X-ray visible implantable scaffolds

Abstract

The present invention relates to the field of implantable scaffolds which can be visualised using X-rays and which are therefore useful in surgical procedures, in particular surgical methods for repairing a tissue defect or reconstructing tissue, or for guiding resection or ablation of a tumour. Specifically, the present invention is directed to an implantable scaffold, wherein (a) the scaffold comprises a plurality of protein molecules, and (b) at least one of said plurality of protein molecules comprises at least one aromatic amino acid residue that is substituted with iodine on its aromatic moiety. The present invention is further directed to methods of using such implantable scaffolds in surgery, and to methods of preparing an iodinated scaffold.

Description

[0001] X-RAY VISIBLE IMPLANTABLE SCAFFOLDS

[0002] Field of the invention

[0003] The present invention relates to the field of implantable scaffolds which can be visualised using X-rays and which are therefore useful in surgical procedures, in particular surgical methods for repairing a tissue defect or reconstructing tissue, or for guiding resection or ablation of a tumour. Specifically, the present invention is directed to an implantable scaffold, wherein (a) the scaffold comprises a plurality of protein molecules, and (b) at least one of said plurality of protein molecules comprises at least one aromatic amino acid residue that is substituted with iodine on its aromatic moiety. The present invention is further directed to methods of using such implantable scaffolds in surgery, and to methods of preparing an iodinated scaffold.

[0004] Background to the invention

[0005] The use of protein biomaterials is widespread in surgery and regenerative medicine (see e.g. Zhang et al.. Bioactive Materials, 2022, 10, 15-31), with collagen-based products clinically established to repair urogenital damage, skin wounds, bone defects, hernia, post- surgical reconstruction, and cardio-vascular disease (see Stenzel et al., Annual Review of Biophysics and Bioengineering, 1974, 3(1), 231-253; Freedman et al., Adv Mater, 2019, 31(19): el806695; Sourushanova et al., Adv Mater, 2019, 3 l(l):el801651). Close matching of protein-based materials to the needs of a given therapy, target tissue or implantation site can be achieved either through the endogenous variety and complexity of various decellularised donor tissues (dermis, aortic valves, lung, heart, etc.), or by synthetic bottom -up engineering to allow complete design of the material.

[0006] Artificial control of degradation rate, structure, and patterning across multiple length scales can be achieved with advanced manufacturing techniques including 3D printing, electrospinning, moulding, microfluidic encapsulation, acoustic cell-patterning, crosslinking, and composite and modular assembly (see Walters et al. , Acta Biomaterialia, 2014, 10(4), 1488-1501; O et al., Adv Mater, 2012, 24(31), 4311-4316; Lee et al., Science, 2019, 265(6452), 482-487; Armstrong et al., Advanced Healthcare Materials, 2022, 1 l(24:e2200481). Furthermore, multiple protein sources contribute to good availability, with porcine, bovine, marine, and recombinant collagen remaining suitable for human use due to low immunogenicity.

[0007] Despite being one of the most well-established biomaterials, the research on collagen for tissue engineering applications has experienced a remarkable three-fold growth in the past 20 years. This growth can be attributed, in part, to collagen’s versatile role as a scaffold for emerging therapeutics. In this way, unmet needs in conditions including spinal cord damage, diabetes, cartilage and connective tissue defects, tooth and heart regeneration are being approached through its combination with stem cells, differentiated cells, growth factors, nanoparticles, and advanced patterning and fabrication technologies.

[0008] However, in order for clinical products comprising collagen (or other structural protein) scaffolds to have utility, it is crucial to validate their behaviour and safety postimplantation. This is a challenge best addressed through the use of non-invasive imaging (see e.g. Scarfe et al.. NP J Regenerative Medicine, 2017, 2:28; Armstrong et al., Science Translational Medicine , 2020, 12(572):eaaz2253). However, this task remains challenging due to the inherent resemblance between protein-based implants and native tissue, with both generating similar signals on commonly used medical imaging modalities such as MRI, CT, and ultrasound. Consequently, detection of critical events such as successful delivery and integration, mis-delivery, mechanical failure, or detachment proves unachievable in both preclinical models and patient populations, primarily due to the limited availability of imageable scaffolds.

[0009] There is consequently a need for protein-based scaffolds which are readily visible using routinely available medical imaging technology. There is, in particular, a need to develop protein-based scaffolds which are visualisable using X-rays (in e.g. a CT scanner), because many patients with tissue defects that require treatment using such scaffolds, e.g. patients with hernias, are too obese to fit inside an MRI machine. The present invention addresses this problem and provides implantable protein scaffolds that are X-ray visible and which can therefore be effectively visualised after implantation within a patient. Summary of the invention

[0010] The present inventors have surprisingly discovered that it is possible to prepare implantable protein scaffolds which are X-ray visible through bulk iodine labelling of aromatic amino acid residues within the protein. The degree of iodination of the scaffold achieved causes a sufficient increase in the radiopacity of the scaffold such that it is clearly identifiable against a patient’s native protein-containing tissue using X-ray CT scanning. These scaffolds have wide applicability in a range of surgical methods in which visualisation and monitoring of the scaffold in situ is critical.

[0011] The iodine labelling of protein scaffolds is advantageously found to be a fast, simple, and cost-effective route to label pre-formed protein-based biomaterials. The labelling method is furthermore achievable under mild and scalable conditions, maintaining the biocompatibility and strength of the starting material.

[0012] The present invention accordingly provides an implantable scaffold, wherein:

[0013] (a) the scaffold comprises a plurality of protein molecules; and

[0014] (b) at least one of said plurality of protein molecules comprises at least one aromatic amino acid residue that is substituted with iodine on its aromatic moiety.

[0015] The present invention further provides a method of using an implantable scaffold of the invention for repairing a tissue defect or reconstructing tissue, or for resection or ablation of a tumour, wherein said method comprises positioning the implantable scaffold in or on a subject such that the scaffold extends across, or adjacent to, all or a proportion of the tissue defect or tissue to be reconstructed, or the tumour to be resected or ablated .

[0016] The present invention further provides a protein comprising at least one aromatic amino acid residue that is substituted with iodine on its aromatic moiety, for use in a method of repairing a tissue defect or reconstructing tissue, or a method for resection or ablation of a tumour, comprising:

[0017] (i) incorporating said protein into an implantable scaffold;

[0018] (ii) positioning said implantable scaffold in or on a subject such that the scaffold extends across, or adjacent to, all or a proportion of the tissue defect or tissue to be reconstructed or the tumour to be resected or ablated; and (iii) visualising the implanted scaffold using a computed tomography (CT) scan or a planar X-ray scan.

[0019] The present invention further provides a method of iodinating a protein scaffold, wherein said protein scaffold comprises a plurality of protein molecules, comprising:

[0020] (i) contacting the scaffold with a buffer solution; and

[0021] (ii) incubating said solution with KI3 or IC1 under suitable conditions to result in iodination of the aromatic moiety of at least one aromatic amino acid residue in at least one of the plurality of protein molecules, optionally wherein the KI3 or IC1 reagent comprises a non-radioactive isotope of iodine.

