Protein-based hydrogel and therapeutic uses thereof

Abstract

The present invention relates generally to a protein-based hydrogel and its use as a tissue adhesive and vehicle for therapeutic agents. In particular, the invention relates to a hydrogel comprising crosslinked serum albumin methacryloyl polymer and having a desirable tissue adhesion strength. The invention also relates to a process for preparing a hydrogel comprising crosslinked serum albumin methacryloyl using a blue-light photoinitiator capable of inducing dityrosine crosslinking when activated.

Description

[0001] PROTEIN-BASED HYDROGEL AND THERAPEUTIC USES THEREOF The present invention relates generally to a protein-based hydrogel and its use as a tissue adhesive and vehicle for therapeutic agents. In particular, the invention relates to a hydrogel comprising crosslinked serum albumin methacryloyl polymer and having a desirable tissue adhesion strength. The invention also relates to a process for preparing a hydrogel comprising crosslinked serum albumin methacryloyl polymer and a hydrogel obtained or obtainable from the process, as well as therapeutic uses of the hydrogel. BACKGROUND

[0002] According to reports by the British Heart Foundation, every 5 minutes a patient is admitted to a hospital in the UK with a heart attack (myocardial infarction, Ml). The prolonged ischemia caused by limited flow of blood and oxygen leads to cell death (up to one billion cardiomyocytes in the affected area) and partial loss of heart function, leading to death or prolonged loss of cardiac function. A bioengineered, minimally invasive, efficient approach to regenerate ischemic heart tissue after Ml is necessary to promote the regain of heart function and improve the prognosis of patients that have suffered Ml. Current interventions are mainly focussed on targeting the prevention of blood clots rather than tissue regeneration, or operate by direct injection of cells and therapeutics into the affected tissues, which approaches can suffer from cell death, poor engraftment, and undesirable burst release.

[0003] Cardiovascular surgical adhesives are known in the art but currently are mainly used as sealants for artery burst and vascular surgery. Other heart adhesives described in the literature include gelatin methacryloyl based hydrogel heart adhesives to reduce scar formation that do not, however, deliver therapeutics (Ptaszek et al., Journal of the American Heart Association, Volume 9, Issue 11, 2 June 2020). Alginate hydrogels have also been used as injectable carriers of therapeutics (dendritic cell-derived exosomes), although these hydrogels are incapable of adhering to the heart tissue itself (Zhang et al., J Nanobiotechnol 19, 271 (2021)). Lin et al (Nat Biomed Eng 3, 632-643 (2019) describe a viscoelastic adhesive epicardial gel-point adhesive patch, composed of a waxy starch

[0004]

[0005] treating myocardial infarction by increasing mechanical integrity of left ventricular tissues to reverse left ventricular remodelling and restore heart function after both acute and subacute myocardial infarction in rats.

[0006] There remains a need for a biocompatible, degradable material capable of adherence to tissues, such as the tissues of the heart, for targeted delivery of therapeutics in a controlled release manner.Photopolymerization of protein-derived polymers functionalized with methacryloyl groups has been reported in the fabrication of three-dimensional tissue constructs for biomedical applications. Ferracci et al., ACS Applied Bio Materials Vol 3, Issue 2, 13 January 2020 describe the preparation of photocurable bovine serum albumin methacryloyl with different degrees of substitution. Similarly, Yoon et al., ACS Omega, Vol 6, Issue 49, 2021 report the preparation of colloidal hydrogels made up of randomly packed human serum albumin-based photo-cross-linkable microparticles. In both cases, the material is prepared using a UV-curing system employing a common UV photoinitiator, namely 2-hydroxy-1-(4-(hydroxyethyl) phenyl)-2-methyl-1 -propanone (Irgacure 2959). The materials described in Farracci et al and Yoon et al have not, however, been investigated for their adhesion properties to heart tissues. Nor have different curing methods been investigated with a view to enhancing adhesion strength and other properties of the hydrogels.

[0007] The present invention is based on the finding that hydrogels formed from crosslinked serum albumin methacryloyl photocured under visible-light activation, in the presence of visible light photoinitiators capable of promoting dityrosine crosslinking, produce particularly advantageous hydrogels. These hydrogels have been found to exhibit particularly desirable tissue adhesion strength that makes them especially useful in adhering to tissues, for example cardiac tissues, as a means for delivering therapeutics for treating pathologies, such as Ml.

[0008] SUMMARY OF INVENTIOIN

[0009] In one aspect, the present invention provides a process for preparing a hydrogel comprising crosslinked serum albumin methacryloyl polymer, said process comprising:

[0010] a) forming an aqueous mixture of serum albumin methacryloyl and a visible-light photoinitiator, wherein the visible-light photoinitiator is capable of inducing dityrosine crosslinking in the serum albumin methacryloyl when activated; and b) subjecting the mixture formed in step a) to visible light of wavelength from 380 nm to 500 nm to activate the visible-light photoinitiator and form the crosslinked polymer.

[0011] In another aspect, the present invention provides a hydrogel obtained, or obtainable, by a process described herein.

[0012] In a further aspect, the present invention provides a hydrogel comprising crosslinked serum albumin methacryloyl polymer, wherein the crosslinked polymer comprises dityrosine crosslinks, and wherein the hydrogel has a uniaxial pull-off adhesion strength from a collagen substrate of at least 5 kPa,wherein the uniaxial pull-off adhesion strength is assessed using a dynamic mechanical analyser with a circular hydrogel sample of 2 mm thickness and 6 mm diameter which has been crosslinked directly on top of one surface of a collagen sheet substrate to form a coated substrate, wherein the uncoated surface of the coated substrate is adhered to a lower platen of the dynamic mechanical analyser with an adhesive and the crosslinked hydrogel coating at the opposing surface of the coated substrate is adhered to the upper platen of the dynamic mechanical analyser with an adhesive, and wherein the dynamic mechanical analyser is operated to separate upper and lower platens at a rate of 1mm / min until adhesion failure at the hydrogel-collagen substrate interface and stress at adhesion failure recorded.

[0013] In a yet further aspect, the present invention provides a hydrogel as described herein (for example, obtained or obtainable by the process described herein) for use in therapy, wherein the hydrogel encapsulates a therapeutic active and the hydrogel is provided on the surface of a tissue of the subject to be treated.

[0014] In a still further aspect, the present invention provides a method of treating a subject in need thereof; said method comprising providing a hydrogel as described herein (for example, obtained or obtainable by the process described herein), on the surface of a tissue of the subject, wherein the hydrogel encapsulates an active therapeutic.

[0015] In a still further aspect, the present invention provides a hydrogel as described herein (for example, obtained or obtainable by the process described herein) for use in treating cardiac disease in a subject, wherein the hydrogel encapsulates a therapeutic active, and wherein the hydrogel is formed on the surface of a cardiac tissue of the subject in vivo.

[0016] In a still further aspect, the present invention provides a method for treating cardiac disease in a subject in need thereof, said method comprising providing a hydrogel as described herein (for example, obtained or obtainable by the process described herein), on a surface of a cardiac tissue of the subject, wherein the hydrogel encapsulates a therapeutic active, and is formed on the cardiac tissue surface in vivo.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 depicts the application of an injectable adhesive protein hydrogel of the present invention for cardiac recovery post myocardial infarction (Ml);

[0019] Figure 2 depicts the chemical synthesis of bovine serum albumin methacryloyl (BSAMA) from BSA and methacrylic anhydride;

[0020] Figure 3 depicts graphs of free the amine content [mmol / g] (left) and degree of modification [%] (right) in unmodified BSA (BSA) and BSA methacryloyl (BSAMA_L and BSAMA_H; n = 4);Figure 4 depicts 1H NMR spectra of unmodified BSA (BSA) and BSA methacryloyl (BSAMA_L and BSAMA_H) (top). The 1H NMR spectra for BSAMA_L and BSAMA_H show peaks in the 5.9 to 5.1 ppm region which correspond to the methacryloyl groups. The 5.9 to 5.1 ppm region has been highlighted (bottom);

[0021] Figure 5 depicts the circular dichroism spectra of unmodified BSA (BSA) and BSA methacryloyl (BSAMA_L and BSAMA_H).

[0022] Figure 6 depicts macroscopic images (scale bar = 6 mm) of BSAMA hydrogels prepared by varying the BSAMA concentration (10, 15, and 20% w / V), the degree of modification (BSAMA_L and BSAMA_H), and the Ru / SPS photoinitiator amount (1 / 10 and 2 / 20 mM);

[0023] Figure 7 depicts representative rheological curves of the hydrogels during the crosslinking and rheological properties after crosslinking (n = 4). Top = BSAMA_L Complex Modulus G* [Pa], Second from top = BSAMA_H Complex Modulus G* [Pa], Second from bottom = BSAMA_L Storage Modulus G’ [Pa], Bottom = BSAMA_H Storage Modulus G” [Pa];

[0024] Figure 8 depicts the weight variation [%] of BSAMA_L hydrogels (top), the weight variation [%] of BSAMA_H hydrogels (middle), and percentage swelling [%] of the both the BSAMA_L and BSAMA_H hydrogels (bottom) at plateau (n = 3);

[0025] Figure 9 depicts representative stress-strain curves [Pa] of BSAMA_L hydrogels during compression tests (top), stress-strain curves [Pa] of BSAMA_H hydrogels during compression tests (middle), and the Elastic Moduli [kPa] of swollen crosslinked BSAMA_L and BSAMA_H hydrogels (n = 5) (bottom);

[0026] Figure 10 depicts representative live / dead images of cells embedded in the BSAMA hydrogels (viable cells are shown in white; scale bar = 250 pm);

[0027] Figure 11 depicts a side view schematic of the tensile strength test apparatus (top), representative stress-strain [kPa] curves of BSAMA hydrogels during tensile tests (second from top), hydrogel tensile stress [kPa] at break (second from bottom), and hydrogel tensile strain [%] at break (bottom);

[0028] Figure 12 depicts an isometric schematic view of the adhesion tests to collagen sheet model apparatus (top), representative stress-strain curves [kPa] of hydrogels pulled off a collagen sheet (middle), maximum strain [%] achieved by the hydrogels adhered to the collagen sheet when pulling them off (bottom left) (n = 5; **: p<0.01 ; ***: p<0.001 ; ****: p<0.0001), and maximum stress [kPa] achieved by the hydrogels adhered to the collagen sheet when pulling them off (bottom right) (n = 5; **: p<0.01; ***: p<0.001; ****: p<0.0001);Figure 13 depicts the percentage viability [%] of adipose derived stem cells cultured with culture medium previously in contact with the hydrogels (n = 3);

