WO2020240034A1

WO2020240034A1 - Hyaluronic acid-based hybrid hydrogel

Info

Publication number: WO2020240034A1
Application number: PCT/EP2020/065102
Authority: WO
Inventors: Wenxin Wang; Sigen A; Qian Xu
Original assignee: University College Dublin
Priority date: 2019-05-31
Filing date: 2020-05-30
Publication date: 2020-12-03
Also published as: EP3976123A1

Abstract

A hydrogel comprising methacryloyl functionalised hyaluronic acid (HA-MA) and hyperbranched PEG-based multi acrylate (HB-PEG) polymer is described. Further described are methods of making the hydrogel. The hydrogel or a device comprising the hydrogel have applications in tissue engineering and regenerative medicine.

Description

Title of the Invention

Hyaluronic acid-based hybrid hydrogel Field of the Invention

The current invention relates to a hydrogel comprising methacryloyl functionalised hyaluronic acid (HA-MA) and hyperbranched PEG-based multi acrylate (HB-PEG) polymer. The current invention also relates to devices comprises the hydrogel and applications in tissue engineering and regenerative medicine.

Background of the Invention

Hydrogels comprise a three-dimensional network of polymers made up of natural or synthetic materials. Hydrogels were discovered in 1960 by Wichterle and Lim and since that time they have been extensively studied for a wide range of biomedical applications. Under physiological conditions, hydrogels can absorb and retain large amount of water or biological fluids, allow nutrients, gasses, wastes products and bioactive agents to exchange within the hydrogels, and offer delivery functions for drugs or cells, all of which make them ideal for a variety of applications.

Hyaluronic acid (HA) is a non-sulphated glycosaminoglycan (GAG) and an essential component of the extracellular matrix (ECM) of most tissues. It is composed of alternating units of D-glucuronic acid and N-acetyl-D-glucosamine, linked together via alternating b-1 ,4 and b-1 ,3 glycosidic bonds. It is synthesized at the inner wall of the plasma membrane by HA synthase and is extruded to the ECM space without any further modifications. In the ECM of most tissues, the high molecular weight HA (up to several million Daltons), along with other structural macromolecules, contributes to the mechanical integrity of the network. HA regulates many cellular processes through its binding with cell surface receptors. HA can be rapidly degraded in the body by hyaluronidase and reactive oxygen species, with tissue half- lives ranging from minutes in the blood to hours or days in skin and joints.

HA is an attractive building block for the fabrication of matrices for tissue engineering because it is biocompatible, biodegradable, bioactive, non-immunogenic, non-thrombogenic and high reproducibility. In physiological solutions, HA assumes an expanded random coil structure that occupies a very large domain that facilitates solute diffusion.

Naturally existing HA, however, does not have long lasting mechanical integrity, as HA can be degraded easily by hyaluronidase enzymes. Therefore, to provide HA-based hydrogels with tailored mechanical properties and degradation rates, while maintaining their native biological functions, controlled chemical modification and covalent crosslinking are often necessary. By varying the molecular weight of HA raw materials, the degree of modification and the concentration of the reactive HA precursors, hydrogels with varying stiffness, pore size and degradation rate can be readily produced. Over the past few decades, researchers have accumulated significant knowledge on HA as a unique biomacromolecule that is involved in various cell signalling processes, and at the same time, have created a range of HA-based hydrogel materials with increasing complexity and diverse functions.

Methacryloyl HA (HA-MA) is one of the most commonly used chemically modified HA derivatives. HA-MA hydrogels have been extensively studied and used in tissue engineering applications. Generally, methacryloyl group can be conjugated to a HA chain using methacrylic anhydride or glycidyl methacrylate.

A UV crosslinked biodegradable hybrid hydrogel based on methacrylated hyaluronic acid (HA MA) and methacrylated gelatin (GelMA) was developed by G. Eke ( Biomaterials 2017, 129, 188-198). The focus of the study was to design a dermal substitute containing adipose derived stem cells (ADSC) that could be used to improve the regeneration of skin on hard-to-heal wounds. This was achieved by first synthesizing methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (HA-MA) precursors. Hydrogel precursors were then dissolved in media (in 15: 1 ratio), ADSCs added together with the photoinitiator and crosslinked with 40s of UV. The results showed that the hydrogels degraded by 50% over 3 weeks in an in vitro environment. The study concluded that the GelMA/HA-MA hydrogel formed and appeared very promising when combined with ADSCs as an approach to provide a well vascularized dermis to improve the engraftment of tissue engineered skin. However, the substitution degree of GelMA in this hydrogel is limited and the MA content is low. A low substitution degree means that the functional group density is low, resulting in a compromised crosslinking degree and low gel or mechanical stiffness.

A study by M.T. Poldervaart ( PLoS One 2017, 12 (6), 1-15) reported another photocrosslinkable methacrylated hyaluronic acid (MeHA) hydrogel for 3D printing application. This hydrogel was reported to have increased mechanical stiffness, long-term stability, and minimal decrease in cytocompatibility. Visco-elastic properties of MeHA gels, measured by rheology and dynamic mechanical analysis, showed that irradiation of the hydrogels with UV light led to increased storage moduli and elastic moduli, indicating increasing gel rigidity. Subsequently, human bone marrow derived mesenchymal stromal cells (MSCs) were incorporated into MeHA hydrogels, and cell viability remained 64.4% after 21 days of culture. However, owing to the poor solubility of MeHA, the hydrogel strength is low. Hyperbranched PEG-based multi acrylate (HB-PEG) polymers synthesis was reported by Wang’s group (Sigen et ai, “Hyperbranched PEG-based multi-NHS polymer and bioconjugation with BSA”, Polym Chem, 2017, 8, 1283-1287; Zhao et ai,“Controlled multi vinyl monomer homopolymerization through vinyl oligomer combination as a universal approach to hyperbranched architectures”, Nature Communications, 2013,4, 1873; Zheng et ai,“Controlled homopolymerization of multi-vinyl monomers: dendritic polymers synthesized via an optimized ATRA reaction”, Chemical Communications, 2013,49, 10124) by controlled/living radical polymerization of poly(ethylene glycol) diacrylate (PEGDA) via deactivation enhanced atom transfer radical polymerization (DE-ATRP). By introducing a high initiator-to-monomer ratio, the obtained HB-PEG polymer is composed of extremely short carbon-carbon backbones interconnected together by the long PEG chains as well as pendent photocrosslinkable acrylate moieties. This work indicated that HPEGDA materials are capable of bonding effectively to soft tissue after rapid crosslinking upon UV cure under physiological conditions which allows them to be used as adhesive tissue engineered matrixes, wound dressings, and sealants.

