EP3523020A1

EP3523020A1 - Flexible and multi-functional coacervates and hydrogel materials

Info

Publication number: EP3523020A1
Application number: EP17787885.7A
Authority: EP
Inventors: Henrik Birkedal; Vicki NUE; PHAT Tan HUYNH
Original assignee: Aarhus Universitet
Current assignee: Aarhus Universitet
Priority date: 2016-10-04
Filing date: 2017-10-04
Publication date: 2019-08-14
Also published as: WO2018065450A1

Abstract

The present invention relates to methods of forming coacervates, methods of forming hydrogels and coacervates using light sensitive groups as well as uses of said coacervates and hydrogels. The invention also provides coacervates and composition for forming coacervates and use of the coacervates and compositions in medical treatment such as in treating tissue injuries.

Description

Flexible and multi-functional coacervates and hydrogel materials Technical field

Background

The blue mussel has evolved the ability to stick to all kinds of surfaces underwater. An essential component in mussel adhesion is the special amino acid L-3,4- dihydroxyphenylalanine (L-DOPA) that as a jack-of-all-trades allows attaching to all types of material surfaces using either covalent or non-covalent bonds (M. Krogsgaard, M. A. Behrens, J. S. Pedersen, H. Birkedal, Biomacromolecules 2013, 14, 297-301 ). Several research groups have pioneered a platform of mussel-inspired materials to be used as bio-inspired adhesives (D. S. Hwang, H. Zeng, A. Srivastava, D. V. Krogstad, M. Tirrell, J. N. Israelachvili, J. H. Waite, Soft Matter 2010, 6, 3232-3236). This was achieved by combining features of the mussel foot proteins in synthetic polymers (M. Krogsgaard, M. A. Behrens, J. S. Pedersen, H. Birkedal, Biomacromolecules 2013, 14, 297-301 ) Thus catechols and analogues thereof are extremely useful in materials design (M. Krogsgaard, V. Nue, H. Birkedal, Chem. Eur. J. 2015, 22, 844-857). While great strides have been made in harnessing blue mussel chemistry in materials, previous efforts fall short in facile and efficient delivery to a surface underwater, without which these materials are difficult to use in real-life applications.

Mussel adhesive is applied in the form of complex fluids that spread spontaneously and exhibit strong reversible interfacial bonding and tunable cross-linking (B. P. Lee, P. B.

Messersmith, J. N. Israelachvili, J. H. Waite, Annu. Rev. Mater. Res. 201 1 , 41, 99-132). The complex fluids are coacervates (C. G. d. Kruif, F. Weinbreck, R. d. Vries, Curr. Opin. Colloid Interface Sci. 2004, 9, 340-349) - mixtures of polyelectrolytes that form a phase separated from the aqueous solution. Coacervates have excellent spreading abilities. Thus the mussel maintains fluid glue that upon meeting the surface spreads and presents DOPA to the target surface for attachment and then cures.

Coacervate films can be formed by injection of polyelectrolyte solution in dimethyl sulfoxide (DMSO) into water. Coacervation happens based on exchange of DMSO by water molecules leading to deprotonation of catechol-bearing poly acrylic acid and dissociation of bis(trifluoromethane-sulphonyl)imide / quaternized chitosan ion pairs. However, the main drawback of this method is release of DMSO - a harmful organic solvent - into water, limiting the use of this method for real application. Thus, a method for the formation of coacervates that do not apply toxic additives, while leading to the formation of coacervates with unique adhesive and cohesive properties is needed.

Summary

The inventors of the present invention have succeeded in inventing a novel method of forming coacervates by electrostatic-driven complexation between polycations and polyanions. The method of the present invention provides coacervates with improved mechanical properties. For example, the coacervates obtained by the method of the present invention have improved shear strength and display surprisingly high adhesion properties. Further, the method of the present invention allows the formation of compositions with very high concentrations of polymer and compound that only forms coacervates when the pH is changed. This allows for easy injections in the body for example to treat injured tissues or wounds. Moreover, the coacervates provided herein are biocompatible and biodegradable with a consistently high adhesive strength.

Accordingly, one aspect of the present invention provides a method of forming coacervates comprising

- providing a composition having a first pH comprising

• a polymer backbone comprising one or more backbone monomer unit(s) and

• a compound

wherein said polymer backbone is charged and said compound is uncharged or wherein said polymer backbone is uncharged and said compound is charged - adjusting the first pH of said composition to obtain a second pH such that the uncharged polymer backbone or the uncharged compound become charged at said second pH,

whereby coacervates are formed.

In one embodiment the second pH value is obtained by addition of a base comprising an OH-group, such as KOH or NaOH. In another embodiment the second pH value is obtained by addition of an acid, such as HCI or acetic acid. In a particular embodiment the second pH value is obtained by delivering said composition to a location in the body whereby pH changes to a second pH and coacervates are formed.

The polymer backbone can for example comprises one kind of backbone monomer units, only, said backbone monomer units comprising one or more chemical groups selected from amino groups, carboxylates, phosphonates, phosphates and sulfonates.

In another embodiment the polymer backbone comprises more than one kind of backbone monomer units, wherein at least one kind of monomer units comprising one or more chemical groups selected from amino groups, carboxylates, phosphonates, phosphates and sulfonates.

In one embodiment of the present invention the polymer backbone is charged and the compound is uncharged. The polymer backbone can for example be cationic at said first pH value. Alternatively, the polymer backbone is anionic at said first pH value.

The polymer backbone may comprise one or more backbone monomer unit(s), which is/are thermo-responsive. In another embodiment the polymer backbone comprises one or more backbone monomer unit(s), which is/are light- responsive. In yet another embodiment polymer backbone comprises one or more backbone monomer unit(s), which is/are soluble in polar solvents such as water.

In one embodiment of the present invention the polymer backbone comprises one or more backbone monomer unit(s) which is/are selected from the group consisting of:

- amino-Ci-io-alk(an/en/yn)yl which is optionally substituted; - amino acids such as of the formula H₂N-CHR¹-COOH wherein R¹ is an organic substituent;

- amides of the formula -(CO)-(NH2)-Ci_-6-alk(an/en/yn)yl-NH2;

- aminostyrenes which are optionally substituted; and

- amino sugar(s) such as of the generic formula 0=CR⁶-CH(NHR⁷)- (CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein

^■ R³, R⁴, R⁵ and R⁶ are independently selected from the group consisting of -H, -OH and Ci_-6-alk(an/en/yn)yl wherein Ci_-6- alk(an/en/yn)yl is optionally substituted with one or more -OH groups; and

- R⁷ is selected from the group consisting of -H and -(CO)-Ci_-6- alk(an/en/yn)yl wherein -(CO)-Ci_-6-alk(an/en/yn)yl is optionally substituted with one or more -OH groups;

in their open chain form or in the a-form or β-form thereof.

The polymer backbone may for example comprise one or more backbone monomer unit(s) of the generic formula amino-Ci-i₀-alk(an/en/yn)yl which is optionally substituted with one or more substituents.

In a preferred embodiment the polymer backbone comprises one or more backbone monomer unit(s) of the formula:

In one embodiment the polymer backbone comprises one or more backbone monomer unit(s) which is/are selected from the group consisting of amino-Ci-i₀-alk(an/en/yn)yl, amino-Ci-6-alk(an/en/yn)yl, amino-C₂-4-alk(an/en/yn)yl and amino-C₃-alk(an/en/yn)yl is substituted with one or more substituent(s) selected from the group consisting of -SH, - OH, -COOH, -NH₂, -S-CH₃, -0-CH₃, -CH(OH)-CH₃ and -CH(SH)-CH₃.

The polymer backbone may coomprise one or more backbone monomer unit(s) selected from the group of amino acids which occur either naturally or which do not occur naturally. In one embodiment said amino acid(s) is/are of the generic formula H₂N-CHR¹-COOH wherein R¹ is an organic substituent. In an embodiment thereof said amino acid(s) is/are of the formula H₂N-CHR¹-COOH;

wherein R¹ is selected from the group consisting of -H, -Ci_-6-alk(an/en/yn)yl, -Ci_-6- alk(an/en/yn)yl-R²,

wherein R² is selected from the group consisting of -SH, -COOH, C₃H₃N2, -NH₂, -S- CH₃, -CO-NH₂, -NH-C(NH)NH₂, - OH, -CH(OH)-CH₃, -SeH, -C₈H₆N, -C₆H₅ -C₆H₄OH, and -C₆H₃(OH)₂.

In a specific embodiment said amino acid(s) of the formula H₂N-CHR¹-COOH is a naturally occurring amino acid which is selected from the group consisting of DOPA, ornithine, lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine and valine.

In one embodiment the polymer backbone comprises one or more polymer monomer unit(s) of the formula -(CO)-(NH₂)-Ci_-6-alk(an/en/yn)yl-NH₂. In another embodiment of the present invention the polymer backbone comprises one or more backbone monomer unit(s) comprising styrene which is substituted, such as with one or more substituents selected from the group consisting of -NH₂ and -Ci_-6- alk(an/en/yn)yl-NH₂. In a preferred embodiment thereof the polymer backbone comprises one or more backbone monomer unit(s) comprising styrene which is substituted with NH₂.

In another preferred embodiment thereof the polymer backbone comprises one or more backbone monomer unit(s) comprising styrene which is substituted with Ci_-6- alk(an/en/yn)yl-NH₂.

Styrene can be further substituted with one or more substituents selected from the group consisting of -SH, - OH, -COOH, -NH₂, -S-CH₃, -0-CH₃, -CH(OH)-CH₃ and - CH(SH)-CH₃.

In one embodiment the polymer backbone comprises one or more backbone monomer unit(s) comprising styrene which is un-substituted. In one embodiment of the present invention the polymer backbone comprises one or more backbone monomer unit(s) selected from the group of amino sugars.

In one embodiment thereof said amino sugar(s) is/are of the generic formula 0=CR⁶- CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein

- R³, R⁴, R⁵ and R⁶ are independently selected from the group consisting of -H, -OH and Ci_-6-alk(an/en/yn)yl wherein Ci_-6-alk(an/en/yn)yl is optionally substituted with one or more -OH groups; and

- R⁷ is selected from the group consisting of -H and -(CO)-Ci_-6- alk(an/en/yn)yl wherein - -(CO)-Ci_-6-alk(an/en/yn)yl is optionally substituted with one or more -OH groups;

in their open chain form or in the a-form or β-form thereof.

In a particular embodiment thereof said amino sugar(s) is/are of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein R³, R⁴, R⁵ and R⁶ are independently selected from the group consisting of -H, -OH, -(CH₂)-OH and -COO^" and -CH(OH)-CH(OH)-CH₂-OH.

In another particular embodiment thereof said amino sugar(s) is/are of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein R³ is -OH.

In another specific embodiment said amino sugar(s) is/are of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein R⁴ is selected from the group consisting of -H and -OH, such as wherein R⁴ is -H, or wherein R⁴ is -OH.

In yet another specific embodiment said amino sugar(s) is/are of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein R⁵ is selected from the group consisting of -(CH₂)-OH and -COO^".

In yet another specific embodiment said amino sugar(s) is/are of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein R⁶ is selected from the group consisting of -OH and -CH(OH)-CH(OH)-CH₂-OH. In a further specific embodiment said amino sugar(s) is/are of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein R⁷ is selected from the group consisting of -H and -(CO)-CH₃. In another embodiment said amino sugar(s) is/are selected from the group consisting of glucosamine, acetylglucosamine, galactosamine and sialic acid in their open chain form or in any a-form or β-form thereof.

In one embodiment of the present invention the polymer backbones are amino- functionalized polymers with a pKa ranging from approximately 4 to 10, such as from 5 to10.

In a preferred embodiment of the present invention the monomer unit(s) comprises one or more catechol and/or catechol analogue group(s).

In preferred embodiment thereof the monomer unit(s) comprises one or more catechol and/or catechol analogue group(s) and further comprising one or more carboxylate- group and/or one or more amino group(s). In another embodiment of the present invention the monomer unit(s) comprises one or more pyrogallol and/or pyrogallol analogue group(s).

In preferred embodiment thereof the monomer unit(s) comprises one or more pyrogallol and/or pyrogallol analogue group(s) and further comprising one or more carboxylate- group and/or one or more amino group(s).

In a more preferred embodiment the monomer unit(s) comprises a compound of the formula:

wherein:

- R¹⁰ is selected from the group consisting of (Ci_-6-alk(an/en/yn)yl)_q- COOH and (Ci_-6-alk(an/en/yn)yl)_r-R¹³ wherein: - R¹³ is selected from the group consisting of NH₂, H, COOH, SH, HCO and OH; and

- q and r are independently selected from the group consisting of 0 and 1 ;

R¹¹ and R¹² are selected from the group consisting of OH, H, N0₂, F, CI, Br, I, SH, (Ci_-6-alk(an/en/yn)yl);

A is selected from the group consisting of C, N, O and S; and when q or r is 1 then Ci_-6-alk(an/en/yn)yl is optionally substituted with one or more substituents selected from the group consisting of -CH₃, - SH, -HCO, -OH, -NH₂ and -CO-C=C-R¹⁴ wherein:

• R¹⁴ is of the formula:

In a particular embodiment thereof R¹⁰ is selected from the group consisting of (C_2-4- alk(an/en/yn)yl)_q-COOH and (C_2-4-alk(an/en/yn)yl)_r-NH₂.

In another particular embodiment thereof R¹⁰ is selected from the group consisting of (C_2-4-alk(an/en/yn)yl)-COOH and (C_2-4-alk(an/en/yn)yl)-NH₂.

In a preferred embodiment R¹⁰ is -COOH or -NH₂. In another preferred embodiment R¹⁰ is (C_2-4-alk(an/en/yn)yl)-COOH. In yet another preferred embodiment R¹⁰ (C_2-4- alk(an/en/yn)yl)-NH₂.

In one preferred embodiment R¹¹ is -OH or H.

R¹¹ of the monomer unit(s), with which the polymer backbone is functionalized, may for example comprise at least one catechol functional group and further comprises at least one amino group and/or one carboxylate-group is positioned as indicated in the below formula:

In another embodiment R¹¹ of said monomer unit(s) with which the polymer backbone is functionalized comprises at least one catechol functional group and further comprises at least one amino group and/or one carboxylate-group is positioned as indicated in the below formula:

R11

In yet another embodiment R¹¹ of said monomer unit(s) with which the polymer backbone is functionalized comprises at least one catechol functional group and further comprises at least one amino group and/or one carboxylate-group is positioned as indicated in the below formula:

In one embodiment of the present invention the monomer unit(s) is/are selected from the group consisting of 2,4,5-trihydroxybenzoic acid, 3-(3,4-dihydroxyphenyl)propanoic acid, 3-(3,4-Dihydroxyphenyl)-2-methyl-alanine, 2,4,5-Trihydroxy-phenylalanine, 2- amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropanoic acid, (2R)-3-(3,4-dihydroxyphenyl)- 2-{[(2E)-3-(3,4-dihydroxyphenyl)-2-propenoyl]oxy}propanoic acid (Rosmarinic acid), dopamine and L-DOPA and D-DOPA; or any isomers or mixtures thereof.

In a preferred embodiment of the present invention said compound is a polymer comprising at least one chemical group selected from -COOH, -OP0₃H, -OS0₃H and - S0₃H. In another preferred embodiment said compound is a polymer comprising glutamic acid and/or aspartic acid and/or analogues thereof.

In yet another preferred embodiment said compound is a polymer comprising a sugar, said sugar comprising at least one chemical group selected from -COOH, -S0₃ and - POsH.

In a specific preferred embodiment said compound is selected from the group consisting of poly(acrylic acid), poly(glutamic acid), poly(aspartic acid), hyaluronic acid, chondroitin sulfates, keratan sulfates and alginate.

In one particular embodiment thereof said compound is poly(acrylic acid).

The composition used in the method of the present invention may comprise further additives. Said composition may for example further comprise a photoacid and/or a photobase

In a preferred embodiment said composition further comprises a soluble metal of formula Mⁿ⁺, or any complex thereof, wherein n is 2, 3, 4, 5 or 6.

For example, M designates iron, aluminium, titanium, vanadium, manganese, copper, chromium, magnesium, calcium, silicon, zinc, gallium, indium or boron.

Preferably, the stoichiometric ratio of catechol :metal is above 3:1 for octahedrally coordinated metals, such as Fe(lll), Al(lll), Mn(ll), V(lll), Cr(lll), and Ti(IV), and 2:1 for tetrahedrally coordinated metals.

In particular, the stoichiometric ratio of catechokmetal can be below or at 3:1 for octahedrally coordinated metals and 2:1 for tetrahedrally coordinated metals.

In another embodiment said composition further comprises carbon nanomaterials.

In one embodiment said additive is selected from the group consisting of proteins, enzymes, small drug molecules, DNA, RNA, lipids and stabilizers. In another embodiment said composition further comprises additional polymers.

Another aspect of the present invention relates to a coacervate obtained by the method as defined herein.

A further aspect relates to a coacervate as defined above for use as a medicament.

Another aspect relates to a coacervate as defined above for use in a medical device or use of the coacervates in a medical device.

Yet another aspect of the present invention relates to a coacervate as defined above for use in the treatment of tissue injuries.

Yet another aspect of the present invention relates to a composition comprising at least 0,25 wt% of a polymer backbone as defined herein and at least 0,25 wt% a compound as defined herein.

In a preferred embodiment said polymer backbone is functionalized with at least one type of monomer units selected from the group consisting of DOPA, 1 -(2'- carboxyethyl)-2-methyl-3-hydroxy-4(1 H)-pyridinone, phenylalanine. Preferably, said DOPA is L-DOPA.

In a preferred embodiment said polymer backbone comprises at least one type of polymer selected from the group consisting of polyallylamine, chitosan,

In one embodiment said compound is selected from the group consisting of polyacrylic acid, tannic acid, phytic acid and osteopontin.

In one embodiment said composition comprises at least 0,5 wt%, such as at least 1 wt% or such as at least 5 wt% of said polymer backbone.

In one embodiment said composition comprises at least 0,5 wt%, such as at least 1 wt% or such as at least 5 % of said compound. In one embodiment the polymer backbone to compound ratio by weight is from 3:1 to 1 :2.

In one embodiment the polymer backbone to compound ratio by weight is 1 :1.

A further aspect relates to a composition as defined above for use as a medicament.

Another aspect relates to a composition as defined above for use in a medical device. Yet another aspect of the present invention relates to composition as defined above for use in the treatment of tissue injuries.

A kit comprising a composition according to any of claims 77-81 and a pH adjustable component for adjusting the pH of said composition to obtain a second pH, whereby coacervates are formed.

