WO2010099818A1 - Thermoreversible polysaccharide hydrogel - Google Patents

Abstract

The present invention relates to a crosslinked or grafted copolymer composition suitable for in situ formation of a hydrogel in aqueous solution comprising a hydrophilic polysaccharide and a thermoreversible polyamide grafted or crosslinked via a 'click chemistry' reaction, its method of production and its use in in vitro cell studies and medical applications.

Description

Thermoreversible polysaccharide hydrogel

Field of the Invention

The present invention describes an injectable, in situ gelling hydrogel based on a polysaccharide, preferably hyaluronic acid, and a thermoreversible polymer, preferably poly (N- isopropylacrylamide) , its method of production and its use in in vitro cell studies and medical applications.

Background

Hydrogels have become an important material for biomedical applications due to their high water content, tissue-like elasticity, tunable diffusion characteristics, and their favorable tolerance by the body. From a material perspective, polysaccharides are a naturally derived hydrogel . Polysaccharides are relatively complex carbohydrates made from monosaccharides joined by glycosidic bonds. In nature they have critical roles in cell signaling, extracellular matrix structure, lubricious fluids, and as energy sources. Due to their important roles in organ development and cell signaling, in addition to their tolerance and clearance in the body, polysaccharides have long been viewed as candidates for biomaterials. Of particular interest in this area are the polysaccharides chitosan, alginate, agarose, and gycosaminoglycans such chondroitin sulfate, heparin, and hyaluronic acid.

Hyaluronic acid (HA) is an endogenous linear heteropolysaccharide composed of D-glucuronic acid and D-N- acetylglucosamine, which are linked together via alternating β-1,4 and β-1,3 glycosidic bonds. This polysaccharide can be composed of up to 25,000 disaccharide repeats, and range in size from 5,000 to 20,000,000 Da depending on the origin. Each repeat unit bears a carboxylic chemical group on the D- glucuronic acid motif. It is a chief component of the extracellular matrix in connective, epithelial, and neural tissues, and is known to play an important role in organ development, cell proliferation and migration. Additionally, HA contributes to the lubrication and maintenance of cartilage, where it is a major component of synovial fluid and forms a coating around chondrocytes .

The first commercial use of HA was as a material for eye surgery in the 1970' s by Pharmacia. Since then several other companies have developed HA based products for viscosupplementation, osteoarthritis, wound healing, tissue fillers, and cell scaffolds for tissue engineering. In many of these products, the HA is synthetically modified to alter its mechanical properties and slow its degradation when implanted in the body. The most common approach is to crosslink the linear polysaccharide to avoid rapid degradation and clearance from the implant site of unmodified polysaccharide. This may be accomplished by e.g. thiolating the carboxylic acid groups present on the HA molecule and subsequently crosslinking the gel by exposure to oxygen through ambient air exposure or by soaking in hydrogen peroxide (see Shu, X. et al Biomacromolecules 2002 3(6), 1304-1311) . While this method can generate adequate gels for cell studies, the long polymerization time (3 days) and exposure to hydrogen peroxide limits their use for cell encapsulation or in situ polymerization. Another method includes addition of polyethylene glycol diacrylate (PEG-DA) , whereby crosslinks are formed via spontaneous reaction of free thiols on the HA molecule with acrylate groups on the PEG-DA crosslinker. This strategy accelerates polymerization to 9 minutes, making them more suitable for in situ applications (see Zheng, S., et al Biomaterials 2004, 25(7-8), 1339-1348) . However, the PEG-DA:HA gels require blending of different components immediately prior to use, at which point polymerization begins immediately and the pot time is limited to under 10 minutes. A further method includes direct methacrylation of HA, making it amenable to in situ crosslinking by exposure to a suitable free radical initiator (see Bencherif, S. A. et al Biomaterials 2008, 29(12), 1739-1749.) . Yet another method includes use of a UV activated initiator to increase the control over polymerization timing, such that a monomer solution can be introduced to a site and subsequently cured by exposure to a UV source (see Masters, K. S. et al Biomaterials 2005, 26(15), 2517-2525) . However, in the case of blocked absorbance of UV light, homogenous polymerization may be difficult to achieve in situ. The above shows that while polymerization kinetics and crosslinking density can be controlled in HA based hydrogels, there are still technical drawbacks to overcome. In addition, biological entities, such as cells, active agents, and the like, encapsulated in these monomer solutions are exposed to reactive moieties, such as free radicals and highly reactive acrylate groups during polymerization, which may be detrimental to the biological entities .

Thus there is a need for a versatile, easily modified polymer that can be tailored to examine a wide range of variables on cell behavior or used for in vivo delivery of therapeutic elements .

One means of introducing crosslinks or functional grafts to the polysaccharide backbone is via the addition of reactive groups suitable for 'click chemistry' . The term 'click chemistry' refers to a variety of chemical reactions that are characterized by their high yield, regioselectivity, large thermodynamic driving force, stability, and ability to proceed without the generation of offensive byproducts (see KoIb, H. C. et al Chem. Int. Ed Engl. 2001, 40(11), 2004-2021) . Importantly, these reactions can occur in a benign solvent, are insensitive to water and oxygen, and have a thermodynamic driving that is usually greater than 20 kcal.mol^"1. The most common examples of these reactions are the carbon heteroatom bond formation resulting from cycloadditions of unsaturated species, nucleophilic substitution chemistry, non-aldol carbonyl chemistry, and additions to carbon-carbon multiple bonds. Of particular interest are the cycloaddition reactions, including the Diels-Alder and, most importantly, the 1,3 dipolar cycloaddition. The rise of 'click chemistry' has been important to quickly and efficiently generate molecular libraries to probe structure-activity relationships (SAR) in the pharmaceutical industry. Additionally, 'click chemistry' has been exploited in the pursuit of telechelic polymers. A telechelic polymer is one which carries functionalized endgroups . By combining functionalized polymers with distinct properties, a wide variety of block or graft copolymers can be quickly and easily created.

Importantly, "click chemistry' has been applied to depart unique properties to polysaccharides. For example, HA has been functionalized with azide and alkyne groups, which are subsequently reacted together with a copper catalyst in order to crosslink the polysaccharide via formation of a triazole ring (WO 2008/031525, Di Meo et al, Macromol . Biosci . 2008, 8(7) , 670-681) . Furthermore, HA has been similarly modified for the attachment of carborane rings intended for the cellular delivery of boron atoms in boron neutron-capture anti-cancer therapy (BNCT) (Crescenzi et al Biomacromolecules . 2007, 8(6), 1844-1850.) . The benefit of modifying a polysaccharide backbone with azide or alkyne groups is manyfold. For example, by introducing an azido functionalized thermoreversible or hydrophobic polymer, the polysaccharide will form a physical network via association of hydrophobic domains. The specificity and efficiency of "click chemistry' assures that grafting of these functional polymer groups will occur at nearly 100% efficiency and can occur in an aqueous medium .

In recent years, reversible addition-fragmentation chain transfer polymerization (RAFT) has become popular as a method to generate highly controlled molecular weight polymers with low polydispersity (PDI) . This polymerization can occur under mild reaction conditions, in an aqueous solvent, and with high fidelity. By functionalizing the endgroups of the trithiocarbonate based chain transfer agent (CTA) used in RAFT polymerizations, it is possible to generate highly specific polymer architectures composed of 2 or more different polymers. Using RAFT to generate specific molecular weight polymers combined with the efficient coupling mechanism of Λ click chemistry' allows highly controlled formation of complex, functional network architectures consisting of 2 or more polymers with distinct physical properties.