[0029] Figure 14 depicts the mesenchymal stem cell (MSC) viability measured via CCK-8 assay of cells embedded in the 3D hydrogels and cultured in vitro up to 7 days (n = 4);

[0030] Figure 15 depicts representative confocal images of the F-Actin of adipose derived stem cell cultured inside the 3D BSAMA hydrogels for 7 days (scale bar = 100 pm);

[0031] Figure 16 depicts the shear viscosity [Pa*s] of hydrogel precursor solutions based on BSAMA (H_1 10 15% w / V) with increasing amount of carboxymethylcellulose as thickening agent (0.25, 0.50, 0.75, and 1% w / V CMC);

[0032] Figure 17 depicts the elastic modulus [kPa] of BSAMA (H_1 10) and BSAMA / CMC (H_1 10 / CMC_1) during cyclic compression tests;

[0033] Figure 18 depicts in vitro enzymatic degradation [%] tests in Proteinase K and PBS, as control, as measured by residual weight overtime;

[0034] Figure 19 depicts a photo of the hydrogel adhered to the murine heart surface ex v / vo;

[0035] Figure 20 depicts a photo of a surgical application of the injectable adhesive hydrogel in an in vivo mouse model;

[0036] Figure 21 depicts a scan of the right and left ventricle of the mouse model over 14 days post-application of the hydrogel;

[0037] Figure 22 depicts a graph of the Ejection Fraction [%] of mice with hydrogels applied on the heart for 14 days (top) and a graph of the Cardiac Index [ml min-1cm-2] of mice with hydrogels applied on the heart for 14 days (bottom);

[0038] Figure 23 depicts a graph of the hydrogel volume [mm3] over 14 days to demonstrate the degradation of the hydrogel in vivo adhered to the mouse heart;

[0039] Figure 24 depicts circular dichroism spectra of BSA and BSAMA (top) compared to HSA and HSAMA (bottom).

[0040] Figure 25 depicts 1 H NMR spectra of BSA and BSAMA (top) as compared to HSA and HSAMA (bottom). The characteristic alkene peak of the methacryloyl group is highlighted;

[0041] Figure 26 depicts a graph quantifying the degree of modification [%] of the BSA, BSAMA, HSA, and HSAMA;

[0042] Figure 27 depicts the rheological characterisation of the crosslinking of BSAMA (left) as compared to HSAMA (right);

[0043] Figure 28 depicts the rheological properties after exposure to blue light of the crosslinking of BSAMA (left) as compared to HSAMA (right);Figure 29 depicts a comparison of the complex modulus G* [pa] of the BSAMA as compared to HSAMA;

[0044] Figure 30 depicts the frequency dependence of the BSAMA (top left) as compared to HSAMA (top right), as well as an estimation of the hydrogel mesh size based on their rheological properties (bottom);

[0045] Figure 31 depicts the weight variation [%] after immersion in PBS at 37 °C of BSAMA (left) and HSAMA (right);

[0046] Figure 32 depicts the weight loss [%] during the degradation in proteinase K vs PBS of BSAMA (left) and HSAMA (right);

[0047] Figure 33 depicts representative images of BSAMA (left) and HSAMA (right) exposed to Proteinase K or PBS over the course of 24 hours;

[0048] Figure 34 depicts a graph of the stress [Pa] vs strain [%] of the BSAMA and HSAMA (left), the elastic modulus (E) [kPa] of the BSAMA and HSAMA (centre), and a side view photo of the stress test apparatus;

[0049] Figure 35 depicts a side view photo of the stress test apparatus before (left) and after (right) the stress test;

[0050] Figure 36 depicts graphs of the adhesion force [N] (left) and the adhesion energy [mJ / m2] of the BSAMA and HSAMA;

[0051] Figure 37 depicts graphs of stress [Pa] vs displacement curves of the BSAMA (top) and HSAMA (bottom);

[0052] Figure 38 depicts in vitro cytocompatibility of mesenchymal stem cell-laden BSAMA (left) and HSAMA (right) analysed via live dead staining of cells embedded 3D in the hydrogels (viable cells are shown in white; scale bar = 250 .m);

[0053] Figure 39 depicts graphs of the adhesion stress [kPa] of the BSAMA vs BSAMA containing carboxymethylcellulose (CMC) (top), adhesion stress [kPa] of BSAMA and HSAMA hydrogels vs a hydrogel prepared in accordance with Ferracci, G. et al. Photocurable Albumin Methacryloyl Hydrogels as a Versatile Platform for Tissue Engineering. ACS Appl Bio Mater 3, 920-934 (2020) (“Irgacure”) (middle), the adhesion strain [%] of BSAMA and HSAMA hydrogels (bottom); and

[0054] Figure 40 depicts the results of experiments to test contraction force in living myocardial slices (LMSs) contacted with BSAMA vs BSAMA loaded with Verapamil, for precursor solutions and hydrogels at various times post initiation of cross-linking via blue light irradiation.DETAILED DESCRIPTION OF THE INVENTION

[0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the skilled person with a general dictionary of many of the terms used in this disclosure. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary.

[0056] This invention is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this invention. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the appended claims.

[0057] The description of embodiments of the invention is not intended to be exhaustive or to limit the invention to the precise form disclosed. While specific embodiments of, and examples for, the invention are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the invention, as those skilled in the relevant art will recognize. The various embodiments described herein can be combined to provide further embodiments. These and other changes can be made to the invention in the light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0058] In certain aspects, the present invention provides a hydrogel, a process for preparing a hydrogel, and uses of the hydrogel in therapy. The term “hydrogel” used herein refers to a biphasic material comprising a three-dimensional network of polymers constituting a solid phase and an aqueous liquid phase. The solid phase of the hydrogels of the present invention comprises crosslinked serum albumin methacryloyl. As a consequence, the hydrogel constitutes a chemical hydrogel, defined by the presence of covalent crosslinks.

[0059] The hydrogel of the invention may be utilised in therapy, for example as a drug carrier, or extended / controlled release, targeted drug delivery vehicle. Serum albumin, from which the hydrogel is ultimately derived, is the most abundant protein in blood plasma, having good mechanical properties, biocompatibility and degradability.Consequently, serum albumin is highly compatible with biomedical applications, and has also been known to reduce drug cytotoxicity when utilised as a drug delivery vehicle.

[0060] The particular utility of the present invention is not only related to the ability of the hydrogel to effectively encapsulate active therapeutics it is also the adhesion strength exhibited by the crosslinked serum albumin methacryloyl to tissue which makes the hydrogel especially useful extended / controlled drug release applications. The adhesion strength of the hydrogel and its other physical properties (e.g. tunable elastic modulus) also mean that the hydrogel can provide mechanical support to a tissue and have a therapeutic value, even in the absence of an active therapeutic encapsulated in the hydrogel. Lin et al., Nat Biomed Eng 3, 632-643 (2019) demonstrated that a viscoelastic adhesive epicardial gel-point adhesive patch can be used for treating myocardial infarction by increasing mechanical integrity of left ventricular tissues to reverse left ventricular remodelling and restore heart function after both acute and subacute myocardial infarction in rats. The inventors believe that the hydrogels of the present invention also offer mechanical support which can find utility in the treatment of certain disorders, even in the absence of the delivery of an active therapeutic, as demonstrated in the in vivo experiment described in the examples.

[0061] The term “tissue” as used herein refers to any human or animal tissue to which treatment with the hydrogel may be applied, including by means of surgical intervention. The tissue may be external (e.g. epithelial tissue) or inside the body (e.g. organ, muscle, or connective tissues). In some embodiments, the tissue may be mammalian or avian, preferably the tissue is mammalian tissue. In preferred embodiments, the tissue corresponds to myocardial tissue where the invention is deployed, for instance, in the treatment of a cardiac disease, such as myocardial infarct.

[0062] The hydrogel of the present invention may therefore be formed in situ at a particular tissue target site to which mechanical support and / or controlled delivery of an active therapeutic is desired, with no concern over premature detachment of the hydrogel from the tissue, particularly in the case of muscle or connective tissue where continuous movement or distortion of the tissue can be expected.

[0063] The term “active therapeutic” as used herein refers to a chemical or biological substance capable of utility in a therapeutic application. In some embodiments the active therapeutic is a pharmaceutical agent, or pharmaceutically acceptable salt or prodrug thereof, or a biologic. In some embodiments the active pharmaceutical agent is selected from the group consisting of: small molecule chemical compounds, or pharmaceutically acceptable salts or prodrug thereof, and biologies. Examples of biologies include cell-based biologies (e.g. a stem cells), antigens, growth factors, peptides, antibodies (e.g. monoclonal and humanised monoclonal antibodies), allergenics, blood components, gene therapies, cell therapies; somatic cells, vaccines, and exosomes. Biologies also include any other biomaterials compatible with encapsulation in the hydrogel of the invention.

[0064] In some embodiments the active therapeutic has a molecular weight (MW) of < 20000 Daltons. In some embodiments the active ingredient has a molecular weight (MW) of < 15000 Daltons. In some embodiments the active ingredient has a molecular weight (MW) of < 10000 Daltons, preferably < 6000 Daltons, more preferably < 900 Daltons, most preferably < 500 Daltons. A wide range of different classes of active agent may be encapsulated by the hydrogel, and these may have a wide range of molecular weights. For example, small molecules may typically have molecular weights of < 500 Daltons, but biologies, for example peptides, may typically have molecular weights of < 10000 Daltons. The wide variety of different active agents that are tolerated by the invention is a significant benefit.

[0065] In preferred embodiments, the pharmaceutical agent is selected from those capable of promoting myocardial repair and regeneration; those capable of promoting cardioprotection (e.g. FSTL1); those capable of inhibiting pathways associated with excessive fibrosis after myocardial infarction; anti-inflammatory agents, anti-apoptotic agents.

[0066] The term “prodrug” as used herein refers to a pharmacological derivative of a parent molecule that requires biotransformation, either spontaneous or enzymatic, within the organism to release the active drug. For example, prodrugs are variations of derivatives of an active pharmaceutical ingredient that have groups cleavable under certain conditions, for example metabolic conditions, which when cleaved become the active pharmaceutical ingredient. Such prodrugs then are pharmaceutically active in vivo, when they undergo solvolysis under physiological conditions or undergo enzymatic degradation. Prodrugs may offer advantages of solubility, tissue compatibility, or delayed release in the human or animal organism.