A further aspect of the present invention relates to kit comprising

- a composition having a first pH comprising

• a polymer backbone comprising one or more backbone

monomer unit(s) and

• a compound

wherein said polymer backbone is charged and said compound is uncharged or wherein said polymer backbone is uncharged and said compound is charged

- a pH adjustable component for adjusting the first pH of said composition to obtain a second pH such that the uncharged polymer backbone or the uncharged compound become charged at said second pH

Preferably, polymer backbone is as defined herein. It is also preferred that the compound is as defined herein..

Another aspect of the present invention relates to use of the coacervate as defined herein and above as an adhesive, bioadhesive, glue, joining, attachment, coating and/or cohesive agent, for dry applications and/or for joining dissimilar substrates such as skin/bandaging and/or rubber/metal.

Yet another aspect of the present invention relates to use of the coacervate as defined herein and above, as a biomedical device such as a drug delivery vesicle, a wound care product or a burn care product.

The present invention further provides a method of forming a coacervate or a hydrogel comprising

- providing a composition comprising

- compound comprising one or more light sensitive catechol- group(s), said compound having the formula :

wherein:

^■ R¹ is selected from the group consisting of amine,

carboxylic acid, amide, carbamate, carbonate, ester, halogen, thiol and H;

^■ R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is selected from the group consisting of halogen, alk(an/en/yn)yl, hydroxyl, carboxylic acid, ester, ether, amine, amide and thiol; and

^■ A is selected from O and S

- a soluble metal of formula Mⁿ⁺, or any complex thereof, wherein n is 2, 3, 4, 5 or 6;

- a functionalized polymer backbone comprising one or more backbone monomer unit(s) comprising at least one functional group selected from amino, thiol and amino-oxy groups - exposing said composition to light whereby alcohol is converted to aldehyde

whereby a coacervate or a hydrogel is obtained and wherein said soluble metal and said polymer can be added to the composition either before or after exposure to light.

M may for example designate iron, aluminium, titanium, vanadium, manganese, copper, chromium, magnesium, calcium, silicon, zinc, gallium, indium, boron, gadolinium, samarium or europium.

In a preferred embodiment said compound is DNBA.

Preferably said polymer backbone is as defined herein. Thus, in a preferred

embodiment the polymer backbone comprises one or more backbone monomer unit(s) of the formula:

In another preferred embodiment the polymer backbone comprises one or more backbone monomer unit(s) comprising a chemical group having the formula: wherein:

R² and R³ are selected from the group consisting of H and Ci_-8-alk(an/en/yn)yl). In a preferred embodiment thereof R² and R³ are selected from the group consisting of H and Ci-8-alkanyl.

The present invention further provides a hydrogel or a coacervate obtained by the method as described above.

Another aspect of the present invention relates to a hydrogel or a coacervate as described herein and above for use as a medicament.

Yet another aspect of the present invention relates to a hydrogel or a coacervate as described herein and above for use in the treatment of tissue injuries. The present invention also relates to a composition comprising a compound comprising one or more light sensitive catechol- group(s), said compound having the formula

wherein:

- R¹ is selected from the group consisting of amine, carboxylic acid, amide, carbamate, carbonate, ester, halogen, thiol and H; - R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is selected from the group

consisting of halogen, alk(an/en/yn)yl, hydroxyl, carboxylic acid, ester, ether, amine, amide and thiol; and

- A is selected from O and S. It is preferred that said composition further comprises a metal of formula Mⁿ⁺, or any complex thereof, wherein n is 2, 3, 4, 5 or 6. It is further preferred that said composition further comprises a polymer backbone comprising one or more backbone monomer unit(s) comprising at least one functional group selected from amino, thiol and amino- oxy groups. Preferably, said polymer backbone is as defined herein.

Description of Drawings

Figure 1 . Wet adhesion through mussel-inspired proto-coacervates. Photographs showing the process of (a) byssal thread formation of a mussel foot at the point of injection of mussel foot protein containing material and (b) retraction of the foot leaving the formed thread exposed to the higher pH of the surrounding seawater resulting in attachment and subsequent curing (c) Mussel-inspired proto-coacervate including a charged polycation and uncharged polyanion secreted in a pipette tip in a manner analogous to the situation in (a), and (d) coacervate plaque and thread formation on a silicon wafer less than a minute after injection of the proto-coacervate. The pipette tip is 50 mm long and 1 mm in diameter. The efficient and simple formation of coacervates can be achieved through formulation of a high-concentration proto- coacervate at a pH where one of the polyions is neutral, in this case under acidic condition where polyallylamine (PAAm) is charged while PAA is not (scheme below Figure 1 c). Upon injection into neutral pH water, coacervates form via pH equilibration with the solvent (scheme below Figure 1 d).

Figure 2. Characterization of coacervation between DOPA-PAAm and PAA using traditional approach, (a) Upper panel: images of the titration stages of the B1 solution using NaOH. Four titration stages were observed (I) pre-coacervation, (II)

coacervation, (III) precipitation, and (IV) quinone oxidation or tanning. Lower panel: optical image of coacervate B1 dispersed in solution, (b-d) Dependence of

coacervation measured by turbidity on (b) pH of B1 , (c) (DOPA-PAAm): PAA ratio, and (d) concentration (or degree of dilution) of polyion at 3:1 (DOPA-PAAm): PAA ratio. In Figures 2c and 2d, filled and unfilled symbols stand for turbidity and pH values, respectively.

Figure 3. Coacervate formation using mussel-inspired proto-coacervate. (a) A coacervate film formed on a glass slide underwater by the present proto-coacervate injection method. A part of the coacervate diffused from the film and was diluted by water during coacervation forming classical water-dispersed coacervate droplets. The solution (B10, 100 μΙ_) was injected using a pipette, (b) ln-situ tracking of the pH change of a proto-coacervate during coacervation in buffer (pH 7) using a solid pH meter onto which the proto-coacervate was deposited, (c) (Water-background)- subtracted IR absorption spectra of B10 (7) proto-coacervate and (2) coacervate film. Figure 4. Rheology of coacervates and proto-coacervate. (a) Storage (filled symbols) and loss modulus (open symbols) of coacervate 1 %Ci :i ,_COntroi> B10, B5i, AO.25, A1 , B1 , and proto-coacervate B10 solution, (b) Storage and loss modulus of different coacervates and proto-coacervate B10 solution at an angular frequency of 2.5 s^' Figure 5. Dry and wet adhesion of coacervates prepared from traditional coacervation method and proto-coacervate. Shear strength of overlapped glass substrates using different coacervates as adhesives. All samples were cured at 53 °C for 20 h before testing, except wet B10 which was tested as a wet (uncured) adhesive. The B10 proto- coacervate is the only non-coacervate sample.

Figure 6. Dependence of underwater shear strength of overlapped glass substrates using B10 coacervate as adhesive on (a) coacervation time, (b) gluing time, and (c) temperature, (d) Swelling of B10 coacervate after 5 h injection on glass substrate at pH 9 at room temperature 22 °C; and therefore, no adhesion test was performed for B10 coacervate at pH 9.

Figure 7. Catechols are pre-coordinated with aluminum ions. Then light is shined on the system, converting the alcohol to an aldehyde. The aldehyde then reacts with the available amino-oxy groups cross-linking the polymers. Top: the reaction on a molecular level, bottom: the reaction on a gel level.

Figure 8. Coacervate on surface of a solid pH meter as deposited as a proto- coacervate while the pH meter was embedded in acetate buffer pH 7. The images show different views (a) in buffer pH 7 and (b and c) in air showing the resulting coacervate.

Figure 9. (Water-background)-substracted IR absorption spectra of solutions of (7) DOPA-PAAm, (2) PAA at pH 2, and (3) PAA at pH 7.

Figure 10. (a) Electrical conductivity of the CNT-coacervate composite as a function of the weight ratio of CNT:coacervate. The inset illustrates a sample prepared for the electrical conductivity measurement. A wet composite was packed into a 1 -mm in diameter glass tube and then dried in an oven at 53 °C for 12 h. (b) Demonstration of the CNT:coacervate (1 :8, w:w) composite conductivity in which the composite is a conductor to light up an LED.

Figure 1 1 . Underwater fabrication of CNT-coacervate composites. CNT-coacervates are formed efficiently by injecting into water a high-concentration CNT-protocoacervate at a pH where one of the polyions is neutral. In the present study, this is ensured under acidic conditions, implying that chitosan is charged while PAA is not (left scheme). Upon injection into water, complex coacervates form as pH is increased and

equilibrium is obtained with the solvent (right scheme). Figure 12. (a) TEM images with scale bars of 5 μηη and (b) storage G' (fill) and loss modulus G" (no fill) of (1 ) (DOPA-chitosan)-PAA coacervate and (2-7) composites of (DOPA-chitosan)/PAA coacervate and CNT at different weight ratio of CNT:coacervate varying from 1 :4 to 1 :48 (w:w). The arrow in Figure 2a represents decrease of protocoacervate concentrations.

Figure 13. (a) Load-displacement plot of a representative indent performed on the CNT-coacervate (1 :30, w:w) film, (b) modulus (fill) and (unfill) hardness of pure coacervate and CNT-coacervate composite films of different CNT:coacervate ratio. The films were coated onto glass slides.

Figure 14. (a) CNT-coacervate composites formed underwater at a volume ratio (and concentration) of CNT: protocoacervate (left) 1 :30 (2.7wt%), (middle) 1 :20 (1 .9wt%), (right) 1 :12 (1.22wt%). The injection volume is 20 μΙ_ and the coacervation time is 10 min. (b) The cross-section SEM image of a composite 20:500 film. For sample preparation, please, refer to the Experimental section.

Figure 15. (a) Modulus and (b) hardness heat map of 10x10-indentation for coacervate and CNT-coacervate films coated on glass slides. The distance between indents is 5 μηη. The ratio numbers stand for CNT:coacervate ratios (see Table 4).

Detailed description Definitions

The expression "C_1-10-alk(en/yn)yl" means Ci-i₀-alkyl, C_2-8-alkenyl or C_2-io-alkynyl;

wherein:

• The term "Ci-i₀-alkyl" refers to a branched or unbranched alkyl group having from one to ten carbon atoms, including but not limited to methyl, ethyl, prop-1 -yl, prop- 2-yl, 2-methyl-prop-1 -yl, 2-methyl-prop-2-yl, 2,2-dimethyl-prop-1 yl, but-1 -yl, but-2-yl, 3-methyl-but-1 -yl, 3-methyl-but-2-yl, pent-1 -yl, [rho]ent-2-yl, pent-3-yl, hex-1 -yl, hex-

2- yl, hex-3-yl, 2-methyl-4,4-dimethyl-pent-1 -yl and hept-1 -yl;

• The term "C₂-io-alkenyl" refers to a branched or unbranched alkenyl group having from two to ten carbon atoms and one double bond, including but not limited to ethenyl, propenyl, and butenyl; and

• The term "C₂-io-alkynyl" refers to a branched or unbranched alkynyl group having from two to ten carbon atoms and one triple bond, including but not limited to ethynyl, propynyl and butynyl. The term "Ci-₆-alk(en/yn)yl" means Ci_-6-alkyl, C_2-6-alkenyl or C_2-6-alkynyl; wherein:

• The term "C_1-6-alkyl" refers to a branched or unbranched alkyl group having from one to six carbon atoms, including but not limited to methyl, ethyl, prop-1 -yl, prop-2 - yl, 2-methyl- prop-1 -yl, 2-methyl-prop-2-yl, 2,2-dimethyl-prop-1 -yl, but-1 -yl, but-2-yl,

3- methyl-but-1 -yl, 3-methyl-but-2-yl, pent-1 -yl, pent-2-yl, pent-3-yl, hex-l-yl, hex-2-yl and hex-3-yl;

• The term "C₂-₆-alkenyl" refers to a branched or unbranched alkenyl group having from two to six carbon atoms and one double bond, including but not limited to ethenyl, propenyl, and butenyl; and

• The term "C₂-₆-alkynyl" refers to a branched or unbranched alkynyl group having from two to six carbon atoms and one triple bond, including but not limited to ethynyl, propynyl and butynyl.

The term "C₂-₄-alk(en/yn)yl" means C_2-4-alkyl, C_2-4-alkenyl or C_2-4-alkynyl; wherein:

• The term "C_2-4-alkyl" refers to a branched or unbranched alkyl group having from two to four carbon atoms, including but not limited to prop-1 -yl, prop-2 -yl, 2-methyl- prop-1 -yl, 2-methyl-prop-2-yl, but-1-yl and but-2-yl;

• The term "C_2-4-alkenyl" refers to a branched or unbranched alkenyl group having from two to four carbon atoms and one double bond, including but not limited to ethenyl, propenyl, and butenyl; and

· The term "C_2-4-alkynyl" refers to a branched or unbranched alkynyl group having from two to four carbon atoms and one triple bond, including but not limited to ethynyl, propynyl and butynyl.

The term "C₃-alk(en/yn)yl" means C₃-alkyl, C₃-alkenyl or C₃-alkynyl; wherein: • The term "C₃-alkyl" refers to a branched or unbranched alkyl group having three carbon atoms, including but not limited to prop-1 -yl and prop-2-yl;

• The term "C₃-alkenyl" refers to a branched or unbranched alkenyl group having from three carbon atoms and one double bond, including but not limited to propenyl; and

• The term "C₃-alkynyl" refers to a branched or unbranched alkynyl group having from two to four carbon atoms and one triple bond, including but not limited to propynyl. The terms "amino-Ci-io-alk(an/en/yn)yl", "amino-Ci-₆-alk(en/yn)yl", "amino-C₂-4- alk(en/yn)yl" and "amino-C₃-alk(en/yn)yl" as used herein refers to "CMO- alk(an/en/yn)yl", "C_1-6-alk(en/yn)yl", "C₂-4-alk(en/yn)yl" and "C₃-alk(en/yn)yl" as defined herein above which is substituted with an amino group (-NH₂), preferably in a terminal position of said alk(an/en/yn)yl.

The term "amino group" or amine as used herein includes primary, secondary and tertiary amines such as -NH₂, -NH(R') and -N(R')₂, but also quartenary ammonium ion of the type -N⁺(R')_3, guanidinium, imidazole, indole, pyridine, pyridinium or other nitrogen containing compounds that either are charged and can become charged at some value of pH.

The inventors of the present invention have succeeded in inventing a novel method of forming coacervates by electrostatic-driven complexation between polycations and polyanions. The method of the present invention provides coacervates with improved mechanical properties. For example, the coacervates obtained by the method of the present invention have improved shear strength and display surprisingly high adhesion properties. Further, the method of the present invention allows the formation of compositions with very high concentrations of polymer and compound that only forms coacervates when the pH is changed. This allows for easy injections in the body because coacervates are formed upon injection that results in hydration of the composition and thereby a pH change. Injection of coacervates into the body can for example be used to treat injured tissues or wounds. Moreover, the coacervates provided herein are not only biocompatible and biodegradable, but also waterproof with a consistently high adhesive strength. One aspect of the present invention provides a method of forming coacervates comprising

- providing a composition having a first pH comprising

• a polymer backbone comprising one or more backbone monomer unit(s) and

• a compound

- adjusting the first pH of said composition to obtain a second pH such that the uncharged polymer backbone or the uncharged compound become charged at said second pH,

whereby coacervates are formed. As used herein the term "coacervates" refers to electrostatically-driven liquid-liquid phase separation, resulting from association of oppositely charged macro-ions.

It is preferred that the composition is not phase-separated. Thus, the term composition preferably refers to the stage where coacervates have not formed. The components of the composition can be referred to as proto-coacervates.

Thus, the term "proto-coacervates" or "protocoacervates" refers to the composition comprising polymer backbone and compound, wherein coacervates have not been formed. The proto-coacervates, comprising the polymer backbone and the compound will form coacervates when the first pH is changed to the second pH.

As used herein, the term 'polymer backbone' refers to the series of covalently bonded atoms that together create a continuous chain of a polymer molecule.

In one embodiment the polymer backbone is charged. Thus, in one embodiment the polymer backbone is cationic at said first pH value. In another embodiment the polymer backbone is anionic at said first pH value. Preferably, the polymer backbone is charged and the compound is uncharged. For example, the polymer backbone is cationic at said first pH value, whereas the compound is uncharged. When pH changes, the compound becomes anionic whereby coacervates are formed. In another embodiment the polymer backbone is uncharged and the compound is charged. Thus, when changing pH the charge of the polymer backbone or the compound changes such that the polymer and the compound become oppositely charged, whereby coacervates are formed. In one preferred embodiment the second pH value is higher than the first pH value. The second pH value can for example be obtained by addition of a base, such as a base comprising an OH-group, such as KOH or NaOH.

In another embodiment the second pH value is lower than the first pH value. Thus, the second pH value can be obtained by addition of an acid, such as HCI or acetic acid.

In a further embodiment the second pH value is obtained by delivering said

composition to a location in the body whereby the first pH value changes to the second pH value whereby coacervates are formed. In one embodiment the pH of the composition is below the pH of location in the body where the composition is delivered. The composition can for example be delivered to the location in body by injection. This will, in one embodiment, lead to an increase of the pH whereby coacervates are formed. The location may for example be a location of tissue injuries. In another embodiment the second pH is obtained by using an enzyme such as for example urease that increases pH by catalysing the hydrolysis of urea into carbon dioxide and ammonia.

The second pH value may also be obtained by exposing the composition to light leading to a chemical change that changes pH. This can be performed by the addition of photoacids and/or a photobases to the compostion. Thus, in one embodiment of the present invention, said composition further comprises a photoacid and/or a photobase.

In one preferred embodiment the first pH value is below 7, such as for example below 6, such as below 5, or for example below 4, such as below 3. In a more preferred embodiment the first pH value is below 2. In another preferred embodiment the first pH value is below 1.

Coacervates are formed when the second pH value of the composition is obtained. Formation of coacervates may for example happen at pH at or above 2, such as at or above 3, such as for example at or above 4, such as at or above 5, such as for example at or above 6 or such as at or above 7.

Thus, in one embodiment the second pH is at least 2, such as at least 3, at least 4, for example at least 5, such as for example at least 6 or at least 7.

In another embodiment the second pH has a value in the interval from 2 to 7, such as from 2 to 6, or more preferred from 2 to 5, such as for example from 2 to 4 or such as from 2 to 3.

In another embodiment the second pH has a value in the interval from 3 to 7, such as from 3 to 6, such as from 3 to 5, or such as for example from 3 to 4.

Increasing the concentration of polymer and compound may elevate the pH at which coacervates are formed (see example section).

THE POLYMER BACKBONE

Within the context of the present invention, a polymer backbone comprises backbone monomer units, which may be identical or different from each other, and of which at least one comprises one or more chemical groups selected from amino groups, carboxylates, phosphates and sulfonates.