[0022] The present invention further provides a method of preparing an iodinated protein scaffold, wherein said protein scaffold comprises a plurality of protein molecules, comprising:

[0023] (i) contacting the plurality of protein molecules with a buffer solution;

[0024] (ii) incubating said solution with KI3 or IC1 under suitable conditions to result in iodination of the aromatic moiety of at least one aromatic amino acid residue in at least one of the plurality of protein molecules; and

[0025] (iii) assembling the plurality of protein molecules into a protein scaffold.

[0026] The present invention further provides a method of iodinating a protein scaffold, wherein said protein scaffold comprises a plurality of protein molecules, comprising:

[0027] (i) contacting the scaffold with a buffer solution; and

[0028] (ii) incubating said solution with an acylating reagent X-(C=O)-R under suitable conditions to result in acylation of the primary amino group of at least one lysine residue in at least one of the plurality of protein molecules, wherein:

[0029] - X is a leaving group under addition-elimination reaction conditions; and

[0030] - R is Ce-Cio aryl or Ce-Cio aralkyl, which is optionally substituted with a hydroxy group on the aromatic ring; and

[0031] (iii) incubating said solution with KI3 or IC1 under suitable conditions to result in iodination of the aromatic moiety of at least one acylated lysine residue in at least one of the plurality of protein molecules; optionally wherein the iodinating reagent comprises a non-radioactive isotope of iodine. The present invention further provides a method of iodinating a protein scaffold, wherein said protein scaffold comprises a plurality of protein molecules, comprising:

[0032] (i) contacting the scaffold with a buffer solution; and

[0033] (ii) incubating said solution with an acylating reagent X-(C=O)-R under suitable conditions to result in acylation of the primary amino group of at least one lysine residue in at least one of the plurality of protein molecules, wherein:

[0034] - X is a leaving group under addition-elimination reaction conditions; and

[0035] - R is Ce-Cio aryl or Ce-Cio aralkyl, which is substituted with one or more iodine atoms (preferably, one or two iodine atoms) on the aromatic ring, and which is further optionally substituted with a hydroxy group on the aromatic ring; optionally wherein the iodinating reagent comprises a non-radioactive isotope of iodine.

[0036] The present invention further provides a method of preparing an iodinated protein scaffold, wherein said protein scaffold comprises a plurality of protein molecules, comprising:

[0037] (i) contacting the plurality of protein molecules with a buffer solution;

[0038] (ii) incubating said solution with an acylating reagent X-(C=O)-R under suitable conditions to result in acylation of the primary amino group of at least one lysine residue in at least one of the plurality of protein molecules, wherein:

[0039] - X is a leaving group under addition-elimination reaction conditions; and

[0040] - R is Ce-Cio aryl or Ce-Cio aralkyl, which is optionally substituted with a hydroxy group on the aromatic ring;

[0041] (iii) incubating said solution with KI3 or IC1 under suitable conditions to result in iodination of the aromatic moiety of at least one acylated lysine residue in at least one of the plurality of protein molecules; and

[0042] (iv) assembling the plurality of protein molecules into a protein scaffold; optionally wherein the iodinating reagent comprises a non-radioactive isotope of iodine.

[0043] The present invention further provides a method of iodinating a protein scaffold, wherein said protein scaffold comprises a plurality of protein molecules, comprising:

[0044] (i) contacting the plurality of protein molecules with a buffer solution; and (ii) incubating said solution with an acylating reagent X-(C=O)-R under suitable conditions to result in acylation of the primary amino group of at least one lysine residue in at least one of the plurality of protein molecules, wherein:

[0045] - X is a leaving group under addition-elimination reaction conditions; and

[0046] - R is Ce-Cio aryl or Ce-Cio aralkyl, which is substituted with one or more iodine atoms (preferably, one or two iodine atoms) on the aromatic ring, and which is further optionally substituted with a hydroxy group on the aromatic ring; and

[0047] (iii) assembling the plurality of protein molecules into a protein scaffold; optionally wherein the iodinating reagent comprises a non-radioactive isotope of iodine.

[0048] Preferred embodiments of the invention are described in further detail below.

[0049] Brief description of the figures

[0050] Figure 1. A. Schematic of collagen labelling showing colour-coded collagen fibrils and chains respectively, with iodination of a tyrosine residue following reaction with KI3 or IC1. B. Increased radi opacity of porcine collagen scaffolds (XenmatrixTM) vs unlabelled controls at 50kVp, following Potassium Tri-iodide (Lugol’s) reaction or Iodine monochloride reaction (IC1) in the indicated buffers (unpaired, 2 -tail t-test). C. Crosssample intensity profile shows relative homogeneity of sampling for each of the reaction conditions. D. Increased radiopacity was retained up to 16 days of incubation at 37°C in human serum. E. Representative X-ray CT sections showing unlabelled and labelled (potassium tri-iodide reaction in PBS) hernia mesh sample following incubation in human serum. F.-J. 3D rendered X-ray CT images of unlabelled (left tube) and labelled (right tube, potassium tri-iodide reaction in PBS) Hernia mesh, nerve guide, egg shell matrix, jellyfish collagen hydrogel, and silk suture.

[0051] Figure 2. A. Surface morphology is comparable between control (unlabelled) collagen scaffolds and those labelled using Potassium triiodide and Iodine monochloride methods, as seen with scanning electron micrographs at two levels of magnification. B. Stress strain curves of labelled and unlabelled meshes, one line per replicate. C. No significant difference was found between labelled and unlabelled scaffolds in elongation percentage at point of fracture (2 -tailed t-test). D. Labelled meshes required a significantly higher max force (ultimate tensile strength, UTS) to induce fracture in primary tests, but not retests (p<0.05, 2-tailed t-test), indicating increased strength. N=5, lines show the mean, error bars show the standard deviation.

[0052] Figure 3. Iodinated hernia mesh samples (Xenmatrix) show increased visibility on X-ray CT vs unlabelled samples and muscle over 3 months post implantation on A. 3D reconstructions, B. axial CT cross sections, and C. region of interest quantification of x-ray absorbance (HU). Holm-Sidak multiple comparison tests show significantly higher radioapacity of labelled meshes vs muscle (paired), and vs unlabelled samples (unpaired) at all time points. N>3 per time point, error bars show standard deviation. D. CT renderings showed lateral mesh migration in one animal post implantation.

[0053] Figure 4. Labelled meshes retained their increased visibility vs muscle and control mesh with decreasing scan times, as shown here with A. axial CT sections at 3 months postimplantation, and region of interest quantification of radiopacity at B. 2 months, and C. 3 months post-implantation, with Holm-Sidak multiple comparison tests. N>3 per time point, error bars show SD. D. Laterally migrated mesh visualised on a 3D rendered 18 second CT scan at 3 months post-implantation using a clinically relevant X-ray dose of 16.4 mGy.

[0054] Figure 5. A. Macroscopic H and E stained sections of explanted meshes at 2, 4, and 12 weeks post-implantation. Scale bar = 1mm. Representative sections showing comparable infiltration of endogenous tissue at the mesh boundary at 12 weeks for B. labelled, and C. unlabelled meshes. D. No significant difference was found between mean neutrophils and lymphocytes counts at 40x magnification in H and E stained tissue sections at 2, 4, and 12 weeks post implantation (2 -tailed unpaired T-test, error bars show SD). Labelled meshes showed comparable collagen coverage to unlabelled meshes up to 3 months postimplantation, as seen with E. Threshold quantification and F. representative images of the collagen-stained area (p>0.05 for all time points, 2-tailed unpaired t-test, n>5 regions of interest).