Sun Wenxu et al. ,“Strong dual-crosslinked hydrogels for ultrasound-triggered drug delivery”, Nano Research, Tsinghua University Press, CN, vol.12 no. 1 , 2018, discloses a gel based on methacrylic hyaluronic acid mixed with four-armed polyethylene glycol acrylate (4-arm-PEG- Aclt). This type of polymer is termed a star-shaped PEG polymer in the art, having a confirmed core structure and four arms radiating from the core. This is not a highly branched, i.e. “hyperbranched”, structure. This type of polymer is synthesized with at least three reaction steps and have a low functional group density. Its complex and sophisticated synthetic methods, as well as high costs, have significantly impeded its wide application.

Previously reported hyaluronic acid hydrogels of the prior art, such as those outlined above, can be regulated only by changing the substitution degree and concentration of HA, resulting in a limited adjustable property. Mixing with another crosslinking polymer can broaden the controlled behaviour of the mechanical property. However, there is a need to provide a biocompatible hydrogel with adjustable properties that can be easily tailored.

The current invention serves to alleviate the problems associated with prior art hydrogels.

Summary of the Invention

The current invention provides the combination of methacryloyl functionalised hyaluronic acid (HA-MA) and hyperbranched PEG-based (HB-PEG) polymer, preferably hyperbranched PEG- based multi-acrylate polymer, for fabrication of a biocompatible hydrogel. The hydrogel of the invention overcomes the drawbacks of prior art hydrogels. For example, the current hydrogel overcomes the low solubility of naturally occurring HA polymer and low cell adhesion ability of synthetic PEG polymer. It has increased mechanical, or gel, stiffness compared to prior art hydrogels. It also has increased biocompatibility which cannot be achieved by PEG only hydrogels.

The solubility and crosslinking point of HB-PEG of the hydrogel of the current invention is higher than GelMA of the prior art. As a result, a higher crosslinking density can be achieved by the hydrogel of the current invention. This results in a higher gel strength and longer degradation time. The HB-PEG of the hydrogel of the current invention also provides more reactive functional groups, such as acrylate groups. Its high solubility allows up to about 40% by weight of the HB-PEG in the hydrogel. This in turn allows the hydrogel to be more easily tailored or“tunable”. Furthermore, the HB-PEG of the current invention is easier to synthesize, using a one-step reaction rather than a three-step reaction of some prior art PEG based polymers. The gelation time of a hydrogel made using the HB-PEG of the current invention is very short.

The polymer concentration, storage modulus, mechanical properties, pore size, swelling and degradation ratios can all be regulated on demand and in a controlled manner by changing the ratio of HA-MA and HB-PEG polymers and/or by altering the degree of substitution of the HA-MA. One aspect of the invention provides a hydrogel comprising crosslinked methacryloyl functionalised hyaluronic acid (HA-MA) and hyper-branched poly(ethylene)glycol based multi functional group (HB-PEG) polymer.

In an embodiment of the invention the hydrogel comprises crosslinked methacryloyl functionalised hyaluronic acid (HA-MA) and hyper-branched poly(ethylene)glycol based multi acrylate (HB-PEG) polymer.

In a preferred embodiment, the HA-MA is hydrazide-modified methacrylated hyaluronic acid (HA-MA-HDZ).

In an embodiment, HA-MA-HDZ has the following structure.

HA-MA

Typically, the HA-MA has a tailored substitution degree (SD). In one embodiment, the degree of substitution (SD) of the HA-MA is at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or about 90%. In one embodiment, the degree of substitution (SD) of the hydrazide-modified HA-MA is from about 10%, 15%, 20%, 25% to about 90%. In one embodiment, the degree of substitution (SD) of the hydrazide-modified HA-MA is from about 30% to about 90%. In one embodiment, the degree of substitution (SD) of the hydrazide-modified HA-MA is from about 40% to about 90%. In one embodiment, the degree of substitution (SD) of the hydrazide-modified HA-MA is from about 50% to about 90%. In one embodiment, the degree of substitution (SD) of the hydrazide- modified HA-MA is from about 60% to about 90%.

In one embodiment, the hydrogel has a HB-PEG concentration of from about 10% to about 40% (w/v), typically about 20% (w/v). In one embodiment, the hydrogel has a HA-MA concentration of from about 0.5% to about 4% (w/v), typically about 2%.

In an embodiment, the HA-MA may be anhydride modified methacrylic HA (HA-AMA), or a glycidyl methacrylate HA (HAGM).

In an embodiment, the glycidyl methacrylate HA (HAGM) has the following structure:

The structure of HAGM.

In an embodiment, anhydride modified methacrylic HA has the following structure.

(Biomacromolecules 2006, 7, 1302-1310) In an embodiment, HB-PEG comprises at least 12 functional groups in the terminal chain of the polymer structure. The HB-PEG may comprise from about 12 to about 30 functional groups in the terminal chain of the polymer structure. In an embodiment, there are from about 8 to about 100 functional groups in the terminal chain of the polymer structure. In an embodiment, there are from about 20 to 30 functional groups in the terminal chain of the polymer structure. In one embodiment, the HB-PEG comprises 12 to 100, 12 to 70, 12 to 50, or 12 to 30 functional groups.

The functional groups may be selected from the group comprising an acrylate group and a methacrylate group.

In a preferred embodiment, HB-PEG comprises from about 12 to about 30 acrylate groups in the terminal chain of the polymer structure. In an embodiment, there are from about 8 to about 100 acrylate groups in the terminal chain of the polymer structure. In an embodiment, there are from about 20 to 30 acrylate groups in the terminal chain of the polymer structure.

In one embodiment, the HB-PEG comprises 12 to 100, 12 to 70, 12 to 50, or 12 to 30 acrylate groups. In an embodiment, HB-PEG has the following structure:

In one embodiment, the HB-PEG is obtainable by controlled/living radical polymerization of poly(ethylene glycol) via deactivation enhanced atom transfer radical polymerization (DE- ATRP).

In one embodiment, the HB-PEG is obtainable by controlled/living radical polymerization of poly(ethylene glycol) diacrylate (PEGDA) via deactivation enhanced atom transfer radical 5 polymerization (DE-ATRP).

In one embodiment, the HBPEG polymer is composed of extremely short carbon-carbon backbones interconnected together by the long PEG chains as well as pendent photo crosslinkable functional moieties, such as acrylate moieties.