[0067] Prodrugs commonly known in the art include well-known acid derivatives, such as, for example, esters prepared by reaction of acid compounds with a suitable alcohol, amides prepared by reaction of acid compounds with an amine, and basic groups reacted to form an acylated base derivative. Other prodrug derivatives may be combined with other features disclosed herein to enhance bioavailability. As such, those of skill in the art will appreciate that certain of the presently disclosed compounds having, for example, free amino or hydroxy groups can be converted into prodrugs. Prodrugs include compounds having an amino acidresidue, or a polypeptide chain of two or more (e.g. two, three or four) amino acid residues which are covalently joined through peptide bonds to free amino, hydroxy or carboxylic acid groups of the presently disclosed compounds. The amino acid residues include the 20 naturally occurring amino acids commonly designated by three letter symbols and also include 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvalin, betaalanine, gamma-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine and methionine sulfone. Prodrugs also include compounds having a carbonate, carbamate, amide or alkyl ester moiety covalently bonded to moieties of the parent molecule.

[0068] The hydrogel of the present invention may comprise (e.g. encapsulate) a broad range of different active therapeutics. It is also possible for the hydrogel to comprise (e.g. encapsulate) a plurality of different active therapeutics. For example, both a pharmaceutical agent and a biologic may be used together as a combination therapy. In other examples, multiple different pharmaceutical agents may be used, each with different functionality (e.g. repair / regeneration, inhibition of apoptosis, inhibition of fibrosis, and anti-inflammatory effects). The skilled person is readily able to achieve encapsulation of active therapeutics in the hydrogel by including (e.g. dissolving or dispersing) the active therapeutic in the serum albumin methacryloyl precursor mixture prior to, or during, crosslinking.

[0069] The hydrogel may also comprise (e.g encapsulate) one or more pharmaceutically acceptable excipients. Those can be any such excipient known in the art and compatible with the invention including those described in, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The pharmaceutically acceptable excipient may include a detectable agent, label, adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like.

[0070] As discussed herein, since the hydrogel is capable of providing mechanical support to a tissue to be treated, the hydrogel may also be used in therapeutic applications in the substantial or complete absence of an active therapeutic.

[0071] Preparation of the hydrogel

[0072] The hydrogels of the present invention may be prepared by a) forming an aqueous mixture of serum albumin methacryloyl and a visible-light photoinitiator, wherein the visible-light photoinitiator is capable of inducing dityrosine crosslinking in the serum albumin methacryloyl when activated; and b) subjecting the mixture formed in step a) to visible light of wavelength from 380 nm to 500 nm to activate the visible-light photoinitiator and form the crosslinked polymer.Optionally, the process may include the preceding step of modifying serum albumin to replace one or more amine groups with methacrylate groups to form serum albumin methacryloyl (i.e. a preceding step of methacryloylation). Serum albumin is highly soluble in water and it is convenient to perform methacryloylation of serum albumin in an aqueous solution with a tunable pH, preferably a buffer solution, as reported, for instance, in Ferracci, G. et al. Photocurable Albumin Methacryloyl Hydrogels as a Versatile Platform for Tissue Engineering. ACS Appl Bio Mater 3, 920-934 (2020). Examples of suitable buffer solutions include a carbonate-bicarbonate buffer solution, for example having a molarity of 0.1 to 1.0 M, preferably from 0.1 to 0.5 M, more preferably from 0.2 to 0.3 M. Serum albumin may be present over a variety of concentrations in the aqueous solution. For example, serum albumin may be present in the solution at a concentration of 1 to 20 % w / v, preferably 5 to 20 % w / v, more preferably 5 to 15 % w / v, for example 8 to 12 % w / v.

[0073] Methacryloylation of serum albumin may also be performed as reported by Ferracci, G. et al by contacting the aqueous solution of serum albumin with methacrylic anhydride in the presence of a base (e.g. sodium hydroxide). Suitable temperatures over which the methacryloylation reaction may be conducted are, for instance, from 20 to 60 °C (i.e. below serum albumin’s denaraturion temperature of 62 °C, preferably from 25 to 50 °C, more preferably from 30 to 40 °C (e.g. 37 °C ). Suitable molar ratios of the methacrylic anhydride to serum albumin may be selected as reported in Ferracci, G. et al based on the amount of lysine groups present in the serum albumin and the desired extent of methacryloylation, which may be determined quantitatively by 2,4,6-trinitrobenzenesulfonic acid (TNBSA) assay described herein.

[0074] In some embodiments, the degree of methacryloylation (also referred to herein as “degree of modification”) in the serum albumin methacryloyl produced is at least 25%, preferably at least 50%, more preferably at least 60%, even more preferably 70%, still more preferably at least 80%. A higher degree of methacryloylation / modification has been found to reduce the capacity of the hydrogel to swell, since there is a denser network of crosslinks, although elastic modulus (i.e. stiffness) in the resulting hydrogel is increased with increasing degree of methacryloylation. Strain in the hydrogel may nevertheless be readily tailored based on the degree of methacryloylation and the corresponding extent of crosslinking that is performed of those available crosslinking sites. The extent of crosslinking may principally be modified by selecting different concentrations of the photoinitiator and exposure time and intensity of the activating blue light.

[0075] Degree of methacryloylation / modification (DoM) may be quantitatively determined by 2,4,6-trinitrobenzenesulfonic acid (TNBSA) assay and qualitatively verified throughnuclear magnetic resonance (NMR) spectroscopy, as described in Ferracci, G. et al. Photocurable Albumin Methacryloyl Hydrogels as a Versatile Platform for Tissue Engineering. ACS Appl Bio Mater 3, 920-934 (2020). Example 1 of the application describes the determination of DoM values for different serum albumin methacryloyl samples using the TNBSA assay. In some embodiments, the degree of modification is at least 50%, at least 75%, at least 90% or at least 95%. Alternatively, the degree of modification may be 100%. As will be appreciated, the skilled person is readily able to tune the degree of modification by extending the contact time between serum albumin and the methacrylating agent (e.g. methacrylate anhydride) and / or concentration of methacrylating agent until complete functionalisation of available reactive groups may be achieved.

[0076] A step in the process of preparing a hydrogel of the invention is to form an aqueous mixture of serum albumin methacryloyl and a visible-light photoinitiator (also referred to herein as a “precursor solution”), wherein the visible-light photoinitiator is capable of inducing dityrosine crosslinking in the serum albumin methacryloyl when activated. The aqueous solvent in which the serum albumin methacryloyl and a visible-light photoinitiator are suspended or dissolved may be any suitable aqueous solution, preferably of physiological pH, e.g. a buffer solution, that stabilises serum albumin. A preferred buffer solution is phosphate buffered saline (PBS). The concentration of serum albumin methacryloyl in the aqueous solution may suitably be from of 1 to 20 % w / v, preferably 5 to 20 % w / v, more preferably 5 to 15 % w / v, for example 8 to 12 % w / v. The concentration of photoinitiator (e.g. Ru(ll) complex or riboflavin of derivative thereof) is from 0.1 to 2% w / v, preferably 0.2 to 1% w / v. Where an oxidant (e.g. sodium persulfate, ammonium persulfate, or potassium persulfate) accompanies the photoinitiator (i.e. in the case where an Ru(ll) complex is used) the concentration of the oxidant is 0.5 to 5% w / v, preferably 1 to 2% w / v.

[0077] As discussed herein, in preferred embodiments the hydrogel comprises a therapeutic which may be incorporated into the precursor solution (e.g. dissolved or suspended). The precursor solution may also comprise one or more pharmaceutically acceptable excipients incorporated into the mixture formed in step a). In some embodiments, the one or more pharmaceutically acceptable excipients comprises alum (aluminium hydroxide) and ovalbumin to enhance / trigger the immune response of the subject to be treated. In other preferred embodiments, the one or more pharmaceutically acceptable excipients comprises one or more rheology modifiers, which can be useful inoptimising the viscosity / thickness of the precursor solution for disposal and retention on a tissue surface until crosslinking and adhesion to the surface occurs.

[0078] Suitable rheology modifiers that may be incorporated into the precursor solution include cellulose derivatives, alginic acid or salts thereof, poly(a-hydroxy acids), polyethylene glycol, polypropylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, and mixtures or copolymers thereof. Preferably, the rheology modifier is; i) a cellulose derivative selected from carboxymethylcellulose, hydroxyethylcellulose, hyaluronic acid, and mixtures or copolymers thereof; or ii) a poly(a-hydroxy acids) selected from poly(D,L-lactic acid), poly(D,L-lactic-co-glycolic acid), poly(E-caprolactone), and mixtures or copolymers thereof. The one or more rheology modifier may be present in the precursor solution in an amount from 0.1 to 2 % w / v, preferably 0.5 to 1 % w / v. The blue-light radiation (380 nm to 500nm) used for cross linking suitably has i) an intensity of from 10 to 50 mW / cm2, preferably from 20 to 30 mW / cm2, ii) a distance from light source of 1 to 10 cm, preferably 2 to 5 cm; and iii) a time period of exposure 2 to 10 minutes, preferably from 4 to 8 minutes. The blue light may be provided by conventional LED, lamp or laser. An example of suitable apparatus is a lamp fitted with radiometer and filter, as well as a fibre light guide for precision direction of light (e.g. OmniCure® S2000 Spot Curing System). In some embodiments, crosslinking to form the hydrogel is performed inside the human or animal body through the use of appropriate surgical instruments (such as a thoracoscope, laparascope or transcatheter system) capable of delivering the precursor solution inside a cavity of the human or animal body and also capable of providing blue-light radiation, for example, inside the chest or abdominal cavities by means of fibre optic.

[0079] Thus, as illustrated in Figure 1, a hydrogel may be formed in situ through injection of a precursor solution comprising serum albumin methacryloyl and a visible-light photoinitiator, preferably in combination with an active therapeutic, or pharmaceutically acceptable salt or prodrug thereof, as well as any pharmaceutically acceptable excipients, wherein the precursor solution comes into contact with a target tissue. Subsequently, subjecting the precursor solution to blue light radiation to induce crosslinking to form and adhere the hydrogel at the tissue target site inside the body can take place inside the body (e.g in the chest or abdominal cavities) using a thoracoscope, laparascope or transcatheter system with blue-light radiation functionality (e.g. via fibre optic). Alternatively, open surgery can be used allowing for the use of other instruments capable of generating blue light (e.g. a lamp).

[0080] Adhesion to tissue and properties of hydrogelAs discussed herein, in order to achieve cross-linking to form the chemical hydrogel of the invention, it is necessary to introduce methacrylate groups in the serum albumin (i.e. by replacing amine groups therein) to form a serum albumin methacryloyl polymer, or source serum albumin methacryloyl from a third-party supplier. The serum albumin methacryloyl may then be mixed with a visible-light photoinitiator as mentioned above and optionally an active therapeutic and / or one or more pharmaceutically acceptable excipients.