[0055] Figure 6. Fluorescence spectroscopy showing decreased tyrosine peak at 300 nm following labelling of Xenmatrix decellularised porcine dermis with either the potassium tri-iodide reaction in PBS (pH 7.4), or the iodine monochloride method in glycine buffer (pH 8.6). Samples were excited at 230nm, and emission intensity recorded for 200 ms in 2 nm steps using a fluorescence spectrometer (Thermo-Fisher Varioskan Lux). Spectra show the average of n=3 independently labelled samples or control unlabeled samples. Intensity at 300 nm was significantly reduced in KE / PBS (0.65 ± 0.25 SD) and IC1 / Glycine (0.63 ± 0.07 SD) vs control samples (upper curve) (30.6 ± 1.8 SD) for both labelling methods (2 -tailed t-test, p<0.005).

[0056] Figure 7. A. Reaction kinetics of the KI3 labelling method in PBS, as measured on decellularised porcine dermis (Xenmatrix) following different incubation times using x-ray CT at 50 kVp. After the indicated reaction times samples were washed in saline for 48 hours to remove unreacted iodine. B. X-ray CT image of representative samples from A, at the indicated times in minutes. Surface modification, with low penetration of the reagents to the inside of the mesh can be seen in 3 and 10 minute reaction samples. The 0 minute (unlabelled control sample) is not visible due to its similar radiopacity to the surrounding saline solution. Labelling was found to be rapid, with 80% of the peak radiopacity achieved following a 45 minute reaction period.

[0057] Figure 8. A. Decellularised porcine dermis (Xenmatrix) labelled with the KI3 / PBS reaction retains its contrast above background over 17 months (510 days), as measured at intervals using X-ray CT at 50 kVp. B. Maximum intensity projection of a representative labelled (left) and unlabelled (right) sample of Xenmatrix suspended in saline at 510 days post labelling.

[0058] Figure 9. Labelled Neuragen collagen tubes retain their signal over 15 months in saline solution (0.9 % w / v NaCl) at room temperature. Signal was quantified following CT acquisition at 50 kVp and ROI analysis.

[0059] Figure 10. Labelled Egg-shell membrane retains its signal over 15 months in saline solution (0.9 % w / v NaCl) at room temperature. Signal was quantified following CT acquisition at 50 kVp and ROI analysis.

[0060] Figure 11. A. Energy Dispersive X-Ray Spectroscopy showing the absence of iodine in stock unmodified mesh (Xenmatrix, Bard). B. Energy Dispersive X-Ray Spectroscopy showing the absence of iodine in control (sham) labelled mesh (Xenmatrix, Bard). C. Energy Dispersive X-Ray Spectroscopy showing the presence of iodine in ICl / Glycine labelled mesh (Xenmatrix, Bard). D. Energy Dispersive X-Ray Spectroscopy showing the presence of iodine in KI3 / PBS labelled mesh (Xenmatrix, Bard).

[0061] Figure 12. FTIR spectra for A. KI3 / PBS labelled mesh, and B. control (unmodified) mesh samples.

[0062] Figure 13. Differential Scanning calorimetry of for A. KI3 / PBS labelled mesh, and B. control (unmodified) mesh samples cycled from 25°C to 400°C at 10°C / min.

[0063] Figure 14. Surface morphology of silk sutures (Ethicon Permahand, 3-0) is comparable between control (unlabelled) samples and those labelled using Potassium triiodide and Iodine monochloride methods (2 day labelling time), as seen with scanning electron micrographs at A. low and B. high magnification.

[0064] Figure 15. Stress-strain curves of silk sutures (Ethicon Permahand 3-0) show no change in mechanical behaviour following labelling with either KI3 (Lugols’ Iodine) or Iodine Monochloride (IC1) reactions, one line per replicate.

[0065] Figure 16. Ultimate tensile strength of silk sutures (Ethicon Permahand 3-0) showed no significant change following labelling with either KI3 (Lugols’ Iodine in PBS with 2 hour or 2 day labelling time) or Iodine Monochloride (IC1 in Glycine, 2 day incubation time), (n=3, error bars show SD, p-value>0.05).

[0066] Figure 17. Volume of labelled meshes showed no significant trend decrease over time (R2=0.0186), as obtained via CT-based semi-automatic region of interest segmentation (n>5).

[0067] Figure 18. CT section showing an unlabelled hernia mesh (Xenmatrix, Bard) (right) and a hernia mesh that was labelled on the primary amino group lysine residues with 3 -(4- hydroxy-3 -iodophenyl )propionate (left). Samples were acquired at 50 kVp. The sample was washed for 72 hours with 3 changes of saline before imaging. Detailed description of the invention

[0068] Implantable scaffolds

[0069] The present invention is concerned with an implantable scaffold, which is suitable for application to a patient undergoing a surgical procedure. The type of surgical procedure for which the scaffold may be useful is not particularly limited, but is typically a method for repairing a tissue defect or reconstructing tissue, or for resection or ablation of a tumour (wherein the scaffold helps to guide the resection or ablation procedure). A method for repairing a tissue defect or reconstructing tissue may, for example, be a method for the repair of urogenital damage, skin wounds, bone defects, hernia, nerve damage, vascular damage, post-surgical reconstruction or cardiovascular disease.

[0070] As defined herein, the term “scaffold” refers to a three-dimensional structure to which biological material is able to adhere and grow around once the structure is implanted into a living body, in particular a human patient. The structural nature of the scaffold is not particularly limited, and may include a sheet, a tube, a mesh, a tubular structure (e.g. a nerve guide tube), a suture, a cardiac patch, a vascular graft, an injectable hydrogel or a decellularized tissue or organ. Thus, in some embodiments, the scaffold is selected from a sheet, a tube, a mesh, a tubular structure, a suture, a cardiac patch, a vascular graft, and an injectable hydrogel. Alternatively, the scaffold is a decellularized tissue or organ. In some embodiments, the scaffold is a micropatterned or a nanopattemed structure. In some preferred embodiments, the scaffold is a mesh.

[0071] A “mesh” is typically a porous three-dimensional structure comprising a network of struts and pores. Meshes find particular surgical use in repairing a tissue defect such as a hernia, skin wound or bone defect. In some embodiments, the scaffold is a porous 3-dimensional matrix, in particular a 3-dimensional protein matrix.

[0072] A “hydrogel” is a biphasic material, a mixture of porous, permeable solids and at least 10% by weight or volume of interstitial fluid composed completely or mainly by water. Hydrogels may be natural or synthetic and typically comprise polymeric materials. In particular, hydrogels for use in implanted scaffolds may comprise a polymer selected from hyaluronic acid, chitosan, heparin, alginate, gelatin, fibrin, polyvinyl alcohol, poly(ethylene glycol), sodium polyacrylate, acrylate polymers, poly(vinyl pyrrolidine) and copolymers thereof. The polymer chains in hydrogels may be chemically cross-linked (so- called chemical hydrogels) or may be non-covalently cross-linked by ionic bonding and / or hydrogen bonding (so-called physical hydrogels).