Applicants have found that a polysaccharide modified with a thermoreversible polyamide, e.g. poly (N-isopropylacrylamide) (pNIPAM) , designed to be a flowing, deformable liquid at room temperature will form a stable, robust, physical gel when exposed to in vivo or body temperature. By using a polysaccharide as the backbone of the polymer composition, it is possible to retain the favorable biocompatibility and degradation inherent to a natural material. By covalently grafting or crosslinking a synthetic, thermoreversible polyamide onto the polysaccharide backbone through 'click chemistry' , it is possible to tailor the degradation and gelation characteristics of the polysaccharide.

Brief Description of Figures

Figure 1. Exemplary grafting of an azide functionalized R group to the backbone of an alkyne functionalized HA polysaccharide. In this embodiment, R can equal a pNIPAM polymer and/or a bioactive agent.

Figure 2. A multi-arm configuration of pNIPAM using a central linkage and multiple 'click' reactive sites can be used to increase the stiffness of the gel and decrease the degradation rate .

Figure 3. Schematic representation of thermally induced gelation of a polysaccharide using pNIPAM grafts via 'click chemistry' . Figure 4. Schematic representation showing the diversity of groups that could occupy 'click chemistry' reactive sites on the polysaccharide backbone.

Figure 5. Rheological behavior of pNIPAM grafted HA showing temperature induced gelation. Gel point is represented by the intersection of G' and G' ' .

Figure 6. Imaging of (A) azide-modified fluorescein mixed with water and non-functionalized HA; (B) fluorescein covalently grafted to a HA-pNIPAM-copolymer composition of the invention; and (C) HA-pNIPAM-copolymer composition of the invention without fluorescein as control .

Summary of the Invention

In a first aspect, the present invention provides a copolymer composition based on a hydrophilic polysaccharide and a thermosensitive polyamide that are crosslinked or grafted by a click chemistry reaction.

In a specific embodiment the copolymer composition of the invention is obtained by reacting an alkyne modified polysaccharide, preferably HA, with an azido-terminated pNIPAM. Upon solubilization in an aqueous solvent, the copolymer composition may form a thermosensitive hydrogel showing a liquid-to-gel phase transition with temperature change.

Thus in a further aspect, the present invention provides a thermoreversible hydrogel which is biodegradable and biocompatible .

In one embodiment the thermoreversible biodegradable hydrogel is in solution or liquid state at or around room temperature or lower and in a gelled state at or around physiological (or in vivo) temperature or higher.

It is another object of this invention to provide a thermosensitive biodegradable hydrogel having utility in many medical applications such as a bioactive agent or cell delivery system, in tissue engineering, cell culture systems, and the like.

In one embodiment the copolymer composition or hydrogel of the invention further comprises a bioactive agent, which is either dispersed in or covalently attached to the copolymer composition. Thus, in a further aspect the present invention is directed towards the use of a thermoreversible biodegradable hydrogel as a bioactive agent delivery system.

In a specific embodiment the bioactive agent delivery system is easily administrable, e.g. can be injected parenterally. Yet still another object of this invention is a method of preparation of a copolymer composition or hydrogel of the invention.

In yet a further aspect, the invention relates to a kit comprising the copolymer composition of the invention, e.g. in a lyophilized form. Additional objects and advantages of this invention will become apparent from the following detailed description of the various embodiments making up this invention.

Detailed Description of the Invention

The present invention relates to copolymer compositions based on a polysaccharide and a thermoreversible polyamide, an injectable, in situ gelling hydrogel obtained thereof as well as their methods of production and their use in various medical applications, e.g. delivery systems and in vitro cell studies .

Unless specified otherwise, the following definitions apply:

As used herein the term "grafting density" or "crosslinking density" is defined as the ratio of polysaccharide (units or mol%) that carry a functional group that has undergone a reaction with a complementary functional group to total polysaccharide (units or mol%) .

The term "degree of crosslink or graft conversion" as used herein means the quantity of pairs of functional groups converted into crosslinking or grafting bonds relative to the total quantity of pairs of functional groups initially present on the polysaccharide and thermoreversible polyamide, expressed as a percentage .

The term "degree of conversion" as used herein with respect to the polysaccharide means the quantity of carboxylate groups converted into functional groups suitable to undergo crosslinking or grafting via 'click chemistry' relative to the total quantity of carboxylate groups initially present on the polysaccharide, expressed as a percentage. The term "biodegradable" refers to the ability of the compositions of the present invention to be degraded and broken down in vivo into non toxic substances, which are excreted. The term "biocompatible" refers to the ability of the compositions of the present invention to be applied to tissues without eliciting adverse tissue responses, such as significant inflammation, fibrosis, and the like. The term of "liquid-to-gel-phase transition" in the present invention refers to a composition that is present as a flowable liquid below a lower critical solution temperature (LCST) and is changed into a gel above said critical temperature. Such a transition may occur reversibly. The terms "gelation temperature" , "gelling temperature" or "LCST" may be used interchangeably to describe the temperature at which the liquid-to-gel-phase transition occurs. This temperature (or temperature range) depends on the nature of the composition. In the case of a hydrogel formed of a copolymer composition, this includes factors such as molecular weight, ratios of the co-monomers, graft and/or crosslinking density of the copolymer composition, the concentration of the copolymer composition in the aqueous solution to form a liquid hydrogel, the absence or presence of one or more bioactive agents, additives, etc. The terms "hydrogel in liquid state", "liquid hydrogel" and "liquid copolymer solution" may be used interchangeably to describe the flowable liquid below the LCST. The terms "hydrogel in gelled state", "stable hydrogel" or "robust hydrogel" may be used interchangeably to describe the gelled state above the LCST.

The term "thermosensitive" refers to a physical property of a composition in which the composition can undergo a physical change of a liquid state to a gel state (i.e. a liquid-to-gel- transititon) repeatedly when exposed to an increase in temperatures. For purposes of the present invention, the terms "thermosensitive" , "thermoreversible" , and "thermoresponsive" may be used interchangeably.

The term "room temperature" refers to a temperature range from about 18 ⁰C to 28 ⁰C, with a preferred ambient temperature of about 25 ⁰C. The term "in vivo temperature" refers to a temperature range from about 28 ⁰C to 41 ⁰C, with a preferred in vivo temperature of about 35 ⁰C to 39 ⁰C. The term "physiological conditions" refers to a temperature range of from 35 ⁰C to 39 ⁰C, with a preferred temperature of a human body of 37 ⁰C.

As used herein the term "graft" refers to two compounds (e.g. two polymers or a polymer and a bioactive agent, etc) that are covalently bound two each other via ^l click chemistry' type reactions.

As used herein the term "crosslink" refers to any covalent, ionic, or physical association of grafted polymers to link the polymer segments together.

As used herein the term chain transfer agent (CTA) refers to a thiocarbonylthio compound containing at least one weak chemical bond by which monomers can be polymerized from in a controlled, living polymerization with end group specificity.