In one embodiment the polymer backbone comprises one kind of backbone monomer units, only, said backbone monomer units comprising one or more chemical groups selected from amino groups, carboxylates, phosphonates, phosphates and sulfonates. In another embodiment the polymer backbone comprises more than one kind of backbone monomer units, wherein at least one kind of monomer units comprising one or more chemical groups selected from amino groups, carboxylates, phosphonates, phosphates and sulfonates.

The polymer backbone can comprise one or more backbone monomer unit(s), which is/are stimuli-responsive. By the term "stimuli-responsive" as used herein is meant that the polymers respond/change conformation when being actuated by a stimuli e.g. pH, temperature, humidity, light etc. In a further embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are thermo- responsive. By the term "thermo-responsive" as used herein is meant that the polymers undergo a physical change when external thermal stimuli are presented. In yet one further embodiment the polymer backbone comprises one or more backbone monomer unit(s), which is/are light-responsive. By the term "light-responsive" as used herein is meant that the polymers undergo a physical change when exposed to light. In yet another embodiment of the invention, the polymer backbone comprises one or more backbone monomer(s) which is/are soluble in polar solvents. The polar solvents can for example be selected from water, DMSO and DMF. For example the polar solvent is water.

In yet one further embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are selected from the group consisting of:

- amino-Ci-io-alk(an/en/yn)yl which is optionally substituted;

- amino acids such as of the formula H₂N-CHR¹-COOH wherein R¹ is an organic substituent;

- amides of the formula -(CO)-(NH2)-Ci_-6-alk(an/en/yn)yl-NH2;

- aminostyrenes which are optionally substituted; and

in their open chain form or in the a-form or β-form thereof.

Accordingly, said polymer backbone may comprise any of the backbone polymer units disclosed herein and it may comprise any mixture thereof with other monomer units known to the skilled person. In one preferred embodiment, the backbone monomer units comprised in the polymer backbone are identical. In another embodiment, two different kinds of backbone monomer units are comprised in the polymer backbone. In yet another embodiment, three different kinds of backbone monomer units comprised in the polymer backbone, such as in a polymer backbone made of chitosan or polyallylamine.

In a particular embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) with a pKa ranging from 4 to 10, such as from 5-10, such as for example from 6-10 or such as from 8-10. In a specific embodiment thereof, said pKa ranges from 6-9, such as from 7-8. In another em bodiment said pKa ranges from 4-8, such as from 5-7. In the present context the pKa value of the polymer is taken to be the same as the pH of the polymer defined as the pH where half the polymer pH-responsive groups are charged and the other half is uncharged.

The method of the present invention is applicable to polymer backbones which in addition to backbone monomer unit(s) comprising one or more chemical groups selected from amino groups, carboxylates, phosphonates, phosphates and sulfonates may further comprise any other monomer unit(s) known to the skilled person. Based on the intended use of the hydrogel obtained, the skilled person will know which backbone monomer units to be incorporated into the backbone polymer. Essential for a purposive selection of backbone monomer units to be incorporated into the backbone polymer is the pKa of the resulting backbone polymer.

Accordingly, the pKa of the resulting backbone polymer shall preferably be within the range of a pH which is appropriate for the intended use of the obtained hydrogel.

Accordingly, for a coacervate for human applications, the pKa of the backbone polymer shall preferably be between 6 and 10, such as about 7. In particular, for a hydrogel for a drug delivery matrix, the pKa of the backbone polymer shall preferably be below 7. In another embodiment the pKa of the backbone polymer is in the interval from 6 to 7 or in the interval from 8.5 to 9.5, such as from 8.5 to 9. In particular, for a coacervate for a tissue adhesive, the pKa of the backbone polymer shall preferably be about 7. In particular, for a coacervate to stop bleedings, the pKa of the backbone polymer shall preferably be between 6 and 8, such as about 7.

Furthermore, for a coacervate for under-water applications, the pKa shall preferably be between 1 and 9, such as about 8. Accordingly, in one embodiment of the invention, the polymer backbone has a pKa between 7 and 9, such as a pKa of about 8. In another embodiment of the invention, the polymer backbone has a pKa of below 7. In one further embodiment of the invention, the polymer backbone has a pKa between 6 and 8, such as at a pKa of about 7.

Furthermore, in one embodiment of the invention, the functionalized polymer backbone has a pKa between 7 and 9, such as a pKa of about 8. In another embodiment of the invention, the functionalized polymer backbone has a pKa of below 7. In one further embodiment of the invention, the functionalized polymer backbone has a pKa between 6 and 8, such as at a pKa of about 7.

In one embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are thermo-responsive and one or more backbone monomer unit(s) selected from the group consisting of:

• amino-Ci-io-alk(an/en/yn)yl which is optionally substituted;

• amino acids such as of the formula H₂N-CHR¹-COOH wherein R¹ is an organic substituent;

• aminostyrenes which are optionally substituted; and

· amino sugar(s) such as of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-

(CHR⁴)-(CHR⁵)-OH; wherein

- R³, R⁴, R⁵ and R⁶ are independently selected from the group consisting of -H, -OH and Ci_-6-alk(an/en/yn)yl wherein Ci_-6-alk(an/en/yn)yl is optionally substituted with one or more -OH groups; and - R⁷ is selected from the group consisting of -H and -(CO)-Ci-6- alk(an/en/yn)yl wherein -(CO)-Ci_-6-alk(an/en/yn)yl is optionally substituted with one or more -OH groups;

in their open chain form or in the a-form or β-form thereof. In one specific embodiment of the present invention, the polymer backbone comprises one or more backbone monomer unit(s) of the generic formula amino-Ci-i₀- alk(an/en/yn)yl which is/are optionally substituted with one or more substituents.

In one particular embodiment thereof, the polymer backbone comprises one or more backbone monomer unit(s) which is/are thermo-responsive and one or more backbone monomer unit(s) selected from the group consisting of amino-Ci-i₀-alk(an/en/yn)yl which is optionally substituted. In one specific embodiment thereof, DOPA is a functionalizing catechol monomer unit which is grafted to the polymer backbone. In one further specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) of the generic formula amino-Ci_-6- alk(an/en/yn)yl which is optionally substituted with one or more substituents. In yet one further specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) of the generic formula amino-C₂-4-alk(an/en/yn)yl which is optionally substituted with one or more substituents. In yet one further specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) of the generic formula amino-C₃-alk(an/en/yn)yl which is optionally substituted with one or more substituents.

The backbone polymer unit allylamine has a pKa value of about 9.3.

In an even more specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are selected from the group consisting of amino-Ci-i₀-alk(an/en/yn)yl, amino-Ci_-6-alk(an/en/yn)yl, amino-C₂-₄- alk(an/en/yn)yl and amino-C₃-alk(an/en/yn)yl is substituted with one or more substituent(s) selected from the group consisting of -SH, - OH, -COOH, -NH₂, -S-CH₃, -O-CH3, -CH(OH)-CH₃ and -CH(SH)-CH₃. In an embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) selected from the group of amino acids which occur naturally and which do not occur naturally.

In a particular embodiment thereof, said amino acid(s) is/are of the generic formula H₂N-CHR¹-COOH wherein R¹ is an organic substituent. In a more specific embodiment thereof, said amino acid(s) is/are of the formula H₂N-CHR¹-COOH; wherein R¹ is selected from the group consisting of -H, -Ci_-6-alk(an/en/yn)yl, -Ci_-6-alk(an/en/yn)yl-R², wherein R² is selected from the group consisting of -SH, -COOH, C₃H₃N₂, -NH₂, -S- CH₃, -CO-NH₂, -NH-C(NH)NH₂, - OH, -CH(OH)-CH₃, -SeH, -C₈H₆N, -C₆H₅ -C₆H₄OH, and -C₆H₃(OH)₂. In one embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) of the formula H₂N-CHR¹-COOH which is/are a naturally occurring amino acid. In one particular embodiment thereof, said amino acid(s) of the formula H₂N-CHR¹-COOH is a naturally occurring amino acid which is selected from the group consisting of DOPA, ornithine, lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine and valine.

In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N-CHR¹-COOH thus being ornithine having a pKa value of about 10.75. In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N-CHR¹-COOH thus being lysine. In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N-CHR¹-COOH thus being arginine. In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N-CHR¹-COOH thus being histidine. In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N- CHR¹-COOH thus being aspartic acid. In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N-CHR¹-COOH thus being glutamic acid. In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N-CHR¹-COOH thus being serine. In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N-CHR¹-COOH thus being threonine. In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N-CHR¹-COOH thus being DOPA. In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N- CHR¹-COOH thus being asparagine. In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N-CHR¹-COOH thus being glutamine. In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N-CHR¹-COOH thus being cysteine. In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N-CHR¹-COOH thus being

selenocysteine. In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N- CHR¹-COOH thus being glycine. In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N-CHR¹-COOH thus being alanine. In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N-CHR¹-COOH thus being isoleucine. In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N-CHR¹-COOH thus being leucine. In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N-CHR¹-COOH thus being methionine. In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N- CHR¹-COOH thus being phenylalanine. In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N-CHR¹-COOH thus being tryptophan. In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N-CHR¹-COOH thus being tyrosine. In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are of the formula H₂N-CHR¹-COOH thus being valine.

In one embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are amide(s). In a particular embodiment thereof, said amide(s) is/are of the formula -(CO)-(NH₂)-Ci_-6-alk(an/en/yn)yl-NH₂.

In one embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) which is/are comprising styrene which is optionally substituted. In an embodiment thereof, said styrene is not substituted. In another embodiment thereof, said styrene is substituted with one or more substituents selected from the group consisting of NH₂-Ci_-6-alk(an/en/yn)yl- and -NH₂. In one embodiment, the polymer backbone comprises one or more backbone monomer unit(s) selected from the group of styrene which is substituted with an amino group. In one embodiment, the polymer backbone comprises one or more backbone monomer unit(s) selected from the group of styrene which is substituted with NH₂-Ci-6-alk(an/en/yn)yl-. In one particular embodiment thereof, said styrene is further substituted with one or more substituents selected from the group consisting of -SH, - OH, -COOH, -NH₂, -S- CH₃, -O-CH3, -CH(OH)-CH₃ and -CH(SH)-CH₃. In one specific embodiment thereof, said substituted styrene is para- NH₂-Ci-6-alk(an/en/yn)yl-styrene. In another specific embodiment thereof, said substituted styrene is meta- NH₂-Ci-6-alk(an/en/yn)yl-styrene. In another specific embodiment thereof, said substituted styrene is ortho- NH₂-Ci_-6- alk(an/en/yn)yl-styrene. In one specific embodiment thereof, said substituted styrene is para-amino-styrene. In another specific embodiment thereof, said substituted styrene is meta-amino-styrene. In another specific embodiment thereof, said substituted styrene is ortho-amino-styrene, such as e.g. 4-aminostyrene having a pKa value of about 4.5.

In one embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) selected from the group of amino sugars. In one particular embodiment thereof, said amino sugar(s) is/are of the generic formula 0=CR⁶- CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein R³, R⁴, R⁵ and R⁶ are independently selected from the group consisting of -H, -OH and Ci_-6-alk(an/en/yn)yl wherein Ci_-6- alk(an/en/yn)yl is optionally substituted with one or more -OH groups; and R⁷ is selected from the group consisting of -H and Ci_-6-alk(an/en/yn)yl-(CO)- ; wherein Ci_-6- alk(an/en/yn)yl-(CO)- is optionally substituted with one or more -OH groups; said amino sugar(s) being in its/their open chain form or in the a-form or β-form thereof.

In one specific embodiment of the invention, the polymer backbone comprises one or more backbone monomer unit(s) selected from the group of amino sugars of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein R³, R⁴, R⁵ and R⁶ are independently selected from the group consisting of -H, -OH, HO-(CH₂)- and - COO^" and HO-CH₂-CH(OH)- CH(OH)-. In one further embodiment, the polymer backbone comprises one or more backbone monomer unit(s) selected from the group of amino sugars, said amino sugar(s) is/are of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein R³ and R⁴ are independently selected from the group consisting of -H and -OH. In yet a further embodiment, the polymer backbone comprises one or more backbone monomer unit(s) selected from the group of amino sugars, said amino sugar(s) is/are of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein R³ is -OH. In yet a further embodiment, the polymer backbone comprises one or more backbone monomer unit(s) selected from the group of amino sugars, said amino sugar(s) is/are of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein R⁴ is selected from the group consisting of -H and -OH, such as wherein R⁴ is -H, or wherein R⁴ is -OH.

In yet a further embodiment, the polymer backbone comprises one or more backbone monomer unit(s) selected from the group of amino sugars, said amino sugar(s) is/are of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein R⁵ is selected from the group consisting of -(CH₂)-OH and -COO^".

In yet a further embodiment, the polymer backbone comprises one or more backbone monomer unit(s) selected from the group of amino sugars, said amino sugar(s) is/are of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein R⁶ is selected from the group consisting of -OH and -CH(OH)-CH(OH)-CH₂-OH.

In yet a further embodiment, the polymer backbone comprises one or more backbone monomer unit(s) selected from the group of amino sugars, said amino sugar(s) is/are of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein R⁷ is selected from the group consisting of -H and -(CO)-CH₃.

In yet a further embodiment, the polymer backbone comprises one or more backbone monomer unit(s) selected from the group of amino sugars, said amino sugar(s) is/are selected from the group consisting of glucosamine, acetyl-glucosamine, galactosamine and sialic acid in their open chain form or in any a-form or β-form thereof.

In yet a further embodiment, the polymer backbone comprises one or more backbone monomer unit(s) selected from the group of amino sugars, said amino sugar(s) is/are selected from the group consisting of glucosamine and acetyl-glucosamine in their open chain form or in any a-form or β-form thereof. In yet a further embodiment, the polymer backbone comprises one or more backbone monomer unit(s) selected from the group of amino sugars, said amino sugar(s) is/are of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein: R³ is -OH, R⁴ is -OH, R⁵ is HO-CH₂-, R⁶ is -OH, R⁷ is -H, in its open chain form or in any a-form or β-form thereof.

In yet a further embodiment, the polymer backbone comprises one or more backbone monomer unit(s) selected from the group of amino sugars, said amino sugar(s) is/are glucosamine(s) of the formula:

said glucosamine being in its open chain form or in any a-form or β-form thereof. The backbone polymer unit glucosamine has a pKa value of about 6.3-6.5.

In yet a further embodiment, the polymer backbone comprises one or more backbone monomer unit(s) selected from the group of amino sugars, said amino sugar(s) is/are galactosamine(s) of the formula:

said galactosamine being in its open chain form or in any a-form or β-form thereof.

In yet a further embodiment, the polymer backbone comprises one or more backbone monomer unit(s) selected from the group of amino sugars, said amino sugar(s) is/are formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein: R³ is -OH, R⁴ is -OH, R⁵ is -CH2-OH, R⁶ is -OH, R⁷ is -(CO)-CH₃; in its open chain form or in any a-form or β-form thereof.

In yet a further embodiment, the polymer backbone comprises one or more backbone monomer unit(s) selected from the group of amino sugars, said amino sugar(s) is/are acetylglucosamine(s) of the formula:

said acetylglucosamine being in its open chain form or in any a-form or β-form thereof.

In yet a further embodiment, the polymer backbone comprises one or more backbone monomer unit(s) selected from the group of amino sugars, said amino sugar(s) is/are formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein: R³ is -OH, R⁴ is -H, R⁵ is -COO^", R⁶ is -CH(OH)-CH(OH)-CH₂-OH., R⁷ is -CO-CH3, in its open chain form or in any a-form or β-form thereof.

In yet a further embodiment, the polymer backbone comprises one or more backbone monomer unit(s) selected from the group of amino sugars, said amino sugar(s) is/are sialic acid of the formula:

a-anomer β-anomer

said sialic acid being in its open chain form or in any a-form or β-form thereof.

In yet a further embodiment, the polymer backbone comprises at least 3 monomer units selected from the group of amino sugars, wherein 3 amino sugar(s) comprised in the polymer backbone together form chitosan having the formula:

in its open chain form or in any a-form or β-form thereof.

In yet a further embodiment, said amino sugar(s) comprised in the polymer backbone together from chitosan with varying degrees of de-acetylation in its open chain form or in any a-form or β-form thereof.

In a preferred embodiment, the polymer backbone comprises at least 3 backbone monomer units selected from the group of amino sugars, wherein said 3 amino sugars together form chitosan which is functionalized with DOPA catechol monomer unit(s).

Accordingly, chitosan is a specific example of a cationic polymer. Chitosan is a linear co-polymer formed from the -acetyl-D-glycosamine and D-glycosamine:

One such chitosan backbone co-polymer has a pKa value of about 6.3-6.5.

In chitosan, the D-glycosamine and N-acetyl-D-glycosamine are linked through β- glycosidic bonds:

Chitosan is both thermo- and pH-responsive owing to the ionisable primary amines on the chitosan chain that have a pKa value of about 6.5. Further to the close-to physiological pH, chitosan also has the advantage that it is biocompatible and biodegradable. Hydrogels comprising chitosan as the only backbone monomer unit is thus highly advantageous.

Polyallylamine is another specific example of a cationic polymer. Polyallylamine has a pKa value around 9.5. Polyallylamine is a linear homo-polymer formed through polymerization of allylamine.

In one embodiment, polyallylamine is used as the free base. In another embodiment, polyallylamine is used as the hydrobromide. In one further embodiment, polyallylamine is used as the hydrochloride, which is highly soluble in water as the amines along the polymer backbone are already protonated:

Chemical structure of poly(allylamine hydrochloride)

Within the context of the present invention, preferred are such polymer backbones being amino-functionalized polymer backbones with a pKa ranging from approximately 4 to 10, preferred 5-10.

In one embodiment of the present invention the polymer backbone has a molecular weight in the range of from 2 to 1000 kiloDalton (kDa) such as from 2 to 500 kDa, such as from 2 to 100 kD or such as for example from 2 to 10 kDa or for example from 4 to 6 kDa.

In another embodiment the polymer backbone has a molecular weight in the range of from 100 to 1000 kDa. In a preferred embodiment the polymer backbone has a molecular weight in the range of from 100 to 400 kDa such as from 190 to 310 kDa. In another preferred embodiment, the polymer backbone has a molecular weight in the range of from 250 to 650 kDa such as for example in the range of from 350 to 550 kDa. CATECHOL MONOMERS

Within the context of the present invention, a 'catechol monomer unit used for functionalizing the polymer backbone' is used synonymous with the term a

'functionalizing catechol monomer unit' and refers to a catechol monomer unit which is grafted to a backbone monomer unit of the polymer backbone, i.e. bound to a backbone monomer unit of the polymer backbone via a covalent bond, said catechol monomer unit comprising at least one catechol or catechol analogue functional group and further comprising at least one amino group and/or one carboxylate group. Such polymer backbone may be functionalized with one or more catechol monomer unit(s). In one embodiment the catechol monomer unit(s) are identical to each other. In another embodiment they are not identical to each other. In one embodiment of the present invention the polymer backbone comprises monomer unit(s) comprising one or more catechol and/or catechol analogue group(s). In one further embodiment thereof, said one or more functionalizing catechol monomer unit(s) comprise(s) at least one carboxylate-group. In a particular embodiment, said functionalizing catechol monomer unit(s) comprise(s) exactly one carboxylate-group. In yet one further embodiment, said one or more functionalizing catechol monomer unit(s) comprise(s) at least one amino group and at least one carboxylate-group.