[0073] A “decellularized” tissue (or organ) is tissue (or organ) from which the extracellular matrix of a tissue (or organ) has been isolated from its inhabiting cells, leaving an extracellular matrix scaffold of the original tissue (or organ). This matrix can be used as a scaffold, particularly in artificial organ and tissue regeneration. Such extracellular matrices may typically comprise proteins that have undergone post-translational modifications, e.g. glycosylation.

[0074] The scaffold may optionally comprise a small molecule drug, cells or nanoparticles. Thus, in some embodiments, the scaffold comprises a small molecule drug. Alternatively, the scaffold comprises cells. Alternatively, the cell comprises nanoparticles or microparticles.

[0075] As used herein, the term “small molecule drug” refers to a chemical compound which has known biological effect on an animal, such as a human. Typically, drugs are chemical compounds which are used to treat, prevent or diagnose a disease. Preferred small molecule drugs are biologically active in that they produce a local or systemic effect in animals, preferably mammals, more preferably humans. The small molecule drug may be referred to as a “drug molecule” or “drug”. Typically, the drug molecule has Mw less than or equal to about 5 kDa. Preferably, the drug molecule has Mw less than or equal to about 1.5 kDa. A more complete, although not exhaustive, listing of classes and specific drugs suitable for use in the present invention may be found in “Pharmaceutical Substances: Syntheses, Patents, Applications” by Axel Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999 and the “Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals”, edited by Susan Budavari et al., CRC Press, 1996.

[0076] As used herein, the term “cells” refers to any cellular material that may have been extracted from an animal, in particular a human, or which have been cultured in vitro or ex vivo. The cells may be derived from any tissue or organ, including but not limited to cells derived from endothelium, blood, cartilage, muscle, tendon, ligament, lymph nodes, gastrointestinal tract, stomach, eosophagus, lung, trachea, larynx, heart, eye, nasal passage, throat, oral cavity, ear drum, brain, nerves, breast, cervix, uterus, ovary, vagina, prostate, colon, rectum, testes, penis, thyroid, kidney, pancreas, bone, spleen, liver, or bladder.

[0077] As used herein, the term “nanoparticle” refers to any particle between 1 and 100 nm in size. As used herein, the term “microparticle” refers to any particle between 0.1 and 100 pm in size. Suitable nanoparticles or microparticles include polymersomes, liposomes, synthosomes, latex, micelles, nanocrystals, quantum dots, metallic nanoparticles, oxide nanoparticles, silica nanoparticles, protein cages, nano and micro gels, dendrimers, virus-like particles, protein, polymers or any other colloidal materials that fall within the aforementioned size range. Typically, however, the nanoparticles or microparticles for use in the present invention are polymersomes, liposomes, synthosomes or micelles. Typically, the nanoparticles or microparticles are self-assembled structures. Nanoparticles and microparticles may be of any feasible geometry, e.g. substantially spherical, ellipsoidal, cylindrical or bilayer form, but typically they are substantially spherical. A typical (largest) diameter of a nanoparticle or microparticle of the present invention is in the range 50 to 5000 nm when measured by transmission electron microscopy (TEM).

[0078] The scaffolds of the present invention comprise a plurality of protein molecules. As used herein, the term “protein” refers to a biological molecule comprising polymers of amino acid monomers which are distinguished from peptides on the basis of size, and as an arbitrary benchmark can be understood to contain approximately 50 or more amino acids. Proteins consist of one or more polypeptides arranged in a biologically functional way, often bound to ligands such as coenzymes and cofactors, or to another protein or other macromolecule (DNA, RNA, etc.) or to complex macromolecular assemblies.

[0079] Typically, the scaffold will comprise a fibrous protein. Fibrous proteins are made up of elongated polypeptide chains which form filamentous and / or sheet-like structures. These types of protein are particularly suitable for generating 3D scaffolds because of their elongate nature and because (in contrast to globular proteins) they typically have low solubility in water. A fibrous protein typically forms an aggregate due to hydrophobic amino acid side chains that protrude from the molecule. Fibrous proteins are also typically more resistant to denaturation than globular proteins. In nature, fibrous proteins serve protective and structural roles by forming connective tissue, tendons, bone matrices and muscle fiber.

[0080] Preferably, the fibrous protein is selected from collagen, keratin, silk, eggshell membrane, elastin, fibrin, extracellular matrix proteins, and the protein components of decellularized tissue or organs. Most preferably, the fibrous protein is collagen.

[0081] Collagen is the most abundant protein in mammals (making up 25-35% of the whole-body protein content) and is the main structural protein in the extracellular matrix found in the body’s various connective tissues. It is mostly found in connective tissue such as cartilage, bones, tendons, ligaments, and skin. Collagen consists of amino acids bound together to form a triple helix of elongated fibril known as a collagen helix. Many different types of collagen have been identified. The five most common types are:

[0082] Type I (fibrous): This is the most abundant collagen of the human body. It is found in tendons, skin, artery walls, cornea, the endomysium surrounding muscle fibers, fibrocartilage, and the organic part of bones and teeth. It is also present in scar tissue, the end product when tissue heals by repair.

[0083] Type II (fibrous): This makes up 50% of all cartilage protein, and is also present in the vitreous humour of the eye.

[0084] Type III (fibrous): This is the collagen of granulation tissue and is produced quickly by young fibroblasts before the tougher type I collagen is synthesized. It is also the main component of reticular fibres. It is also found in artery walls, skin, intestines and the uterus.

[0085] Type IV (non-fibrous): This type of collagen forms basal lamina, the epithelium- secreted layer of the basement membrane. It is also found in eye lens, and serves as part of the filtration system in capillaries and the glomeruli of nephron in the kidney.

[0086] Type V (fibrous): Found in interstitial tissue, and often associated with type I collagen. It is found in cell surfaces, hair and placenta.

[0087] In some embodiments, collagen may be type I collagen. Alternatively, collagen may be type II collagen. Alternatively, collagen may be type III collagen. Alternatively, collagen may be type IV collagen. Alternatively, collagen may be type V collagen. Alternatively, collagen may be a mixture of different types of collagen.

[0088] Keratin is one of a family of structural fibrous proteins also known as scleroproteins. Alpha-keratin (a-keratin) is a type of keratin found in vertebrates. It is the key structural material making up scales, hair, nails, feathers, horns, claws, hooves, and the outer layer of skin among vertebrates. Keratin also protects epithelial cells from damage or stress. Keratin is insoluble in water and organic solvents. Keratin monomers assemble into bundles to form intermediate filaments, which are tough. There are two main forms of keratin, the primitive, softer forms found in all vertebrates and harder, derived forms found only among sauropsids (i.e. reptiles and birds).

[0089] Silk is a natural protein fibre, some forms of which can be woven into textiles. The protein fibre of silk is composed mainly of fibroin and is produced by certain insect larvae to form cocoons. The best-known silk is obtained from the cocoons of the larvae of the mulberry silkworm Bombyx mori. Silk comprises twisted P-pleated sheets incorporated into fibers wound into larger supermolecular aggregates. The properties of silk fibers depend on the organization of multiple adjacent protein chains into hard, crystalline regions of varying size, alternating with flexible, amorphous regions where the chains are randomly coiled.