In one aspect, the current invention provides a composition comprising a hydrogel of the invention. In an embodiment, a pharmaceutically or biologically active agent is contained within a matrix of the hydrogel. An aspect of the invention provides a curable composition comprising methacryloyl functionalised hyaluronic acid (HA-MA) and hyper-branched poly(ethylene)glycol based (HB- PEG) polymer. The HA-MA and HB-PEG may be as described herein.

In an embodiment, the curable composition comprises methacryloyl functionalised hyaluronic acid (HA-MA) and hyper-branched poly(ethylene)glycol based multi-acrylate (HB-PEG) polymer.

One aspect of the invention provides use of the hydrogel for tissue engineering applications, regenerative medicine applications and methods, cell-based technologies, 3D cell and drug delivery and 3D bio-printing. The invention also provides use of the hydrogel as a hygiene product, contact lens(es), tissue engineering scaffold, a biomedical implant, a tissue adhesive material, a sealant material, a drug delivery system, bioadhesive and wound dressings.

One aspect of the invention provides a medical device comprising the hydrogel of the invention. The device may be a scaffold or an implant. The device may be an implantable device.

Typically, the hydrogel is UV-crosslinked.

One aspect of the invention provides a method of tailoring the physical properties of a hydrogel. In another aspect, the invention provides a method of making a hydrogel, comprising the steps of: providing an aqueous solution of a methacryloyl functionalised hyaluronic acid and a hyperbranched PEG-based (HB-PEG) polymer and crosslinking the aqueous solution of polymer to obtain the hydrogel.

The method involves providing a methacryloyl functionalised hyaluronic acid and a hyperbranched PEG-based (HB-PEG) polymer, mixing the polymer with water to provide an aqueous solution of polymer and crosslinking the aqueous solution of polymer to obtain the hydrogel.

The HA-MA and HB-PEG may be any as described herein.

The invention provides a method of making a hydrogel, comprising the steps of: providing a methacryloyl functionalised hyaluronic acid and a hyperbranched PEG- based multi acrylate (HB-PEG) polymer mixing the polymers with water to provide an aqueous solution of polymer; and crosslinking the aqueous solution of polymer to obtain the hydrogel.

In one embodiment, the polymer is UV-crosslinked. Definitions

Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

As used herein, the term "comprise," or variations thereof such as "comprises" or "comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open-ended and does not exclude additional, unrecited integers or method/process steps.

As used herein, the term“disease” is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition or syndrome in which physiological function is impaired irrespective of the nature of the aetiology (or indeed whether the aetiological basis for the disease is established). It therefore encompasses conditions arising from infection, trauma, injury, surgery, radiological ablation, poisoning or nutritional deficiencies.

As used herein, the term "treatment" or "treating" refers to an intervention (e.g. the administration of an agent to a subject) which cures, ameliorates or lessens the symptoms of a disease or removes (or lessens the impact of) its cause(s) (for example, a decrease in fibrotic area, an increase muscle volume fraction in ischaemic zones, an improvement in preservation of small vessels, or a decreased pro-inflammatory response in the infarct zone). In this case, the term is used synonymously with the term“therapy”.

Additionally, the terms "treatment" or "treating" refers to an intervention (e.g. the administration of an agent to a subject) which prevents or delays the onset or progression of a disease or reduces (or eradicates) its incidence within a treated population. In this case, the term treatment is used synonymously with the term“prophylaxis”. As used herein, an“effective amount” or a“therapeutically effective amount” of an agent defines an amount that can be administered to a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio, but one that is sufficient to provide the desired effect, e.g. a clinically significant repair of cardiac muscle evidenced by at least one or more of a clinically relevant decrease in fibrotic area, an increase muscle volume fraction in ischaemic zones, an improvement in preservation of small vessels, or a decreased pro-inflammatory response in the infarct zone, and typically within 10, 20 or 30 days of administration. The amount will vary from subject to subject, depending on the age and general condition of the individual, mode of administration and other factors. Thus, while it is not possible to specify an exact effective amount, those skilled in the art will be able to determine an appropriate "effective" amount in any individual case using routine experimentation and background general knowledge. A therapeutic result in this context includes eradication or lessening of symptoms, reduced pain or discomfort, prolonged survival, improved mobility and other markers of clinical improvement. A therapeutic result need not be a complete cure.

In the context of treatment and effective amounts as defined above, the term subject (which is to be read to include "individual", "animal", "patient" or "mammal" where context permits) defines any subject, particularly a mammalian subject, for whom treatment is indicated. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; and rodents such as mice, rats, hamsters and guinea pigs. In preferred embodiments, the subject is a human.

The term“tissue engineering” when used herein is the use of cells, engineering methods and biochemical and physiochemical factors to improve, repair or replace biological tissue, for instance, a tissue scaffold.

The term “methacryloyl hydrazide” refers to the reagent shown in Scheme 4 and has a chemical formula of NH₂-NH-C(0)-C(CH₂)-CH₃.

The term“molar equivalent” refers to the amount of a first substance that reacts with an arbitrary amount of a second substance in a chemical reaction. For example, reacting 2.0 molar equivalents of methacryloyl hydrazide with activated hyaluronic acid means reacting 2 moles of methacryloyl hydrazide with 1 mole of hyaluronic acid. The term“degree of substitution” or“SD%” as applied to a polymer refers tothe number of vinyl-hydrazide groups per 100 monomer units on a polymer chain. In the case of methacrylated HA, it refers to the number of methacryloyl groups per 100 disaccharide units on HA chain.

The term “hyaluronan” or “hyaluronic acid” or “HA” refers to the anionic non-sulphated glycosaminoglycan that forms part of the extracellular matrix in humans and consists of a repeating disaccharide 4)^-d-GlcpA-(1 3)^-d-GlcpNAc-(1 . Hyaluronan is the conjugate base of hyaluronic acid, however the two terms are used interchangeably. When a salt of hyaluronic acid is employed, the sale is generally a sodium salt, although the salt may be employed such a calcium or potassium salts. The hyaluronic acid or hyaluronan may be obtained from any source, including bacterial sources. Hyaluronic acid sodium salt from Streptococcus equi is sold by Sigma-Aldrich under the product reference 53747-1 G and 53747-10G. Microbial production of hyaluronic acid is described in Liu et al (Microb Cell Fact. 201 1 ; 10:99). The term also includes derivatives of HA, for example HA derivatised with cationic groups as disclosed in US2009/0281056 and US2010/0197904, and other types of functionalised derivatives, such as the derivatives disclosed in Menaa et al (J. Biotechnol Biomaterial S3:001 (201 1)), Schante et al (Carbohydrate Polymers 85 (201 1)), EP0138572, EP0216453, EP1095064, EP0702699, EP0341745, EP1313772 and EP1339753.