As used herein the term reversible addition-fragmentation chain transfer (RAFT) polymerization refers to a living polymerization technique by which polymers of controlled molecular weight, end group specificity, and low polydispersity can be formed by use of a chain transfer agent

(CTA) .

The term "carboxylic acid reactive group" refers to any group that undergoes a reaction with carboxylic acids and includes halogen, hydroxy or alkoxy groups, amines, carboxylic acid derivatives, such as an acid chloride or an acid anhydride and the like.

The term "alkyl" used alone or in combination with other groups, e.g. alkoxy, refers to saturated, straight or branched-chain hydrocarbon radicals containing between one and ten carbon atoms including, but not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl and neopentyl . Preferably, alkyl is limited to 1-4 carbons.

The term "halogen" or "halo" refers to fluorine, chlorine, bromine and iodine . In a first aspect, the present invention relates to a crosslinked or grafted copolymer composition suitable for in situ formation of a hydrogel in aqueous solution comprising a hydrophilic polysaccharide and a thermoreversible polyamide grafted or crosslinked via a "click chemistry' reaction, hereinafter also called copolymer composition of the invention.

As used herein the term "click chemistry" refers to a selection of chemical reactions characterized by their rapidity, regioselectivity, high yield, and high thermodynamic driving force. They are powerful and efficient chemical reactions that are modular and wide in scope, and have the benefit of being insensitive to solvents and pH conditions. Common features of 'click chemistry' reactions include i) stable linkages; ii) minimal cross-reactivity with other functional groups; iii) high conversions/yields that react to completion; iv) absence of appreciable amounts of side product (s); and v) proceed under relatively benign reaction conditions. Thus 'click chemistry' reactions provide stereoselective conversion with high to very high yields. Generally, click reactions are driven by a high thermodynamic force (e.g. > 20 kcal.rn.ol^~1), which gives rise to highly modular and stereospecific reactions with high yields. It is understood that 'click chemistry' is not limited to a specific type of reaction but rather includes a range of different reactions, with different reaction mechanisms. These include, but are not limited to, a [1, 3] -dipolar cycloaddition (Huisgen) ; a [2,4] -cycloaddition (Diels-Alder) ; nucleophilic substitution reaction-ring opening reactions; carbonyl reactions of the non- aldol type; and addition to carbon-carbon multiple bonds. Preferred reactions are pericyclic reactions, preferably a cycloaddition reaction, preferably a 1,3 -dipolar cycloaddition reaction or a Diels-Alder reaction.

Thus in a specific embodiment the present invention relates towards a copolymer composition of the invention, wherein the 'click chemistry' reaction is a 1,3 -dipolar cycloaddition reaction between at least one first functional group present on a hydrophilic polysaccharide capable of participating in a 'click chemistry' reaction and at least one second complementary functional group present on a thermoreversible polyamide capable of participating in a 'click chemistry' reaction with the first functional group.

In a specific embodiment, the first functional group is introduced by reacting a group RG-L-FG, wherein RG is a carboxylic acid reactive group, L is a linker and FG is the first functional group, with a carboxylic acid group present on the hydrophilic polysaccharide. The linker may be selected from the group consisting of a covalent bond, straight or branched C(I- 8)alkyl groups and polyethers of the formula -(CH₂-CH₂-O)_n- wherein n is 1 to 20, preferably 1 and 8. In a specific embodiment 0.1 to 100%, more preferably 5 to 70%, more preferably 15 to 50% of the at least one carboxylic acid groups present on the hydrophilic polysaccharide are subjected to conjugation to a group RG-L-FG.

In another specific embodiment 0.1 to 100%, preferably 5 to 50 % of the functional group FG present on the hydrophilic polysaccharaide have been subjected to reaction with the complementary functional group present on the thermoreversible polyamide. The functional groups involved in 'click chemistry' include e.g. a diene and/or a dienophile as used in the Diels Alder reaction, and an azide and/or alkyne group as used in a 1,3 cycloaddition .

Thus in one preferred embodiment the invention is directed towards copolymer compositions of the invention, wherein the at least one first functional group is an alkyne group and the at least one second functional group is a terminal azide group. In another preferred embodiment the invention is directed towards copolymer compositions of the invention, wherein the at least one first functional group is an azide group and the at least one second functional group is a terminal alkyne group .

Thus in a specific embodiment the copolymer compositions of the invention have a grafting and/or crosslinking density between the polysaccharide and the thermoreversible polyamide of from 0.01 to 100 % preferably 5 to 50 % more preferably 10 to 30 %.

Thus, in a preferred embodiment the present invention is directed towards a copolymer composition of the invention comprising a hydrophilic polysaccharide and a thermoreversible polyamide grafted or crosslinked via a Huisgen 1,3 -dipolar cycloaddition. This reaction is well described in the literature (see e.g. WO 03/101972, incorporated herein by reference in its entirety) and includes the reaction between an azide and an alkyne group to form a 1, 2, 3-triazole ring (Huisgen et al . , Chem. Bex. 1967, 100, 2494-2507) . It may proceed in water alone or in the presence of a catalyst, such as a copper (I) catalyst, to enhance its efficiency, regioselectivity, and/or reaction rate.

As used herein the term "polysaccharide" includes any complex carbohydrate made of monosaccharides joined by glycosidic bonds. Typical monosaccharide units include arabinose, fructose, galactose, galactopyranosyl , galacturonic acid, guluronic acid, glucuronic acid, glucose, glucoside,N- acetylglucosamine, mannuronic acid, mannose, pyranosyl sulfate, rhamnose, or xylose. Polysaccharides containing the foregoing units include cyclodextrins, starch, chitosan, trehalose, cellobiose, maltotriose, maltohexaose, chitohexaose, agarose, chitin 50, amylose, glucans, xylan, pectin, galactan, dextran, aminated dextran, cellulose, hydroxyalkylcelluloses, carboxyalkylcelluloses, fucoidan, sulfate polysaccharides, mucopolysaccharides, gelatin, zein, collagen, alginic acid, agar, carrageean, guar gum, gum arabic, gum ghatti, gum karaya, gum konjak, gum tamarind, gum tara, gum tragacanth, locust bean gum, pectins, xanthan gum, and the glycosaminoglycans such as HA, heparin, chondroitin sulphate. Preferred polysaccharides include chitosan and members of the glycosaminoglycan family, such as HA, heparin, chondroitin sulphate, most preferably HA. Preferably, the hydrophilic polysaccharide of choice has a molecular weight of between 1 kDa to 10 MDa, preferably between 10 kDa to 5 MDa. It is understood that the molecular weight of choice will depend on the intended function of the copolymer composition of the invention and hydrogel formed thereof (and thus by such factors as the desired copolymer composition degradation rate and the desired release rate of a bioactive agent optionally incorporated therein, i.e. the therapeutic condition to be treated) . For example, a polysaccharide with a higher molecular weight will exhibit increased stiffness of a hydrogel formed thereof and decreased clearance rate when implanted in vivo.

In a specific embodiment, the present invention relates to a copolymer composition of the invention comprising a HA and a thermoreversible polyamide grafted or crosslinked via a 'click chemistry' reaction, such as the Huisgen 1,3-dipolar cycloaddition .