[0090] Eggshell membrane is a composition of fibrous protein, in particular collagen type I, but further comprises glycosaminoglycans (e.g. dermatan sulfate, chondroitin sulfate), and sulfated glycoproteins (e.g. glucosamine), and optionally components such as hyaluronic acid, sialic acid, desmosine, isodesmosine, ovotransferrin, lysyl oxidase, lysozyme and / or P-7V-acetylglycosaminidase.

[0091] Elastin is a protein which is a key component of the extracellular matrix in gnathostomes (jawed vertebrates). It is highly elastic and present in connective tissue allowing many tissues in the body to resume their shape after stretching or contracting, e.g. elastin helps skin to return to its original position when it is poked or pinched. Elastin is also an important load-bearing tissue in the bodies of vertebrates and used in places where mechanical energy is required to be stored. Elastin is rich in hydrophobic amino acids such as glycine and proline, which form mobile hydrophobic regions bounded by crosslinks between lysine residues. Fibrin (also known as Factor la) is a fibrous, non-globular protein involved in the clotting of blood. It is formed by the action of the protease thrombin on fibrinogen, which causes it to polymerize. The polymerized fibrin forms long strands of tough insoluble protein; these bind to platelets to form a hemostatic plug or clot over a wound site. Factor XIII completes the cross-linking of fibrin so that it hardens and contracts. The cross-linked fibrin forms a mesh atop the platelet plug that completes a blood clot.

[0092] The protein may be soluble or insoluble in water. However, typically, the protein is insoluble in water. Such insoluble proteins are typically prepared or isolated from a solution phase under conditions which have promoted the precipitation or aggregation of the protein from the solution in a manner that is not readily reversible. In this context, “insoluble in water” typically refers to a protein with solubility below 5 mg / mL at room or body temperature and atmospheric pressure in solutions between pH 5 and 9.

[0093] The implantable scaffold typically comprises at least 30% (w / w) protein molecules. Preferably, the implantable scaffold comprises at least 40% (w / w) protein molecules.

[0094] More preferably, the implantable scaffold comprises at least 50% (w / w) protein molecules. Even more preferably, the implantable scaffold comprises at least 60% (w / w) protein molecules. Yet more preferably, the implantable scaffold comprises at least 70% (w / w) protein molecules. Still more preferably, the implantable scaffold comprises at least 75% (w / w) protein molecules. Most preferably, the implantable scaffold comprises at least 80% (w / w) protein molecules. In some embodiments, the implantable scaffold may comprise at least 85% (w / w) protein molecules, at least 90% (w / w) protein molecules, or at least 95% (w / w) protein molecules.

[0095] The implantable scaffold may additionally comprise any other biocompatible materials, i.e. materials that are non-toxic and do not typically cause any biological reaction in vivo.

[0096] Such materials may be biodegradable or non-biodegradable. Materials that may be present in the implantable scaffold include metals (e.g. stainless steel; iron, gold, Co-Cr or titanium alloys), ceramic materials (e.g. hydroxyapatite, tricalcium phosphate, calcium sulfate, aluminium oxide or zirconium oxide) and synthetic polymers (e.g. polyethylene, polyamide, polymethylmethacrylate, polytetrafluoroethylene or polyurethane). The implantable scaffold may comprise a mixture of further biocompatible materials in addition to the plurality of protein molecules. Typically, the plurality of protein molecules in the scaffold are overlapping. That is to say that protein molecules are able to interact with adjacent protein molecules in the scaffold either covalently or non-covalently. Thus, in some embodiments, covalent bonds are formed between adjacent protein molecules in the scaffold. Preferably, a covalent bond between adjacent protein molecules is a disulfide bond between two cysteine residues or a glutaraldehyde cross-link between two amino acid residues with a nucleophilic side chain (e.g. lysine or arginine). In other embodiments, non-covalent interactions are formed between adjacent protein molecules between adjacent protein molecules in the scaffold. Non-covalent interactions typically include ionic bonds, hydrogen bonds and van der Waals interactions. Thus, typically, the plurality of protein molecules are assembled into a scaffold via non-covalent and / or covalent interactions between adjacent protein molecules.

[0097] In the implantable scaffolds of the present invention, at least one of said plurality of protein molecules comprises at least one aromatic amino acid residue that is substituted with iodine on its aromatic moiety.

[0098] As used herein, the term “aromatic amino acid” refers to any natural or synthetic amino acid (that is, an organic compound comprising both amino (-NH2) and carboxylic acid (-COOH) functional groups) which contains an aromatic moiety. In a typical protein molecule, the aromatic moiety is present in the amino acid side chain, and not in the backbone of the protein polymer. Typically, the at least one amino acid is an a-amino acid. Alternatively, the at least one amino acid is a P-, y- or 6-amino acid. Typically the at least one aromatic amino acid is a naturally occurring amino acid selected from tryptophan, tyrosine, histidine, phenylalanine and combinations thereof. Alternatively, the amino acid is a synthetic amino acid selected from 4-aminobenzoic acid, hydroxytryptophan, 4-acetylphenylalanine, 4-azidophenylalanine, 4- azidomethylphenylalanine, 4-ethynylphenylalanine and 4-propargyloxyphenylalanine. Alternatively still, the amino acid is a synthetic (i.e. non-natural) amino acid which is a modified lysine or ornithine, preferably a modified lysine, wherein the primary amino group in the side chain of the naturally occurring amino acid residue is covalently bonded, via an amide bond (i.e. via a -(C=O)- linker) to a Ce-Cio aryl or Ce-Cio aralkyl group, which is substituted with one or more iodine atoms (preferably, one or two iodine atoms) on the aromatic ring and which may be further optionally substituted with a hydroxy group on the aromatic ring. In other words, the naturally occurring lysine or ornithine residue has been modified by replacement of one of the hydrogen atoms on the side chain -NH2 moiety with the group -(C=O)R, wherein R is a Ce-Cio aryl or Ce-Cio aralkyl group, which is substituted with one or more iodine atoms (preferably, one or two iodine atoms) on the aromatic ring and which may be further optionally substituted with a hydroxy group on the aromatic ring. An amino acid which possess a stereogenic centre may be present as a single enantiomer or as a mixture of enantiomers (e.g. a racemic mixture). Preferably, if the amino acid is an a-amino acid, the amino acid has L stereochemistry about the a- carbon stereogenic centre.

[0099] As defined herein, the term “alkyl” refers to a linear or branched saturated monovalent hydrocarbon radical. Preferably, an alkyl group is a C1-C12 alkyl group, more preferably a C1-C4 alkyl group, e.g. methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, and the like.

[0100] As defined herein, the term “aryl” refers to a monovalent monocyclic or bicyclic aromatic hydrocarbon radical of 6 to 10 ring atoms, e.g. phenyl or naphthyl, and the like.

[0101] As defined herein, the term “alkylene” refers to a linear saturated divalent hydrocarbon radical or a branched saturated divalent hydrocarbon radical having the number of carbon atoms indicated in the prefix, e.g. methylene, ethylene, propylene, 1 -methylpropylene, 2- methylpropylene, butylene, pentylene, and the like. Preferably, an alkylene group is a Ci- C4 alkylene group, and more preferably a C2 alkylene group.