In one embodiment of the invention, the one or more functionalizing catechol monomer or catechol analogue monomer unit(s) comprise(s) at least one amino group and/or one carboxylate-group of the below formula:

R¹⁰ is selected from the group consisting of (Ci_-6-alk(an/en/yn)yl)_q- COOH and (Ci_-6-alk(an/en/yn)yl)_r-R¹³ wherein:

- R¹³ is selected from the group consisting of NH₂, H, COOH, SH, HCO and OH; and

- q and r are independently selected from the group consisting of 0 and 1 ;

• R is of the formula:

In an embodiment, q is 0. In another embodiment, q is 1 . In an embodiment, r is 0. In another embodiment, r is 1. In an embodiment, R¹⁰ is selected from the group consisting of optionally substituted (C2-4-alk(an/en/yn)yl)_q-COOH and (C2-4-alk(an/en/yn)yl)_r-NH₂. In another embodiment, R¹⁰ is selected from the group consisting of optionally substituted (C_2-4-alk(an/en/yn)yl)- COOH and (C₂-4-alk(an/en/yn)yl)-NH₂. In one further embodiment, R¹⁰ is -COOH. In another embodiment, R¹⁰ is -NH₂. In yet another embodiment, R¹⁰ is optionally substituted (C₂-₄-alk(an/en/yn)yl)-COOH. In yet another embodiment, R¹⁰ is optionally substituted (C_2-4-alk(an/en/yn)yl)-NH₂. In an embodiment, R¹¹ is -OH. In another embodiment, R¹¹ is H.

In yet another embodiment, R¹¹ is ositioned as indicated herein: yet another embodiment, R¹¹ is positioned as indicated herein

R11

In yet another embodiment, R¹¹ is positioned as indicated herein:

In a specific embodiment of the invention, the one or more functionalizing catechol monomer unit(s) is/are selected from the group consisting of 2,4,5-trihydroxybenzoic acid, 3-(3,4-dihydroxyphenyl)propanoic acid, 3-(3,4-Dihydroxyphenyl)-2-methyl-alanine, 2,4,5-Trihydroxy-phenylalanine, 2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropanoic acid, (2R)-3-(3,4-dihydroxyphenyl)-2-{[(2E)-3-(3,4-dihydroxyphenyl)-2- propenoyl]oxy}propanoic acid (Rosmarinic acid), dopamine and L-DOPA and D-DOPA; or any isomers or mixtures thereof.

In an even more specific embodiment of the invention, the one or more functionalizing catechol monomer unit(s) is/are selected from the group consisting of 3,4- dihydroxyphenylacetic acid (DHPAA),3,4-dihydroxycinnamic acid (DHCA ) and DOPA; or any isomers or mixtures thereof.

In one further embodiment, the one or more functionalizing catechol monomer unit(s) is/are 2,4,5-trihydroxybenzoic acid of the formula:

In yet one further embodiment, the one or more functionalizing catechol monomer unit(s) is/are 3,4-dihydroxyphen lacetic acid of the formula:

In yet one further embodiment, the one or more functionalizing catechol monomer unit(s) is/are 3-(3,4-dihydroxyphenyl)propanoic acid of the formula:

In yet one further embodiment, the one or more functionalizing catechol monomer unit(s) is/are 3,4-dihydroxycinnamic acid (DHCA ) of the formula:

In yet one further embodiment, the one or more functionalizing catechol monomer unit(s) is/are 3-(3,4-dihydroxyphenyl)-2-methyl-alanine of the formula:

yet one further embodiment, the one or more functionalizing catechol

unit(s) is/are 2,4,5-ttrihydroxy-phenylalanine of the formula:

In yet one further embodiment, the one or more functionalizing catechol monomer unit(s) is/are 2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropanoic acid of the formula

In yet one further embodiment, the one or more functionalizing catechol monomer unit(s) is/are (2R)-3-(3,4-dihydroxyphenyl)-2-{[(2E)-3-(3,4-dihydroxyphenyl)-2- propenoyl]oxy}propanoic acid (Rosmarinic acid) of the formula:

In yet one further embodiment, the one or more functionalizing catechol monomer unit(s) is/are dopamine(s) which is/are linked to the backbone polymer via a di- carboxylic acid or other intermediate linker molecule of the formula:

In yet one further embodiment, the one or more functionalizing catechol monomer unit(s) is/are DOPA of the formula:

In yet one further embodiment, the one or more functionalizing catechol monomer unit(s) is/are L-DOPA of the formula:

In yet one further embodiment, the one or more functionalizing catechol monomer unit(s) is/are D-DOPA of the formula:

DOPA (i.e. L-DOPA and/or D-DOPA) is a specific example of such monomer unit(s) used for functionalizing the polymer backbone. DOPA is an amino acid, which is widely distributed in nature. It is produced from the amino acid tyrosine through

posttranslational modifications by the enzyme tyrosine hydroxylase. DOPA has two different forms; an unoxidized catechol form and an oxidized quinone form:

Catediol-DOPA Quinonc-DOPA

Chemical structures of the two forms of DOPA; Catechol-DOPA and Quinone-DOPA.

In an embodiment of the invention, the one or more catechol monomer unit(s) is/are attached to the polymer backbone via covalent bond(s). In an embodiment thereof, said covalent bonds are ester bonds or peptide bonds or a mixture of ester bonds and peptide bonds. In a particular embodiment thereof, said covalent bonds are a mixture of ester bonds and peptide bonds. In another particular embodiment thereof, said covalent bond is/are peptide bonds. In another particular embodiment thereof, said covalent bond(s) is/are ester bond(s).

A specific embodiment relates to DOPA functionalized polyallylamine and/or chitosan as shown below:

Chemical structure of A) DOPA functionalized chitosan and B) DOPA functionalized polyallylamine

In one embodiment, DOPA, such as L-DOPA, is a functionalizing monomer unit.

In one embodiment the polymer backbone to functionalizing monomer unit molar ratio is in the interval from 0.1 :99 to 1 :60 such as for example 1 :40

In one embodiment, suitable the reaction conditions for the synthesis of DOPA to chitosan is determined to be a DOPA to chitosan molar ratio of 0.75-3.0 : 0.5-1 .5 such as 1.5-2.5 : 0.5-1 .5 or such as for example DOPA to chitosan molar ratio of 1.8-2.2 : 0.8-1 .2. In a specific embodiment the DOPA to chitosan molar ratio is 2:1 . Preferably the pH of the reaction mixture is 6.. THE COMPOUND

The compound that is used in the method of the present invention forms a coacervate with the polymer backbone upon a change in pH. For example, when adjusting pH of the composition to obtain the second pH the compound become charged. At the second pH value, the charge of the compound is opposite the charge of the polymer backbone. This will result in the formation of coacervates. At the second pH, the compound is for example negatively charged. In another embodiment the compound is positively charged at the second pH. In one embodiment the compound is a polyion.

The compound can for example be a small molecule or an oligomer such as citric acid. In one embodiment the compound is selected from pyrophosphate and triphosphate. The compound can also be a higher polyphosphate.

In another embodiment, the compound is a peptide or protein with groups that can change charge with pH so that the overall charge of the molecules is negative or positive. In one such embodiment, the charge of the protein is negative. In a further such embodiment, the protein is osteopontin.

In one embodiment of the present invention the compound is a polymer. The compound may for example comprise backbone monomers as described above.

Preferably, the compound is a polymer comprising at least one chemical group selected from -COOH, -OP0₃H, -OS0₃H and -S0₃H. In another preferred

embodiment the compound is a polymer comprising glutamic acid and/or aspartic acid and/or analogues thereof. In yet another embodiment the compound is a polymer comprising a sugar, said sugar comprising at least one chemical group selected from - COOH, -S0₃ and -P0₃H. In another embodiment of the present invention the compound is selected from the group consisting of poly(acrylic acid), poly(glutamic acid), poly(aspartic acid), hyaluronic acid, chondroitin sulfates, keratan sulfates and alginate. In yet another embodiment, the compound is selected from the group consisting of poly(arginine), poly(lysine), polyallylamine and chitosan.

In a particular embodiment the compound is poly(acrylic acid) (PAA). A specific embodiment thereof relates to DOPA functionalized polyallylamine wherein the compound is PAA.

FURTHER ADDITIVES

In one embodiment the composition used in the method of the present invention further comprises a a soluble metal of formula Mⁿ⁺, or any complex thereof, wherein n is 2, 3, 4, 5 or 6.

Thus, metals suitable within the present invention are metals that can engage in coordinate bonding. Included are those metals which can have more than one oxidation state.

In one embodiment of the invention, the metal comprised in the composition is a transition metal. In another embodiment, the metal used in the method of the invention is selected from metals which have a high affinity for coordinate binding to 3,4- dihydroxyphenylalanine. In yet another embodiment, the metal used in the method of the invention is selected from metals which have a high affinity for coordinate bonding to dihydroxybenzene derivatives. In one further embodiment, the metal is ionic, that is, it does not form a covalent bond with a carbon atom such as would be present in a bond between a platinum atom and a phenyl derivative.

In one further embodiment, the soluble metal of formula Mⁿ⁺ designates such metals wherein n is selected from the group consisting of 2, 3, 4, 5 or 6. In another

embodiment thereof, n is selected from the group consisting of 2, 3 and 4. In yet another embodiment thereof, n is 2. In yet another embodiment thereof, n is 3. In yet another embodiment thereof, n is 4. In one further embodiment, M designates iron, aluminium, titanium, vanadium, manganese, copper, chromium, magnesium, calcium, silicon, zinc, gallium, indium or boron.

In one preferred embodiment thereof, M designates iron. In further embodiments, M designates iron, aluminium, titanium, vanadium, manganese, copper, gallium, indium, boron or chromium. In one preferred embodiment thereof, M designates boron. In a specific embodiment, boron is provided as B(OH)₃

In a further embodiment, M designates Ga, Fe, Al, Mn, V, Cr, Ti, Cu, Zn, Mg, Ca, Ag, Au, Ni or Co. In a further embodiment, M designates Ga, Fe, Al, Mn, V, Cr, Ti, Cu, Zn, Mg or Ca. In a further embodiment, M designates Ga, Fe, Al, Mn, V, Cr, Ti, Cu, Zn, Mg, Ca, In, Ln or Hf, where Ln represents a member of the Lanthanides that includes La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu. In a further embodiment, M designates Ga, Fe, Al, Mn, V, Cr, Ti, Cu, Zn, Mg, Ca, Ag or Au. In a further embodiment, M ⁿ⁺ designates Ga(lll), Fe(lll), Al(lll), Mn(ll), V(lll), Cr(lll), Ti(IV), Cu(ll), Zn(ll), Mg(ll), Ca(ll), B(lll) or In(lll). In one yet further embodiment, M designates an octahedrally coordinated metal, such as Ga(lll), Fe(lll), Al(lll), Mn(ll), V(lll), Ti(IV), Zn(ll), Mg(ll), Ca(ll) or Cr(lll), e.g. such as Ga(lll), Fe(lll), Al(lll), Mn(ll), V(lll) or Cr(lll), In one yet further embodiment, M designates a tetrahedrally

coordinated metals, such as Cu(ll) or B(lll). In a specific embodiment, M designates B, Fe, Al, Mn, Mg or Ca. In a further embodiment, M designates B, Fe, Al, or Ca. In a preferred embodiment, M designates B, Fe or Al.

In further embodiments, M designates iron, titanium, manganese, boron or copper, such as e.g. wherein M ⁿ⁺ designates B(lll), Fe(lll), Cu(ll), Ti(IV) or Mn(ll). In a specific embodiment thereof, M designates iron, such as wherein Mⁿ⁺ designates Fe(lll). In a further embodiment thereof, M designates aluminium, such as wherein Mⁿ⁺ designates Al(lll). In yet a further embodiment thereof, M designates titanium, such as wherein Mⁿ⁺ designates Ti(IV). In yet a further embodiment thereof, M designates vanadium, such as wherein Mⁿ⁺ designates V(lll). In yet a further embodiment thereof, M designates manganese, such as wherein Mⁿ⁺ designates Mn(ll). In yet a further embodiment thereof, M designates copper, such as wherein Mⁿ⁺ designates Cu(ll). In yet a further embodiment thereof, M designates chromium, such as wherein Mⁿ⁺ designates Cr(lll). In yet a further embodiment thereof, M designates boron, such as wherein Mⁿ⁺ designates B(lll). In yet a further embodiment thereof, M designates indium, such as wherein Mⁿ⁺ designates In(lll).

In one further embodiment, at the second pH, the metal of formula Mⁿ⁺ is coordinated to one, two or three catechol groups comprised in the one or more monomer(s) comprising an amino group and a catechol functional group. In one further

embodiment, at the second pH, the metal of formula Mⁿ⁺ is coordinated to one catechol group comprised in the one or more monomer(s) comprising an amino group and a catechol functional group. In one further embodiment, at the second pH, the metal of formula Mⁿ⁺ is coordinated to two catechol groups comprised in the one or more monomer(s) comprising an amino group and a catechol functional group. In one further embodiment, at the second pH, the metal of formula Mⁿ⁺ is coordinated to three catechol groups comprised in the one or more monomer(s) comprising an amino group and a catechol functional group. In one embodiment the stoichiometric ratio of catechokmetal is above 3:1 for octahedrally coordinated metals, such as Fe(lll), Al(lll), Mn(ll), V(lll), Cr(lll), and Ti(IV), and 2:1 for tetrahedrally coordinated metals.

In a particular embodiment the stoichiometric ratio of catechokmetal is below or at 3:1 for octahedrally coordinated metals and 2:1 for tetrahedrally coordinated metals.

In a preferred embodiment, the hydrogel of the present invention is for human applications and Mⁿ⁺ preferably designates Fe(lll). In another preferred embodiment, the hydrogel of the present invention is for human applications and Mⁿ⁺ preferably designates Fe(lll), Mg(ll), Gd(lll), B(lll) or Al(lll).

In another preferred embodiment, the hydrogel of the present invention is for non- human applications and then the order of preference for Mⁿ⁺ is: Fe(lll), Al(lll), Ti(IV), Mn(ll), Cu(ll), V(lll), Cr(lll), Hf(IV), Ln. In all embodiments, the metals may also be able to coordinate to amine groups on the polymer backbone; the tendency for a given metal to do this depends on pH, the relative binding strength of metal to catechol and metal to amine as well as the molar ratios of amine to catechol in the polymer. This adds to the flexibility of the present invention. Note that interactions between metals and amines can effectively lower the pKa value of the amine. Metals particularly prone to bind amines include but is not restricted to Al(lll), Fe(lll), Ga(lll), Cr(lll), Mg(ll), Ca(ll), Ag(l), Au(lll).

In another embodiment of the present invention the composition further comprises carbon nanomaterials. Carbon nanomaterials can for example include carbon nanotubes, fullerenes and graphene. In one embodiment the carbon nanomaterials are selected from graphene, graphene oxide and carbon nanotubes. In a particular preferred embodiment, the carbon nanomaterials are carbon nanotubes (CNTs).

Carbon nanotubes are allotropes of carbon with a cylindrical nanostructure and they include single-walled nanotubes and multi-walled nanotubes.

Thus, the present invention also provides a CNT-coacervate composite obtained by the method of the present invention. In a preferred embodiment, the CNT-coacervate composite has electrical conductivity.

In a preferred embodiment the CNT-coacervate composite is obtained by using a polymer backbone comprising or consisting of DOPA functionalized polyallylamine and/or DOPA functionalized chitosan as described herein and above. In one embodiment said DOPA is L-DOPA.

In one embodiment the CNT's are reacted with COOH. This is done to functionalize the CNT's. It is preferred that an acid is added to the CNT solution before mixing with polymer backbone and compound. This is to prevent coacervation. In one embodiment the acid is HCI.

The CNT:protocoacervates ratio by weight is for example at least 1 :12, such as at least 1 :10 or more preferably at least 1 :8. In one embodiment the CNT:protocoacervate ratio by weight is in the interval from 1 :12 to 1 :2, such as from 1 :12 to 1 :4, or preferably from 1 :10 to 1 :4 or more preferably from 1 :8 to 1 :4.

Protocoacervate refer to the solution comprising polymer and compound before coacervation is initiated due to a change in pH. The polymer backbone and the compound are as defined herein.The proto-coacervate solution may for example comprise DOPA functionalized chitosan and PAA. In another embodiment the proto- coacervate solution comprises DOPA functionalized PAAm and PAA.

The polymer backbone:compound ratio by weight is a defined herein above.

Preferably, the polymer backbone:compound ratio is 1 :1 . In one embodiment, suitable the reaction conditions for the synthesis of DOPA to chitosan is determined to be a DOPA to chitosan ratio of 0.75-3.0 : 0.5-1 .5 such as 1 .5- 2.5 : 0.5-1 .5 or such as for example DOPA to chitosan molar ratio of 1.8-2.2 : 0.8-1 .2. In a specific embodiment the DOPA to chitosan ratio is 2:1. Preferably the pH of the reaction mixture is 6..

An example for producing CNT-coacervate composites is shown in the example section. The example shows that CNT-coacervate composites are electrically conductive. Thus, in one embodiment the CNT-coacervate composite is electrically conductive.

It is clear from the example that the method of the present invention provides an extremely simple method for producing CNT- coacervate composites with surprising properties such as a tunable crosslinked network and electrical conductivity. Thus, another aspect of the present invention relates to use of a CNT-coacervate as described above for an organic field-effect transistor, a chemical sensor or a conductive tape. In one embodiment the composition further comprises at least one additive. Said additive may for example be selected from the group consisting of proteins, enzymes, small drug molecules, DNA, RNA, lipids and stabilizers. In another embodiment of the present invention the composition further comprises additional polymers.

MECHANICAL PROPERTIES OF THE COACERVATES Another aspect of the present invention relates to a coacervate obtained by the method as defined herein above. Thus, the coacervate is obtained by providing a composition having a first pH comprising

• a polymer backbone comprising one or more backbone monomer unit(s) and

· a compound

wherein said polymer backbone is charged and said compound is uncharged or wherein said polymer backbone is uncharged and said compound is charged adjusting the first pH of said composition to obtain a second pH such that the uncharged polymer backbone or the uncharged compound become charged at said second pH.