[0081] The visible-light photoinitiator used in accordance with the present invention is both capable of activation under exposure of visible light of wavelength of from 380 nm to 500 nm (also referred to herein as “blue light”), but also be capable of inducing dityrosine crosslinking in the serum albumin methacryloyl when activated. As the skilled person will be aware, serum albumin naturally contains tyrosine residues which can take part in crosslinking in the presence of an appropriate crosslinker. The presence of dityrosine crosslinking in the hydrogels of the present invention is believed to be beneficial in contributing to adhesion mechanisms between the hydrogel and the tissue surface, discussed in more detail below.

[0082] Photoinitiators capable of inducing dityrosine crosslinking are reported in the literature and are known to the skilled person. These include photoinitiators based on a ruthenium (II) complex in combination with an oxidant; or ii) riboflavin or a derivatives thereof (e.g. riboflavin 5'-phosphate). Preferably, the visible-light photoinitiator used in the preparation of the hydrogels of the invention comprises tris(bipyridine)ruthenium(ll) chloride (Ru(bpy)3Cl2) in combination with an oxidant, preferably selected from sodium persulfate, ammonium persulfate, or potassium persulfate.

[0083] A particular advantage of the process of the present invention is that visible-light activation of the photoinitiators used in the present invention does not exacerbate degradation of active therapeutics, or induce tissue damage or inflammation, as can arise with exposure to UV. Furthermore, the process for the preparation of the hydrogel in accordance with the present invention has been found to produce a hydrogel with particularly advantageous properties in terms of adhesion strength and elongation (tensile strain at break). In some embodiments, the hydrogel has a tensile strain at break of at least 50%, preferably at least 75%, more preferably at least 100%, measured for example in line with ISO 527-4:2023.

[0084] The hydrogels of the present invention can react with functional groups on the tissue surface, forming covalent bonds such as amides, imines, and carbon-nitrogen bonds, thereby achieving wet adhesion. Myocardial tissue, for instance, possesses reactivefunctional groups such as sulfhydryl and hydroxyl groups on its surface, the latter also capable of hydrogen bonding to the hydrogel thereby also contributing to a secondary adhesion mechanism, supplementing that arising through covalent bonding. Hydrophobic interactions between the hydrogel and the tissue may also contribute to yet further adhesion mechanisms.

[0085] It has been found that adhesion properties in the hydrogels of the present invention are superior to those obtained in cross-linked serum albumin methacryloyl formed with conventional UV photoinitiators (e.g. Irgacure 2959) following UV activation. Thus, it is apparent that cross-linking in the manner of the present invention - by subjecting a serum albumin methacryloyl precursor mixture including a visible-light photoinitiator capable of inducing dityrosine crosslinking in the serum albumin methacryloyl to visible light of wavelength from 380 nm to 500 nm - gives rise to a particular hitherto unknown architecture in the resulting hydrogel. This architecture includes dityrosine crosslinking, as a consequence of both the presence of tyrosine residues in the serum albumin and the ability of the photoinitiator to induce such crosslinking in those residues. The particular 3D polymer network of the hydrogel that may be produced in accordance with the present invention is believed to take part in superior adhesion mechanisms with tissues, such as myocardial tissue.

[0086] Thus, in a further aspect, the present invention provides a hydrogel comprising crosslinked serum albumin methacryloyl polymer, wherein the crosslinked polymer comprises dityrosine crosslinks, and wherein the hydrogel has a uniaxial pull-off adhesion strength from a collagen substrate of at least 5 kPa,

[0087] wherein the uniaxial pull-off adhesion strength is assessed using a dynamic mechanical analyser (e.g. Electroforce 5500, TA Instruments) with a circular hydrogel sample of 2 mm thickness and 6 mm diameter which has been crosslinked directly on top of one surface of a collagen sheet substrate (optionally pre-wetted with PBS) to form a coated substrate, wherein the uncoated surface of the coated substrate is adhered to a lower platen of the dynamic mechanical analyser with an adhesive (e.g. with Loctite Super Glue) and the crosslinked hydrogel coating at the opposing surface of the coated substrate is adhered to the upper platen of the dynamic mechanical analyser with an adhesive (e.g. with Loctite Super Glue), optionally applying a 10 g preload for 1.5 min on to the adhered sample before the dynamic mechanical analyser is operated to separate upper and lower platens at a rate of 1mm / min until adhesion failure at the hydrogel-collagen substrate interface and stress at adhesion failure recorded (determined quantitatively based on force-displacement curve assessment). As the skilled person will appreciate, any form ofadhesive can be used to secure the collagen sheet substrate and hydrogel to lower and upper platens, provided the strength of adhesion form is stronger than that which may be achieved between the interface of the collagen substrate and the hydrogel.

[0088] In some embodiments, the uniaxial pull-off adhesion strength is at least 20 kPa, preferably at least 40 kPa, more preferably at least 50 kPa, still more preferably at least 60 kPa. In further embodiments, the uniaxial pull-off adhesion strength is from 5 to 75 kPa, preferably from 10 to 70 kPa, more preferably from 20 kPa to 60 kPa.

[0089] As discussed above, in some embodiments, the hydrogel includes (e.g. encapsulates) one or more excipients which are selected from rheology modifiers. Although the presence of rheology modifiers is not essential for adhesion of the crosslinked serum albumin methacryloyl, the ability to control the precursor solution viscosity to allow it to be retained in position upon application to a target surface can be particularly useful in certain applications of the invention. Increasing the concentration of the rheology modifier has also been found to increase shear viscosity and elastic modulus, without at the same time increasing degradation of the resulting hydrogel.

[0090] In some embodiments, the viscosity of the precursor solution is from 0.1 to 10 Pa.s, preferably from 0.5 to 55 Pa.s.

[0091] In some embodiments, the elastic modulus of the hydrogel is from 2 to 20 kPa, preferably from 5 to 20 kPa.

[0092] Therapeutic Uses

[0093] The therapeutic uses of the hydrogel of the invention are defined by its ability to encapsulate active therapeutics effectively, as well as its particularly advantageous adhesion properties, which allow it to adhere effectively to tissues to which the active therapeutic may be delivered in a controlled release manner. The hydrogel of the present invention includes pores of various sizes, depending on the degree of cross-linking. The porous nature of the hydrogel is beneficial at the site of adhesion to the tissue, allowing rapid transfer of nutrients and oxygen to cells in, for instance, the infarct microenvironment, facilitating cell attachment, expansion and maturation. The porous nature of the hydrogel also allows passive diffusion of active therapeutics dissolved or dispersed in the aqueous phase of the hydrogel to the tissue to which the hydrogel may be adhered in use. Degradation of the hydrogel also allows active release of active therapeutics at the target site over the lifetime of the hydrogel.

[0094] Additionally, the therapeutic uses of the hydrogel of the invention may derive principally from the mechanical support that the hydrogel can provide to the tissue to which it may be adhered. For instance, the hydrogel may offer mechanical support to cardiactissue, such as that which is assessed to be damaged or in need of mechanical support, for example the left ventricle surface of the epicardium following myocardial infarction.

[0095] In embodiments where the hydrogel further comprises an active therapeutic, for example a pharmaceutical agent or a biologic, the active therapeutic is stable in aqueous solution at temperatures up to 40 °C, preferably up to 50 °C, more preferably up to 60 °C. It may be the case that being encapsulated in the hydrogel provides an additional stabilising effect to the active therapeutic, making it more resistant to degradation. “Stable” in this context may be used to refer to the proportion of the active agent that has degraded. As one of ordinary skill in the art will be aware, a number of analytical techniques may be used to determine the purity of a sample. These analytical techniques include, but are not limited to: NMR, for example1H NMR and19F NMR; HPLC; SFC; GC; LC-MS; colourimetry; titration; and IR.

[0096] The term “treatment” (and related terms, such as "treat", "treated", "treating") as used herein includes one or more of: inhibiting a disease or disorder; slowing or arresting the development of clinical symptoms of a disease or disorder; and / or relieving a disease or disorder (i.e. , causing relief from or regression of clinical symptoms). The term covers both complete and partial reduction of the condition or disorder, and complete or partial reduction of clinical symptoms of a disease or disorder. In the context of the present invention, treatment incorporates the formation of the crosslinked hydrogel on the surface of a tissue of the subject to be treated so that the hydrogel become adhered to the tissue surface. In some embodiments, the nature of the hydrogel may alone offer therapeutic benefits (e.g. in the case of certain dermatological conditions or through mechanical support that may be provided, for instance, to myocardial tissue damaged as a result of myocardial infarction). In other preferred embodiments, the hydrogel encapsulates an active therapeutic which may be delivered in a controlled manner (e.g. extended release).

[0097] The extended release of an active therapeutic is a significant benefit of the present invention. As a person skilled in the art would appreciate, a slower release of the active therapeutic means that a reduction in dosage frequency is possible in certain applications of the invention. Additionally, a slower release of the active pharmaceutical results in a lower peak concentration and avoids a “burst release”. The effect of this may be that the hydrogel of the invention encapsulating an active therapeutic results in fewer side effects in patients and / or improved therapeutic response of the patient.

[0098] The terms "patient," "individual," and "subject" as used herein refer to a human or animal. Preferably, the patient is a mammal including, for example, mice, rats, dogs, cats, pigs, sheep, horses, cows and humans. In some embodiments, the patient or subject is ahuman, for example a human that has been or will be the object of treatment, observation or experiment. The hydrogel described herein can be useful in both human therapy and veterinary applications.

[0099] Thus, in one aspect the present invention also provides a hydrogel as described herein for use in therapy, preferably wherein the hydrogel encapsulates a therapeutic active, wherein the hydrogel is provided on the surface of a tissue of the subject to be treated.

[0100] The hydrogels comprising an active therapeutic are particularly useful in treating cardiovascular diseases, wherein the hydrogel is formed in situ on a cardiac tissue. In some embodiments, the cardiovascular disease is selected from congestive heart failure, stroke, acute coronary artery disease, arrhythmia, asymmetric septal hypertrophy (e.g., left ventricular hypertrophy with resultant diastolic dysfunction), cardiomyopathy, valvular dysfunction, pericarditis, atherosclerosis, or, preferably, myocardial infarction.

[0101] Thus, in one aspect, the present invention provides a hydrogel as described herein for use in treating cardiac disease in a subject, preferably wherein the hydrogel encapsulates a therapeutic active, wherein the hydrogel is formed on the surface of a cardiac tissue of the subject in vivo, for example wherein the cardiac disease is myocardial infarction. In some embodiments, the hydrogel is at least partially formed at a site of the cardiac tissue which is assessed to be damaged or in need of mechanical support, for example the left ventricle surface of the epicardium (e.g. following myocardial infarction). As will be appreciated, where the intended therapeutic effect of the hydrogel derives primarily from the mechanical support provided to the tissue, the hydrogel need not necessarily include an encapsulated active therapeutic, although an active therapeutic may nevertheless be included in preferred embodiments.