HA is a polymer based on disaccharide units (composed of D- glucuronic acid and D-N-acetylglucosamine linked together via alternating β-1,4 and β-1,3 glycosidic bonds) each having one carboxylate function, wherein the carboxylate function of the disaccharide unit of HA may be conjugated to a functional group suitable to undergo ' click chemistry' reactions, such as the Huisgen 1,3-dipolar cycloaddition. Thus in one embodiment an alkyne- (or azide-) functionalized

HA may be used to undergo a 1,3 -dipolar cycloaddition with a thermosensitive polyamide carrying the corresponding azide-

(or alkyne-) group. More specifically, the carboxylic acid group on the disaccharide D-glucuronic acid subunit of HA may be functionalized with propargylamine, whereby the percentage of carboxylic acid groups functionalized may be controlled by stoichiometric addition of propargylamine.

As used herein the term "thermosensitive polyamide" may include any polyamide capable of forming a hydration shell in an aqueous solution and capable to undergo significant changes in hydration in response to a change of temperature of said solution. More specifically, the polyamide may be selected from the group polyacrylamides , polyvinylamides, polyalkyloxazolines, polyvinylimidazoles, and their copolymers and blends. Specific examples for such polymers are poly (N- isopropylacrylamide) (pNIPAM) , poly (N-vinylcaprolactam) , poly

(N-isopropyloxazoline) , poly (vinylimidazole) and poly (N- acryloyl-pyrrolidin) , most preferably poly (N- isopropylacrylamide) (pNIPAM) or a copolymer or derivative thereof .

Thus, in a specific embodiment, the present invention relates to a copolymer composition of the invention comprising HA and a pNIPAM polymer grafted or crosslinked via a 'click chemistry' reaction, such as the Huisgen 1,3 -dipolar eye1oaddition .

More specifically, an alkyne- (or azide-) functionalized HA may be used to undergo a 1,3 -dipolar cycloaddition with a pNIPAM polymer functionalized with the corresponding azide- (or alkyne-) group. In one embodiment, a pNIPAM polymer may be functionalized with a single azide group. These azide- functionalized pNIPAM polymers may then be grafted onto an alkyne-functionalized polysaccharide backbone (such as HA) , where R is equal to the pNIPAM polymer (Figure 1) . In yet another embodiment , thermoreversible synthetic polymers containing more than one group functionalized for 'click chemistry' can be introduced to covalently crosslink the polysaccharide. In one example, this can be achieved by polymerization of NIPAM using a chain transfer agent that has been modified to include 2 or more azide groups. The azide/alkyne crosslinking of the polysaccharide would form a weak gel, and further gelation of the material would occur via association of hydrophobic domains in the crosslinker. This would result in a stiffer hydrogel with slower degradation and slower drug release kinetics than a hydrogel formed from a polysaccharide grafted with pNIPAM containing only one azide group .

In another embodiment, the crosslinker could be a pNIPAM polymer in a branched or multi-armed configuration, with multiple sites activated for coupling to the polysaccharide via * click chemistry' . Figure 2 shows e.g. a four-arm pNIPAM configuration connected through a central linkage, whereby each "pNIPAM-arm" is linked through a 'click' reactive site to one or more polysaccharides. A branched or multi-armed thermoreversible crosslinker of this sort may reduce the viscosity of the liquid copolymer solution at room temperature or produce a greater increase in stiffness during temperature induced formation of a hydrogel . The molecular weight of the polyamide, e.g. the pNIPAM polymer is 1 to 200 kDa, preferably 5 to 100 kDa. In one specific embodiment, the molecular weight is chosen below 30 kDa, e.g. preferably from 5 to 30 kDa, to facilitate clearance from the body. In another specific embodiment, the molecular weight may be chosen higher than 30 kDa, e.g. from 30 to 100 kDa, to facilitate formation of a more robust hydrogel. It is understood that if a polyamide, e.g. a pNIPAM polymer comprising a degradable linkage is used, its molecular weight may be chosen higher than 3OkDa without impact on its clearance from the body. Thus, the molecular weight of the copolymer compositions of the invention is 1 kDa - 1 GDa, more preferably 5 kDa to 500 MDa, most preferably between 1 MDa to 500 MDa. As indicated above, it is understood that the molecular weight of choice will depend on the intended function of the copolymer composition of the invention and hydrogel formed thereof (and thus by such factors as the desired copolymer composition degradation rate and the desired release rate of a bioactive agent optionally incorporated therein, i.e. the therapeutic condition to be treated) . In one embodiment, the molecular weight of the copolymer composition degradation products may be chosen sufficiently low, preferably below 30 kDa, to facilitate clearance from the body via the renal system. In another embodiment, the molecular weight of the copolymer compositions of the invention may be chosen higher, preferably higher than 30 kDa, to facilitate formation of a more robust hydrogel, e.g. a hydrogel with a higher modulus, or to slow the degradation of the material .

For the purposes of the present invention the copolymer composition of the invention is designed such that upon dissolution in an aqueous solvent it forms a hydrogel, which is in a flowing, deformable liquid state (i.e. a copolymer solution) at a lower temperature, such as room temperature (e.g. below the LCST), and will undergo a transition into a stable, robust, gelled state (i.e. a stable hydrogel) when exposed to temperatures at or around in vivo temperature (e.g. above the LCST) , such as mammalian body temperature of 37 ⁰C. Thermally induced gelation of the liquid copolymer solution, i.e. the hydrogel in liquid state, will occur as the material warms from room temperature (at or around 20 ⁰C) to in vivo temperature, (e.g. 37°C ) . Dehydration of the thermoresponsive polymer segments occurs above the LCST and will lead to association of hydrophobic domains and the formation of a physical gel network, resulting in a stable hydrogel. Figure 3 gives a schematic representation of a thermally induced gelation of a polysaccharide using pNIPAM grafts via 'click chemistry' : at temperatures below body temperature, e.g. < 37 ⁰C, the two pNIPAM polymers occupying each a click reactive site on the hydrophilic polysaccharide backbone are in an extended, hydrophilic state. Upon increasing temperature to body temperature or higher, e.g. ≥ 37⁰C the two pNIPAM polymers are in a coiled, hydrophobic state. Association of the hydrophobic domains leads to gelation.

Thus, in a further aspect the invention is directed to a hydrogel which is characterized by showing a liquid-to-gel phase transition around body temperature, i.e around 18 ⁰C to

42⁰C, preferably around 30 ⁰C to 38 ⁰C. Below said transition temperature the hydrogel of the invention is in a liquid state, and above said transition temperature the hydrogel is in a gelled state.

Preferably, the hydrogel may be a solution based on 1 to 50 wt%, more preferably from 1 to 20 wt%, more preferably between 5 to 15 wt% of the copolymer composition of the invention dissolved in an aqueous solvent. The aqueous solvent may be any of the commonly used aqueous solvents, which are all well known by those of ordinary skill in the art. This may be selected from the group consisting of water (including water for injection) , buffer solutions, acid solutions, basic solutions, salt solutions, saline solution, and glucose salt solution, for example sodium acetate, Tris, sodium phosphate, MOPS, PIPES, MES and potassium phosphate (e.g. in the range of 25 mM to 500 mM and in the pH range of 4.0 to 8.5) .