The polymer backbone and the compound are as defined herein above. Also, the composition is as defined herein above. Thus, the composition may comprise metal ions, nanomaterials and/or other additives as defined herein above.

The coacervates obtained by the methods of the present invention display unique properties related to adhesion. The method leads to the formation of coacervates having a surprisingly strong adhesion. The coacervates provided herein are not only biocompatible and biodegradable, but also waterproof with a consistently high adhesive strength. The adhesive strengths of various coacervates of the present invetion are shown in Table 2. Moreover, the coacervates provided herein are capable of forming strong under-water bonds to a wide range of surfaces, from inorganic to organic materials, and even in wet environments. The inventors of the present invention have found that parameters such as the concentration of coacervates, coacervation time, temperature and pH influence the adhesive properties of the resulting coacervates. Accordingly, one embodiment of the invention provides control of the adhesive properties of the coacervates provided herein.

For example, when the concentration of polymer backbone and compound in the composition is increase, the strength of adhesion of the resulting coacervates increases. In one embodiment the invention the concentration of polymer backbone and compound in the composition is at least 0.25 % by weight, such as at least 0.5 % by weight, such as for example at least 1 % by weight, at least 2 % by weight, such as at least 5 % by weight, such as for example at least 7 % by weight or such as for example at least 10 % by weight.

In a preferred embodiment the concentration of polymer backbone and compound in the composition is at least 5 % by weight. In a more preferred embodiment the concentration of polymer backbone and compound in the composition is at least 10 % by weight.

When the polymer backbone is functionalized with monomer units, the weight of the polymer backbone refers to the polymer backbone and the monomer units.

Raising the coacervation time leads to an increase in the adhesion strength of the resulting coacervates. The term "coacervation time" as used herein refers to the reaction time wherein compound and polymer backbone are allowed to assemble into coacervates. In one embodiment the coacervation time is at least 10 minutes, such as for example at least 30 minutes, such as for example at least 1 hour, such as for example at least 5 hours, such as at least 10 hours, such as for example at least 15 hours, such as at least 20 hours or such as for example at least 24 hours. Decreasing the temperature may in one embodiment lead to an increase in the adhesion strength of the coacervates. For example, the adhesion strength of the coacervates are increased when the temperature is decreased to a temperature below 37 °C. Thus, in a preferred embodiment the temperature of the coacervates is below 37 °C, such as for example below 35 °C, such as below 30 °C, such as for example below 25 °C, or such as below 20 °C.

In one embodiment the weight ratio of polymer backbone:compound is in the interval from 4:1 to 1 :1 , such as for example from 3:1 to 1 :1. In one embodiment the stoichiometric ratio of polymer backbone:compound is 3:1. In another embodiment the stoichiometric ratio of polymer backbone:compound is 2:1. In yet another embodiment the stoichiometric ratio of polymer backbone:compound is 1 :1 . In another embodiment the weight ratio of polymer backbone:compound is in the interval from 1 :1 to 1 :4, such as for example from 1 :1 to 1 :3, or such as from 1 :1 to 1 :2.

Further, the presence of a catechol or a catechol monomer unit may increase the adhesive strength of the resulting coacervates. Thus, in a preferred embodiment the polymer backbone comprises a catechol or a catechol monomer unit as described herein above. Preferably, said catechol is DOPA.

In a particular embodiment DOPA is grafted onto a polymer backbone comprising polyallylamine. In a specific embodiment the composition comprises polyallylamine comprising DOPA as a functional monomer. In a particular embodiment thereof, the compound is poly(acrylic acid).

In one embodiment of the present invention the strength of adhesion of the

coacervates is at least 0.5 MPa, such as for example at least 1 MPa. In a preferred embodiment the strength of adhesion of the coacervates is at least 1.5 MPa. In an even more preferred embodiment the strength of adhesion of the coacervates is at least 2 MPa. It is preferred that the strength of adhesion is measured using a lap shear test. This is explained in detail in the example section under "Adhesion

measurements". COMPOSITION

The present invention also provides a composition comprising at least 0,25 wt% of a polymer backbone according as defined herein and at least 0,25 wt% a compound as defined herein.

In a preferred embodiment the polymer backbone is functionalized. The polymer backbone can be functionalized in accordance with the embodiments described herein and above.

Preferably, said polymer backbone is functionalized with at least one type of monomer units selected from the group consisting of DOPA, 1 -(2'-carboxyethyl)-2-methyl-3- hydroxy-4(1 H)-pyridinone, phenylalanine. Said DOPA can be L-DOPA or D-DOPA as described above. It can also be a mixture of L-DOPA and D-DOPA. It is preferred that said DOPA is L-DOPA.

In one preferred embodiment said polymer backbone comprises polyallylamine, In another preferred embodiment said polymer backbone comprises chitosan. In a particular embodiment the polymer comprises or consists of polyallylamine and at least one type of monomer with which the polymer backbone is functionalized. In another particular embodiment the polymer comprises or consists of chitosan and at least one type of monomer with which the polymer backbone is functionalized.

In one embodiment the compound is selected from the group consisting of PAA, tannic acid, phytic acid, and osteopontin. The composition may further comprise Fe.

In a specific embodiment said composition comprises DOPA functionalized

polyallylamine and at least one compound selected from the group consisting of PAA, phytic acid and osteopontin. In one preferred embodiment the composition comprises DOPA functionalized polyallylamine and PAA. In one embodiment thereof, the composition further comprises Fe.

In another specific embodiment the composition comprises DOPA functionalized chitosan and at least one compound selected from the group consisting of PAA, tannic acid, alginate, phytic acid and osteopontin. The composition may also comprise chitosan and tannic acid. For example the composition comprises polyallylamine and tannic acid. The composition may in one embodiment comprise HOPO and at least one compound selected from the group consisting of osteopontin, phytic acid and tannic acid.

In one embodiment the invention the concentration of polymer backbone and compound in the composition is at least 0.25 % by weight, such as at least 0.5 % by weight, such as for example at least 1 % by weight, at least 2 % by weight, such as at least 5 % by weight, such as for example at least 7 % by weight or such as for example at least 10 % by weight.

In another embodiment the concentration of polymer backbone and compound in the composition is in the interval from 1 % by weight to 30 % by weight, such as from 1 % by weight to 20 % by weight, such as from 1 % by weight to 15 % by weight, or such as from 1 % by weight to 10 % by weight.

In a preferred embodiment the concentration of polymer backbone and compound in the composition is in the interval from 5 % by weight to 30 % by weight, such as from 5 % by weight to 20 % by weight, such as from 5 % by weight to 15 % by weight, or such as from 5 % by weight to 10 % by weight.

In a particular embodiment the concentration of polymer backbone and compound in the composition is 10 % by weight.

In one embodiment said composition comprises at least 0,5 % by weight, such as at least 1 % by weight or such as at least 5 % by weight of said polymer backbone. In a preferred embodiment said composition comprises at least 0,5 % by weight, such as at least 1 % by weight or such as at least 5 % by weight of said compound.

For example, the polymer backbone to compound ratio by weight is from 3:1 to 1 :2. Preferably, the polymer backbone to compound ratio by weight is 1 :1 .

In a preferred embodiment the composition comprises at least 5 % by weight of DOPA functionalized polyallylamine and PAA. In another preferred embodiment the composition comprises at least 10 % by weight of DOPA functionalized polyallylamine and PAA. In yet another preferred embodiment the concentration of DOPA

functionalized polyallylamine and PAA is in the interval from 5 % by weight to 10 % by weight.

In a particular embodiment the concentration of DOPA functionalized polyallylamine and PAA is 10 % by weight. The DOPA-polyallylamine to PAA ratio by weight is as defined herein above for polymer and compound. Preferably, the DOPA-polyallylamine to PAA ratio by weight is 1 :1.

In another embodiment of the present invention the composition further comprises carbon nanomaterials (CNTs). The CNTs are as defined herein and above.

The CNT:protocoacervates ratio by weight is as described herein and above. Thus, preferably, the CNT:protocoacervate ratio by weight is in the interval from 1 :8 to 1 :4. It is preferred that the composition comprising CNT comprises DOPA functionalized chitosan and PAA. In another embodiment the composition comprising CNT comprises DOPA functionalized PAAm and PAA.

An example for producing the CNT-composition is shown in the example section. Thus, another aspect of the present invention relates to use of a composition comprising CNT as described above for an organic field-effect transistor, a chemical sensor or a conductive tape.

One aspect of the invention relates to a composition as described above for use as a medicament. The composition can be applied to injured tissues inside the body by injection. In one preferred embodiment the composition as defined above is injected into the body at a location where tissue is damaged. Thus, preferably, proto-coacervates is injected into the body. When the composition enters the body, pH of the composition changes resulting in the formation of coacervates. The coacervates will then form a bioadhesive or a glue that promotes healing of the tissue injury.

Another aspect of the invention relates to a composition as described above for use in the treatment of tissue injuries.

Yet another aspect of the invention relates to use of the composition as a as an adhesive, bioadhesive, glue, joining, attachment, coating and/or cohesive agent. In further embodiment the composition may be used for dry applications and/or for joining dissimilar substrates such as skin/bandaging and/or rubber/metal.

Yet another aspect of the invention relates to use of the composition as a biomedical device such as a drug delivery vesicle, a wound care product or a burn care product.

USE OF COACERVATES

A further aspect of the present invention relates to a coacervate as defined herein above for use as a medicament.

Another aspect relates to use of the coacervate as defined herein in a medical device or as a medical device.

Yet another aspect of the present invention relates to a coacervate as defined herein above for use in the treatment of tissue injuries.

In yet another aspect, the present invention relates to a method for treating and/or ameliorating tissue injuries of an individual by applying coacervates as defined herein above to injured tissue. Tissue injuries may include damaged or injured tissue inside the body and wounds located at the surface of the body of a human or animal. For example, the tissue injury is a wound or a gash. The tissue injury can for example be in the intestine of a human or an animal. When the coacervates or composition are used for treating tissue injuries the coacervates preferably functions as a glue or a bioadhesive.

Thus, the present invention also relates to a coacervates or a composition as defined herein for use as a bioadhesive. The coacervates can be applied to injured tissues inside the body by injection. In one preferred embodiment the composition as defined above is injected into the body at a location where tissue is damaged. Thus, preferably, proto-coacervates is injected into the body. When the composition enters the body, pH of the composition changes resulting in the formation of coacervates. The coacervates will then form a bioadhesive or a glue that promotes healing of the tissue injury.

Yet another aspect of the present invention relates to use of the coacervate as defined herein as an adhesive, bioadhesive, glue, joining, attachment, coating and/or cohesive agent, for dry applications and/or for joining dissimilar substrates such as skin or bandaging and/or rubber or metal.

Yet another aspect of the present invention relates to use of the coacervate as defined herein as a biomedical device such as a drug delivery vesicle, a wound care product or a burn care product.

In one embodiment, the hydrogels coacervates provided herein are not only biocompatible and biodegradable, but also waterproof with a consistently high adhesive strength. Moreover, the coacervates provided herein are capable of forming strong under-water bonds to a wide range of surfaces, from inorganic to organic materials, and even in wet environments.

In certain embodiments, the formed coacervate may overtime swell in the solvent to become a hydrogel and/or highly viscous liquid that remains separate from the solvent. In a specific embodiment, hydrogels are formed at pH above 5, such as above 8, such as above 9.

Thus, a further aspect of the present invention relates to the use of coacervates in under-water applications. Thus, in one embodiment the coacervates are for use underwater.

KIT Another aspect of the present invention relates to a kit comprising

- a composition having a first pH comprising

• a polymer backbone comprising one or more backbone

monomer unit(s) and

• a compound

A further aspect of the present invention relates to a kit comprising a composition as defined herein and a pH adjustable component for adjusting the pH of said composition to obtain a second pH, whereby coacervates are formed.

It is appreciated that the polymer backbone is as defined herein above. Also, the compound and the composition is as defined herein above.

METHOD USING LIGHT SENSITIVE GROUPS

Another aspect of the present invention relates to a method of forming a coacervate or a hydrogel comprising

- providing a composition comprising

- compound comprising one or more light sensitive catechol- group(s), said compound having the formula:

wherein:

^■ R¹ is selected from the group consisting of amine,

carboxylic acid, amide, carbamate, carbonate, ester, halogen, thiol and H;

^■ R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are selected from the group consisting of H, halogen, alk(an/en/yn)yl, hydroxyl, carboxylic acid, ester, ether, amine, amide and thiol; and

^■ A is selected from O and S

- a functionalized polymer backbone comprising one or more backbone monomer unit(s) comprising at least one functional group selected from amino, thiol and amino-oxy groups

- exposing said composition to light

When the composition is exposed to light alcohol is converted to aldehyde leading to the formation of coacervates or a hydrogel.

In one embodiment A is S. In preferred embodiment A is O. In one embodiment R² is H and R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are selected from the group consisting of H, halogen, alk(an/en/yn)yl, hydroxyl, carboxylic acid, ester, ether, amine, amide and thiol. In another embodiment R⁵ is H and R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are selected from the group consisting of H, halogen, alk(an/en/yn)yl, hydroxyl, carboxylic acid, ester, ether, amine, amide and thiol.

In another embodiment R² and R⁵ are H and R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are selected from the group consisting of H, halogen, alk(an/en/yn)yl, hydroxyl, carboxylic acid, ester, ether, amine, amide and thiol.

In one embodiment R² and R³ are H. In another embodiment R⁵ and R⁶ are H. In one embodiment R², R³ and R⁴ are H. In yet another embodiment R⁵, R⁶ and R⁷ are H. In yet another embodiment R², R³, R⁵ and R⁶ are H.

In one particular embodiment R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are selected from the group consisting of H, halogen, Ci_-6alk(an/en/yn)yl, hydroxyl, carboxylic acid, ester, ether, amine, amide and thiol.

In a more specific embodiment said compound has the formula:

In one specific embodiment said compound is 4,5-dihydroxy-2-nitrobenzyl alcohol with the formula

In one embodiment M designates iron, aluminium, titanium, vanadium, manganese, copper, chromium, magnesium, calcium, silicon, zinc, gallium, indium, boron, terbium, erbium, gadolinium, samarium or europium.

In one embodiment Mⁿ⁺ designates Fe(lll), Al(lll), Ga(lll), Mn(ll), V(lll), Cr(lll), Ti(IV), Cu(ll), Zn(ll), Mg(ll), Ca(ll), B(lll), Si(IV), Gd(lll), Sm(ll), Eu(ll), Tb(IV), Er(lll) or In(lll).

In another embodiment, the hydrogel of the present invention is for non-human applications and then the order of preference for Mⁿ⁺ is: Fe(lll), Al(lll), Ti(IV), Mn(ll), Cu(ll), V(lll), Cr(lll), Hf(IV), Ln.

In a preferred embodiment M designates iron, aluminium, boron, gadolinium, terbium or erbium. Thus, in one preferred embodiment Mⁿ⁺ designates Fe(lll), Al(lll), B(lll), Gd (III), Tb(IV) or Er(lll).

embodiment thereof, n is selected from the group consisting of 2, 3 and 4. In yet another embodiment thereof, n is 2. In yet another embodiment thereof, n is 3. In yet another embodiment thereof, n is 4.

In one embodiment the polymer backbone is as defined herein above.

In one preferred embodiment th e comprises one or more backbone monomer unit(s) of the formula:

In another preferred embodiment the polymer backbone comprises one or more backbone monomer unit(s) comprising a chemical group having the formula:

wherein:

R² and R³ are selected from the group consisting of H and Ci_-8-alk(an/en/yn)yl). In one embodiment R² and R³ are selected from the group consisting of H and Ci_-8- alkanyl.

Another aspect of the present invention relates to hydrogel or a coacervate obtained by the method defined above, i.e. a hydrogel or a coacervate obtained by the method comprising

- providing a composition comprising

^■ R¹ is selected from the group consisting of amine,

carboxylic acid, amide, carbamate, carbonate, ester, halogen, thiol and H;

^■ A is selected from O and S

- a soluble metal of formula Mⁿ⁺, or any complex thereof, wherein n is 2, 3, 4, 5 or 6; - a functionalized polymer backbone comprising one or more backbone monomer unit(s) comprising at least one functional group selected from amino, thiol and amino-oxy groups

- exposing said composition to light

In yet another aspect, the present invention relates to a hydrogel or a coacervate as defined above for use as a medicament.

In yet another aspect, the present invention relates to a hydrogel or a coacervate as defined above for use in the treatment of tissue injuries.

A further aspect of the present invention relates to a composition comprising a compound comprising one or more light sensitive catechol- group(s), said compound having the formula:

- R¹ is selected from the group consisting of amine, carboxylic acid, amide, carbamate, carbonate, ester, halogen, thiol and H;

- R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is selected from the group

- A is selected from O and S. In one preferred embodiment the composition further comprises a metal of formula Mⁿ⁺, or any complex thereof, wherein n is 2, 3, 4, 5 or 6. Preferably, the metal is as defined above. In another preferred embodiment the composition further comprises a polymer backbone comprising one or more backbone monomer unit(s) comprising at least one functional group selected from amino, thiol and amino-oxy groups.

Preferably, said polymer backbone is as defined herein above.

Examples

Chemicals

PAA (M_w = 450000), NaOH (> 98%), HCI (≥ 37%), NaCI, and Na₂B₄0₇ (anhydrous) were purchased from Sigma. Glass slides were supplied from Hounisen

Laboratorieudstyr. Solution of PAAm 40% (for control experiment) was bought from PolyScience. DOPA-PAAm was synthesized according to previous published procedure (Krogsgaard, M.,et al. Biomacromolecules 14, 297-301 (2013)). Buffer pH 9 was prepared from 100 mL of 0.025 M Na₂B₄0₇ and 9.2 mL of 0.1 M HCI.

Coacervation

Different solutions of DOPA-PAAm polycation and PAA polyanion for studying coacervation were prepared and are presented in Table 1. DOPA-PAAm and PAA of AO.25, A1 , B1 , and B1 Control were dissolved in 0.2 mM HCI under vigorous stirring condition for 1 h. B1 Control is the same as B1 but the polycation is pristine PAAm (without functionalization with DOPA). Components of B5 and B10 were dissolved in demineralized water at 60 °C under vigorous stirring for 1 h. Coacervation of A0.25, A1 , B1 , and Control was observed by adding dropwise NaOH 2M and then NaOH 0.1 M solutions and pH simultaneously monitoring by a solid-state pH meter (FieldScout SoilStik). Coacervation of B5 and B10 was carried out by slow injection of their solution into distilled water using a 100 μΙ_ pipette tip.

Table 1. Composition of different solutions prepared for coacervation. Here, A and B stand for 3:1 and 1 :1 ratio of (DOPA-PAAm):PAA by weight, respectively. The numbers next to A or B are mass concentrations. Control represents for replacement of DOPA-PAAm by PAAm.