As used herein, the term “hydrazide-modified methacryloyl functionalised hyaluronic acid” refers to hyaluronic acid that has been functionalised with methacryloyl hydrazide or a derivative thereof. An example is provided in Scheme 4, in which hyaluronic acid and in which the degree of substitution is 50% as an example.

As used herein, the term“methacryloyl functionalised hyaluronic acid” refers to a hyaluronic acid that has been functionalised with methacryloyl or a methacryloyl derivative.

As used herein, the term“anhydride modified methacrylic hyaluronic acid (HA-AMA)” refers to a hyaluronic that has been functionalised with methacrylic anhydride or a derivative thereof. An example is provided in Scheme 3.

As used herein, the term“glycidyl methacrylate hyaluronic acid (HAGM)” refers to a hyaluronic that has been functionalised with glycidyl methacrylate or a derivative thereof. An example is provided in Scheme 1.

As used herein, the term “hyperbranched PEG-based multi functional group (HB-PEG) polymer” refers to a highly branched PEG based polymer with multi functional groups in the chain end. (Qian Xu.,“A hybrid injectable hydrogel from hyperbranched PEG macromer as a stem cell delivery and retention platform for diabetic wound healing”, Acta Biomaterialia 75 (2018) 63-74). The functional groups may be any suitable functional group and such functional groups are known the art.

An example of a HB-PEG with methacrylate groups is in the below scheme (Scheme 6):

As used herein, the term“hyperbranched PEG-based multi acrylate (HB-PEG) polymer” refers to a highly branched PEG based polymer with multi acrylate groups in the chain end (Acta Biomaterialia 75 (2018) 63-74). An example is provided in Scheme 2 below.

Scheme 2: The term“hydrogel” refers to means a three-dimensional network of hyaluronan polymers in a water dispersion medium. In one embodiment, the hyaluronan polymers are crosslinked to form the three-dimensional network. In one embodiment, the network is formed with a homopolymer.

As used herein, the term“cross-linked” as applied to the polymers of the hydrogel of the invention means that the polymer chains are cross-linked to form a three-dimensional network. Crosslinking can be achieved by thermal treatment, irradiation (i.e. UV-curing), or using a crosslinking agent. Cross-linked HA hydrogels are described in the literature, for example in Kenne et al (Carbohydrate Polymers, Vol. 91 , Issue 1 (2011)), Segura et al (Biomaterials, Vol. 26, Issue 4 (2005)), Yeom et al (Bioconjugate Chem, Vol. 21 (2) 2010), US8124120, and US6013679. The term“cross-linking agent” generally means a molecule containing two or more functional groups that can react with HA. Examples of cross-linking agents include ethylene glycol crosslinking agents, including functionalised polyethylene glycol (PEG), for example PEG-amine and PEG diglycidylether (EX810), 1-ethyl-3-(3-dimethylaminopropyl) carboimide (EDC), divinyl sulfone (DVS) and ethylene glycol diacrylates and dimethacrylates, derivatives of methylenebisacrylamide (Sigma-Aldrich). Methods of crosslinking HMW HA with PEG-amine are described below and in Isa et al. (Biomacromolecules 2015, 16, 1714-1725).

The term“UV-curing” refers to crosslinking a polymer by exposing the polymer to UV radiation. The process is described in detail in Biomacromolecules 2012, 13, 1818-1827 or Sigen A, et al., Chemical Communications, 2018ln one embodiment, the UV crosslinked hydrogel is prepared as follows: Photo initiator 2959 was dissolved in Dl water with a concentration of 0.5% (w/v). HA-A was dissolved in the prepared solution (2%, 4%, and 6% w/v). The hydrogel was crosslinked by 5 min of UV irradiation (365 nm).

As used herein the term“pharmaceutically or biologically active agent” refers generally to an agent or component that has a pharmaceutical or biological effect in a mammal. Examples include cells, cell components, polysaccharides, proteins, peptides, polypeptides, antigen, antibody (monoclonal or polyclonal), antibody fragment s(for example an Fc region, a Fab region, a single domain antibody such as a nanobody or VHV fragment), a conjugate of an antibody (or antibody fragment) and a binding partner such as a protein or peptide, a nucleic acid (including genes, gene constructs, DNA sequence, RNA sequence, miRNA, shRNA, siRNA, anti-sense nucleic acid), cellular products such as growth factors (i.e. EGF, HGF, IGF- 1 , IGF-2, FGF, GDNF, TGF-alpha, TGF-beta, TNF-alpha, VEGF, PDGF and an interleukin), drugs, for example, a drug to relieve pain such as non-steroidal anti-inflammatory drug (such as Ibuprofen, Ketoprofen or Naproxen), aspirin, acetaminophen, codeine, hydrocodone, an anti-inflammatory agent such as a steroidal anti-inflammatory agent, an anti-depressant, an anti-histamine, or an analgesic. The cell may be autologous, allogenic, xenogenic. The cell may be a stem cell. The stem cell may be selected from the group comprising a side population, embryonic, germinal, endothelial, hematopoietic, myoblast, placental, cord-blood, adipocyte and mesenchymal stem cells. The cells may be engineered to express a biological product, for example a therapeutic biological product such as a growth factor.

Other aspects and preferred embodiments of the invention are defined and described in the other claims set out below.

Brief Description of the Figures

The invention will be described with reference to the Figures wherein:

Figure 1 Rheometric analysis in a time sweep mode of HB-PEG-10%, HB-PEG-15%, HB- PEG-20% & HB-PEG-40% with HA and HA-MA-0.5%, 1%, 2% & 4% to examine gelation kinetics of HB-PEG-HA-MA hydrogels.

Figure 2 Represents swelling and degradation of polymer concentration, (i) HB-PEG-10%- HA-MA, (ii) HB-PEG-15%-HA-MA, (iii) HB-PEG-40%-HA-MA and (iv) HB-PEG-20%-HA-MA (describing clockwise).

Figure 3 Comparative displays pore size of hydrogels.

Figure 4 Quantitative cell viability evaluation by MTT assay using fibroblast-3T3 cells.

Figure 5 (A) 1 H-NMR results of HA, MH, HA-MA-Hs and (B) 1 H-NMR results of HA-MA- GMA25% and HA-MA-H26%.

Detailed Description of the Invention

All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.