[0102] The active therapeutic may be encapsulated by the hydrogel, for example, dissolved or dispersed in the aqueous phase of the hydrogel entrained by the 3D network of crosslinked serum albumin methacryloyl polymer. The porous nature of the hydrogel allows a varying degree of passive diffusion of an active therapeutic out of the hydrogel which may in turn be absorbed by the tissue the hydrogel is contact with. This allows an extended or slow release of an active therapeutic, the extent of which may be tailored depending on the particular active therapeutic in question and the desired rate of delivery to the target tissue. For instance, the skilled person is readily able to modify the degree of methacryloylation (i.e. the extent of replacement of amine groups with methacrylate groups), thereby influencing the extent or density of crosslinking possible, which is believed to control pore size and therefore passive diffusion out of the hydrogel. This inturn is believed to control encapsulation efficiency. Swelling of the hydrogel can also be used to adjust pore size. Additionally, the hydrogel of the invention is degradable and therefore the active therapeutic can be actively released at the target site on degradation. Complete degradation of the hydrogels of the invention may, for instance, occur within 3 months, preferably within 3 to 4 weeks. The invention is thus particularly compatible with the use of a thoracoscope, laparascope, or transcatheter system and surgical methods comprising in situ crosslinking to form the hydrogel at a tissue target site.

[0103] Serum albumin is the most abundant protein in blood plasma and therefore may be readily harvested from human or animal blood products by known processes. Mammalian serum albumin, particularly bovine serum albumin and human serum albumin is also commercially available for purchase. Nevertheless, in some embodiments, the serum albumin is harvested from a blood product obtained from a subject for preparation of a hydrogel in accordance with the invention, which hydrogel is in turn used in the treatment of the same subject (i.e. autologous therapy). This autologous approach offers advantages both in terms of availability and biocompatibility in the resulting hydrogel that is prepared.

[0104] The adhesive properties of the hydrogel, as well as the tunable viscosity properties of the precursor solution, also allow for targeted implementation of an active therapeutic to a particular patient tissue. In some embodiments, the invention provides a method of treatment comprising the application of a hydrogel precursor solution to a patient tissue and crosslinking of the hydrogel in situ. As will be appreciated by the skilled person, prior to curing, a mixture comprising serum albumin methacryloyl and a visible-light photoinitiator may be provided in the form of a mobile liquid (e.g. a solution or suspension). The serum albumin methacryloyl and visible-light photoinitiator may be dissolved or dispersed in an aqueous solution, for example a buffer solution (e.g. a carbonatebicarbonate buffer solution).

[0105] In some embodiments, the active therapeutic agent is a pharmaceutical agent. In some embodiments the pharmaceutical agent is selected from the group of general drug categories consisting of: antiarrhythmics; anti-inflammatories; and beta-blockers. Examples of pharmaceutical agents include antiarrhythmic drugs of classes (l-IV), as defined by the Vaughan Williams system, such as sodium channel blockers (class I), beta blockers (class II), potassium-channel blockers (class III), and calcium-channel blockers (class IV). Other examples include immunomodulatory agents, metabolic modulators, epigenetic / transcriptional modulators, matrix-modifying and tissue-remodelling agents, hormones, and RNA / DNA-based therapeutics.The invention will now be described by reference to the following non-limiting Examples.

[0106] EXAMPLES

[0107] Materials

[0108] Bovine Serum Albumin (BSA), Methacrylic Anhydride (MA), Tris(2,2'-bipyridyl)dichlororuthenium(ll) hexahydrate(Ru), Sodium Persulfate (SPS), Sodium Carbonate, Sodium Bicarbonate, dialysis membranes (Molecular Weight Cut Off 10 kDa), 2,4,6-Trinitrobenzene Sulfonic Acid (TNBSA), Dodecyl sulfate sodium salt (SDS) were purchased from Merck; ActinRe (RTM) 555 ReadyProbe (RTM) from Thermo Fisher. ASC52telo, hTERT immortalized adipose derived Mesenchymal Stem Cells (ATCC (RTM) SCRC-4000 (RMT); MSCs) were cultured at 37 °C, 5% CO2and maintained in DMEM (1X) + GlutaMAX-1 Media (GIBCO), supplemented with 10% FBS and 1% Penicillinstreptomycin (P / S).

[0109] Statistical Analysis

[0110] Data are presented as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism software; data normal distribution was evaluated via Shapiro-Wilk test; differences between data group was tested by one-way ANOVA, applying Tukey’s multiple comparison. Statistical significance between data groups was set for p < 0.05.

[0111] EXAMPLE 1 - Synthesis and Characterisation of Bovine Serum Albumin

[0112]

[0113] Bovine serum albumin methacryloyl (BSAMA) was synthesised adapting a previous protocol published in Ferracci, G. et al. Photocurable Albumin Methacryloyl Hydrogels as a Versatile Platform for Tissue Engineering. ACS Appl Bio Mater 3, 920-934 (2020).

[0114] BSA was dissolved 10% w / V in a carbonate-bicarbonate buffer at 37 °C, and the pH was adjusted to 9 by addition of NaOH. Methacrylic anhydride (MA) was added dropwise to the BSA solution under stirring to achieve a low (0.444 mmol MA per g BSA; BSAMA_L) and high (1.332 mmol MA per g of BSA; BSAMA_H) degree of modification (DoM). After 1 h of reaction under stirring, the pH was adjusted to 7.4 by adding HCI and the solutions were filtered by paper filters. The solution was dialysed against Milli-Q water for 3 days at 37 °C and eventually freeze-dried to obtain BSAMA_L and BSAMA_H powders.The functionalisation of BSAMA_L and BSAMA_H was qualitatively confirmed via proton nuclear magnetic resonance (1H NMR) spectroscopy (Bruker Avance I (RTM)). The DoM was quantitatively determined by TNBSA assay, which is described in Ferracci, G. et al. Photocurable Albumin Methacryloyl Hydrogels as a Versatile Platform for Tissue Engineering. ACS Appl Bio Mater 3, 920-934 (2020). BSA or BSAMA samples (0.25 mL; 0.2 mg / mL; n = 3) were incubated with 0.01% w / V TNBSA solution (0.25 mL) at 37 °C for 2 h. Then, 10% w / V SDS (0.25 mL) and 1M HOI (0.125 mL) were added to each sample, and absorbance was measured (wavelength X = 335 nm). The DoM was calculated following Equation (1):

[0115] Equation (1)

[0116]

[0117] Where MBSAMA and MBSA are the molarity of the free primary amines of BSAMA and BSA respectively. The secondary structure of unmodified BSA, BSAMA_L, and BSAMA_H (n = 3) was evaluated via circular dichroism (190-270 nm, step resolution 0.2 nm, response time 1 s, bandwidth 1 nm; Applied Photophysics Chirascan (RTM)); CD spectra were analysed to estimate the secondary structure using the BeStSel (Beta Structure Selection) web server, using the method described in Smith, P. T. et al. Additive Manufacturing of Bovine Serum Albumin-Based Hydrogels and Bioplastics. Biomacromolecules 21, 484-492 (2020).

[0118] Preparation and characterisation of BSAMA hydrogels

[0119] A library of BSAMA hydrogels was prepared by crosslinking BSAMA in presence of Ru / SPS photo-initiators via blue light irradiation (wavelength = 400-500 nm; intensity = 26 mW / cm2; time = 6 min; distance = 3 cm) performed with the OmniCure® S2000 Spot Curing System fitted with radiometer and filter, as well as a fibre light guide, for precision delivery of light of the desired spectral output.

[0120] Table 1. Hydrogel library prepared by varying the BSAMA DoM (BSAMA_L, and BSAMA_H), the BSAMA concentration (10, 15, and 20% w / V), and the photoinitiator concentration (Ru / SPS 1 / 10 and 2 / 20 mM). Each sample name (n = 12) reports this information in the same order (e.g., L_10_1 10 is a 10% BSAMA_L solution crosslinked in presence of Ru / SPS 1 / 10 mM).

[0121]

[0122] The cross inking of the hydrogel was evaluated via rheological tests. Hydrogel precursors were placed on the transparent plate of a rheometer (Netzsch Kinexus Ultra+), tested via oscillatory strain tests (parallel plate geometry, diameter 0 = 22 mm; 1 Hz; temperature T = 37 °C; 0.3% oscillatory strain; n = 4), and after 120 sec of oscillatory strain the blue light was activated to crosslink the hydrogel and measure the evolution of their rheological properties (Complex Modulus, G*). The rheological properties of the hydrogels at the end of the test (Storage Modulus, G’; Loss Modulus, G”) were characterised to compare the rheological response of the crosslinked hydrogel formulations. The swelling of the crosslinked hydrogels was investigated by measuring the weight variation in time after immersing freeze-dried samples in phosphate-buffered saline (PBS) and storing them at 37 °C. The compressive mechanical properties of swollen crosslinked hydrogels were tested with a dynamic mechanical analyser (Electroforce 5500, TA Instruments (RTM)); samples (n = 4) were tested via a hysteresis cycle comprised of a loading (30%) and unloading phase, and the Elastic Modulus was calculated (slope on linear interpolation in the 0-5% strain range, R2> 0.95). The cytocompatibility of the hydrogels was evaluated by loading adipose-derived human mesenchymal stem cell line (MSCSs) in the hydrogels (1x106cells mL-1). Cell-laden hydrogels were prepared and cell viability after hydrogel crosslinking was investigated via live / dead staining and confocal microscopy (n = 3, 2 images per sample, Leica SP8 (RTM) Confocal Microscope).

[0123] Investigating adhesive properties of BSAMA hydrogels

[0124] The tensile properties of selected hydrogel formulations (i.e., fixed concentration = 15% w / V) was assessed via uniaxial tensile tests (Mach-1, Biomomentum (RTM)). Hydrogel samples (n = 3) were prepared in dog bone-shaped moulds (ISO 527-4:2023) andstretched at a crosshead speed of 4 mm min-1. Force and displacement were recorded, and the maximum stress and strain at break of the samples were calculated.