It is understood that the ratios of hydrophilic polysaccharide to thermosensitive polyamide in the copolymer compositions of the invention (which includes their grafting or crosslinking density) are chosen such that they show retention of the desired water solubility and gelling properties of the hydrogel formed. For the purposes of the present invention the copolymer compositions of the invention should show sufficient water solubility at temperatures below the LCST, and instant gelation under physiological conditions (e.g. pH 7.0 and 37°C) . In one embodiment, the hydrophilic polysaccharide is present in the copolymer compositions of the invention from about 2 % to 90 wt%, preferably from 10 to 50 wt% of the copolymer compositions of the invention. In another embodiment, the hydrophilic polysaccharide is present in hydrogels of the invention (i.e. the hydrated copolymer compositions of the invention) from about 1 % to 20 wt%, preferably from 2 to 10 wt% of the hydrogels of the invention. In yet another embodiment, the thermosensitive polyamide graft or crosslinks are present in the hydrogels of the invention from about 2 to 50 wt%, preferably 5 to 20 wt% of the hydrogels of the invention (or the hydrogels formed thereof) . It is understood that the exact ratios will also depend on the nature of the polyamide, i.e. linear vs. branched vs. star shape polyamide and degree of graft vs. crosslinking. The concentration at which the copolymer compositions of the present-invention remain soluble below the LCST and form an instant gel at physiological conditions is typically up to about 60% by weight, with a preferred concentration range from about 1% to 50%, more preferred from about 2% to 10%. It is understood that those concentrations also depend on the exact nature of the copolymer composition.

Both the copolymer compositions of the invention as well as the hydrogel formed thereof are bioresorbable, cytocompatible, nontoxic, and biocompatible and are therefore particularly useful in various medical applications.

Thus, in a further aspect the invention is directed towards the use of the copolymer compositions and hydrogels of the invention in in vivo drug delivery, in vitro cell culture, tissue filler, cosmetic, and tissue engineering applications.

In one embodiment the copolymer compositions and hydrogels of the invention may be used as a bioactive agent delivery system for various bioactive agents ranging from small molecule compounds to macromolecular compounds to cells.

More specifically, the present invention provides a bioactive agent delivery system comprising a hydrogel of the invention and at least one bioactive agent dissolved in an aqueous solvent. Figure 4 illustrates schematically a hydrophilic polysaccharide backbone having two click reactive sites occupied by pNIPAM polymers and two click reactive sites occupied by a bioactive agent, drug depot or the like as well as one free click reactive site. Thus, the copolymer compositions of the invention or the hydrogel of the invention may further comprise at least one bioactive agent . The term "bioactive agent" is used throughout the specification to describe ay agent with biological activity to be incorporated into a copolymer composition of the invention. The at least one bioactive agent may be natural, synthetic, semi-synthetic or derivatives thereof and may include both hydrophobic and hydrophilic, soluble and insoluble compounds. More specifically, the at least one bioactive agent may be any bioactive agent useful for the treatment and/or prevention and/or diagnosis of conditions in any therapeutic area known in mammals, such as animals and humans, particularly humans, which include, but are not limited to, infectious disease (anti-bacterial, anti-fungal and anti-viral activity, vaccines,), inflammatory disease (including arthritis, and hypertension) , neoplastic disease, diabetes, osteoporosis, pain management, general cardiovascular disease and lung disease (e.g. asthma, emphysema, lung cancer, chronic obstructive pulmonary disease (COPD) , bronchitis, influenza, pneumonia, tuberculosis, respiratory distress syndrome, cystic fibrosis, sudden infant death syndrome (SDKs) , respiratory synctial virus (RSV) , AIDS related lung diseases, sarcoidosis, sleep apnea, acute respiratory distress syndrome (ARDS) , bronchiectasis, bronchiolitis, bronchopulmonary dysplasia, coccidioidomycosis, hantavirus pulmonary syndrome, histoplasmosis, pertussis and pulmonary hypertension) , as well as in general conditions caused by growth of harmful or pathogenic organisms, including, but not limited to bacteria, yeast, viruses, protozoa or parasites, conditions to be treated by gene therapy or diagnosed by imaging means .

The at least one bioactive agent may be selected from a macromolecular compound or a small molecule compound, such as peptides, proteins, oligo- and poly-nucleotides, antibiotics, antimicrobials, growth factors, enzymes, antitumoral drugs, anti-inflammatory drugs, antiviral drugs, antifungal drugs, anesthetics, anti neoplastic drugs, antimitotic drugs, analgesics, narcotics, antithrombotic drugs, anticoagulants, haemostatic drugs. More specifically the at least one bioactive agent may be selected from the group consisting of (a) proteins or (poly) peptides, such as erythropoietin (EPO) , interferon-alpha, interferon-beta, interferon- gamma, growth hormone (human, pig, cow, etc.), growth hormone releasing factor, nerve growth factor (NGF) , granulocyte-colony stimulating factor (G-CSF) , granulocyte macrophage-colony stimulating factor (GM-CSF) , macrophage-colony stimulating factor (M-CSF) , blood clotting factor, insulin, oxytocin, vasopressin, adrenocorticotropic hormone, epidermal growth factor, platelet-derived growth factor (PDGF) , prolactin, luliberin, luteinizing hormone releasing hormone (LHRH) , LHRH agonists, LHRH antagonists, somatostatin, glucagon, interleukin-2 (IL-2) , interleukin-11 (IL-Il) , gastrin, tetragastrin, pentagastrin, urogastrone, secretin, calcitonin, enkephalins, endorphins, angiotensins, thyrotropin releasing hormone (TRH) 5 tumor necrosis factor (TNF) 5 tumor necrosis factor related apoptosis inducing ligand (TRAIL) 5 heparinase, bone raorphogenic protein (BMP) , human atrial natriuretic peptide (hANP) , glucagon-like peptide (GLP-I) 5 renin, bradykinin, bacitracins, polymyxins, colistins, tyrocidine, gramicidins, cyclosporins and synthetic analogs thereof, monoclonal antibody, and cytokines; and (b) vaccines; and (c) genes such as small interference RNA (siRNA) , plasmid DNA, and antisense oligodeoxynucleotide (AS- ODN) ; and (d) hormones, such as testosterone, estradiol, progesterone, prostaglandins and synthetic analogs thereof; and (e) an anti-cancer drug, such as paclitaxel, doxorubicin, 5-fluorouracil, cisplatin, carboplatin, oxaliplatin, tegafur, irinotecan, docetaxel, cyclophosphamide, cemcitabine, ifosfamide, mitomycin C, vincristine, etoposide, methotrexate, topotecan, tamoxifen, vinorelbine, camptothecin, danuorubicin, chlorambucil, bryostatin-1, calicheamicin, mayatansme, levamisole, DNA recombinant interferon alfa-2a, mitoxantrone, nimustine, interferon alfa-2a, doxifluridine, formestane, leuprolide acetate, megestrol acetate, carmofur, teniposide, bleomycin, carmustine, heptaplatin, exemestane, anastrozole, estramustine, capecitabine, goserelin acetate, polysaccharide potassium, medroxypogesterone acetate, epirubicin, letrozole, pirarubicin, topotecan, altretamine, toremifene citrate, BCNU, taxotere, actinomycin D, polyethylene glycol conjugated protein, and synthetic analogs thereof; and (f) an angiogenesis inhibitor, such as Clodronate, Doxycycline,

Marimastat , 2-Methoxyestradiol, Squalamine, Thalidomide,

Combretastatin A4, Soy Isoflavone, Enzastaurin, CC 5013

(Revimid; Celgene Corp, Warren, NJ) , Celecoxib, Halofuginone hydrobromide , interferon-alpha, Bevacizumab, Interleukin-12, VEFG-trap, Cetuximab, and synthetic analogs thereof.