The current invention provides a hydrogel comprising crosslinked methacryloyl functionalised hyaluronic acid (HA-MA) and hyper-branched poly(ethylene)glycol based, e.g. multi acrylate, (HB-PEG) polymer. Crosslinking can be achieved by any suitable means known in the art, for example including but not limited to, thermal treatment, UV-curing or using a crosslinking agent. Preferably, crosslinking is UV-curing. The crosslinking may occur in the presence of thiol-crosslinker. The polymers can be crosslinked rapidly to form a biocompatible hydrogel. The properties of the hydrogel of the invention formed from these two polymers by crosslinking can be tuned (tailored) or modified by altering the amount, and properties of the component polymers. These properties include storage modules, pore size, mechanical properties, swelling and degradation, compress modules, Young’s modules etc. This has not been achieved with hydrogels of the prior art. Naturally, it will be appreciated that the preferred or desired physical properties of the hydrogel of the invention will depend on the intended use of the hydrogel.

It will be appreciated that the hydrogel of the invention can comprise any suitable HA-MA known in the art. Examples include but are not limited to anhydride modified methacrylic HA (HA-AMA), and glycidyl methacrylate HA (HAGM).

Scheme 1 below is methacrylation of HA with GM to yield photopolymerizable HAGM by a competition reaction between ring opening and transesterification (Biomaterials 29, 2008).

Scheme 1 : An example of the reaction between HA and methacrylic anhydride (AMA) to provide HA-AMA derivative is shown in Scheme 3.

Scheme 3: (Biomacromolecules 2006, 7, 1302-1310)

Generally, methacryloyl group can be conjugated to a HA chain using methacrylic anhydride or glycidyl methacrylate. Methods are known in the art and any HA-MA produced by said methods may be used.

Rachel Auzely-Velty et al. reported methacrylate conjugation method using a mix solution of H2O and dimethylformamide (DMF) for dissolving HA and methacrylic anhydride. Similarly, Spencer L. Fenn and Rachael A. Oldinski utilized an ion exchange resin and a mixed solution of H2O and dimethyl sulfoxide to increase the solubility of HA and methacrylic anhydride. As for glycidyl methacrylate method, the methacrylate group is conjugated to HA chain through two competing reactions, namely, reversible transesterification and irreversible ring opening. High SD (90%) can be achieved by using a high amount of glycidyl methacrylate (100 eq.) and a mixed solvent (H2O/DMF). In one embodiment, the HA-MA is hydrazide-modified methacrylated hyaluronic acid (HA-MA- HDZ).

The current inventors have identified that synthesis of HA-MA-HDZ does not require a high amount of modification reagents or any organic solvent. As a result, HA-MA-HDZ may significantly minimizes the risk of hazardous residue in the hydrogel of the invention and increases the biocompatibility of the hydrogel. HA-MA-HDZ can also be crosslinked by UV irradiation to form the hydrogel in less than 30s. Also, the resulting hydrogels hold a broad visco-elastic region. By adjusting the SD as well as the concentration of the new HA-MA, the gel strength can be regulated from 250 Pa to over 30 kPa.

A tuneable degradation rate of the hydrogel (e.g. from days to a month) was also achieved by changing the SDs of the HA-MA. The hydrogel produced exhibits a good biocompatibility.

In one approach to the synthesis of a methacrylicated hyaluronic acid (HA-MA), hydrazide groups can be conjugated with carboxyl group in HA at a very high efficiency with N-(3- dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDCI) as the activating agent. Hence, HA-MA can be synthesized in an environmentally friendly manner using a water soluble modification agent, methacryloyl hydrazide. Thus, a toxic organic solvent for reagent mixing can be omitted and the high amount of modification agent can also be reduced significantly. The approach offers a more facile strategy to tailor the substitution degree of methacryloyl group comparing with the conventional method of HA-MA synthesis. As an example, the synthesis of hydrazide-modified methacryloyl functionalised hyaluronic acid is shown in Scheme 4 below.

Scheme 4:

Typically, the HA-MA has a tailored substitution degree (SD). In one embodiment, the degree of substitution (SD) of the HA-MA is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or about 90%. HB-PEGs may be one synthesized by a controlled/living radical polymerization approach. This is a one-step reaction. It may be by any known method of controlled/living radical polymerization of PEG, e.g. PEGDA, including but not limited to deactivation enhanced atom transfer radical polymerization (DE-ATRP), reversible addition fragmentation chain transfer polymerisation (RAFT) and stable free radical polymerisation (SFRP), e.g. nitroxide mediated polymerisation (NMP).

An example scheme, Scheme 5, via RAFT and resulting polymer is provided below:

HB-PEG with acrylate groups are described in Qian Xu.,“A hybrid injectable hydrogel from hyperbranched PEG macromer as a stem cell delivery and retention platform for diabetic wound healing”, (Acta Biomaterialia 75 (2018) 63-74).

The HB-PEG is a polymer of interconnected PEG chains with functional group moieties at the end of each chain. The HB-PEG contains at least 12 functional group moieties in each macromolecule. It does not have a single central core in contrast to the star-shaped PEG polymers of the prior art. The HB-PEG is a highly branched three-dimensional (3D) structure. The HB-PEG polymer possesses a high amount of functional groups in the terminal chain of the polymer structure. The functional group may be an acrylate group or a methacrylate group. The hyperbranched multi-acrylated poly(ethylene glycol) (HB-PEG) polymer possesses a high amount of acrylate groups in the terminal chain of the polymer structure. Advantageously this makes the gelation time of this type of polymer very short with thiolated biopolymers (< 10 mins) or exposed to UV light (< 1 min).

In an embodiment, HB-PEG has the following structure:

The example HB-PEG polymer from RAFT polymerization is synthesized using PEGDA575 with a final Mw of 20 kDa. There are about 17 acrylate groups in each macromer. This is the polymer used in the examples described herein.

The acrylate groups and high solubility of the HB-PEG used in the hydrogel of the current invention differentiates it from UV-curable PEG biopolymers of the prior art. Other PEG biopolymers contain less than 8, or equal to 8, acrylate groups in each polymer chain. The HB-PEG of the current invention is multi-branched or armed, e.g. has between 12 to 100 arms with functional groups as disclosed herein. Preferably, the HB-PEG polymer of the hydrogel of the current invention comprises between 12 and 30 acrylate groups.

HB-PEG of the current invention is water miscible. The polymer can be dissolved with more than 60% w/v, typically between 60% and 95% (w/v), between 60% and 80% (w/v), between 70% and 80% (w/v), it may be more than 70%, or 80% (w/v). It can form a thick solution.