Name Composition

A0.25 0.25 wt% (3:1 , (DOPA-PAAm): PAA)

A1 1 wt% (3:1 , (DOPA-PAAm):PAA)

B1 1 wt% (1 :1 , (DOPA-PAAm): PAA)

B5 5 wt% (1 :1 , (DOPA-PAAm): PAA)

B10 10 wt% (1 :1 , (DOPA-PAAm): PAA)

B1 (Control) 1 wt% (1 :1 , PAAm: PAA)

Characterization of coacervates

Coacervation of DOPA-PAAm and PAA (Table 1 , except B5 and B10) at different conditions (pH, concentration, ratio) was examined by turbidimetry at 600 nm using an UV-vis spectrophotometer. The absorbance of non-coacervated polyions was negligible at 600 nm. The turbidity [mm^"1] is defined as r = Aid, where A is absorbance and d is sample or cuvette thickness (d = 10 mm).

Microscopy was performed on a Zeiss LSM700 inverted confocal microscope (CLSM) operated in bright-field mode. All images and videos were captured with a 63x objective and analyzed using Zen 2012 software (Zeiss). Samples were prepared by dropping coacervate solutions onto a microscope glass slide. Rheology

Rheology of the coacervates was investigated by performing dynamic oscillatory rheology experiments using an Anton Parr MCR 501 Rheometer equipped with a parallel plate geometry (diameter of rotating top plate: 8 mm) and an evaporation hood at 20 °C. Rheological properties were assessed by performing frequency sweeps from 0.1 to 100 rad/s in the linear viscoelastic range at a strain of 1 % and 0.2 mm gap between the two plates. Coacervate solutions of AO.25, A1 , B1 , and B1 Control were centrifuged at 10000 rpm for 10 min and then coacervates were collected from bottom of the eppendorf tube. Coacervates of B5 and B10 were obtained directly from water. Adhesion measurements

The adhesion ability of the formed materials was investigating using glass substrates. Before start, glass slides were cleaned with demineralized water and then ethanol under ultrasound for 1 h, and later dried in an oven at 53 °C for 30 min. Samples for adhesion tests were prepared in two different ways. With coacervates A0.25, A1 , B1 , and B1 Control solution, 50 μΙ_ of each solution was drop-cast on one glass slide. Then another glass slide was placed on top of the first to result in an overlap area of 0.95 cm². ForB5 and B10, 50 μΙ_ of the protocoacervates were drop-cast underwater on glass slides and allowed to wait 1 h for coacervation. Then two glass slides are placed on top of each other with an overlap area of 0.35 cm². Samples were cured in oven at 53 °C for 20 h. Overlap shear tests were performed with an ElectroForce 5500 (Bose) using WinTest progam. It features a 200 N maximum force. The pulling speed was 1 N/s. Before testing, one end of the sample was mounted gently in the upper grip of the tester leaving the other end free. The lower grip of the tester held a glass slide. Then the lower grip was slowly adjusted to approach the other end of the sample and superglue (Loctite) was used to glue them together followed by at least a 1 h cure times at room temperature (Figure 2). In this way, failure of samples during mounting was avoided.

For adhesion measurement underwater, samples were prepared by placing two (B10 coacervate )-coated glass slides underwater tot 30 min (gluing time). The influence of different parameters (coacervation time, gluing time, temperature, and pH) on the shear strength was studied. The samples were mounted in the tester as described above.

Example 1: Coacervation of DOPA-PAAm and PAA

Herein, we introduce a new organic-solvent-free method for preparation of coacervate materials, such as for example coacervates films, underwater by injection of a mussel- inspired proto-coacervate. The unexpected proto-coacervate was formulated with L- DOPA-polyallylamine (DOPA-PAAm) and PAA dissolved in water at acidic pH were PAA was uncharged but still water soluble due to its highly hydrophilic nature. This pre-complexation polyelectrolyte proto-coacervate formulation also provided the new way of preparation of pre-complex fluids in acidic condition which was not achieved (precipitated due to insolubility) by using polyampholytes. For application as an underwater adhesive, a high concentration proto-coacervate solution was injected into water by using a 100-μΙ_ pipette tip. The concept is illustrated schematically in Figures 1 c-d. Interestingly, this proto-coacervate could intimately mimic not only the material constituent but also the process of mussel attachment. Prior to attachment, the proto- coacervate remains a complex liquid while inside the pipette (Figure 1 c), as is the case for the mussel before it ejects material. Upon injection on the target surface, a plaque is formed and when retracting the pipette tip, a thread is formed (Figure 1 d).

The coacervation is driven by electrostatic complexation between the polycationic DOPA-PAAm and polyanionic PAA resulting in phase separation (Figure 1 c-d). This phenomenon depends strongly on the pH of the medium and the p _a of the polyions. The traditional approach (Table 1 ) of studying the coacervation behavior of the present system is that polyion solutions were prepared under acidic conditions (pH -1 ) in order to protonate both amine groups of DOPA-PAAm (p _a -9.5) and carboxylic groups of PAA (p _a -4.5) so that only the DOPA-PAAm is charged while PAA is neutral resulting in a homogenous uncomplexed and non-coacervated solution (Figure 2a, cuvette I). At this stage (I), DOPA-PAAm and PAA completely dissolved in water yielding a transparent solution without any sign of phase separation. Next, base (NaOH) was slowly added to the solution under vigorous stirring to avoid localized complexation or coacervation. Stage (I I) of Figure 2a shows that coacervation happened at pH = 2 to 2.5 as observed from the solution turning highly turbid (Figure 2a, cuvette II and Figure 2b). This is because the carboxylic groups of PAA became deprotonated following the increase of pH allowing interactions between the negatively charged unprotonated carboxylate groups and the positively charged amine groups of DOPA-PAAm inducing phase separation. Coacervation is a phase separation phenomenon; therefore, it is possible to observe coacervates in an optical microscope if the size of the coacervate is larger than 0.2 μηη. A water-dispersed coacervate droplet, e.g. 1 wt% (DOPA- PAAm):PAA of 1 : 1 (w.w) ratio named B1 (Table 1 ), is large enough to be captured by optical microscopy. Sphere-like non-aggregated coacervate droplets -5-10 μηη in diameter were observed (Figure 2a, below scheme). Due to the small diameter of the coacervate droplet, high diffusivity of Brownian motion is observed

as the diffusivity is inversely proportional to the coacervate radius.

The apparent turbidity of the suspension starts to decrease above pH 3 due to precipitation of abundant coacervates (third cuvette in inset of Figure 2b). Finally, the solution turned orange above pH -9 due to oxidation (tanning) of DOPA to quinone (Figure 2a, cuvette IV). The highest-turbidity coacervates formed at pH 2 to 2.5 (Figure 2a, cuvette I I) were highly stable in suspension and could easily be made in large quantities for further studies of the influence of (DOPA-PAAm )/PAA ratio,

concentration, and ionic strength (or NaCI concentration) on coacervation. The ratio and concentration of polycation DOPA-PAAm and polyanion PAA influenced coacervation and shifted the coacervation pH or isoelectric point of coacervation. For example, at the same concentration (1 %), increasing DOPA-PAAm concentration elevated the coacervation-onset pH value from 2.2 to 3.1 (Figure 2c). Decrease the polyion concentration (at a fixed ratio of 3:1 ) produced dramatic change in coacervation pH-onset value from 3.1 up to 6.0 (Figure 2d). Therefore, it is more efficient to manipulate the isoelectric point of coacervation by dilution. Moreover, dilution does not seem to influence the coacervate percentage in solution since the same turbidity was observed for varying concentration coacervates at their maximal turbidity pH

(Figure 2d). It is worth mentioning that coacervation pH correlates to adhesion (at low pH) and cohesion (at higher pH); thus one could optimize coacervation pH to produce either strong adhesive or cohesive coacervates. Ionic strength also has effect on coacervation, e.g., the turbidity of the solution increases dramatically at low salt concentration (<50 mM) and starts decreasing at high salt concentration (>0.5 M).

Surprisingly, high concentration (>5%) solutions were acidic and crowded enough to hinder DOPA-PAAm and PAA forming coacervates (the first two vials in Figure 4a). These solutions were named proto-coacervates. Surprisingly, coacervation was observed during dilution of highly concentrated (14%) stock solution of (DOPA- PAAm):PAA (1 :1 , w.w) (the last three vials in Figure 3a). Simply injecting high concentration (10%) solution B10 underwater and waiting -20 min for coacervation obtains a coacervate that strongly sticks to the glass substrate (Figure 3b). The mechanism of the coacervation was found to be mainly diffusion-controlled hydration of the injected proto-coacervate solution. The resulting coacervate film is strong enough to survive water blasting. This technique of producing coacervate film is environmently friendly since no acid or organic solvent is added and simple to perform by injection from a syringe.

Figure 3b illustrates the measured pH during coacervation, recorded by a solid pH meter in pH 7 buffer onto which a proto-coacervate B10 was deposited. Coacervation and pH change was recorded simultaneously over time following the transition from proto-coacervate to coacervate. The pH of the initially injected proto-coacervate was low and increased slowly, which is also observed in blue mussels. Importantly, the pH of the formed coacervate film (Figure 3b) remained much lower than the buffer medium (pH 7). This allows the coacervate film to adhere strongly to the electrode surface (Figure 8). This result is a solid contribution to the understanding of mussel byssus formation and provides strong support to the hypothesis of a similar process occurring during this biological process.

The proto-coacervate and coacervate films were characterized by IR spectroscopy (Figure 3c). Most of the peaks, e.g. at 1700, (-C=0 stretch of -COOH), 1628 (-NH₂ bend), 1559 (-NH₃ ⁺ bend), 1457 (-CH₂- bend), and 1256 cm^"1 (-C-0 of -COOH) (curve 1 in Figure 3c and more details in curve 1 and 2 in Figure 9) shifted to lower wavenumber (higher wavelength and lower energy) (curve 2 in Figure 3c) due to complexation as well as unprotonation of PAA. Moreover, an increased relative intensity of the 1542 (asymmetric COO-) as well as appearance of 1401 cm^"1 (COO-) in the coacervate spectrum (curve 2 in Figure 3c) which was not observed in B10 proto-coacervate solution (curve 1 in Figure 3c) proving that the -COOH group of PAA are unprotonated (see curve 3 in Figure 9).

Example 2: Rheology

Generally, DOPA-functionalized coacervates A0.25, A1 , and B1 formed by pH- triggered were gel-like crosslinked materials because G' > G" (elastic behavior dominated). Both moduli tended toward a constant limiting value at lower frequencies (Figure 4). Surprisingly, without DOPA, the coacervate, i.e., B1 Control, become a hard and unlinked (G" > G') material, with a much higher modulus than those of DOPA- containing systems. The rheological behavior depends strongly on solution formation. AO.25, A1 , and B1 were more elastic, tan5 < 1 (tan5 = G'VG') than B1 Control for which tan > 1 . Coacervate B5 is much stiffer than coacervates AO.25, A1 , and B1 with at least one order-of-magnitude higher modulus (Figure 4) due to both the higher concentration and improved cohesion (B5 formed under neutral pH). The modulus of coacervate B10 is one order-of-magnitude higher than coacervates B5 with and more viscous in behavior (G" = G'). To highlight the importance of mechanical property of this material, rheology of pre-coac.B10 (proto-coacervate solution) was measured. Surprisingly, it behaved like a liquid even though there were up to 10 wt% of polymers in solution (Figure 4).

Example 3: Adhesion of coacervates on glass

DOPA plays a very important role in adhesion of coacervates even on glass substrate that was chosen herein as a challenging substrate due to its smoothness and hydrophobicity. Coacervate B1 is three time higher in shear strength than that of coacervate B1 Control (without DOPA) (Figure 5). There is no difference in shear strength between 3:1 and 1 :1 ratio of (DOPA-PAAm):PAA of A1 and B1. As discussed above, the maximal turbidity and pH of coacervation can be manipulated by dilution of polyion concentration. Coacervate AO.25 at pH 6 had much lower (fifteen-times) shear strength than coacervate A1 at pH 3 even though their turbidity is the same (Figure 2C). Most interesting are the high-concentrated coacervates B5 and B10 prepared from hydration-triggered coacervation of proto-coacervates because they provide the greatest shear strength. Indeed, when the concentration of polyions increased to 5 and 10%, the shear strength of the corresponding coacervates improved to 1 .48 and 2.39 MPa, respectively (Figure 5). This result is highly comparable to the performance of commercial glues on the same glass substrate (Table 2) and even surpasses them for B10. Moreover, huge advantages of this B10 coacervate glue include the fact that it is free of organic solvent and can perform underwater adhesion. Indeed commercial glues were found to fail dramatically in underwater adhesion, as demonstrated by injecting them onto glass slides underwater and following the same procedure of treatment (curing at 53 °C for 20 h) (Table 2). Both commercial glues failed to work as adhesives due to the interference of the water medium.

The secrets behind the strong adhesion of these coacervates are (1 ) phase separation (coacervation) by hydration creates a very dense population of coacervates on glass substrate; (2) the high concentration of DOPA in the coacervates provides strong adhesion. Therefore, even uncured (wet) coacervate B10underwater manifests remarkable underwater adhesion (0.14 MPa). To highlight the importance of coacervate usage as adhesive, a non-coacervated B10 - pre-coac.B10 was prepared in the same way as B10 (but without coacervation) for overlap shear experiments. The shear strength of pre-coac.B10 (0.76 MPa) was three times smaller than that of B10. This is because coacervation contributed greatly to cohesion due to complexation between DOPA-PAAm and PAA which cannot be attained by pre-coac.B10.

Table 2. Comparison of some commercial epoxy adhesives with the coacervate B10 on the same glass substrate and overlap shear test. Glue Strength [[MPa]

3M Scotch-Weld™ DP125 Epoxy Adhesive 1 .72

3M Scotch-Weld™ DP105 Epoxy Adhesive 1 .37

3M Scotch-Weld™ DP100 plus Epoxy 1 .72

Adhesive

3M Scotch-Weld™ EPX™ DP 105 Epoxy 1 .40

Adhesive

Coacervate B10 2.39

Coacervate B5 1 .49

For underwater applications, the wet (uncured) coacervate wet B10 demonstrates remarkable underwater adhesion of 0.15±0.03 MPa which is comparable to mussel adhesion of 0.17±0.07 MPa on the same glass substrate. The influence of different parameters (coacervation time, gluing time, temperature, and pH) on shear strength was studied (Figure 6). Increase of coacervation time resulted in asymptotic growth of adhesion strength that reached a plateau after -24 h (Figure 6a). This behavior results from extended time for coacervate assembly resulting in improved wetting and bonding onto the target glass surface. Raising the gluing time and temperature tend to decrease the adhesion strength of B10 coacervate (Figure 6b and 6c) because adhesion between coacervate and glass surface was reduced by compression and heat, respectively.

Interestingly, when B10 coacervate was injected in pH 9 buffer, two phenomena were observed (Figure 6d). First, coacervation at pH 9 appeared much faster than at lower pH. This is due to efficient deprotonation of PAA at pH 9 leading to more rapid complexation between DOPA-PAAm and PAA. Therefore, there was no coacervate dispersed in solution after injection as shown in Figure 2b. Second, after complete coacervation, the pH 9 coacervate swelled turning into a transparent substance with an 8.5 times increases in weight. Upon contact with water the sodium ions are hydrated, which reduces their attraction to the carboxylate ions (due to the high dielectric constant of water). This allows the sodium ions to move freely within the network, which contributes to the osmotic pressure within the gel. The mobile positive sodium ions however, cannot leave the gel because they are still weakly attracted to the negative carboxylate ions along the polymer backbone and so behave like they are trapped by a semi-permeable membrane. So the driving force for swelling is the difference between the osmotic pressure inside and outside the gel. Increasing the level of sodium outside of the gel will lower the osmotic pressure and reduce the swelling capacity of the gel. Swelling of B10 coacervate appeared more dramatically at high temperature, 37 °C, and at this temperature the mass increase was 25 times. Example 4: Test of glue formulation for leather

Two pieces of tanned leather were cut in similar sizes, and glued together in dry conditions. The protocoacervate was applied to one of the leather pieces, and the other piece mounted on top immediately after. The glued leather pieces were left over night in room temperature. It was not possible to separate the two leather pieces manually either with shear force or using nails to test the peel force. The formulations tested were HOPO-PAAm-PAA and DOPA-PAAm-PAA.

Example 5: Test of biocompatibility - using ex-vivo human skin model

An ex-vivo human skin model was used to assess the biocompatibility of the DOPA- PAAm-PAA (10 wt%) formulation. The human skin was obtained from skin reduction surgery, and collected the same day as the surgery is performed. The fat was removed from the skin, and skin samples cut out. A 2 mm diameter hole was pinched out through the -4 mm thick skin, making a cylindrical hole in the skin -12.6 mm³. The hole was filled with glue, by injecting the proto-coacervate formulation into the hole, immediately below the skin surface. The skin (including glue) was placed onto a pre- made collagen gel, submerged in buffer (William^'s medium) , and incubated at 37°C according to the setup described by Knowles et al. (Knowles NG, Miyashita Y, Usui ML, et al. A model for studying epithelial attachment and morphology at the interface between skin and percutaneous devices. (J Biomed Mater Res - Part A. 2005;74:482- 488) The Williams^'medium is changed daily throughout the study. Samples were extracted and fixated in formalin at two timepoints, 1 and 7 days after starting the experiment. N = 3 samples were harvested at each time point, as well as a control sample at t = 0. After fixation, the samples were embedded in PMMA, section into 5 μηη thin sections and stained with H&E. A resulting example of a histological section is shown below, with the top of the skin facing downwards in the lower end of the image. The central hole is seen to be partially filled with cured glue (white/grey). No cell death is observed around the glue at either time points, demonstrating that there does not appear to be an immediate cytotoxic effect of the glue. Also, there is no misalignment of the cells, and very good skin-glue contact is observed. It should be noted, that this way of applying the glue, exaggerates the effect of the glue, as in the real life application of a topical glue, the wound edges will be aligned manually, and the glue applied on the surface of the skin.

Example 6: Testing protocoacervates formulations

The protocoacervates were made by dissolving the individual components in water (or a combination of water and hydrochloric acid). The components were dissolved using a magnetic stirrer with or without heat (up to 50 °C), followed by sonification if needed. The dissolved solutions were combined and mixed using a magnetic stirrer. If coacervates formed spontaneously upon mixing, the pH was adjusted with HCI until coacervates were dissolved.

All combinations in table 3 have been tested for 1 :1 weight ratio of polyanion and polycation. Hence, the reported results on whether they coacervate or not upon injection into liquid medium are valid for this ratio, and do not imply that other mixing ratios would not lead to a different outcome. A blank space in table 3 means that that the entry was not tested.