The at least one bioactive agent may also be a (therapeutic) cell and may be selected from the group consisting of preosteoblast , chondrocyte, umbilical vein endothelial cell (UVEC) , osteoblast, adult stem cell, Schwann cell, oligodendrocyte, hepatocyte, mural cell (used in combination with UVEC), myoblast, insulin-secreting cell, endothelial cell, smooth muscle cell, fibroblast, [beta] -cell, endodermal cell, hepatic stem cell, juxraglomerular cell, skeletal muscle cell, keratinocyte, melanocyte, langerhans cell, merkel cell, dermal fibroblast, and preadipocyte .

In one embodiment the at least one bioactive substance may be present in an amount of between about 0.01 to 50 wt%, preferably about 0.1 to 20 wt% based on the total amount of the copolymer composition or the hydrogel of the invention.

The bioactive agent may be dispersed within the copolymer composition or the hydrogel of the invention. Alternatively, the bioactive agent may be conjugated to the copolymer composition or the hydrogel of the invention via a ^λ click chemistry' reaction, e.g. by conjugation to functional groups on the polysaccharide that have not been coupled to a polyamide . In a specific embodiment the ^λ click chemistry' reaction is 1,3 -dipolar cycloaddition reaction between a first functional group on the hydrophilic polysaccharide and a third complementary functional group capable of participating in a 'click chemistry' reaction with the first functional group on the at least one bioactive agent. Methods for introducing functional groups capable of participating in a ^λ click chemistry' reaction are known in the art. Thus in a preferred embodiment the third complementary functional group is an azide group when the first functional group is an alkyne group. In another preferred embodiment the third functional group is an alkyne group when the first functional group is an azide group (see e.g. Figure 1, wherein the R group in may act as a bioactive agent) .

In a further embodiment the copolymer compositions of the invention or the hydrogel of the invention may further comprise at least one additive to modulate their properties (and thereby increasing the efficacy thereof), e.g. by modulating the water content and/or ionic charge thereof and/or by altering the solidity and gelling temperature thereof and/or by modulating the degradation rate and/or release rate thereof as well as by modulating the stability of one or more bioactive agents optionally incorporated therein.

Thus in a further embodiment , the present invention provides a bioactive agent delivery system comprising a hydrogel of the invention and at least one bioactive agent and at least one additive dissolved in an aqueous solvent. These additives may include, but are not limited to, cationic polymers; anionic polymers; sugars; polyols, sugar-containing polyols and polymer-containing polyols, amino acids and sugar- containing amino acids, other bioavailable materials, sugar- containing ions, surfactants, organic solvents, preservatives, etc. More specifically the at least one additive may be selected from the group consisting of: (a) cationic polymers having the molecular weight from 200 to 750,000), such as, poly-L-arginine, poly-L-lysine, poly (ethylene glycol), polyethylenimine , chitosan, protamin, and the like; (b) anionic polymers such as poly (N-vinyl-2 -pyrrolidone) , polyvinylacetate (PVA) , alginate, and the like; (c) bioavailable materials such as amiloride, procainamide, acetyl-beta- methylcholine, spermine, spermidine, lysozyme, fibroin, albumin, collagen, growth factors such as transforming growth factor-beta (TGF-beta) , fibroblast growth factor (bFGF) , vascular endothelial growth factor (VEGF) , and the like, bone morphogenetic proteins (BMPs) , dexamethasonfibronectin, fibrinogen, thrombin, proteins, dexrazoxane, leucovorin, ricinoleic acid, phospholipid, small intestinal submucosa, vitamin E, polyglycerol ester of fatty acid, Labrafil, Labrafil Ml 944CS, citric acid, glutamic acid, hydroxypropyl methylcellulose, gelatin, isopropyl myristate, Eudragit, tego betain, dimyristoylphosphatidylcholine, scleroglucan, and the like; (d) sugars, such as, starch, cyclodextrin and derivatives thereof, lactose, glucose, dextran, mannose, sucrose, trehalose, maltose, ficoll, and the like; (e) polyols, such as, innositol, mannitol, sorbitol, and the like, sugar-containing polyols, such as, sucrose-mannitol, glucose-mannitol, and the like, and polymer-containing polyols, such as, trehalose-PEG, sucrose-PEG, sucrose-dextran, and the like; (f) amino acids (including sugar-containing amino acids), such as, alanine, arginine, glycine, sorbitol- glycine, sucrose-glycine, and the like,- (g) sugar-containing ions, such as, trehalose-ZnS04, maltose-ZnSO4, and the like; and bioacceptable salts, such as, silicate, NaCl, KCl, NaBr, NaI, LiCl, n-BvuNBr, n-Pr4NBr, Et4NBr, Mg (OH) 2, Ca (OH) 2, ZnCO3, Ca3(PO4)2, ZnCl2, (C2H3O2)2Zn, ZnCO3 , CdCl2, HgCl2, CoC12, (CaNOs) 2, BaCl2, MgCl2, PbC12, A1C13, FeC12 , FeC13, NiCl2, AgCl, AuCl3, CuCl2, sodium tetradecyl sulfate, dodecyltrimethylammonium bromide, dodecyltrmethylammonium chloride, tetradecyltrimethylammonium bromide, and the like; organic solvents, such as, cremophor EL, ethanol, dimethyl sulfoxide, and the like; (h) preservatives, such as, methylparaben and the like; and (i) surfactants, such as, poloxamer of various molecular weights, tween 20, tween 80, triton X-IOO, sodium dodecyl sulfate (SDS, Brij) and the like.

The at least one additive may be physically incorporated in the copolymer composition or the hydrogel of the invention, e.g. by dispersion, and may be present from about 0.001 to 30 wt%, preferably about 0.1 to 10 wt% based on the total weight of the copolymer composition or the hydrogel of the invention.

Thus, in a preferred embodiment, the invention is directed to a crosslinked or grafted copolymer composition or a hydrogel of the invention comprising HA and pNIPAM grafted or crosslinked via a * click chemistry' reaction between at least one alkyne group present on the HA and at least one terminal azide group present on the pNIPAM optionally further comprising at least one bioactive agent, wherein the bioactive agent is either dispersed within the copolymer composition or the hydrogel or conjugated to the copolymer composition or the hydrogel via a "click chemistry' reaction between at least one alkyne group present on HA and an azide group present on the bioactive agent and optionally further comprising at least one additive .

In a further aspect, the invention is directed to a method of preparing a copolymer composition of the invention comprising grafting or crosslinking a hydrophilic polysaccharide and a thermoreversible polyamide via a ^x click chemistry' reaction as defined hereinabove.

Thus in a specific embodiment the 'click chemistry reaction' is a pericyclic reaction, preferably a cycloaddition reaction, preferably a 1,3 -dipolar cycloaddition reaction or a Diels- Alder reaction.