In an embodiment, HB-PEG comprises at least 12 functional groups. In an embodiment, the HB-PEG comprises from about 12 to 200 functional groups, from about 12 to about 150 functional groups, from about 12 to about 75 functional groups, from about 12 to about 50 functional groups, from about 15 to about 50 functional groups or from about 20 to about 30 functional groups.

The functional group may be an acrylate group. The acrylate group may be methacrylate.

In a preferred embodiment, HB-PEG comprises from about 8 about 100 acrylate groups in the terminal chain of the polymer structure, typically, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 acrylate groups. In an embodiment, there are from about 12 to about 30 acrylate groups in the terminal chain of the polymer structure. In an embodiment, there are from about 20 to 30 acrylate groups in the terminal chain of the polymer structure, typically, 21 , 22, 23, 24, 25, 26, 27, 28 or 29 acrylate groups. Altering the amount of acrylate groups will change a series of hydrogel properties such as the crosslinking degree, gel strength, degradation rate, swelling ratio.

The high solubility enables the use of a very high concentration of HB-PEG polymers (up to 40% w/v) in the pre-gel solution. Therefore, the stiffness of HA-MA and HB-PEG hydrogel of the invention is able to reach the range of from 10² to 10⁸ Pa, for example from 10⁴ to 10⁵ Pa. This type of hydrogel, 10⁴ to 10⁵ Pa, is very suitable for bone tissue regeneration application. This gel strength cannot be achieved by using other biomaterials such as gelatin owing to the low solubility (less than 20% w/v) and the low functional group density.

In one embodiment, the hydrogel has a HB-PEG concentration of from about 5% to about 40% (w/v), typically, from about 10% to about 30%, from about 15% to about 25%, typically, about 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36% or 38% (w/v), or any combination or range thereof.

In one embodiment, the hydrogel has a HA-MA concentration of from about 0.1 % to about 6% (w/v), from about 0.5% to about 5%, from about 1 % to about 4%, from about 2% to about 3%, typically about, 0.1 %, 0.2%, 0.5%, 0.8%, 1 %, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, or 6% (w/v), or any combination or range thereof.

The hydrogel may have a concentration of HA-MA and HB-PEG of any combination of the above disclosed concentrations.

For example, the hydrogel of the invention may have a HB-PEG concentration of about 10% (w/v) and a HA-MA concentration of about 4% (w/v).

For example, the hydrogel of the invention may have a HB-PEG concentration of about 10% (w/v) and a HA-MA concentration of about 2% (w/v).

The HB-PEG concentration may be about 10%, 15% or 20% and the HA-MA concentration may be about 0.5%, 1 %, or 2%, or any combination thereof. The HB-PEG concentration may be about 10%, 15% or 20% and the HA-MA concentration may be about 3%, or any combination thereof. The HB-PEG concentration may be about 30% or 40% and the HA-MA concentration may be about 0.5%, 1 %, or 2%, or any combination thereof. The HB-PEG concentration may be about 30% or 40% and the HA-MA concentration may be about 3%, or any combination thereof.

It will be understood that the concentrations provided depend on the end use or intention of the hydrogel of the invention. One merit of the hybrid hydrogel of the invention is the broad gel strength. For soft tissue regeneration applications, the hydrogel may have a HA-MA concentration of less than 1 % and HB-PEG concentration of less than 15% (w/v). This would be suitable for hydrogel strength around 10³ Pa. For cartilage tissue regeneration applications, the hydrogel of the invention may have a HA-MA concentration from about 1-2%, or 2.5% and HB-PEG concentration from about 15-20% (w/v). This would be suitable for hydrogel strength around 10⁴ Pa.

For bone tissue regeneration, a HA-MA concentration of greater than 3% would be suitable.

The hydrogel of the invention is promising for biomedical engineering and will facilitate the commercial scale process.

Typically, the hydrogel is UV-crosslinked.

The hydrogel of the invention may be in the form of a plurality of microgels.

The hydrogel of the invention may be any defined size or shape.

In one aspect, the current invention provides a composition comprising a hydrogel of the invention. In an embodiment, a pharmaceutically or biologically active agent is contained within a matrix of the hydrogel. The active agent may be a cell or other biological material, a pharmaceutical (drug), or an imaging dye. In one embodiment, the agent is a cell or a plurality of cells.

The composition may be injectable.

In another aspect, the invention provides a method of making a hydrogel, comprising the steps of: providing a methacryloyl functionalised hyaluronic acid and a hyperbranched PEG- based multi acrylate (HB-PEG) polymer mixing the polymers with water to provide an aqueous solution of polymer; and crosslinking the aqueous solution of polymer to obtain the hydrogel.

In one embodiment, the polymer is UV-crosslinked. In one embodiment, the crosslinking step comprises exposing the polymer to UV light for less than 5, 4, 3, 2, or 1 minutes.

A hydrogel produced by the method of the invention is also provided.

Also provided by the current invention is a hydrogel for tissue engineering applications, regenerative medicine applications, cell-based technologies, 3D cell and drug delivery and 3D bio-printing. Regeneration may be tissue regeneration, such as bone tissue regeneration. It will be appreciated that any tissue regeneration is envisaged. The invention provides the use of the hydrogel for cartilage repair.

The invention also provides use of the hydrogel as a hygiene product, contact lens, tissue engineering scaffold, a biomedical implant, a tissue adhesive material, a sealant material, a drug delivery system, bioadhesive and wound dressings.

One aspect of the invention provides a method of tailoring the physical properties of a hydrogel. The physical properties of the hydrogel may be tailored by selecting or providing a particular or desired concentration of HA-MA in the hydrogel of the invention, and/or by providing a particular or desired concentration of HB-PEG in the hydrogel of the invention, and/or by providing a HA-MA with a particular or desired SD, and/or by selecting or providing a HB-PEG with a particular or desired amount of acrylate groups. Changing or selecting these parameters when preparing the hydrogel of the invention provides the hydrogel with the desired properties. Naturally, it will be appreciated that the properties will depend on the intended use of the hydrogel of the invention. Any concentration disclosed herein may be used in the method of tailoring the physical properties of the hydrogel.

Figure 1 of the current application provides gel strength achieved by different concentrations of HB-PEG and HA-MA polymer.

The concentrations may be any concentrations disclosed herein or combinations thereof.

The hydrogel may be made using the method of the invention.

Also provided as an aspect of the invention is a HB-PEG with methacrylate groups, as described herein. In an embodiment, the HB-PEG is as disclosed in scheme 6.