[0102] As defined herein, the term “aralkyl” refers to an -(alkylene)-R’ radical where R is aryl as defined above.

[0103] Preferably, the at least one amino acid is selected from tyrosine, histidine and a combination thereof.

[0104] Thus, in some embodiments, at least one of said plurality of protein molecules in the implantable scaffold comprises at least one tyrosine residue that is substituted with iodine on its aromatic moiety. Tyrosine residues may be substituted with one or two iodine atoms on its aromatic moiety. Typically, an iodinated tyrosine residue comprises two iodine substituents on its aromatic moiety. Alternatively, an iodinated tyrosine residue comprises one iodine substituent on its aromatic moiety. Therefore, at least one of said plurality of protein molecules in the implantable scaffold may comprise at least one tyrosine residue that is substituted with a single iodine substituent on its aromatic moiety. Preferably, at least one of said plurality of protein molecules in the implantable scaffold may comprise at least one tyrosine residue that is substituted with two iodine substituents on its aromatic moiety.

[0105] In such embodiments, typically at least 10% of the tyrosine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, at least 10% of the tyrosine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety. Alternatively, at least 10% of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety. Preferably, at least 20% of the tyrosine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, at least 20% of the tyrosine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety. Alternatively, at least 20% of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety. More preferably, at least 30% of the tyrosine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, at least 30% of the tyrosine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety. Alternatively, at least 30% of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety. Still more preferably, at least 40% of the tyrosine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, at least 40% of the tyrosine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety. Alternatively, at least 40% of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety. Most preferably, at least 50% of the tyrosine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, at least 50% of the tyrosine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety. Alternatively, at least 50% of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety. Typically, up to 100% (e.g. up to 90%, or up to 80%) of the tyrosine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, up to 100% (e.g. up to 90%, or up to 80%) of the tyrosine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety. Alternatively, up to 100% (e.g. up to 90%, or up to 80%) of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety.

[0106] In some embodiments, at least one of said plurality of protein molecules in the implantable scaffold comprises at least one histidine residue that is substituted with iodine on its aromatic moiety. Typically, the histidine residue is substituted with a single iodine substituent.

[0107] In such embodiments, typically at least 10% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, at least 10% of the histidine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety. Preferably, at least 20% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, at least 20% of the histidine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety. More preferably, at least 30% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, at least 30% of the histidine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety. Even more preferably, at least 40% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, at least 40% of the histidine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety. Most preferably, at least 50% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, at least 50% of the histidine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety.

[0108] Typically, up to 100% (e.g. up to 90%, or up to 80%) of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, up to 100% (e.g. up to 90%, or up to 80%) of the histidine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety.

[0109] In some embodiments, at least one of said plurality of protein molecules in the implantable scaffold comprises at least one tyrosine residue that is substituted with iodine on its aromatic moiety and at least one histidine residue that is substituted with iodine on its aromatic moiety. Typically, the iodinated tyrosine residue comprises two iodine substituents on its aromatic moiety and the iodinated histidine residue comprises a single iodine substituent on its aromatic moiety. Alternatively, the iodinated tyrosine residue comprises a single iodine substituent on its aromatic moiety and the iodinated histidine residue comprises a single iodine substituent on its aromatic moiety. Therefore, at least one of said plurality of protein molecules in the implantable scaffold may comprise at least one tyrosine residue that is substituted with a single iodine substituent on its aromatic moiety and at least one histidine residue that is substituted with a single iodine substituent on its aromatic moiety. Preferably, at least one of said plurality of protein molecules in the implantable scaffold may comprise at least one tyrosine residue that is substituted with two iodine substituents on its aromatic moiety and at least one histidine residue that is substituted with a single iodine substituent on its aromatic moiety.

[0110] In such embodiments, typically at least 10% of the tyrosine residues and at least 10% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, at least 10% of the tyrosine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety and at least 10% of the histidine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety. Alternatively, at least 10% of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety and at least 10% of the histidine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety. Preferably, at least 20% of the tyrosine residues and at least 20% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, at least 20% of the tyrosine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety and at least 20% of the histidine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety. Alternatively, at least 20% of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety and at least 20% of the histidine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety. More preferably, at least 30% of the tyrosine residues and at least 30% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, at least 30% of the tyrosine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety and at least 30% of the histidine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety. Alternatively, at least 30% of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety and at least 30% of the histidine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety. Still more preferably, at least 40% of the tyrosine residues and at least 40% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, at least 40% of the tyrosine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety and at least 40% of the histidine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety. Alternatively, at least 40% of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety and at least 40% of the histidine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety. Most preferably, at least 50% of the tyrosine residues and at least 50% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, at least 50% of the tyrosine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety and at least 50% of the histidine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety. Alternatively, at least 50% of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety and at least 50% of the histidine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety.

[0111] Typically, up to 100% (e.g. up to 90%, or up to 80%) of the tyrosine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety and up to 100% (e.g. up to 90%, or up to 80%) of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, up to 100% (e.g. up to 90%, or up to 80%) of the tyrosine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety and up to 100% (e.g. up to 90%, or up to 80%) of the histidine residues in the plurality of protein molecules are substituted with a single iodine substituent on their aromatic moiety. Alternatively, up to 100% (e.g. up to 90%, or up to 80%) of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety and up to 100% (e.g. up to 90%, or up to 80%) of the histidine residues in the plurality of protein molecules are substituted with a single iodine substituent on their aromatic moiety.

[0112] The proportion of tyrosine and / or histidine residues in the protein molecules that are iodinated may depend on the identity of the protein, as different proteins contain different proportions of aromatic amino acid residues. For example, silk is a protein that is rich in amino acids such as tyrosine, and therefore a smaller proportion of the tyrosine residues need to be iodinated in order to achieve the desired level of radiopacity of the scaffold. The skilled person is readily able to determine an appropriate level of aromatic amino acid residues to iodinate for any given protein.

[0113] Alternatively, or in addition, the at least one amino acid may be a synthetic (i.e. nonnatural) amino acid. In particular, the at least one amino acid may be a synthetic amino acid which is a modified lysine or ornithine wherein the primary amino group in the side chain of the naturally occurring amino acid residue is covalently bonded, via an amide bond (i.e. via a -(C=O)- linker) to a Ce-Cio aryl or Ce-Cio aralkyl group, which is substituted with one or more iodine atoms (preferably, one or two iodine atoms) on the aromatic ring and which may be further optionally substituted with a hydroxy group on the aromatic ring. Preferably in this embodiment the synthetic amino acid is a modified lysine. Preferably in this embodiment, the at least one amino acid has formula (I): wherein:

[0114] (i) X is H or OH;

[0115] (ii) n is 1 or 2;

[0116] (iii) m is an integer from 0 to 4; and

[0117] (iv) * andArepresent points of attachment of the aromatic amino acid residue to adjacent amino acid residues in the protein.

[0118] Preferably in this embodiment, the at least one amino acid has formula (la) or formula (lb):

[0119] More preferably in this embodiment, m is 0 or 2. More preferably, when m is 0, X is H.