[0125] The adhesive property of the hydrogel was investigated with uniaxial pull-off tests on a dynamic mechanical analyser (Electroforce 5500, TA Instruments (RTM)). Collagen sheets (Tongmaster (RTM) sausage casing skin) were wetted with PBS and glued to the lower platen with Loctite Super Glue (RTM). Cylindrical hydrogel samples (0 = 6 mm, thickness = 2mm; n = 5) were directly crosslinked onto the collagen sheets using a PDMS mould. A drop of Loctite Super Glue (RTM) was applied on the top surface of the hydrogel samples and immediately brought into contact with the upper platen applying a 10 g preload for 1.5 min. Pull-off tests were performed at 1 mm min-1until adhesive failure occurred at the interface between collagen sheets and hydrogels. Force and displacement were recorded, and the maximum stress and strain at break were calculated, using the method described in Montazerian, H. et al. Stretchable and Bioadhesive Gelatin Methacryloyl-Based Hydrogels Enabled by in Situ Dopamine Polymerization. ACS Appl Mater Interfaces 13, 40290-40301 (2021).

[0126] Adhesive hydrogel indirect cytotoxicity was tested (ISO 10993-12) using MSCSs as a cell model. Culture medium eluates were obtained by incubating hydrogel samples with culture medium for 72 h (n = 3). Culture medium controls were incubated for the same time without hydrogel samples. MSCSs were cultured with culture medium eluates or controls for 24 h and their viability were measured via CCK-8 assay. The percentage cell viability was calculated as ratio of the absorbance (A = 460 nm; CLARIOstar Plate Reader, BMG LABTECH (RTM)) measured for cells cultured with eluates to the absorbance of cells cultured with medium controls. Adhesive hydrogel cytocompatibility was tested by culturing MSCS-laden hydrogels as cell model (1x106cells ml’1). Cell viability was measured up to 7 days of culture via CCK8, and the percentage increase was reported compared to day 1, correcting the hydrogel background absorbance (n = 4). The morphology of encapsulated MSCSs was evaluated after 7 days of cultured via F-actin staining (n = 3, Leica SP8 (RTM) confocal microscope).

[0127] Preparation of Injectable adhesive hydrogels

[0128] Carboxymethyl cellulose (CMC) was added to the optimised BSAMA hydrogels (H_15_1 10) to promote its application and retention on the heart surface. CMC was added to BSAMA precursors with increasing concentrations 0, 0.25, 0.5, 0.75, and 1% w / V. The hydrogel viscosity was evaluated as an indicator of injectability via shear rheological tests (parallel plate geometry 0 = 22 mm; shear rate = 0.1-1000 s’1; time = 120 s; n = 3; Netzsch Kinexus Ultra+ (RTM)).The degradation of the hydrogels was tested in vitro via enzymatic degradation tests, using the method described in Lantigua, D. et al. Synthesis and characterization of photocrosslinkable albumin-based hydrogels for biomedical applications. Soft Matter 16, 9242-9252 (2020). Hydrogel samples were dehydrated in a gradient of ethanol solution and dried in sterile conditions overnight. The initial dry weight (w0) of the samples was measured before incubation in 0.1 mg mL-1proteinase K solution at 37 °C. Samples (n = 3) were freeze-dried at established time points and their weight (wt) recorded to calculate the percentage residual weight wres(Equation 2):

[0129] 100 (Equation 2)

[0130]

[0131] Cyclic compression tests were performed by applying 500 load-unload cycles up to 60% strain (Electroforce 5500, TA Instruments (RTM)). The energy dissipation efficiency (EDE) was calculated as the proportion of the area of the hysteresis loop to the area under the loading curve (n = 3), using the method described in Shi, T. et al. Conductive Hydrogel Patches with High Elasticity and Fatigue Resistance for Cardiac Microenvironment Remodeling. ACS Appl Mater Interfaces (2022) doi:10.1021 / acsami.2c22673.

[0132] Adhesion of the injectable adhesive hydrogels was preliminarily tested ex vivo. Freshly excised mouse hearts were maintained humid in sterile PBS; the hydrogel precursor (4 pL) was applied to the left ventricle of the heart and crosslinked via blue light to qualitatively evaluate the adhesion of the hydrogel to the heart surface. The heart / hydrogel were stored in PBS overnight to qualitatively check the stability after crosslinking.

[0133] In vivo model

[0134] All animal work conformed to the UK Animals (Scientific Procedures) Act, 1986, amended with Regulations 2012 to transpose European Directive 2010 / 63 / EU. All animalbased work was conducted under the procedure project licence (PPL) number PP2659456 and was performed under aseptic conditions.

[0135] In vivo tests were conducted on C57BL / 6j mice (Charles River UK (RTM)) to verify the hydrogel adhesion to the heart and its safety. After induction anaesthesia (4% isoflurane, 1 I min-1O2), the mouse was given a 0.1 mg kg-1subcutaneous injection of buprenorphine and a 5 mg kg-1carprofen injection before returning to the anaesthetic chamber. The mouse was intubated and placed in supine position with eye ointment for thoracotomy under mechanical ventilation (2% isoflurane, 1 I min-1O2). Briefly, 10%povidone-iodine was applied over the thorax (surgical area), an occlusive film dressing was placed over the chest to prevent fur and dander from entering the wound, and three doses (10-20 pl) of bupivacaine 2 mg kg-1were injected along the incision site. After 3 min, a small incision (1-2 cm) was made across the left side of the thorax. The skin and the underlying muscle layer were blunt dissected, a left thoracotomy in the fourth intercostal space was performed, the ribs were retracted, and the left side of the heart exposed after partial removal of the pericardium. The BSAMA H_1 10 / CMC_1 hydrogel (4 pl) was directly applied to the left ventricle surface of the mice (n = 4) and crosslinked via blue light. After hydrogel crosslinking and adhesion (using the OmniCure® S2000 Spot Curing System), the separated ribs were pulled tight with sutures, the muscle and skin layers were closed with appropriate sutures before the animals were allowed to wake up and recover with post-operative care provided and surveillance.

[0136] Cardiac magnetic resonance imaging (MRI) scans were conducted on days 1, 3, 7, 10, and 14 following hydrogel application on mouse hearts. Mice were anaesthetised with 1.5-2% isoflurane adjusted to maintain a respiratory rate of 50-55 breaths min-1; respiration, ECG and body temperature were monitored continuously (SA Instruments (RTM), Stony Brook, NY, USA). MRI was performed on a 9.4 T-BioSpec system (Bruker (RTM) BioSpin, Ettlingen, Germany) equipped with a mouse heart array receiver. Systolic function was assessed using a cine short axis multi-stack sequence with the following parameters: repetition time (TR)=RR interval / number of frames (~6.2 ms for ~20 frames), TReffective=RR interval, echo time (TE)=2.2 ms, flip angle=18°, slice thickness=0.70 mm (continuous slices covering the entire left ventricle), acquisition matrix (136x136), field of view (20x20) mm2, spatial in-plane resolution (147x147) pm2, total scan time 15 min. Frames covered a full cardiac cycle. Indices including ejection fraction (EF), cardiac index (Cl), end diastolic volume index (EDVI), and left ventricular mass index (LVMI) were measured. All MRI metrics were normalized to the body surface area (BSA) using the method described in Gouma, E. et al. A simple procedure for estimation of total body surface area and determination of a new value of Meeh’s constant in rats. Lab Anim 46, 40-45 (2012). A high-resolution segmented gradient echo sequence was used for hydrogel visualisation, with the following parameters: TR=6.2 ms, TE=2.2 ms, slice thickness 0.5 mm, 4 segments, field of view (20x20) mm2, matrix size 222x222, in plane spatial resolution (90x90) pm2, 6 averages, scan time 15 minutes.

[0137] Cardiac MR images were converted in Dicom and analysed in Segment (Segment V2.2 R6190). To measure cardiac function, epicardial and endocardial contours were manually traced to delineate the ventricularwall at end diastole (ED) and end systole (ES).The interventricular septum was regarded as a component of the left ventricle (LV). Papillary muscles and trabeculations were considered part of the blood pool. Ejection fraction was defined as EF = SV / EDV * 100%, where stroke volume (SV) was the difference between ED volume (EDV) and ES volume (ESV). Cardiac output was defined as the product of heart rate (HR) and stroke volume (SV). Ventricular mass was calculated by multiplying the specific myocardial density (1.05 g ml’1) with the ED myocardial volume. Following the contour tracing for hydrogel segmentation, we quantified the hydrogel volume and measured the change in signal intensity over time. A decrease in signal intensity would indicate a reduction in water content, potentially caused by hydrogel degradation.

[0138] Results and Discussion

[0139] Figure 1 depicts the application of an injectable adhesive protein hydrogel of the present invention for cardiac recovery post myocardial infarction (Ml). The hydrogel precursor is applied on the heart surface and cured via blue light irradiation, adhering to the cardiac tissue while crosslinking. In time, the hydrogel degrades promoting cardiac function regain.

[0140] Figure 2 Depicts synthesis of bovine serum albumin methacryloyl (BSAMA) from BSA and methacrylic anhydride. The reactive primary amine groups and the resulting methacryloyl groups are explicitly depicted. BSAMA with low (BSAMA_L) and high (BSAMA_H) degree of modification are depicted. As can be seen, BSAMA_H comprises a greater number of methacryloyl groups.

[0141] Figure 3 depicts quantification of free amine content [mmol / g] and degree of modification [%] in unmodified BSA (BSA) and BSA methacryloyl (BSAMA_L and BSAMA_H; n = 4). As would be expected, modification of the BSA as depicted in figure 2 reduces the amine content as these groups are consumed by reaction with methacrylic anhydride. BSAMA_L undergoes a lesser degree of reaction than BSAMA_H and as expected was found to have a higher remaining amine content. Conversely, the degree of modification was shown to be higher in BSAMA_H than in BSAMA_L. As expected, no modification is detected in unreacted BSA. Figure 4 depicts 1 H NMR spectra of unmodified BSA (BSA) and BSA methacryloyl (BSAMA_L and BSAMA_H). The 1H NMR spectra for BSAMA_L and BSAMA_H show peaks in the 5.8 to 5.6 ppm region which correspond to the methacryloyl groups. As expected, no such peaks are seen in the 1H NMR spectrum of BSA. Figure 5 depicts circular dichroism spectra of unmodified BSA (BSA) and BSA methacryloyl (BSAMA_L and BSAMA_H).Figure 6 depicts macroscopic images (scale bar = 6 mm) of BSAMA hydrogels prepared by varying the BSAMA concentration (10, 15, and 20% w / V), the degree of modification (BSAMA_L and BSAMA_H), and the Ru / SPS photoinitiator amount (1 / 10 and 2 / 20 mM). Figure 7 depicts representative rheological curves of the hydrogels during the crosslinking and rheological properties after crosslinking (n = 4). Top = BSAMA_L Complex Modulus G* [Pa], Second from top = BSAMA_H Complex Modulus G* [Pa], Second from bottom = BSAMA_L Storage Modulus G’ [Pa], Bottom = BSAMA_H Storage Modulus G” [Pa], Figure 8 depicts the weight variation [%] of BSAMA_L hydrogels (top), the weight variation [%] of BSAMA_H hydrogels (middle), and percentage swelling [%] of the both the BSAMA_L and BSAMA_H hydrogels (bottom) at plateau (n = 3. Figure 9 depicts representative stress-strain curves [Pa] of BSAMA_L hydrogels during compression tests (top), stress-strain curves [Pa] of BSAMA_H hydrogels during compression tests (middle), and the Elastic Moduli [kPa] of swollen crosslinked BSAMA_L and BSAMA_H hydrogels (n = 5) (bottom). Figure 9 shows that elastic modulus E increases for the hydrogels as degree of modification (DoM) increases (i.e. as number of available crosslinking sites increases) and as the extent of crosslinking formation increases (resulting from higher photoinitiator concentrations).