A preferred method of the invention comprises the steps of:

(a) providing a hydrophilic polysaccharide having at least one carboxylic acid group which is conjugated to at least one first functional group capable of participating in a * click chemistry' reaction;

(b) providing a thermoreversible polyamide having at least one second complementary functional group capable of participating in a 'click chemistry' reaction with the first functional group ; (c) reacting the at least one first functional group of the hydrophilic polysaccharide with the at least one second complementary functional group of the thermoreversible polyamide via a 'click chemistry' reaction to obtain the copolymer composition; and (d) isolating the copolymer composition.

Further steps may include physically dispersing or covalently linking at least one bioactive agent in the copolymer composition obtained in step d) . More specifically the method may comprise the steps of:

(e) providing at least one bioactive agent having a third complementary functional group capable of participating in a ^λ click chemistry' reaction; and (f) reacting the first functional group of the hydrophilic polysaccharide with the third complementary functional group of the at least one bioactive agent via a * click chemistry' reaction; and

(g) isolating the copolymer composition. Optionally the methods of the invention may further comprise the steps of dispersing at least one additive in the copolymer compositions of the invention (or the hydrogels obtained therefrom) .

Thus in a preferred embodiment the invention provides a method for preparing a copolymer composition of the invention comprising the steps of:

(a) providing a HA having at least one carboxylic acid group which is conjugated to at least one alkyne group capable of participating in a ^λ click chemistry' reaction; (b) providing a pNIPAM having at least one azide group capable of participating in a ^Λ click chemistry' reaction with the alkyne group;

(c) reacting the at least one alkyne group of HA with the at least one azide group of the pNIPAMvia a 'click chemistry' reaction to obtain the copolymer composition; and

(d) isolating the copolymer composition.

In a further aspect the invention also provides a method for preparing a hydrogel comprising dissolving a copolymer composition according to the invention in an aqueous solvent as defined hereinabove . The at least one bioactive agent and optionally the at least one additive may be present in the copolymer composition prior to dissolution in an aqueous solvent to form the hydrogel . Alternatively, the at least one bioactive agent and optionally the at least one additive may be after dissolution of the copolymer composition.

Thus, in one specific embodiment a bioactive agent and optionally an additive are added to a copolymer composition of the invention. The obtained bioactive agent composition may be used immediately by dissolution in an aqueous solution to form a hydrogel, i.e. a bioactive agent delivery system.

Alternatively the obtained bioactive agent composition may be stored, e.g. in a lyophilized state, until further use is required.

In another specific embodiment a bioactive agent and/or an additive are added to a hydrogel of the invention to form a bioactive agent delivery system which may be used immediately. In a further aspect the invention is directed towards further uses of the copolymer compositions and hydrogels of the invention, such as in vitro cell culture, tissue filler, cosmetic, and tissue engineering applications.

Thus in another embodiment, bioactive agents grafted onto a copolymer composition of the invention may be used to investigate the impact of said bioactive agents on cells cultured in vitro.

In another embodiment, the copolymer compositions of the invention (and hydrogels formed thereof) may be used as a cell culture device. Cells may be suspended in the liquid copolymer solution at room temperature and then warmed to create a robust, stable hydrogel for three dimensional cell culture. Retrieval of the cells may be achieved by simply cooling the hydrogel such that it returns to its liquid copolymer solution state .

Due to its thermosensitive nature the hydrogel or bioactive agent delivery system of the invention may be administered to a subject through various routes depending on its final use. These routes of administration may include oral administration, buccal administration, mucosal administration, nasal administration, intraperitoneal administration, hypodermic injection, muscular injection, percutaneous administration, and intratumoral administration, whereby a local administration such as hypodermic injection, muscular injection, or percutaneous administration is preferred.

In one embodiment, a bioactive agent delivery system may be injected as an aqueous solution into a living body (at a temperature below the LCST) . Upon injection the bioactive agent delivery system forms a bioactive agent-containing depot in a gel state at in vivo temperature. Release (or diffusion) of the bioactive agent occurs upon degradation of the copolymer composition or via simple diffusion from the gelled hydrogel depot. In case of a chemically incorporated bioactive agent (i.e. by covalent linkage) the release or diffusion also depends on the degradation of the chemical linkage to the polysaccharide backbone .

Prior to its use as a bioactive agent delivery system, the copolymer composition may be stored in lyophilized form until further use. Thus in a further aspect, the invention provides a single or multi compartment kit comprising a copolymer composition according to the invention in a sterile, lyophilized form. In a specific embodiment, the kit may comprise in separate compartments a defined amount of aqueous solvent for reconstitution of the copolymer composition and/or a defined amount of a bioactive agent and/or a defined amount of an additive.

The following examples are intended to illustrate and not limit the scope of the invention. Other aspects of the invention will be apparent to those skilled in the art to which the invention pertains. Example 1. Synthesis of a thermoreversible hydrogel using HA grafted with pNIPAM

(a) HA modified with alkyne groups (HA-PA) .

HA-PA was prepared using modified procedures already established in the field (WO 2008/031525) : Briefly, 0.3 g of HA sodium salt were dissolved in 30 ml of 100 mM MES buffer, pH=5.6. The following were added to this solution: 0.192 g N- 3,dimethylaminopropyl) -N'ethylcarbodiimide (EDC-HCl 100 mM) , 0.230 g N-hydroxysuccinimide (NHS 200 mM) , and 770 μl propargylamine . The mixture was stirred at room temperature and allowed to react for 24 hours. Following the reaction, the mixture was transferred to dialysis tubing (MWCO= 12-14 kDa) , dialyzed against 0.1 M NaCl in water for 24 hours, and dialyzed against distilled water for an additional 5 days. The solution was then transferred to a 50 ml conical tube, frozen at -80 ⁰C, and lyophilized. The degree of substitution was determined by reacting a portion of the HA-PA with excess sodium azide using copper sulfate (CuSO4) as the catalyst and ascorbic acid as the reducing agent . After dialysis and lyophilization, the material was dissolved in deuterium oxide

(D20) and nuclear magnetic resonance spectroscopy (NMR) was performed. The degree of substitution was determined by comparing the integration of the triazole proton peak (~7.88 ppm) to the integration of the acetyl proton peak (~2.00 ppm) . The degree of substitution varied between 6-28%, depending on the amount of propargylamine, EDC, and NHS used during the reaction.

(b) Polymers of azido terminated N-isopropylacrylamide (N3- pNIPAM) . N3-pNIPAM was prepared according to procedures already established in the field. A chain transfer agent (CTA) consisting of S-1-dodecyl-S' - (α,α' -dimethyl-α" -acetic acid) trithiocarbonate was first prepared according to Lai JT et al , Macromolecules 2002, 35, 6754-6756. This CTA was then modified with an azido group according to Gondi et al (Macromolecules 2007, 40, 474-481) . Finally, N-isopropylacrylamide (NIPAM) was polymerized with this CTA according to Li et al (Macromol . Rapid Commun. 2008, 29, 1172-1176) . Using these established techniques and varying the polymerization time and monomer: CTA ratio, azido terminated pNIPAM polymers were made with Mn ranging between 4-50 kDa.

(c) Grafting of N3-pNIPAM to HA-PA.

