example is to demonstrate one way of providing the adhesive of the present disclosure.

[0160] Methods

[0161] To prepare the Chi-C solution, a 1 mM NaCI solution at pH 3.0 was first prepared. Next, 0.5 g of dried Chi-C powder was added to obtain a 50 mL Chi-C solution at a concentration of 1 wt%. Once fully dissolved, the pH value was readjusted to 3.0 using a 1 M HCL The solution was then transferred to 16 glass vials, each containing 2.5 mL of the solution, for further phase separation testing. Subsequently, a 2.5 mL NaCI solution (pH 3.0) with an ionic strength from 0.2 M to 4 M was separately added to Chi-C solutions. After stirring, the solution was allowed to settle and centrifuged at 6500 rpm / min for 5 minutes.

[0162] Results

[0163] Initially, an investigation into the phase behavior of Chi-C polymers with varying catechol contents in response to increasing concentrations of NaCI was conducted. To achieve this, a solution of Chi-C at a concentration of 1 wt.% in NaCI 1 mM with pH adjusted to 3 was prepared. An equal volume of NaCI solution with a higher concentration was then added to the Chi-C solution. For instance, to attain a Chi-C solution with a NaCI concentration of 100 mM, the Chi-C solution was mixed with NaCI at a concentration of 200 mM. By adopting this approach, it was ensured that the concentration of NaCI in the final solution was the only variable, while the polymer concentration and total volume remained consistent across all combinations.

[0164] The phase diagram in Figure 3 shows the behavior of Chi-C solutions with varying NaCI concentrations and catechol contents. Different phase behaviors were observed based on the concentration of salt and catechol content of Chi-C. When the concentration of salt and catechol content were low (top left corner of the diagram: light grey color), no phase separation occurred and Chi-C remained in solution. Conversely, as the concentration of salt and catechol content increased, phase separation occurred (bottom right corner of the diagram: colors in mid-grey, dark grey, and black). A certain minimum amount of catechol was required to initiate phase separation through the addition of salt. Notably, Chi and Chi- Ci 0 solutions did not show any phase separation even in the presence of 2 M NaCI. Chi- C20 showed no clear phase separation up to 1 M NaCI either, but at 1 .2 M NaCI, the solution turned cloudy, indicating the onset of phase separation, with precipitated material that could be collected by centrifugation. The minimum amount of salt required to initiate phase separation was observed to be dependent on the catechol content, Chi-C with higher catechol contents requiring less salt to undergo phase separation.

[0165] The precipitated material collected at the onset of phase separation (mid-grey areas in figure 3) did not exhibit strong cohesive properties, and shaking could easily re-disperse it into a cloudy mixture. Increasing the salt concentration beyond the onset of phase separation significantly affected the properties of the collected precipitate. Initially, a gel-like material (dark grey areas in figure 3) was formed, followed by a more solid and rubbery material (black areas in figure 3) at higher salt concentrations. Both of these phases rendered proper cohesively and homogeneity, but the gel-like phase showed more favorable flow properties.

[0166] As an additional investigation, Chi-C phase separation was also studied at pH 5 as well (see figure 11). Chitosan, by increasing pH to 5, is expected to become less charged, which can affect the phase diagram compared to that obtained for pH 3. As shown in Figure 11 , it was observed that cloudiness appeared earlier at pH 5 than at pH 3.0 (Figure 3). For instance, Chi-C25% solution exhibited the start of phase separation at 0.4 M of ionic strength at pH 5, while at pH 3.0, it was delayed until 0.9 M. The smaller amount of salt required to initiate phase separation at pH 5 suggests a less charged network or a more complex crosslinking in the Chi-C solution. However, Chi-C phase separation was still shown to occur at pH 5.

[0167] Example 3 - Adhesion testing

[0168] The aim of the present examples is to demonstrate the underwater adhesive properties of the Chi-C phase separated adhesive.