[0142] Figure 10 depicts representative live / dead images of cells embedded in the BSAMA hydrogels (viable cells green, dead cells red; scale bar = 250 pm). As shown, all of the tested hydrogels predominantly kept the cells alive.

[0143] BSAMA adhesive hydrogel patches prepared with 15% BSAMA and by varying BSAMA DoM (low, L, or high, H) and photoinitiator (1:10 or 2:20 = Ru:SPS). Figure 11 depicts a side view of the tensile strength test apparatus (top), representative stress-strain [kPa] curves of BSAMA hydrogels during tensile tests (second from top), hydrogel tensile stress [kPa] at break (second from bottom) (n = 3; *: p<0.05; **: p<0.01; ***: p<0.001), and hydrogel tensile strain [%] at break (bottom) (n = 3; *: p<0.05; **: p<0.01; ***: p<0.001). Figure 12 depicts an isometric view of the adhesion tests to collagen sheet model apparatus (top), representative stress-strain curves [kPa] of hydrogels pulled off a collagen sheet (middle), maximum strain [%] achieved by the hydrogels adhered to the collagen sheet when pulling them off (bottom left) (n = 5; **: p<0.01; ***: p<0.001; ****: p<0.0001), and maximum stress [kPa] achieved by the hydrogels adhered to the collagen sheet when pulling them off (bottom right) (n = 5; **: p<0.01 ; ***: p<0.001 ; ****: p<0.0001).

[0144] Figure 13 depicts the percentage viability [%] of adipose derived stem cells cultured with culture medium in contact with the hydrogels (n = 3). Each of the hydrogels, as well as the positive control showed approximately 100% viability to the cells cultured in contactwith the hydrogel. As would be expected, a low viability was observed for the negative control. Figure 14 depicts the MSC viability measured via CCK-8 assay of cells embedded in the 3D hydrogels and cultured in vitro up to 7 days (n = 4). The viability increased over 7 days for each of the hydrogels, in particular for those using the Ru / SPS photoinitiator in an amount of 1 / 10 mM. Figure 15 depicts representative confocal images of the F-Actin of adipose derived stem cell cultured inside the 3D BSAMA hydrogels for 7 days (scale bar = 100 pm). Adipose derived stem cell cultures can be seen in each of the tested hydrogels. These results suggest that the hydrogels serve as a good medium to promote cell growth.

[0145] Figure 16 depicts the shear viscosity [Pa*s] of hydrogel precursor solutions based on BSAMA (H_1 10 15% w / V) with increasing amount of carboxymethylcellulose as thickening agent (0.25, 0.50, 0.75, and 1% w / V CMC). These results demonstrate that a carboxymethylcellulose additive serves to increase shear viscosity of the hydrogel. Figure 17 depicts the elastic modulus [kPa] of BSAMA (H_1 10) and BSAMA / CMC (H_1 10 / CMC_1) during cyclic compression tests. These results demonstrate that a carboxymethylcellulose additive also serves to increase the elastic modulus. Figure 18 depicts in vitro enzymatic degradation [%] tests in Proteinase K and PBS, as control, as measured by residual weight over time. These results demonstrate that a carboxymethylcellulose additive does not impact the degradation of the hydrogel by proteinase K.

[0146] Figure 19 depicts a photo of the hydrogel adhered to the murine heart surface ex vivo. The hydrogel is visibly adhered to the heart tissue in the same as depicted in Figure 1. This test validates the model of adhesion to cardiovascular tissue. Figure 20 depicts a photo of a surgical application of the injectable adhesive hydrogel in an in vivo mouse model. Figure 21 depicts a scan of the right and left ventricle of the mouse model over 14 days post-application of the hydrogel.

[0147] Figure 22 depicts a graph of the Ejection Fraction [%] of mice with hydrogels applied on the heart for 14 days (top) and a graph of the Cardiac Index [ml min-1cm-2] of mice with hydrogels applied on the heart for 14 days (bottom). Both the Ejection Fraction and the Cardiac Index remained relatively stable over the course of the 14 days.

[0148] Figure 23 depicts a graph of the hydrogel volume [mm3] over 14 days to demonstrate the degradation of the hydrogel adhered to the mouse heart. Minor degradation of the hydrogel was observed after 10 to 14 days.

[0149] EXAMPLE 2 - Synthesis and Characterisation of Human Serum Albumin

[0150]

[0151] The synthesis of human serum albumin methacryloyl (HSAMA) was performed following the same procedure as described in Example 1 for bovine serum albumin methacryloyl (BSAMA - high degree of modification); BSA was substitute with HSA (Sigma Aldrich) for the synthesis. HSAMA hydrogels (HSAMA_H_15_1 10) were prepared following the same procedure used to prepare BSAMA_H_15_1 10. The hydrogel characterisation was performed following the same protocols used for BSAMA; the results are all reported as a comparison between BSAMA and HSAMA.

[0152] Results and Discussion

[0153] The results show comparable properties obtained between serum albumin hydrogels derived from bovine (BSAMA) and human (HSAMA) origin. Specifically, human serum albumin hydrogels show similar degree of modification, swelling and stability after crosslinking, degradability in enzymatic solutions, comparable rheological properties, adhesion to collagen sheets similar to that of BSAMA, and cytocompatibility enabling the fabrication of cell-laden HSAMA hydrogels.

[0154] Figure 24 depicts circular dichroism spectra of BSA and BSAMA (top) compared to HSA and HSAMA (bottom). Figure 25 depicts 1H NMR spectra of BSA and BSAMA (top) as compared to HSA and HSAMA (bottom). The characteristic alkene peak of the methacryloyl group is highlighted. The spectra for BSAMA and HSAMA proved to be very similar which suggested that the modification with methacryloyl groups proceeds in much the same way with human serum albumin as it does in bovine serum albumin. This is further validated by Figure 26 which depicts a graph quantifying the degree of modification [%] of the BSA, BSAMA, HSA, and HSAMA. The degree of modification BSAMA and HSAMA are very similar. A would be expected, unmodified BSA and HAS showed zero degree of modification.

[0155] Figure 27 depicts the rheological characterisation of the crosslinking of BSAMA (left) as compared to HSAMA (right). Figure 28 depicts the rheological properties after exposure to blue light of the crosslinking of BSAMA (left) as compared to HSAMA (right). Figure 29 depicts a comparison of the complex modulus G* [pa] of the BSAMA as compared to HSAMA. These results show that the rheological properties of the HSAMA were very similar to those of the BSAMA.

[0156] Figure 30 depicts the frequency dependence of the BSAMA (left) as compared to HSAMA (right), as well as an estimation of the hydrogel mesh size based on their rheological properties (bottom). Figure 31 depicts the weight variation [%] after immersion in PBS at 37 °C of BSAMA (left) and HSAMA (right). Figure 32 depicts the weight loss [%] during the degradation in proteinase K vs PBS of BSAMA (left) and HSAMA (right). Figure33 depicts representative images of BSAMA (left) and HSAMA (right) exposed to Proteinase K or PBS over the course of 24 hours. These results show that the stability properties of the HSAMA were also very similar to those of the BSAMA.

[0157] Figure 34 depicts a graph of the stress [Pa] vs strain [%] of the BSAMA and HSAMA (left), the elastic modulus (E) [kPa] of the BSAMA and HSAMA (centre), and a side view photo of the stress test apparatus. Figure 35 depicts a side view photo of the stress test apparatus before (left) and after (right) the stress test. Figure 36 depicts graphs of the adhesion force [N] (left) and the adhesion energy [mJ / m2] of the BSAMA and HSAMA. Figure 37 depicts graphs of stress [Pa] vs displacement curves of the BSAMA (top) and HSAMA (bottom). These results show that the adhesive properties of the HSAMA are equal, if not superior to those of the BSAMA.

[0158] Figure 38 depicts in vitro cytocompatibility of mesenchymal stem cell-laden BSAMA (left) and HSAMA (right) analysed via live dead staining of cells embedded 3D in the hydrogels (viable cells are shown in white; scale bar = 250 .m). The BSAMA and HSAMA also showed a similar ability to act as a medium to promote cell growth.

[0159] In conclusion, these data show that the HSAMA may function in much the same way as the BSAMA.

[0160] COMPARATIVE EXAMPLE 3 - Crosslinked BSAMA obtained from a UV system Bovine serum albumin methacryloyl (BSAMA (20 % w / V in PBS) was crosslinked using the method reported in Ferracci etal., ACS Applied Bio Materials Vol 3, Issue 2, 13 January 2020, using 0.5 % w / v of Irgacure 2959. The hydrogel precursor solution was photo-cross-linked by UV-light irradiation (365 nm; 150 mW cm-2) for 6 min at a distance of 3 cm.

[0161] The adhesion performance of the crosslinked polymer was tested as described above in Example 1 and the results are provided in Figure 39 (bottom) plotted against the results for BSAMA hydrogels prepared by the blue-light system in accordance with the invention. Despite the same BSAMA substrate with comparable levels of DoM (see in particular the H_20_1 10 and H_20_2 20 entries), it is clear that adhesion in the present invention is superior to that observed in the UV / Irgacure system. This supports the conclusion that crosslinking using the blue-light system of the present invention gives rise to a different hydrogel architecture, which exhibits different adhesion mechanisms to the tissue compared with the UV / Irgacure 2959 system.

[0162] Figure 39 (bottom) also shows that the adhesion exhibited by the hydrogels of the invention is inherent to the crosslinked hydrogel architecture rather than excipients comprised in the hydrogel (CMC rheology modifier is beneficial for application of theprecursor solution in certain applications, but is shown to have a minor dampening effect on adhesion of the hydrogel, rather than contributing to, or enhancing, adhesion).