The grafting of N3-pNIPAM to HA-PA was performed by dissolving HA-PA in distilled water at 0.5% w/v. In 10 ml of this HA-PA solution was added 0.291 g of N3-pNIPAM with a Mn=IO.3 kDa, corresponding to 25% of all disaccharide subunits being grafted with pNIPAM. A catalyst solution was prepared with 0.3731 g ascorbic acid sodium salt, 0.0470 g CuSO4-5H20, and 2 ml of distilled water. After thorough mixing of both solutions, 200 μl of the catalyst solution was added to the HA-PA and N3 -pNIPAM solution. The reaction was stirred and allowed to proceed overnight, at which point 0.3723 g of ethylenediaminetetraacetic acid (EDTA) was added to chelate copper ions. This solution was then transferred to dialysis tubing (MWCO= 12-14 kDa), dialyzed against 0.1 M NaCl in water for 24 hours, then distilled water for 5 days. The solution was then transferred to a 50 ml conical tube, frozen at -80 ⁰C, and lyophilized to constant weight. The pNIPAM-HA conjugate was then reconstituted with distilled water at 2% w/v. Rheological measurements confirmed a gel point at approximately 36 ⁰C (see Figure 5) .

Example 2. Thermoresponsive Copolymer comprising covalently grafted fluorescein.

A 0.5% w/v solution of HA-PA was prepared as in Example 1. Azido modified fluorescein (0.0047 g) was dissolved in 125 μl of dimethylsulfoxide (DMSO) . To 3 ml of the 0.5% HA-PA solution was added 50 μl of azido fluorescein and 0.45 g of N3 -pNIPAM with a Mn of 29 kDa. Based on these weights, the fluorescein and pNIPAM chains would occupy 10 and 8% of the disaccharide subunits, respectively. The solution was mixed and a catalyst solution consisting of 200 μl water, 0.0047 g CuSO4 , and 0.0373 g ascorbic acid sodium salt was added. The reaction was allowed to proceed and rest overnight, at which time 0.1117 g ethylenediaminetetraacetic (EDTA) was added to chelate copper ions. The solution was then transferred to dialysis tubing (MWCO = 12-14 kDa) and exhaustively dialyzed against distilled water for 5 days. Control materials of native HA (lacking alkyne functionality) and HA-pNIPAM (lacking a fluorescein label) were prepared in parallel. The gels were then imaged using a fluorescent microscope with an excitation of 488 nm. Images obtained in this way revealed that the azido fluorescein was insoluble in the native HA/water solution, resulting in punctuate areas of fluorescence (Figure 6 A) . In contrast, covalent binding of fluorescein to the hydrophilic HA backbone was sufficient to solubilize the fluorescein (Figure 6 B) . The pNIPAM grafted HA without fluorescein does not fluoresce in this wavelength

(Figure 6 C) . The so obtained fluorescein-grafted thermoresponsive copolymer may be used for imaging purposes by itself or in the presence of other bioactive agents coupled to any of the additional active sites on the HA.

Claims

1. A crosslinked or grafted copolymer composition suitable for in situ formation of a hydrogel in aqueous solution comprising a hydrophilic polysaccharide and a thermoreversible polyamide grafted or crosslinked via a 'click chemistry' reaction.

2. A copolymer composition according to claim 1, wherein the

^Λ click chemistry' reaction is a 1,3 -dipolar cycloaddition reaction between at least one first functional group present on the hydrophilic polysaccharide capable of participating in a 'click chemistry' reaction and at least one second complementary functional group present on the thermoreversible polyamide capable of participating in a 'click chemistry' reaction with the first functional group.

3. A copolymer composition according to claim 2 , wherein the first functional group is introduced by reacting a group RG- L-FG, where RG is a carboxylic acid reactive group, L is a linker and F is said first functional group with a carboxylic acid group present on the hydrophilic polysaccharide.

4. A copolymer composition according to claim 3, wherein 0.1- 100%, more preferably 5-70%, more preferably 15-50% of the carboxylic acid groups are conjugated to a group RG-L-FG.

5. A copolymer composition according to anyone of claims 2 to 4, wherein 0.1-100%, preferably 5 to 50 % of the at least one first functional group of the hydrophilic polysaccharide have reacted with the at least one second complementary functional group of the thermoreversible polyamide.

6. A copolymer composition according to anyone of claims 2 to 5, wherein the at least one first functional group is an alkyne group and the at least one second functional group is a terminal azide group, or wherein the at least one first functional group is an azide group and the at least one second functional group is a terminal alkyne group.

7. A copolymer composition according to any preceding claim having a grafting and/or crosslinking density between the polysaccharide and the thermoreversible polyamide of from

0.01 to 100 %, preferably 5 to 50 % more preferably 10 to 30

%.

8. A copolymer composition according to any preceding claim, wherein the hydrophilic polysaccharide is hyaluronic acid.

9. A copolymer composition according to any preceding claim, wherein the thermoreversible polyamide is poly (N- isopropylacrylamide) in linear, branched, or star-shaped form, preferably in linear form.

10. A hydrogel comprising a copolymer composition according to any preceding claim dissolved in at least one aqueous solvent .

11. A hydrogel according to claim 10, wherein the aqueous solvent is selected from the group consisting of water, buffer solution, acid solution, basic solution, salt solution, saline solution, water for injection, and glucose salt solution, and the concentration of the copolymer composition is from 1 to 50 wt %, more preferably from 1 to 15 wt %.

12. A copolymer composition according to anyone of claims 1 to 9 or a hydrogel according to claims 10 or 11, further comprising at least one bioactive agent and optionally at least one additive.

13. A copolymer composition or a hydrogel according to claim 12, wherein the bioactive agent is present in an amount of 0.01 to 50 % based on the total weight of the composition.

14. A copolymer composition or a hydrogel according to claims 12 or 13, wherein the bioactive agent is (i) dispersed within the copolymer composition or (ii) conjugated to the copolymer composition via a 'click chemistry' reaction.

15. A hydrogel according to claims 10 to 14, for use in in vivo drug delivery, in vitro cell culture, tissue filler, cosmetic, and tissue engineering applications.

16. A bioactive agent delivery system comprising a hydrogel according to claims 10 to 15.

17. A method for preparing a crosslinked or grafted copolymer composition suitable for in situ formation of a hydrogel in aqueous solution, the method comprising grafting or crosslinking a hydrophilic polysaccharide and a thermoreversible polyamide via a "click chemistry' reaction.

18. A method according to claim 17, the method comprising the steps of : (a) providing a hydrophilic polysaccharide having at least one carboxylic acid group which is conjugated to at least one first functional group capable of participating in a 'click chemistry' reaction;

(b) providing a thermoreversible polyamide having at least one second complementary functional group capable of participating in a 'click chemistry' reaction with the first functional group;

(c) reacting the at least one first functional group of the hydrophilic polysaccharide with the at least one second complementary functional group of the thermoreversible polyamide via a 'click chemistry' reaction to obtain the copolymer composition; and

(d) isolating the copolymer composition.

19. A method according to claims 17 or 18, further comprising the step of dispersing or covalently linking at least one bioactive agent in the copolymer composition obtained in step d) .

20. A kit comprising a copolymer composition according to claims 1 to 9 in a sterile, lyophilized form.