[0120] More preferably, when m is 2, X is OH.In such embodiments, typically at least 10% of the lysine residues in the plurality of protein molecules are modified lysine residues which are modified as described herein (i.e. wherein the primary amino group in the side chain of the naturally occurring amino acid residue is covalently bonded, via an amide bond (i.e. via a -(C=O)- linker) to a Ce-Cio aryl or Ce-Cio aralkyl group, which is substituted with one or more iodine atoms (preferably, one or two iodine atoms) on the aromatic ring and which may be further optionally substituted with a hydroxy group on the aromatic ring, including preferable embodiments). Thus, at least 10% of the lysine residues in the plurality of protein molecules are modified as described herein and may comprise a single iodine substituent. Alternatively, at least 10% of the lysine residues in the plurality of protein molecules are modified as described herein and comprise two iodine substituents. Preferably, at least 20% of the lysine residues in the plurality of protein molecules are modified lysine residues which are modified as described herein and are substituted with iodine. Thus, at least 20% of the lysine residues in the plurality of protein molecules are modified as described herein and may comprise a single iodine substituent. Alternatively, at least 20% of the lysine residues in the plurality of protein molecules are modified as described herein and comprise two iodine substituents. More preferably, at least 30% of the lysine residues in the plurality of protein molecules are modified lysine residues which are modified as described herein and are substituted with iodine. Thus, at least 30% of the lysine residues in the plurality of protein molecules are modified as described herein and may comprise a single iodine substituent. Alternatively, at least 30% of the lysine residues in the plurality of protein molecules are modified as described herein and comprise two iodine substituents. Still more preferably, at least 40% of the lysine residues in the plurality of protein molecules are modified lysine residues which are modified as described herein and are substituted with iodine. Thus, at least 40% of the lysine residues in the plurality of protein molecules are modified as described herein and may comprise a single iodine substituent. Alternatively, at least 40% of the lysine residues in the plurality of protein molecules are modified as described herein and comprise two iodine substituents. Most preferably, at least 50% of the lysine residues in the plurality of protein molecules are modified lysine residues which are modified as described herein and are substituted with iodine. Thus, at least 50% of the lysine residues in the plurality of protein molecules are modified as described herein and may comprise a single iodine substituent. Alternatively, at least 50% of the lysine residues in the plurality of protein molecules are modified as described herein and comprise two iodine substituents.

[0121] Typically, up to 100% (e.g. up to 90%, or up to 80%) of the lysine residues in the plurality of protein molecules modified as described herein and are substituted with iodine. Thus, up to 100% (e.g. up to 90%, or up to 80%) of the lysine residues in the plurality of protein molecules are modified as described herein and may comprise a single iodine substituent. Alternatively, up to 100% (e.g. up to 90%, or up to 80%) of the lysine residues in the plurality of protein molecules are modified as described herein and comprise two iodine substituent. The proportion of lysine residues in the protein which are modified and iodinated may depend on the identity of the protein, as different proteins contain different proportions of amino acid residues. Modification of lysine residues in the manner described herein is particularly advantageous for proteins having low tyrosine and / or histidine content. Typically, proteins contain a higher number of lysine residues than tyrosine residues (e.g. in human collagen Type I, there is a four-fold higher number of lysine residues than tyrosine residues (UniProt ref: P02452 C01A1 HUMAN). Modification and iodination of lysine residues therefore offers the ability to attach a higher number of iodines per protein (though, this is sequence specific) which can lead to a higher radiopacity of the scaffold. A high level of control can also be exercised over total iodine content of the scaffold when modifying lysine residues, as either mono- or di-iodinated modification (i.e. acylating) reagents can be used.

[0122] Moreover, in the specific case where the modified lysine residue is of formula (la), m is 0 and X is H, it may be possible to enhance stability of the iodinated scaffold (i.e. maintain a high radiopacity for a longer period of time) because compounds such as A-succinimidyl 3-[*I]iodobenzoate have been reported to have greater resistance to de-iodinase in vivo than either e.g. A-succinimidyl-3-(4-hydroxy-3-iodophenyl)propionate or iodo-tyrosine (see Nat. Protoc, 2006, 1 :707-713).

[0123] In some embodiments, a proportion of the lysine residues in the protein molecules may be modified as described herein and are substituted with iodine, and a proportion of the tyrosine and / or histidine residues are substituted with iodine on their aromatic moiety.

[0124] Thus, in these embodiments, at least one of said plurality of protein molecules in the implantable scaffold comprises at least one lysine residue that is modified as described herein and substituted with iodine and (i) at least one tyrosine residue that is substituted with iodine on its aromatic moiety and / or (ii) at least one histidine residue that is substituted with iodine on its aromatic moiety. Typically, the modified lysine residue comprises one iodine substituent, the iodinated tyrosine residue comprises two iodine substituents on its aromatic moiety and the iodinated histidine residue comprises a single iodine substituent on its aromatic moiety. Alternatively, the modified lysine residue comprises two iodine substituents, the iodinated tyrosine residue comprises two iodine substituents on its aromatic moiety and the iodinated histidine residue comprises a single iodine substituent on its aromatic moiety. Alternatively, the modified lysine residue comprises one iodine substituent, the iodinated tyrosine residue comprises a single iodine substituent on its aromatic moiety and the iodinated histidine residue comprises a single iodine substituent on its aromatic moiety. Alternatively, the modified lysine residue comprises two iodine substituents, the iodinated tyrosine residue comprises a single iodine substituent on its aromatic moiety and the iodinated histidine residue comprises a single iodine substituent on its aromatic moiety.

[0125] In such embodiments, typically at least 10% of the lysine residues in the plurality of protein molecules are modified lysine residues which are modified as described herein and are substituted with iodine and (i) at least 10% of the tyrosine residues and / or (ii) at least 10% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Preferably, at least 20% of the lysine residues in the plurality of protein molecules are modified lysine residues which are modified as described herein and are substituted with iodine and (i) at least 20% of the tyrosine residues and / or (ii) at least 20% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. More preferably, at least 30% of the lysine residues in the plurality of protein molecules are modified lysine residues which are modified as described herein and are substituted with iodine and (i) at least 30% of the tyrosine residues and / or (ii) at least 30% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Still more preferably, at least 40% of the lysine residues in the plurality of protein molecules are modified lysine residues which are modified as described herein and are substituted with iodine and (i) at least 40% of the tyrosine residues and / or (ii) at least 40% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Most preferably, at least 50% of the lysine residues in the plurality of protein molecules are modified lysine residues which are modified as described herein and are substituted with iodine and (i) at least 50% of the tyrosine residues and / or (ii) at least 50% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety.

[0126] Typically, up to 100% (e.g. up to 90%, or up to 80%) of the lysine residues in the plurality of protein molecules are modified lysine residues which are modified as described herein and (i) up to 100% (e.g. up to 90%, or up to 80%) of the tyrosine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety and / or (ii) up to 100% (e.g. up to 90%, or up to 80%) of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety.

[0127] The iodine substituent may be any isotopic form of iodine but is preferably a nonradioactive isotope of iodine. Thus, preferably, the iodine substituent is127I. Alternatively, however, the iodine substituent may be a radioactive isotope of iodine selected from108I,109I,110I,mI,112I,113I,114I,115I,116I,117I,1181,119I,120I,1211,122I,123I, 124j 125j 126j 128j 129j 130j 131j 132j 133j 134j 135j 136j 137j 138j 139j 140j 141j 142j 143janj144I, optionally selected from123I,124I,125I and131I.