[0163] EXAMPLE 4 - Ex vivo human model

[0164] Human donor hearts were obtained from the NHS Blood and Transplant INOAR program (IRAS project ID: 189069), approved by the NHS Health Research Authority, in accordance with the Governance Arrangements for Research Ethics Committees. This study is fully compliant with the Standard Operating Procedures for Research Ethics Committees in the UK. Informed consent was obtained from each patient / family involved in this study.

[0165] Human hearts donated after brain death or circulatory death (female and male donors, 66-76 years old) and with no history of cardiac disease were used. Living myocardial slices (LMSs) were prepared as described in Watson, S. A. et al. Preparation of viable adult ventricular myocardial slices from large and small mammals. Nat Protoc 12, 2623-2639 (2017). After heart excision, the organ was stored in cold cardioplegia on ice. A 1-1.5 cm2tissue block from the left ventricular free wall was dissected. LMSs (thickness = 300 um) were obtained using a high-precision vibratome (7000smz-2, Campden Instruments). LMSs were trimmed to align myocardial fibres, and custom-made 3D-printed T-Glase rectangular rings were attached perpendicular to the fibre alignment. LMSs were mounted onto custom-made stretchers and stretched under physiological load to achieve a sarcomere length of 2.2 .m. The LMSs were subsequently cultured in customised culture chambers for 24 h at 37 °C with electrical stimulation (frequency = 1 Hz, voltage = 10 V, pulse width = 10 ms), under carbogen superfusion and continuous circulation of culture medium.

[0166] LMS contraction force was then measured in the presence of BSAMA hydrogels prepared as described in Example 1, with the exception that certain of the BSAMA hydrogels were also loaded with Verapamil (10 mM), a small-molecule calcium channel blocker and suppressor of myocardial contractility (falling in the category of an antiarrhythmic). Before the test, LMSs were removed from the stretchers and placed on a force transducer apparatus (Harvard Apparatus, USA). During the test, LMSs were perfused with warm Tyrode’s solution and electrically stimulated (frequency = 1 Hz, voltage = 40 V, pulse width = 10 ms). The contraction force was measured at unloaded length, followed by measurements at 10% and subsequent 22% strain. Hydrogel precursors (4 uL) were applied onto the surface of the LMSs under exposure to blue light irradiation such that crosslinking of the hydrogels commenced immediately uponapplication to the LMSs and the contraction force of the LMSs was recorded for 30 min. The measurement results are presented in Figure 40.

[0167] The results in Figure 40 shows that, prior to application and cross-linking of the hydrogel (“Pre-light on”), there is little difference in recorded contraction force between the samples. Following application and commencement of crosslinking (“Post-light off”), there is a decrease in contraction force for the Verapamil loaded BSAMA hydrogel, indicating that Verapamil is exerting a suppression effect on the LMS. In contrast, the unloaded BSAMA temporarily increases the contraction force of the LMS following its application and crosslinking has begun (“Post-light off”). Following a period of 10 minutes after crosslinking has been initiated, the Verapamil samples are observed to completely suppress myocardial contraction in the LMSs and no contraction force is recorded (“+10 min crosslinking”), and nor is any contraction force observed in any other Verapamil loaded sample where a longer duration of cross-linking is permitted (“+20 min crosslinking” and “+30 min crosslinking”). These results show a rapid diffusion of the small-molecule active Verapamil to the LMS upon application and initiation of cross-linking to form a crosslinked hydrogel in accordance with the invention. This demonstrates the feasibility of delivering an active therapeutic agent to the tissue by means of a hydrogel according to the invention.

[0168] BSAMA hydrogels without Verapamil loading which have been allowed to crosslink for 10, 20 or 30 minutes post initiation are observed to slightly decrease contraction force compared to that observed for the LMS prior to application and cross-linking of the hydrogel (“Pre-light on”). This demonstrates that contractile function nevertheless remains, despite a substantial mass loading of the hydrogel relative to the mass of the very thin slice of LMS upon which it is deposited. Application of the hydrogel to a surface of an intact heart tissue (e.g. in vivo), where the mass of the hydrogel is trivial in comparison to that of the tissue itself, is expected to support mechanical integrity, as shown in rat models by Lin et al., Nat Biomed Eng 3, 632-643 (2019).

Claims

33Claims:

1. A process for preparing a hydrogel comprising crosslinked methacrylated serum albumin polymer, said process comprising:a) forming an aqueous mixture of serum albumin methacryloyl with a visible-light photoinitiator, wherein the visible-light photoinitiator is capable of inducing dityrosine crosslinking in the serum albumin methacryloyl when activated; andb) subjecting the mixture formed in step a) to visible light of wavelength from 380 nm to 500 nm to activate the visible-light photoinitiator and form the crosslinked polymer.

2. A process according to claim 1, wherein the process further comprises the preceding step of modifying serum albumin to replace one or more amine groups with methacrylate groups to form serum albumin methacryloyl.

3. A process according to claim 1 or claim 2, wherein the serum albumin modified in step a) is mammalian serum albumin, preferably human or bovine serum albumin.

4. A process according to any one of the preceding claims, wherein the visible- light photoinitiator comprises i) a ruthenium (II) complex in combination with an oxidant; or ii) riboflavin or a derivative thereof (e.g. riboflavin 5'-phosphate).

5. A process according to claim 4, wherein the visible-light photoinitiator comprises tris(bipyridine)ruthenium(ll) chloride (Ru(bpy)3Cl2) in combination with sodium persulfate, ammonium persulfate, or potassium persulfate.

6. A process according to any one of the preceding claims, wherein an active therapeutic is incorporated into the mixture formed in step a).

7. A process according to claim 6, wherein the active therapeutic is a pharmaceutical agent, or pharmaceutically acceptable salt or prodrug thereof, preferably selected from the general drug categories consisting of: antiarrhythmics; anti-inflammatories; and beta-blockers; or a biologic, wherein the biologic is preferably selected from a cell-based biologic (e.g. a stem cell), antigen, growth, peptide, antibody, vaccine, and exosome.

8. A process according to any one of the preceding claims, wherein one or more pharmaceutically acceptable excipients is / are incorporated into the mixture formed in step a).

9. A process according to claim 8, wherein the one or more pharmaceutically acceptable excipients comprises one or more rheology modifiers, preferably34selected from cellulose derivatives, alginic acid or salts thereof, poly(a-hydroxy acids), polyethylene glycol, polypropylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, and mixtures or copolymers thereof.

10. A process according to claim 9, wherein the rheology modifier is; i) a cellulose derivative selected from carboxymethylcellulose, hydroxyethylcellulose, hyaluronic acid, and mixtures or copolymers thereof; or ii) a poly(a-hydroxy acids) selected from poly(D,L-lactic acid), poly(D,L-lactic-co-glycolic acid), poly(E-caprolactone), and mixtures or copolymers thereof.

11. A process according to any one of the preceding claims, wherein the mixture formed in step a) is stored and / or transported prior to step b).

12. A process according to any one of the preceding claims, wherein the visible light used to activate the visible-light photoinitiator in step c) is of wavelength from 400 to 500 nm.

13. A hydrogel obtained, or obtainable, by the process of any one of the preceding claims.

14. A hydrogel comprising crosslinked serum albumin methacryloyl polymer, wherein the crosslinked polymer comprises dityrosine crosslinks, and wherein the hydrogel has a uniaxial pull-off adhesion strength from a collagen substrate of at least 5 kPa,wherein the uniaxial pull-off adhesion strength is assessed using a dynamic mechanical analyser with a circular hydrogel sample of 2 mm thickness and 6 mm diameter which has been crosslinked directly on top of one surface of a collagen sheet substrate to form a coated substrate, wherein the uncoated surface of the coated substrate is adhered to a lower platen of the dynamic mechanical analyser with an adhesive and the crosslinked hydrogel coating at the opposing surface of the coated substrate is adhered to the upper platen of the dynamic mechanical analyser with an adhesive, and wherein the dynamic mechanical analyser is operated to separate upper and lower platens at a rate of 1mm / min until adhesion failure at the hydrogel-collagen substrate interface and stress at adhesion failure recorded.

15. A hydrogel according to claim 13 or claim 14, wherein the hydrogel comprises mammalian derived serum albumin, preferably human or bovine derived serum albumin.

16. A hydrogel according to any one of claims 13 to 15, wherein the hydrogel further comprises an encapsulated active therapeutic.

17. A hydrogel according to claim 16, wherein the active therapeutic is a pharmaceutical agent, or a pharmaceutically acceptable salt or prodrug thereof, or a biologic, preferably wherein the biologic is selected from a cell-based biologic (e.g. a stem cell), antigen, growth factor, peptide, antibody, vaccine, and exosome.

18. A hydrogel according to any one of claims 13 to 17, wherein the hydrogel further comprises a pharmaceutically acceptable excipient.

19. A hydrogel according to claim 18, wherein the hydrogel comprises one or more rheology modifiers, preferably selected from cellulose derivatives, alginic acid or salts thereof, poly(a-hydroxy acids), polyethylene glycol, polypropylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, and mixtures or copolymers thereof.

20. A hydrogel according to claim 19, wherein the rheology modifier is; i) a cellulose derivative selected from carboxymethylcellulose, hydroxyethylcellulose, hyaluronic acid, and mixtures or copolymers thereof; or ii) a poly(a-hydroxy acids) selected from poly(D,L-lactic acid), poly(D,L-lactic-co-glycolic acid), poly(E-caprolactone), and mixtures or copolymers thereof.

21. A hydrogel according to any one of claims 13 to 20, wherein the hydrogel has a tensile strain at break of at least 50%, preferably at least 75%, more preferably at least 100%.

22. A hydrogel according to any one of claims 13 to 21 for use in therapy, preferably wherein the hydrogel encapsulates a therapeutic active, wherein the hydrogel is provided on the surface of a tissue of the subject to be treated.

23. A hydrogel according to any one of claims 13 to 21 for use in treating cardiac disease in a subject, preferably wherein the hydrogel encapsulates a therapeutic active, wherein the hydrogel is formed on the surface of a cardiac tissue of the subject in vivo.

24. A hydrogel for use according to claim 23, wherein the cardiac disease is myocardial infarction, preferably wherein the hydrogel is at least partially formed at a site of the cardiac tissue which is assessed to be damaged or in need of mechanical support, for example the left ventricle surface of the epicardium.

25. A hydrogel for use according to any one of claims 22 to 24, wherein the hydrogel is prepared from serum albumin which has been harvested from a blood product obtained from the subject to be treated.

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