[0169] Methods

[0170] An underwater adhesion test (Instron 34SC-2, 50 N load cell) was conducted on the aluminum substrates, immersed in demineralized water. The Chi-C material (0.2 g) was applied onto the bottom cylinder (aluminum, diameter 20 mm) underwater, then the upper cylinder (aluminum, diameter 10 mm) was lowered (vertical approach step) at a constant speed of 1 mm min'1until contact was made (set point: 10 N, duration: 60 s). The upper probe was then retracted with a speed of 1 mm min'1. The adhesion strength was calculated as the maximum recorded force on retraction normalized by the contact area. Data were plotted as mean ± standard deviation, calculated based on five replicates. To confirm the results, an additional independent experiment was performed.

[0171] Results

[0172] The adhesion characteristics of Chi-C with varying catechol concentrations (20%, 25%, 34%, 43%, and 54%), which showed phase separation / precipitation when NaCI was added was explored. The aim was to investigate how altering the catechol content and the amount of salt affected the properties of the precipitate. To evaluate the adhesive strength of the precipitate, a specific amount of Chi-C precipitate (0.2 g) was applied onto an aluminum substrate, submerged it underwater, pressed another aluminum substrate onto it, allowed it to dwell, and measured the maximum recorded force upon retraction using tensile mode. Figure 5 displays the adhesion strength of Chi-C precipitates with varying catechol contents prepared by adding different salt amounts. It was found that the physical properties of the precipitate significantly affected the adhesion strength. The gel-like material in the grey bars had larger adhesion properties, supposedly due to its ability to flow and form good contact between substrates, whereas the solid rubbery material in the black bars was harder to flow and form contact area thus rendered reduced adhesion.

[0173] Furthermore, it was observed that both the catechol content and NaCI concentration (the added amount to prepare phase separated / precipitated Chi-C) affected underwater adhesion strength. For a fixed catechol content, adhesion strength increased with increasing NaCI concentration until an optimum point was reached. Further increase in NaCI concentration led to a decrease in adhesion strength. The optimum NaCI concentration providing maximal adhesion strength decreased with increasing catechol content.

[0174] Example 4 - Preparation of a two-component curable adhesive

[0175] The presence of catechol groups in the Chi-C material allows for a vast range of different curing strategies. Catechol oxidation can be achieved through various methods, including exposure to air, metal ions, heat, UV radiation, and enzymatic catalysis. For instance, it has been shown that UV radiation can be used to oxidize catechol and create cross-linkable curable materials. Herein is described one exemplary way of performing the curing using HRP and H2O2. The HRP-catalyzed oxidation of catechol is a process that involves the use of horseradish peroxidase (HRP) enzyme to catalyze the oxidation of catechol. With the presence of nucleophilic amine groups on Chi-C, an investigation into whether a combination of HRP and H2O2 could be utilized in self-cross-linking Chi-C was conducted. Chi-C25% (precipitated in 1.2 M NaCI) was selected due to its favorable physical properties, including maximal adhesion performance (as shown in Figure 3), ease of application to a surface, and injectability. This material, after optimizing the Chi-C:HRP:H2O2 mixing ratios, was used for demonstration / analysis data provided in Figure 6-10.

[0176] Methods

[0177] A covalent curing strategy was employed using HRP in the presence of H2O2. Phase separated Chi-C25% was mixed with HRP and H2O2 in separate batches, with an optimized weight ratio of 1500:1 (Chi-C25%:HRP both in dried state) and 625:1 (Chi-C25%:H2O2). Specifically, 0.5 g of phase separated Chi-C25% was mixed with 100 pL of 0.1 wt% HRP as the first part, and 0.5 g of phase separated Chi-C25% was mixed with 100 pL of 0.8 wt% H2O2 as the second part. These two constituents were thoroughly mixed in a 1 :1 weight using a spatula to achieve a uniform mixture, after which a color change and gelation occurred within a certain amount of time.

[0178] The following recipe was used for making the curable two-component adhesive:

[0179] • Part A: phase separated Chi-C25% mixed with HRP

[0180] • Part B: phase separated Chi-C25% mixed with H2O2

[0181] Table 3. Curing Chi-C25% adhesive; varying ratios of HRP and H2O2

[0182] After optimizing the Chi-C / HRP / H2O2 mixing ratios as shown in table 3, a two-part adhesive chemistry was developed. The first part involved mixing Chi-C precipitate with HRP in a ratio of 1500: 1 , while in the second part, Chi-C was mixed with H2O2 in a ratio of 625:1 .