Table 3

in Dl water

(HOPO-PAAm)- Yes In both buffers

osteopontin and serum, not

in Dl water

(HOPO-PAAm)-Phyt Yes Yes

(HOPO-PAAm)-TA Yes In both buffers

and serum, not

in Dl water

Example 7: Creating a light curing polymer system

In the present example a hydrogel was created using a polymer component and a catechol component, wherein the catechol component comprises light sensitive groups. In the present example, 4,5-dihydroxy-2-nitrobenzyl alcohol (DNBA) was chosen as the catechol (Scheme 1 left). The polymer component contained a backbone and a reactive amino-oxy group. Polyallylamine (PAAm) was chosen as the backbone and find the simplest possible molecule containing a carboxylic acid and an amino-oxy group which turned out to be amino-oxy acetic acid (AOAA), and graft it onto the backbone (Scheme 1 right).

Scheme 1 : Molecules for light curing system. 4,5-dihydroxy-2-nitrobenzyl alcohol (DNBA) as the catechol component (left) and PAA grafted with amino-oxy acetic acid (AOAA) as the polymer component (right).

Catechols were pre-coordinated with metal ions as shown in Fejl! Henvisningskilde ikke fundet. 7. Subsequently light would transform the primary alcohol into an aldehyde which could react with the amino-oxy group on the polymer. In a hydrogel system this would lead to small complexes floating around inside a solution heavy with polymers until cross-linking was needed at which time shining light on the solution would allow the polymers to cross-link making a stable hydrogel. The polymer was synthesized first from commercially available PAA and tert- butyloxycarbonyl - Amino-Oxy Acetic Acid (boc-AOAA). The polymer was synthesized as described Scheme 2. Since boc-AOAA is not sensitive to base or air and easily dissolvable in demineralized water, no precautions needed to be taken. Thus, no degassing and no Trifluoroacetic acid (TFA) were used, and purification was done using demineralized water. When no TFA is added to the synthesis the pH will be 7, which is closer to the optimal point for the synthesis. After the grafting reaction deprotection was needed in order to free the amino-oxy group. The deprotection was performed in a 5:95 mixture of H₂0 and TFA. The reaction mixture was stirred for 30 mins. Afterwards, the mixture was dialysed against demineralized water until the pH was neutral.

Scheme 2: Polymer synthesis. The grafting and deprotection of boc-protected amino- oxy acetic acid.

The catechol molecule 4,5-dihydroxy-2-nitrobenzyl alcohol can be synthesized as shown in Scheme 3. Either starting with 3,4-dihydroxybenzyl alcohol (a) and nitrating to reach DNBA (b) or starting from 4,5-dimethoxy-2-nitrobenzyl alcohol (MNBA) (c) and deprotecting the phenol groups to reach DNBA (b).

a b c

Scheme 3: Synthesis of the catechol molecule. Either a to b via nitration or c to b via deprotection of phenol groups, a and c are both commercially available.

The light sensitive catechol is synthesized as shown in scheme 4.

Scheme 4.

Example 8: Underwater fabrication of CNT-coacervate composites

Materials and methods

Chemicals

PAA (Mw = 450000) and HCI (≥ 37%) were purchased from Sigma Aldrich (Germany). Glass slides were supplied from Hounisen Laboratorieudstyr (Aarhus, Denmark). The COOH functionalized CNT suspension 10wt% in water was supplied from NanoLab, Inc. (MA, USA). DOPA-chitosan (see below) was synthesized according to previously published procedure (M. Krogsgaard, M. R. Hansen, H. Birkedal, J. Mater. Chem. B 2014, 2, 8292-8297).

DOPA-chitosan

Preparation of CNT-(DOPA-chitosan)/PAA composites by coacervation Preparation of (DOPA-chitosan)/PAA (1:1, w:w) 20% protocoacervate 400 mg DOPA-chitosan was dissolved in 4 mL water and sonicated for 30 min until a yellowish/transparent solution was observed. 400 mg PAA was added to the solution with subsequent continuos stirring for 4 h at 60 °C. The resulting solution has a

i*_tes

concentration of 20 wt%, and was subsequently used as the stock solution for preparing the CNT-coacervate composites.

Preparation of CNT-protocoacervate solutions

Depending on the protocoacervate concentration (Table 4), a small volume of concentrated HCI was spiked with 500 μΙ_ of CNT solution and sonicated for -5 min. The HCI is added to prevent coacervation of the protocoacervate in the following step. Different volumes of the 20 wt% stock solution of (DOPA-chitosan)/PAA

protocoacervates were added slowly to the CNT solution to obtain a range of

CNT:coacervate ratios, and sonicated for 20 min to obtain the final homogeneous inklike solutions. The details of each solution are shown in Table 4.

Table 4. Composition of different solutions prepared for CNT-coacervate composite.

Sample Ratio Protocoacervate CNT Total HCI

CNT:protocoacervate cone. [wt%] cone. cone. cone.

[wt%] [wt%] [mM]

Coacervate 0 48 3.92 - 3.92 -

1 48 3.92 0.082 4.00 -

*Due to fixed CNT weight (10 wt%) in the solution, the total concentrations of solutes are decreasing (from 4 to 0.49%) with decline of protocoacervate weight. The range of CNT:protocoacervate ratios was chosen based on of the possible limits defined by sample preparation.

Formation of CNT-coacervate composites

The CNT-coacervate composites were produced by injecting the corresponding CNT- protocoacervate solutions into Dl water using a 100-μΙ_ pipette tip. Injection volumes vary from 50 to 100 μΙ_ depending on experiments. The coacervation time was kept constant at 10 min. The resulting composites were characterized in the form of film (for SEM and nanoindentation tests) or as bulk materials (for rheology and electrical conductivity measurements). Composite films are prepared by injecting of 10-μΙ_ CNT- protocoacervate solutions onto glass slides underwater using 100-μΙ_ pipette tip and followed by 10-min coacervation. The composite-coated glass slides were then delicately removed from water bath and dried in an oven for 53 °C for 12 h. The procedure for preparing bulk materials is similar to that of film but instead of letting the film stay on glass slide, we used a pipette to scrape off and collect the materials from water.

Characterization

TEM A Talos F200X Transmission electron microscope (FEI, Eindhoven, the Netherlands) operated at 200 kV was used to investigate the CNT-protocoacervate composites. Images were acquired in bright field (BF) mode and recorded with a Ceta 16M pixel CMOS detector. Samples were prepared by dipping TEM copper grids into the CNT- protocoacervate solution for a few seconds. Next, most of the solution was wiped from the grids using lens paper. The CNT-protocoacervate coated grids were dipped in Dl water for 1 min (due to thin film) before drying at 53 °C for 3 h and then under vacuum overnight. Pure CNTs for TEM characterization were prepared by drop casting a diluted CNT solution onto the grid and follow the same procedure as for the composites. SEM

A Versa 3D DualBeam microscope (FEI, Eindhoven, the Netherlands) was used for imaging the thickness of the CNT-coacervate films through cross-sections of the glass- composite interface. The microscope was operated at an accelerating voltage of 5 kV in secondary electron (SE) mode using an Everhart-Thornley SE detector (ETD). The samples were made by cutting and breaking the CNT-coacervate coated glass slides that had been used in nanoindentation experiments.

Rheology

Rheology of the coacervates was investigated by performing dynamic oscillatory rheology experiments using an Anton Parr MCR 501 Rheometer equipped with a parallel plate geometry (diameter of rotating top plate: 8 mm) and an evaporation hood at 20 °C. The rheological properties were assessed by performing frequency sweeps from 0.1 to 100 rad/s in the linear viscoelastic range at a strain of 1 % and 0.2 mm gap between the two plates. The recorded data are plotted in storage modulus (G') and loss modulus (G") versus angular frequency (ω). The composite coacervates were collected 10-min after the injection of the CNT:protocoacervate into Dl water.

Nanoindentation

Nanoindentation experiments were performed using a Hysitron Tribolndenter (Hysitron, Minneapolis, MN) with a Berkovitch tip. The tip area function was calibrated using a quartz standard. Indents were made in depth control using a load-hold-unload function of 10-60-10 s with displacement depth controlled at 300 nm (A. Bruel, J. Olsen, H. Birkedal, M. Risager, T. T. Andreassen, A. C. Raffalt, J. E. T. Andersen, J. S.

Thomsen, Calcif. Tissue Int. 201 1 , 88, 142-152). Indents were placed in grids with 10 μηη spacing; with this indent spacing, interindent effects are negligible. The grids were placed accurately in the desired region of interest using the contact topography mode of the Tribolndenter. The scan mode was also used to verify the precise placement of the indents, by scanning the area immediately after the nanoindentation

measurements. Indents were analyzed using the Oliver-Pharr method (W. C. Oliver, G. M. Pharr, J. Mater. Res. 1992, 7, 1564-1583).

Electrical conductivity characterization

Composites collected from Dl water was packed into a 1 -mm diameter glass tube (inset in Figure 13) and dried in an oven at 53 °C for 12 h. The resistance of the composites was measured using an Alcron digital multimeter. The conductivity of the composites was calculated from the measured resistance, area and length of the packed composites. In order to further extend the properties and applications of coacervates, more functionalities were added to coacervates by preparing coacervate composites. The composites have been presented in the form of either inorganic-organic or organic- organic hybrids. Previously, hybrid systems were accomplished by coacervating metal oxide ((Pb,Bi)(Ti,Fe)0₃ or Ce0₂) micro- or nanoparticles with polyelectrolytes (K. Han, A. Safari, R. E. Riman, J. Am. Ceram. Soc. 1991 , 74, 1699-1702) through

intermolecular interactions. The latter composites were attained by loading a coacervate matrix with different organic active materials (curcumin, baicalin, or plasmid DNA) (D. Chirio, M. Gallarate, E. Peira, L. Battaglia, L. Serpe, M. Trotta, J.

Microencapsulation 201 1 , 28, 537-548). These hybrid materials show diverse physical and chemical characteristics including piezoelectric, coating and anti-biofouling, fluorescent properties or as pharmaceutical carriers. However, formulating coacervate composites containing carbon nanotubes (CNTs) providing electrical conductivity and a means to tune the mechanical properties of the coacervates have not previously been done. In the present example, an injection-coacervation method of coacervate preparation from "protocoacervate" underwater was introduced by using L-3,4- dihydroxyphenylalanine functionalized chitosan (DOPA-chitosan) and polyacrylic acid (PAA) as the polyelectrolytes for the coacervate scaffold of the composites. By scrutinizing the CNT dispersion in protocoacervate, composites of different volume ratios of CNT and protocoacervate could be simply fabricated underwater by injection of CNT-protocoacervate solutions.

The concept is illustrated schematically in Figure 1 1 . Prior to injection, the CNT- protocoacervate remains a complex liquid while inside the vial or pipette. Upon injection onto the target surface, herein glass slides, a plaque is formed by

coacervation - the process where PAA becomes unprotonated and interacts electrostatically with DOPA-chitosan in the presence of CNTs towards, thus forming the CNT-coacervate composite underwater. The formed composite film is stable underwater, allowing for applications underwater as well as enabling material to be removed from water (Figure 1 1 ) for study.

In this study, CNT:protocoacervate ratios in the CNT-coacervate composites was varied from 1 :48 to 1 :4 (Table 4).

The CNT-protocoacervate solutions are homogenous and stable for weeks without ultrasonication because the protocoacervate worked a solubilizing surfactant interacting with the CNTs to result in excellent dispersion of CNTs in the

protocoacervate solution. At protocoacervate concentrations above 2.6wf%, the polyelectrolyte solutions were acidic and entangled enough to prevent DOPA-chitosan and PAA from forming coacervates. When the electrolyte concentration was lower than 2.6 wt%, a small amount of HCI was added to prevent the polyions from coacervating. Hence, the lower the protocoacervate concentration, the higher amount of HCI is needed (Table 4). Upon addition of HCI, the protocoacervate solutions became homogenous, but equally well formed complex coacervates upon injection in water. Coacervation upon injection resulted in a coacervate film forming onto the glass substrate. Because incorporation of CNT into protocoacervate caused in increase in viscosity of solutions, the spreading ability of the injected CNT-protocoacervate fluid is slower than that of the pristine coacervate at the same protocoacervate concentration (-3.9%). However, lowering concentration of total solutes including CNTs and protocoacervates, i.e. decreasing the viscosity of the CNT-protocoacervate solutions brought about a rise in spreading ability (Figure 14a). At sufficient coacervation time, the resulting composites are highly cohesive, allowing them to be collected from the water in form of either films or bulk materials for further testing. Nonetheless, the protocoacervate concentration in the composite cannot be less than 0.4wf% because insufficient coacervate, which we believe as cohesive agent, results in a weak composite that cannot endure further processing. Apart from that limitation, this technique of producing coacervate films is environmentally friendly since no acid or organic solvent added, and efficient to perform by simple injection of CNT-protocoacervate solutions into water using a pipette/syringe only.

The micro- and nanostructures of the fabricated CNT-coacervate composites were studied using different techniques including Transparent Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), and rheometry. As seen on TEM images of Figure 12a, a decrease of protocoacervate amount (arrow direction in Figure 12a, pictures a_2-a₇) tends to reduce CNTs aggregation in the composites; especially, no observed aggregate for the last two composites (Figure 12a, picture a₆ and picture a₇) because the coacervates interacts well with CNTs instead of cohesive binding with each other. At high protocoacervate concentration, due to a rapid phase change (from protocoacervate to coacervate), thus most of the protocoacervates were acting as surfactants, and when transformed to coacervates, this results inaggregation of CNTs. The spherical bubbles observed in TEM images (Figure 12a), are caused by evaporated water and an inevitable consequence of TEM sample preparation. The structural change of the pristine coacervate and its composites at different

CNT:protocoacervate ratio is correlated with the mapped rheological properties of respective structures (Figure 12b). There are two transition points from left to right in the series of modulus-frequency plots (Figure 12b). At first, the pure coacervate of (DOPA-chitosan)-PAA has gel-like structure. Loading CNTs into the coacervate network then induces an immediate composite structural change to a non-crosslinked network, represented by G' > G" (Figures 12b, diagram b₂), where G' and G" stand for storage and loss modulus, respectively, because of bad dispersion (aggregation) of CNT in the coacervate matrix (Figures 12, picture a₂). However, less protocoacervate improves the dispersion of CNTs (Figures 12a, pictures a₃ and a₄), thus the structure became slightly crosslinked, demonstrated by the left shift in the cross point of the G" and G' curves making them turn parallel (Figure 12b, diagrams b₃ and b₄). When the CNT:protocoacervate reach 1 :12, the network is almost gel like (G' > G" and parallel curves) (Figures 12b, diagram b₅) due to improved dispersion of CNT in the coacervate matrix (Figures 12a, picture a₅). This trend remained to the lowest

CNT:protocoacervate ratio, viz. 1 :4 where the system is a perfect gel (G" and G' curves are straight lines and parallel with each other) (Figure 12b, diagram b₇).

The mechanical properties of the composite films coated on glass slides, using nanoindentation were studied. Due to their viscoelastic property (see Figure 12b), the holding time during each indent must be long enough, i.e. 60 s, to avoid creep effects. This effect was also observed by the additional displacement (of the unloading curve compared to that of loading curve) during hold segment (Figure 13a). The

displacement depth of indents varied from 10 nm to 300 nm below composite film surface. This helps to avoid a hit on glass beneath by tip which causes error in load values. The reduced modulus (E_r) and hardness (H) of the coacervate and composite films are obtained by fitting the curves in Figure 13a, and presented in Figure 13b. The error bars of the CNT-coacervate (1 :30 and 1 :48, w.w) composites are much lower than those of the other composites due to a slight difference in the sample preparation of the CNT-coacervate (1 :30 and 1 :48, w.w) composites compared to others. This is because these two sample coatings are rough after coacervation (e.g. CNT-coacervate (1 :30, w.w) in Figure 14a), implying that they cannot be measured directly with nanoindentation. A small manual compression was performed to smooth the surfaces and indirectly reduce defects of the films. Consequently, the mechanical property maps of the CNT-coacervate (1 :30 and 1 :48, w.w) composite films display less heterogeneity than the other samples (Figure 15). E_r and H values of all coacervate and CNT- coacervate films are very high with respect to the reports for some common polymers (e.g. coacervate film is two and five times higher than those of

poly(methylmethacrylate) PMMA and poly(carbonate) PC in H and E_r values, respectively) or CNT-chitosan composite (at least twice higher in H value). Changes in modulus and hardness follow the same trend, i.e. with both the H and E_r values being larger for the coacervate film compared to the CNT:coacervate composite films. This may be caused by higher porosity of the composite films compared to the coacervate one. Apart from lower H and E_r values of the composite films, they adhered well on the glass substrate, i.e. the film was not peeled off after cracking the glass (Figure 14b). Figure 14b shows a cross-section SEM image of the CNT:coacervate (1 :8, w.w) composite film displaying a homogenous distribution of CNTs in the coacervate matrix contributing in their electrical conductivities. Figure 10a shows that the electrical conductivity of the composites depends on the CNT:coacervate ratio. The percolation threshold appears at a CNT:coacervate ratio of 1 :12 (w.w) and the conductivity increases up to seven order-of-magnitude at a ratio of 1 :4 (w.w). The measured boost in conductivity of the composites is caused by the increased CNT content as well as dispersity of the CNTs in the coacervate matrix towards continuous connectivity of CNTs in composite. This is clearly demonstrated in the TEM images in Figures 12a, pictures a_2-a₇. The conductivity of composites with CNT:coacervate ratios lower than 1 :4 (w.w) could not be measured as these low-concentration coacervate composites did not form sufficiently connected films. Moreover, the CNT:coacervate (1 :8, w.w) composite was selected to use as a conductor to light up an LED (Figure 10b).

Conclusions

This example successfully demonstrates that CNTs can be incorporated into coacervates using the injection-coacervation method, which provides an extremely simple technique for fabricating CNT-polymer composites. The key material herein is protocoacervate - the uncomplexed form of the coacervate which enables CNTs to be dispersed homogeneously. The coacervated composites exposed surprising properties, e.g. tunable crosslinked network and electrical conductivity, opening up for applications both in the laboratory and industrial settings including for example organic field-effect transistor (OFET), chemical sensors and conductive tape.

Claims

oZ Claims

A method of forming coacervates comprising

- providing a composition having a first pH comprising

• a polymer backbone comprising one or more backbone monomer unit(s) and

• a compound

whereby coacervates are formed.

The method according to claim 1 , wherein said composition is not phase- separated.

The method according to any of claims 1 and 2, wherein the second pH value obtained by addition of a base comprising an OH-group, such as KOH or NaOH.

The method according to any of claims 1 and 2, wherein the second pH value is obtained by addition of an acid, such as HCI or acetic acid.