The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention. EXAMPLES

Example 1

Method of preparing the hydrogel of the invention UV crosslinked hydrogel:

Photo initiator 2959 was dissolved in Dl water with a concentration of 0.5% (w/v). HA-MA was dissolved in the prepared initiator solution (0.5%, 1 %, 2%, and 4% w/v). HB-PEG was dissolved in the prepared initiator solution (10%, 15%, 20%, and 40%). The pregel solution was prepared by mixing the two solution at a 1 : 1 volume ratio. The hydrogel was crosslinked by 1 min of UV irradiation (365 nm).

Example 2

Time Sweep Test Methodology

The purpose of this study was to determine hydrogel modulus and gelation time.

Rheological characterizations of the materials were performed using an HR-2 Advanced Rheometer (TA Instruments) with an aluminium parallel plate geometry of 8 mm diameter and UV-visible light attachment connected to an Omnicure S1000, consisting of 100 Watt Mercury Arc light source with 365 nm bandpass filter.

Time sweep of HB-PEG and HA-MA was is measured at a frequency of 1.0 Hz and a strain of 1.0% for 120 seconds. UV light was directed on the polymer solution after 30 seconds of putting the solution on the plate.

Results

Figure 1 represents time sweep data of all the polymer concentrations and comparison between the storage modulus (G’) (also called stiffness of hydrogel) of monomer HA and modified HA when cross-linked with HB-PEG.

In Figure 1 , HB-PEG 10% and 15% groups cross-linking with HA did not show much increase in the G’, whereas when crosslinking with HA-MA, significant increase in G’ was seen and the highest concentrated group showed the biggest of G’. With further increase of the concentration, HB-PEG-20% and HB-PEG-40% (both HA and HA-MA groups) showed significant increase in G’. Conclusion

The increased stiffness when crosslinked with HA-MA indicated the HA chains are chemically incorporated into the 3D network, increasing the crosslinking degree.

Example 3 Swelling and Degradation study Methodology

To examine the swelling behaviour, various concentration hydrogels prepared were soaked, respectively, in PBS water at 37 °C until swelling equilibrium had been reached. The hydrogels were then taken out and weighed after the removal of excess water on their surfaces with filter papers. The dried hydrogel was weighed, and the swelling was calculated based on the following equation:

where Wo is the weight of the hydrogel after swelling and W is the weight of the dried hydrogel. All the experiments were done in duplicates and data represents average of two sets. Measurements were taken at day 1 , 7, 14, 21 and 28 respectively.

Results

Swelling and degradation behaviour of UV crosslinked hydrogels is shown in Figure 2.

For HB-PEG-10% group, HA-MA 0.5%, 1% and 2% concentration hydrogels showed the same trend. Mass of the hydrogels increased about 2 folds of its original weight till 14 days, and then a decrease in the weight is observed. Whereas, HB-PEG-10%-HA-MA-4% hydrogels maintained same weight throughout 28 days.

For HB-PEG-15% group, all the concentration of HA-MA showed a similar trend. The hydrogel weight increased by 1.5 times the original weight till day 14. Also, the weight after day 14 did not show significant decrease, the weight was constant till day 28. HB-PEG-20% shows a different curve among all the groups. The weight of the hydrogels increased about 3 times in 24 hours and then decreases. From day 7 to day 28, the hydrogel maintained a constant weight not showing further decrease in the weight. Whereas, in HB- PEG-40% group, all the hydrogels reached the maximum weight till day 14 with 1.5 times increase and further maintained almost the same weight till day 28. Conclusion

The degradation behaviour can be regulated by changing the concentrations of HA-MA or H 13- PEG polymers to meet the specific requirement in different applications. For implantation applications, HB-PEG 10% and HA-MA 4% is advantageous. This provides a gel mass that is relatively stable.

Example 4

Pore Size Calculation Methodology

Pore size of the hydrogels was calculated based on the following equation, which is based on the rubber elastic theory.

Where G ' is the storage modulus, NA is the Avogadro constant, R is the molar gas constant, and T is the temperature. The theory assumes a purely elastic behaviour of hydrogel where all chains contribute to the retraction force after small deformation (affine deformation), neglects end effects of single chains (all chains have fixed ends toward an elastic background), and excludes any influence of physical entanglements.

Results

Figure 3 summarizes the pore size values of all the groups.

The table shows that the pore sizes of hydrogels decreases as the cross-linking polymer (HA- MA) concentration increases. This similar trend is observed in all groups of HB-PEG-10%, HB-PEG-15% and HB-PEG-20%. Also, the pore size is seen to decrease with increasing HB- PEG concentration with the pore size of 11.44 nm of HB-PEG-10%-HA-MA-0.5% and the least being 3.12 nm of HB-PEG-40%-HA-MA-0.5%. But, a different trend is seen in HB-PEG-40% group. As HA-MA concentration was increased from 0.5% to 1 % the pore size decreases, but as the concentration is further increased to 2% the pore size increased.

Example 5

Cell viability determination by MTT assay

Methodology MTT assay was conducted to evaluate the cytotoxicity. Briefly, the cells were cultured in DMEM at 37 °C, 5% CO2 and 95% humidity. The cells were seeded in 96 well plates at the density of 6^c10³ cells per well for 24 h, followed by a change of the medium with polymer solutions in DMEM containing 10% v/v FBS and 1 % v/v P/S at the concentration of 0 pg/ml (control), 50 pg/ml, 100 pg/ml, 250 pg/ml, 500 pg/ml, 1000 pg/ml respectively (n = 4, each concentration repeated in quadruplicates). After 24 h, the polymer solution was changed with 200 pi MTT solution (0.2 mg/ml), the plates were cultured at standard condition for 4 h and then the MTT solution was removed. 100 pi DMSO was added into each well and the plates were placed on a shaker for 15 min to fully dissolve the purple crystal. The absorbance was measured at 570 nm and 630 nm on a plate reader.

Results

Figure 4 shows the cytotoxicity data obtained from cells exposed to the hydrogel extracts. All the groups of 7 day and 21 day hydrogel extract show cell viability of 80% and above, which is considered to be sufficient for polymers to be used in tissue engineering applications. Only HB-PEG-20%-HA-MA-2% 7 day group had less cell viability compared to the other groups.