[0183] Example 5 -Mechanical characterization of adhesive

[0184] In the present example, the mechanical properties of the single component and two component chitosan adhesive described in examples 2 and 4 were compared. Methods

[0185] To evaluate the gelation behavior of Chi-C25%, rheological measurements were performed using a conventional rheometer (RSA II , TA Instruments, USA) with a parallel plate geometry (diameter: 20 mm, gap distance: 0.5 mm). Strain-sweep analysis was conducted at a fixed frequency of 10 Hz across a strain range of 0.01 to 100%, with data points collected within 2 minutes. Samples with HRP / H2O2 treatment were subjected to strainsweep analysis immediately after mixing (Part A with Part B), and 15 minutes after mixing. Similarly, Chi-C samples without HRP / H2O2 treatment (as control samples, no curing reaction occurring) were also examined at time points of 0 and 15 minutes. All experiments were performed at 25 °C in duplicates.

[0186] Results

[0187] In the present example the aim was to investigate the curability of phase separated Chi-C with HRP:H2C>2 treatment using rheological techniques.

[0188] Figure 5a displays storage (G’) and loss (G”) modulus measured for Chi-C samples with and without HRP / H2O2 treatment. The sample with HRP / H2O2 treatment demonstrates increment in G’ with time (when comparing panel a (data collected right after mixing part A with part B) with panel B (data collected 15 minutes after mixing part A with part B)). In contrast, the sample without HRP / H2O2 treatment did not show significant changes in G’ and G” with time. This can indicate that the sample with HRP / H2O2 treatment undergoes curing / gelation.

[0189] Example 6 - Visual characterization of adhesive

[0190] Methods

[0191] The adhesive described in the present examples was prepared as described in example 4.

[0192] Results

[0193] The gel strength and resistance to deformation of phase separated Chi-C was investigated using a visual demonstration.

[0194] Briefly, phase separated Chi-C with or without HRP:H2O2 was cured in room temperature for 15 minutes, followed by compression-release measurements. As shown in the figure 7, a visible change in color from white to brown was observed in the HRP:H2O2 treated Chi-C, indicating the occurrence of catechol oxidative crosslinking. The introduction of HRP:H2O2 enable to increase the rigidity / elasticity of the gel network, resulting in higher compression strength and better resilience. These findings suggest that HRP:H2C>2 curability can endow phase separated Chi-C with a robust polymer network that could exhibit better wet performance.

[0195] Example 7 - Adhesion after curing

[0196] Aim

[0197] In order to further investigate the capabilities of the adhesive, the chemical curability functionality was examined. Specifically, when wet adhesion is necessary, such as in the case of medical tissue adhesives, it is desirable for the mechanical properties to improve and create a stronger bond after initial contact is made. Therefore, the adhesive properties of the curable adhesive as described in previous examples was investigated.

[0198] Methods

[0199] A covalent curing strategy was employed as described in example 4, and the adhesive experiments were conducted as described in example 3.

[0200] Results

[0201] Figure 8 and 9 displays the adhesion strength measurements of phase separated Chi-C with or without HRP:H2C>2 curing underwater. For a given curing time (15 minutes), it was observed that HRP:H2C>2 curability significantly improved adhesion strength to ~ 550 KPa (Figure 9), which was nearly twice as strong as that of Chi-C sample without HRP:H2C>2. In addition to HRP:H2C>2, it was found that curing time also played an important role in adhesion strength. For example, compared to Chi-C sample without HRP:H2C>2, a one-minute curing did not exhibit any significant changes in adhesion strength. As demonstrated in Example 4 though, the curing time of the adhesive is easily adjustable by tuning ratios of Chi-C:H2O2 and Chi-C / HRP.

[0202] Example 8 - Adhesion on pig skin

[0203] Aim

[0204] The aim of the present study is to evaluate the adhesive properties of the cured and noncured adhesives on tissue, compared to available adhesives. Methods

[0205] Tissue adhesion test (Instron 34SC-2, 50 N load cell) was conducted on the aluminum substrates with pig skin on top (skin cut into pieces and then glued onto aluminum substrates). In each case, the adhesive applied onto the bottom cylinder (diameter 20 mm), then the upper cylinder (diameter 10 mm) was lowered (vertical approach step) at a constant speed of 1 mm min-1 until contact was made (set point: 10 N, duration: 60 s). The upper probe was then retracted with a speed of 1 mm min-1 . The adhesion strength was calculated as the maximum recorded force on retraction normalized by the contact area.