The method according to any of claims 1 and 2, wherein the second pH value is obtained by delivering said composition to a location in the body whereby pH changes to a second pH of said location and coacervates are formed.

The method according to any of the preceding claims, wherein the polymer backbone comprises one kind of backbone monomer units, only, said backbone monomer units comprising one or more chemical groups selected from amino groups, carboxylates, phosphonates, phosphates and sulfonates.

7. The method according to any of claims 1 -5; wherein the polymer backbone comprises more than one kind of backbone monomer units, wherein at least one kind of monomer units comprising one or more chemical groups selected from amino groups, carboxylates, phosphonates, phosphates and sulfonates.

8. The method according to any of the preceding claims, wherein the polymer backbone is charged and the compound is uncharged.

9. The method according to any of the preceding claims, wherein the polymer backbone is cationic at said first pH value.

10. The method according to any of the preceding claims, wherein the polymer backbone is anionic at said first pH value.

1 1 . The method according to any of the preceding claims, wherein the polymer backbone comprises one or more backbone monomer unit(s), which is/are thermo-responsive.

12. The method according to any of the preceding claims, wherein the polymer backbone comprises one or more backbone monomer unit(s), which is/are light- responsive.

13. The method according to any of the preceding claims, wherein the polymer backbone comprises one or more backbone monomer unit(s), which is/are soluble in polar solvents such as water.

14. The method according to any of the preceding claims, wherein the polymer backbone comprises one or more backbone monomer unit(s) which is/are selected from the group consisting of:

- amino-Ci-io-alk(an/en/yn)yl which is optionally substituted;

- amides of the formula -(CO)-(NH2)-Ci_-6-alk(an/en/yn)yl-NH2;

- aminostyrenes which are optionally substituted; and - amino sugar(s) such as of the generic formula 0=CR⁶-CH(NHR⁷)- (CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein

in their open chain form or in the a-form or β-form thereof.

15. The method according to any of the preceding claims, wherein the polymer backbone comprises one or more backbone monomer unit(s) of the generic formula amino-Ci-i₀-alk(an/en/yn)yl which is optionally substituted with one or more substituents.

16. The method according to any of the preceding claims, wherein the polymer ses one or more backbone monomer unit(s) of the formula:

17. The method according to any of claims 1 -15, wherein the polymer backbone comprises one or more backbone monomer unit(s) which is/are selected from the group consisting of amino-Ci-i₀-alk(an/en/yn)yl, amino-Ci_-6-alk(an/en/yn)yl, amino-C₂-4-alk(an/en/yn)yl and amino-C₃-alk(an/en/yn)yl is substituted with one or more substituent(s) selected from the group consisting of -SH, - OH, - COOH, -NH₂, -S-CH₃, -O-CH3, -CH(OH)-CH₃ and -CH(SH)-CH₃.

18. The method according to claim 17, wherein the polymer backbone comprises or consists of polyallylamine.

19. The method according to any of claims 1 -14, wherein polymer backbone

comprises one or more backbone monomer unit(s) selected from the group of amino acids. oo

20. The method according to claim 19, wherein said amino acid(s) is/are of the

generic formula H₂N-CHR¹-COOH wherein R¹ is an organic substituent.

21 . The method according to any of claims 19-20, wherein said amino acid(s) is/are of the formula H₂N-CHR¹-COOH;

wherein R¹ is selected from the group consisting of -H, -Ci_-6-alk(an/en/yn)yl, -d. 6-alk(an/en/yn)yl-R²,

wherein R² is selected from the group consisting of -SH, -COOH, C₃H₃N2, -NH₂, -S-CH₃, -CO-NH₂, -NH-C(NH)NH₂, - OH, -CH(OH)-CH₃, -SeH, -C₈H₆N, -C₆H₅- C₆H₄OH, and -C₆H₃(OH)₂.

22. The method according to any of claims 19-21 , wherein said amino acid(s) of the formula H₂N-CHR¹-COOH is a naturally occurring amino acid which is selected from the group consisting of DOPA, ornithine, lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine and valine.

23. The method according to any of claims 1 -14, wherein the polymer backbone comprises one or more polymer monomer unit(s) of the formula -(CO)-(NH₂)- Ci-₆-alk(an/en/yn)yl-NH₂.

24. The method according to any of claims 1 -14, wherein the polymer backbone comprises one or more backbone monomer unit(s) comprising styrene which substituted, such as with one or more substituents selected from the group consisting of -NH₂ and -Ci_-6-alk(an/en/yn)yl-NH₂.

25. The method according to claim 24, wherein the polymer backbone comprises one or more backbone monomer unit(s) comprising styrene which is substituted with NH₂.

26. The method according to claim 24, wherein the polymer backbone comprises one or more backbone monomer unit(s) comprising styrene which is substituted with Ci-₆-alk(an/en/yn)yl-NH₂.

27. The method according to any of claims 25-26, wherein styrene is further substituted with one or more substituents selected from the group consisting of -SH, - OH, -COOH, -NH₂, -S-CH₃, -0-CH₃, -CH(OH)-CH₃ and -CH(SH)-CH₃.

28. The method according to any of claims 1 -14, wherein the polymer backbone comprises one or more backbone monomer unit(s) comprising styrene which is un-substituted.

29. The method according to any of claims 1 -14, wherein polymer backbone

comprises one or more backbone monomer unit(s) selected from the group of amino sugars.

30. The method according to claim 29, wherein said amino sugar(s) is/are of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein

in their open chain form or in the a-form or β-form thereof.

31 . The method according to any of claims 29-30, wherein said amino sugar(s) is/are of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein R³, R⁴, R⁵ and R⁶ are independently selected from the group consisting of -H, -OH, -(CH₂)-OH and -COO^" and -CH(OH)-CH(OH)-CH₂-OH.

32. The method according to any of claims 29-31 , wherein said amino sugar(s) is/are of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein R³ is -OH.

33. The method according to any of claims 29-32, wherein said amino sugar(s) is/are of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein R⁴ is selected from the group consisting of -H and -OH, such as wherein R⁴ is -H, or wherein R⁴ is -OH.

34. The method according to any of claims 29-33, wherein said amino sugar(s) is/are of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein R⁵ is selected from the group consisting of -(CH₂)-OH and -COO^".

35. The method according to any of claims 29-34, wherein said amino sugar(s) is/are of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein R⁶ is selected from the group consisting of -OH and -CH(OH)-CH(OH)- CH₂-OH.

36. The method according to any of claims 29-35, wherein said amino sugar(s) is/are of the generic formula 0=CR⁶-CH(NHR⁷)-(CHR³)-(CHR⁴)-(CHR⁵)-OH; wherein R⁷ is selected from the group consisting of -H and -(CO)-CH₃.

37. The method according to any of claims 29-36, wherein said amino sugar(s) is/are selected from the group consisting of glucosamine, acetylglucosamine, galactosamine and sialic acid in their open chain form or in any a-form or β- form thereof.

38. The method according to any of claims 29-36, wherein 3 such said amino

sugar(s) comprised in the polymer backbone together form chitosan having the formula

in its open chain form or in any a-form or β-form thereof.

39. The method according to any of claims 29-36, wherein said amino sugar(s) comprised in the polymer backbone together from chitosan with varying degrees of de-acetylation in its open chain form or in any a-form or β-form thereof.

40. The method according to any of claims 29-36, wherein the polymer backbones are amino-functionalized polymers with a pKa ranging from approximately 4 to 10, such as from 5 to10.

41 . The method according to any of the preceding claims, wherein the monomer unit(s) comprises one or more catechol and/or catechol analogue group(s).

42. The method according to any of the preceding claims, wherein the monomer unit(s) comprises one or more catechol and/or catechol analogue group(s) and further comprising one or more carboxylate-group and/or one or more amino group(s).

43. The method according to claim 42, wherein the monomer unit(s) comprises a compound of the formula:

wherein:

- R¹⁰ is selected from the group consisting of (Ci_-6-alk(an/en/yn)yl)_q- COOH and (Ci_-6-alk(an/en/yn)yl)_r-R¹³ wherein:

- q and r are independently selected from the group consisting of 0 and 1 ;

- R¹¹ is R¹² selected from the group consisting of OH, H, N0₂, F, CI, Br, I, SH, (Ci_-6-alk(an/en/yn)yl);

- A is selected from the group consisting of C, N, O and S; and

- when q or r is 1 then Ci_-6-alk(an/en/yn)yl is optionally substituted with one or more substituents selected from the group consisting of -CH₃, - SH, -HCO, -OH, -NH₂ and -CO-C=C-R¹⁴ wherein:

• R¹⁴ is of the formula:

44. The method according to claim 43, wherein R¹⁰ is selected from the group consisting of (C₂-4-alk(an/en/yn)yl)_q-COOH and (C2-4-alk(an/en/yn)yl)_rNH₂.

45. The method according to any of claims 43-44, wherein R¹⁰ is selected from the group consisting of (C₂-4-alk(an/en/yn)yl)-COOH and (C₂-4-alk(an/en/yn)yl)-NH₂.

46. The method according to any of claims 43-44, wherein R¹⁰ is -COOH or -NH₂.

47. The method according to any of claims 43-44, wherein R¹⁰ is (C_2-4- alk(an/en/yn)yl)-COOH.

48. The method according to any of claims 43-44, wherein R¹⁰ (C_2-4- alk(an/en/yn)yl)-NH₂.

49. The method according to any of claims 43-46, wherein R¹¹ is -OH or H.

50. The method according to any of claims 43-46, wherein R¹¹ of the monomer unit(s) with which the polymer backbone is functionalized comprises at least one catechol functional group and further comprises at least one amino group and/or one carboxylate-group is positioned as indicated in the below formula:

51 . The method according to any of claims 43-46, wherein R¹¹ of said monomer unit(s) with which the polymer backbone is functionalized comprises at least one catechol functional group and further comprises at least one amino group and/or one carboxylate-group is positioned as indicated in the below formula: 0

R11

52. The method according to any of claims 43-46, wherein R¹¹ of said monomer unit(s) with which the polymer backbone is functionalized comprises at least one catechol functional group and further comprises at least one amino group and/or one carboxylate-group is positioned as indicated in the below formula:

53. The method according to any of claims 43-49, wherein said monomer unit(s) is/are selected from the group consisting of 2,4,5-trihydroxybenzoic acid, 3-(3,4- dihydroxyphenyl)propanoic acid, 3-(3,4-Dihydroxyphenyl)-2-methyl-alanine, 2,4,5-Trihydroxy-phenylalanine, 2-amino-3-(3,4-dihydroxyphenyl)-3- hydroxypropanoic acid, (2R)-3-(3,4-dihydroxyphenyl)-2-{[(2E)-3-(3,4- dihydroxyphenyl)-2-propenoyl]oxy}propanoic acid (Rosmarinic acid), dopamine and L-DOPA and D-DOPA; or any isomers or mixtures thereof.

54. The method according to any of claims 43-49, wherein said monomer unit(s) is

L-DOPA of the below formula:

55. The method according to any of claims 43-49, wherein said monomer unit(s) D-DOPA of the below formula:

56. The method according to any of the preceding claims, wherein the catechol monomer units are attached to the polymer backbone via covalent bonds.

57. The method according to claim 56, wherein said covalent bonds are ester bonds or peptide bonds or a mixture of ester bonds and peptide bonds.

58. The method according to any of claims 56-57, wherein said covalent bonds are a mixture of ester bonds and peptide bonds.

59. The method according to any of claims 56-57, wherein said covalent bonds are peptide bonds.

60. The method according to any of claims 56-57, wherein said covalent bonds are ester bonds.

61 . The method according to any one of the preceding claims, wherein said

compound is a polymer comprising at least one chemical group selected from - COOH, -OPO₃H, -OSO₃H and -SO₃H.

62. The method according to any one of the preceding claims, wherein said

compound is a polymer comprising glutamic acid and/or aspartic acid and/or analogues thereof.

63. The method according to any one of the preceding claims, wherein said

compound is a polymer comprising a sugar, said sugar comprising at least one chemical group selected from -COOH, -S0₃ and -P0₃H.

64. The method according to any one of the preceding claims, wherein said

compound is selected from the group consisting of poly(acrylic acid), poly(glutamic acid), poly(aspartic acid), hyaluronic acid, chondroitin sulfates, keratan sulfates and alginate.

65. The method according to claim 46 wherein said compound is poly(acrylic acid).

66. The method according to any of the preceding claims, wherein said composition further comprises a photoacid and/or a photobase

67. The method according to any of the preceding claims, wherein said composition further comprises a soluble metal of formula Mⁿ⁺, or any complex thereof, wherein n is 2, 3, 4, 5 or 6.

68. The method according to claim 67, wherein M designates iron, aluminium, titanium, vanadium, manganese, copper, chromium, magnesium, calcium, silicon, zinc, gallium, indium or boron.

69. The method according to any of claims 67-68, wherein the the stoichiometric ratio of catechokmetal is above 3:1 for octahedrally coordinated metals, such as Fe(lll), Al(lll), Mn(ll), V(lll), Cr(lll), and Ti(IV), and 2:1 for tetrahedrally coordinated metals.

70. The method according to any of claims 67-68, wherein the stoichiometric ratio of catechokmetal is below or at 3:1 for octahedrally coordinated metals and 2:1 for tetrahedrally coordinated metals.

71 . The method according to any one of the preceding claims, wherein said

composition further comprises carbon nanomaterials.

72. The method according to claim 71 , wherein said carbon nanomaterials are selected from graphene, graphene oxide and carbon nanotubes.

73. The method according to any one of the preceding claims, wherein said

composition further comprises at least one additive.

74. The method according to claim 73, wherein said additive is selected from the group consisting of proteins, enzymes, small drug molecules, DNA, RNA, lipids and stabilizers.

75. The method according to any one of the preceding claims, wherein said

composition further comprises additional polymers.

76. A coacervate obtained by the method according to any one of the preceding claims.

77. A composition comprising at least 0,25 wt% of a polymer backbone according to any of claims 1 to 75 and at least 0,25 wt% a compound according to any of claims 1 to 75.

78. The composition according to claim 77, wherein said polymer backbone is functionalized with at least one type of monomer units selected from the group consisting of DOPA, 1 -(2'-carboxyethyl)-2-methyl-3-hydroxy-4(1 H)-pyridinone, phenylalanine.

79. The composition according to claim 78, wherein said DOPA is L-DOPA.

80. The composition according to any of claims 77-79, wherein said polymer

backbone comprises at least one type of polymer selected from the group consisting of polyallylamine, chitosan,

81 . The composition according to any of claims 77-80, wherein said compound is selected from the group consisting of polyacrylic acid, tannic acid, phytic acid and osteopontin.

82. The composition according to any of claims 77-81 , wherein said composition comprises at least 0,5 wt%, such as at least 1 wt% or such as at least 5 wt% of said polymer backbone.

83. The composition according to any of claims 77-82, wherein said composition comprises at least 0,5 wt%, such as at least 1 wt% or such as at least 5 % of said compound.

84. The composition according to any of claims 77-83, wherein the polymer

backbone to compound ratio by weight is from 3:1 to 1 :2.

85. The composition according to claim 84, wherein the polymer backbone to compound ratio by weight is 1 :1.

86. A coacervate according to claim 76 or a composition according to any of claims 77-81 for use as a medicament and/or for use as a bioadhesive.

87. A coacervate according to claim 76 or a composition according to any of claims 77-81 for use in the treatment of tissue injuries.

88. A kit comprising

- a composition having a first pH comprising

• a polymer backbone comprising one or more backbone

monomer unit(s) and

• a compound

89. The kit according to claim 88, wherein polymer backbone is as defined in any of claims 6 to 62.

90. The kit according to any of claims 88-89, wherein compound is as defined in any of claims 65-69.

91 . The kit according to any of claims 88-90, wherein said composition is as defined in any of claims 66-75.

92. A kit comprising a composition according to any of claims 77-81 and a pH

adjustable component for adjusting the pH of said composition to obtain a second pH, whereby coacervates are formed.

93. Use of the coacervate according to claim 76 or the composition according to any of claims 77-81 as an adhesive, bioadhesive, glue, joining, attachment, coating and/or cohesive agent, for dry applications and/or for joining dissimilar substrates such as skin/bandaging and/or rubber/metal.

94. Use of the coacervate according to claim 76 or the composition according to any of claims 77-81 , as a biomedical device such as a drug delivery vesicle, a wound care product or a burn care product.

95. A method of forming a coacervate or a hydrogel comprising

- providing a composition comprising

^■ R¹ is selected from the group consisting of amine, carboxylic acid, amide, carbamate, carbonate, ester, halogen, thiol and H;

^■ A is selected from O and S

- exposing said composition to light whereby alcohol is converted to

aldehyde

96. The method according to claim 95, wherein M designates iron, aluminium, titanium, vanadium, manganese, copper, chromium, magnesium, calcium, silicon, zinc, gallium, indium, boron, gadolinium, samarium or europium.

97. The method according to any of claims 95-96 wherein said compound is DNBA.

98. The method according to any of claims 95-97 wherein said polymer backbone is as defined in any of claims 6-60.

99. The method according to any of claims 95-98, wherein said one or more

backbone monomer unit(s) comprises -0-NH₂ and/or -0-NH_qR'_rR"_t, wherein

- r and t is selected from 0 and 1 ;

- q is 2 - (r+t); and

- R' and R" are selected from ??

100. The method according to any of claims 95-97, wherein the polymer

ses one or more backbone monomer unit(s) of the formula:

101 . The method according to any of claims 95-97, wherein the polymer

backbone comprises one or more backbone monomer unit(s) comprising a chemical group having the formula: wherein:

R² and R³ are selected from the group consisting of H and Ci_-8-alk(an/en/yn)yl).

102. The method according to claim 101 , wherein R² and R³ are selected from the group consisting of H and Ci_-8-alkanyl.

103. A hydrogel or a coacervate obtained by the method according to claims 95-102.

104. A hydrogel or a coacervate according to claim 103 for use as a

medicament.

105. A hydrogel or a coacervate according to claim 103 for use in the

treatment of tissue injuries.

106. A composition comprising a compound comprising one or more light sensitive catechol- group(s), said compound having the formula:

- R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is selected from the group

consisting of halogen, alk(an/en/yn)yl, hydroxyl, carboxylic acid, ester, ether, amine, amide and thiol; and - A is selected from O and S.

107. The composition according to claim 106, wherein said composition further comprises a metal of formula Mⁿ⁺, or any complex thereof, wherein n is 2, 3, 4, 5 or 6.

108. The composition according to any of claims 106-107, wherein said

composition further comprises a polymer backbone comprising one or more backbone monomer unit(s) comprising at least one functional group selected from amino, thiol and amino-oxy groups.

109. The composition according to any of claims 106-108, wherein said

polymer backbone is as defined in any of claims 6-62.