Study conclusion

Rheometer was used to study the gelation ability of both the polymer by UV light in the presence of photoinitiator. Increasing storage modulus (G’) in time sweep results after directing UV light showed that the polymers can crosslink and form a hydrogel. Also, with increasing concentration of HA-MA within these groups, higher values of G’ were observed. Also, within each group of HB-PEG with increasing concentration of HA-MA the pore size reduced. The swelling and degradation data show a similar trend for HB-PEG 10%, 15% and 40% groups, where the maximum swelling capacity of 1.5 times of its original weight was reached in 14 days and then till day 28 small decrease in weight values was observed. Difference seen in HB-PEG-20% groups were the hydrogels weight was increased 3 times in 1 day and further the weight started to decrease from 7th day. Cell study indicates that HB- PEG and HA-MA hydrogels exhibit a good biocompatibility which was confirmed by cell viability percentage - above 80% for all the groups.

Example 6

Synthesis of HA-MA

Methodology The synthesis approach has been verified, the functional groups of the final product have been proved to cross-link and form hydrogels by UV irradiation and be biocompatible in vitro.

HA modification reactions were conducted at various methacryloyl hydrazide feed ratios. At the beginning of the reaction, 2 eq. of EDCI (with respect to the carboxyl group in HA) was used to activate the carboxyl group in HA chain for 15 min at the pH of 4.75. The 0.3 eq., 0.6 eq., 1.0 eq., and 2 eq. of methacryloyl hydrazide were added in different groups, respectively. The pH was increased slowly, indicating the conjugation reaction between the hydrazide group and the carboxyl group. After purification by dialysis and freeze-drying, the hydrazide modifed methacryloyl HA (HA-MA-H) products with different SD were successfully achieved, confirmed by 1 H-NMR spectra (Figure 5A). The two new peaks at 5.65 and 5.90 ppm were attributed to the conjugated methacryloyl groups. Compared with methacryloyl hydrazide, the vinyl peaks of HA-MA-H were shifted to the low field, owing to the electron withdrawing effect of the conjugated carbonyl group. The SD was calculated by NMR result and it can be tailored by adjusting the molar ratio of methacryloyl hydrazide. HA-MA synthesized by glycidyl methacrylate (HA-MA-G) with a SD of 25% was used as the counterpart for comparison. As shown in Figure 5B, vinyl peaks of HA-MA (GMA) are in the lower field compared with vinyl peaks of HA-MA-H, indicating a higher electron cloud density of methacryloyl group in HA MA. As a result, the vinyl groups in HA-MA-H are more active than that in HA-MA-G under free radical crosslinking reaction. Equivalents

The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.

Claims

1. A hydrogel comprising (a) crosslinked methacryloyl functionalised hyaluronic acid (HA MA) and (b) hyperbranched PEG-based (HB-PEG) polymer comprising from about 12 to about 100 functional groups in the terminal chain(s) of the polymer structure.

2. The hydrogel of Claim 1 , in which HA-MA is hydrazide-modified methacryloyl functionalised hyaluronic acid (HA-MA-HDZ).

3. The hydrogel of Claim 1 , in which HA-MA is anhydride modified methacrylic HA (HA- AMA) or a glycidyl methacrylate HA (HAGM).

4. The hydrogel of any one of the preceding claims, in which the functional group is selected from the group comprising acrylate and methacrylate.

5. The hydrogel of any one of the preceding claims, in which the degree of substitution (SD) of the HA-MA is from about 10% to 90%.

6. The hydrogel of any one of the preceding claims, in which the degree of substitution (SD) of the HA-MA is from about 40% to about 90%.

7. The hydrogel of any one of the preceding claims, in which the HB-PEG comprises from about 12 to about 50 functional groups.

8. The hydrogel of any one of the preceding claims, in which the HB-PEG comprises from about 12 to about 30 functional groups

9. The hydrogel of any one of the preceding claims, comprising a HB-PEG concentration of from about 10% to about 40% (w/v).

10. The hydrogel of Claim 9, in which the concentration is about 20% (w/v).

11. The hydrogel of any one of the preceding claims, comprising a HA-MA concentration of from about 0.5% to about 4% (w/v).

12. The hydrogel of Claim 11 , in which the concentration is about 2%.

13. The hydrogel of any one of the preceding claims, in which the HB-PEG is obtained from a one-step controlled/living radical polymerisation reaction.

14. The hydrogel of any one of the preceding claims, in which the hydrogel is UV- crosslinked.

15. A curable composition comprising methacryloyl functionalised hyaluronic acid (HA MA) and hyperbranched PEG-based (HB-PEG) polymer.

16. The curable composition of Claim 15, in which the HB-PEG is a hyperbranched PEG- based multi acrylate polymer.

17. A device comprising the hydrogel of any one of Claims 1 to 14.

18. The device of Claim 17, which is a scaffold, a drug delivery system, a dressing, a hygiene product, a contact lens, an implant, a tissue adhesive material, a sealant material, or a bioadhesive.

19. A composition comprising a hydrogel of any one of Claims 1 to 14 and a pharmaceutically or biologically active agent contained within a matrix of the hydrogel.

20. The composition of Claim 19, in which the active agent is selected from the group comprising a cell or other biological material, a pharmaceutical (drug), or an imaging dye.

21. The composition of Claim 19 or 20, which is injectable or implantable.

22. A method of making a hydrogel, comprising the steps of:

providing an aqueous solution of a methacryloyl functionalised hyaluronic acid and a hyperbranched PEG-based multi acrylate (HB-PEG) polymer and crosslinking the aqueous solution of polymer to obtain the hydrogel.

23. The method of Claim 22, in which the HB-PEG concentration is of from about 10% to about 40% (w/v).

24. The method of Claim 22 or 23, in which the HA-MA concentration of from about 0.5% to about 4% (w/v).

25. The method of any one of Claims 22 to 24, in which the HA-MA is hydrazide-modified methacryloyl functionalised hyaluronic acid (HA-MA-HDZ).

26. A hydrogel produced by the method of any one of Claims 22 to 25.

27. Use of the hydrogel of any one of Claims 1 to 14, as a hygiene product, a contact lens, tissue engineering scaffold, a biomedical implant, a tissue adhesive material, a sealant material, a drug delivery system, bioadhesive and/or wound dressing.

28. A method of tailoring the physical properties of a hydrogel, comprising the method of any one of Claims 22 to 25.

29. A hydrogel comprising (a) crosslinked methacryloyl functionalised hyaluronic acid (HA MA) and (b) hyperbranched PEG-based multi-acrylate (HB-PEG) polymer.

30. A hydrogel comprising (a) crosslinked hydrazide-modified functionalised hyaluronic acid (HA-MA-HDZ) and hyperbranched PEG-based multi acrylate (HB-PEG) polymer.