[0206] Results

[0207] The present example compares the adhesion strength (against pig skin) of Chi-C adhesive against two commercial tissue glue benchmarks, i.e. , Bio-glue (bovine serum albumin) and 3M Vetbond cyanoacrylate tissue glue (See figure 10).

[0208] It was found that enzymatic curing by HRP:H2O2 increases the adhesion strength against wet tissue (compare to Chi-C without HRP:H2O2).

[0209] The range of adhesion strength for the enzymatically cured Chi-C adhesive is found to be comparable to the commercial bio-based tissue glue (Bio-glue), which is around 30 kPa.

Claims

CLAIMS1. A method for preparing an adhesive, said method comprising providing catechol substituted chitosan in an aqueous solution with a pH in the range of pH 3-5, admixing said aqueous solution with a salt to obtain a polymer-rich phase and a polymer-poor phase; and isolating the polymer-rich phase to obtain said adhesive.

2. A method according to claim 1 , wherein the salt present in an amount in the range of 0.18-2M.

3. A method according to claim 1 or 2, wherein the salt is NaCL4. A method according to any of the preceding claims, wherein the catechol content in the catechol substituted chitosan is in the range of 15-60 % (wt. %).

5. A method according to any of the preceding claims, wherein said chitosan in said chitosan solution has a degree of deacetylation of chitosan is in the range of 70-99%, preferably in the range of 75-85%.

6. A method according to any of the preceding claims, wherein said isolation step comprises a step of centrifugation, filtration, sedimentation, decantation, or flocculation or any combination thereof.

7. A method according to any of the preceding claims, further comprising a step of curing the isolated adhesive.

8. A method according to claim 7, wherein curing includes enzymatic curing.

9. A method according to claim 7 or 8, wherein curing includes enzymatic curing using a mixture of HRP and an oxidative agent, preferably H2O2.

10. A method according to any of preceding claims, wherein the salt is NaCI and the pH of the aqueous solution is about pH 3 and;■ the catechol content in the catechol substituted chitosan is 20%, and the salt content is in the range of 0.1 -3M; or■ the catechol content in the catechol substituted chitosan is 25%, and the salt content is in the range of 0.2-2M; or■ the catechol content in the catechol substituted chitosan is 34%, and the salt content is in the range of 0.1-1 .5M; or■ the catechol content in the catechol substituted chitosan is 43%, and the salt content is in in the range of 0.1-1.5M; or■ the catechol content in the catechol substituted chitosan is 54%, and the salt content is in the range of 0.1-1 M.11 . A method according to any of claims 1-9, wherein the salt is NaCI and the pH of the aqueous solution is about pH 4.5 and;■ the catechol content in the catechol substituted chitosan is 20%, and the salt content is in the range 0.5-2M; or■ the catechol content in the catechol substituted chitosan is 25%, and the salt content is in range of 0.5-2M; or■ the catechol content in the catechol substituted chitosan is 34%, and the salt content is in range of 0.1-1.5M; or■ the catechol content in the catechol substituted chitosan is 43%, and the salt content is in the range of 0.1-1 .5M; or■ the catechol content in the catechol substituted chitosan is 54%, and the salt content is in the range of 0.1-1 M.

12. An adhesive containing catechol substituted chitosan, obtainable by a method according to any of claims 1-11.

13. An adhesive according to any of claims 12, for use as a surgical adhesive.

14. A gel consisting of catechol substituted chitosan, wherein catechol substituted chitosan is present in the gel in a total concentration of from 1 to 25 wt% such as from 5 to 20 wt%, from 10 to 15 wt%, such as about 15 wt%.

15. A kit comprising; a tissue sealant comprising a catechol substituted chitosan with a catechol content in the range of 15-60 %,A curing agent, and optionally, Instructions for use.

Images