[0128] Advantageously, the present invention allows for the first time a sufficient level of iodine labelling of a protein scaffold such that the labelled material is readily visible using in vivo X-ray imaging (e.g. CT scanning or planar X-ray scanning). In particular, the present invention provides an implantable scaffold which typically has a radi opacity of 100 Hounsfield Units (HU) or greater, and therefore is imageable in vivo against background soft tissue, which typically has a radiopacity of from 0 to 50 HU. In some embodiments, the scaffold has a radi opacity of from 100 to 10,000 HU, preferably from 150 to 5,000 HU, and more preferably from 200 to 1,000 HU. Radi opacity is typically measured using a CT scan, preferably using X-rays with a peak potential of 50 kVp. For example, radiopacity can be measured using a PET-CT system (Mediso nanoScan®) with a semi-circular scan at 50 kVp with 170ms exposure, 720 projections, 1 :4 binning, and medium FOV setting. Region of interest analysis can be performed using VivoQuant software (Invicro) to quantify X-ray absorbance. Typically, the radiopacity of the implantable scaffold is such that the scaffold can be visualised in vivo with X-rays having a peak kilovoltage from 30 kVp to 200 kVp, e.g. from 50 kVp to 100 kVp.

[0129] In contrast to radionuclide-based polymer labelling methods, CT imaging offers the advantage of requiring a lower patient radiation dose, while overcoming the limitations associated with label half-life, thereby enabling imaging at significantly later time points post-transplantation. The ability to visualise the labels persists for at least several months, if not years, which advantageously enables long-term tracking of the scaffold materials. This feature is particularly valuable when considering implants like hernia meshes, which need to remain in place for extended periods.

[0130] Iodine labelling is furthermore a minimal structural modification of the protein material and so does not affect its natural function. Thus, the iodine labelled scaffolds described herein exhibit advantages over protein scaffolds labelled with other optical labels, e.g. fluorescent tags or dyes that are conjugated to the protein via bioconjugation / click chemistry methods. These methods have had some success with labelling of small soluble proteins, but have limited utility in implantable scaffolds that have a defined 3D macromolecular structure that is integral to its function.

[0131] The ability to selectively label tyrosine residues, as is possible with the scaffolds of the present invention, also has advantages over other more-established protein labelling strategies which often rely on labelling of cysteine and lysine residues, as these nucleophilic amino acid residues often have an inherent biological reactivity and labelling them may disrupt biological function in a manner which does not occur with the labelling of tyrosine residues. Tyrosine (and / or histidine) labelling is therefore a widely applicable strategy to many types of protein. Nonetheless, where lysine labelling is possible without disrupting natural protein function, modification in the manner discussed herein to introduce iodine substituents to lysine residues can be a beneficial approach for the reasons set out above.

[0132] With hernia meshes being among the most established clinically used biomaterials (~20 million implantations per year globally), their improved radiological visibility demonstrated here could potentially improve healthcare outcomes for a considerable population. Complication rates for implanted meshes often exceed 10%, reaching above 60% where comorbidities such as diabetes, obesity, chronic heart disease, and infection, increase relapse or complication risks. This can include infection, perforation, tearing, detachment, and mesh migration, typically necessitating either removal, reattachment, or repair. The similarity of biological meshes to native tissue frustrates their discrimination when using CT, or indeed visually during surgery. The ability to non-invasively and longitudinally visualise mesh placement in patients over time will likely therefore improve diagnosis of complications, and surgical planning to correct these. Currently, the only commercially available contrast-enhanced hernia mesh is a synthetic (PVDF - polyvinylidene difluoride) material, loaded with iron-oxide nanoparticles that reduce signal intensity on MR imaging (see Lechner et aL, Hernia, 2019, 23(6): 1133- 1140). While this labelling and imaging approach increases visibility of the implant and its shrinkage, MRI is used much less frequently than CT in the clinic for hernia patients. In particular, smaller bores and lower patient weight limits vs CT mean that obese patients, a significant target for mesh therapy, often cannot be imaged with MRI. Furthermore, CT is also generally more available, faster and less expensive.

[0133] Embodiments of the present invention in which the protein molecules are silk may have particular benefits in certain applications. Though silk is most commonly used as a surgical material, a variety of cellular and acellular regenerative therapies are now emerging, including tendon and ligament grafts, cardiac patches, and vascular constructs. Applying the iodine labelling of the present invention, silk may exhibit a 6-fold higher radiopacity than decellularised porcine dermis, in line with the 6-fold higher tyrosine content of silk vs collagen. While previous attempts to produce radiopaque silk have decreased its tensile strength by several fold (see e.g. Francis et al., ACS Biomaterials, 2016, 2(2): 188-196), the present iodine labelling method allows silk to retain its prized mechanical properties.

[0134] Further embodiments in which the protein molecules are egg shell membrane also have great potential in regenerative medicine applications, as egg shell membrane has recently found uses including the repair of damaged ear drums, nerves, cartilage, and cardiovascular defects.

[0135] Methods of using the implantable scaffolds

[0136] The implantable scaffolds of the present invention have a range of applications in medicine and surgery. In particular, the implantable scaffold finds utility in methods of repairing a tissue defect or reconstructing tissue. In such methods, the ability to visualise the scaffolds against background tissue assists in surgery because a medical practitioner can determine whether the scaffold has attached to the correct location in vivo, and corrective action can be taken if not. Thus, the present invention provides a method of using an implantable scaffold as described herein for repairing a tissue defect or reconstructing tissue, wherein said method comprises positioning the implantable scaffold in or on a subject such that the scaffold extends across, or adjacent to, all or a proportion of the tissue defect or tissue to be reconstructed. Thus, in some embodiments, the scaffold extends across all or a proportion of the tissue defect or tissue to be reconstructed. In other embodiments, the scaffold is adjacent to all or a proportion of the tissue defect or tissue to be reconstructed. In some embodiments, the scaffold extends across, or is adjacent to, all of the tissue defect or tissue to be reconstructed. In other embodiments, the scaffold extends across, or is adjacent to, a proportion of the tissue defect or tissue to be reconstructed. Typically “a proportion of’ the tissue defect or tissue to be recovered means that the scaffold extends across, or is adjacent to (as the case may be) at least 5% of the tissue defect or tissue to be reconstructed, preferably at least 10%, more preferably at least 20%, still more preferably at least 30%, even more preferably at least 50%, yet more preferably at least 75%, and most preferably at least 90%.

[0137] Example methods of repairing a tissue defect or reconstructing tissue include methods for the repair of urogenital damage, skin wounds, bone defects, hernia, nerve damage, vascular damage, post-surgical reconstruction and cardiovascular disease. Thus, in some embodiments, the method is a method of the repair of urogenital damage. In other embodiments, the method is a method of the repair of skin wounds. In other embodiments, the method is a method of the repair of bone defects. In other embodiments, the method is a method of the repair of a hernia. In other embodiments, the method is a method of the repair of nerve damage. In other embodiments, the method is a method of the repair of vascular damage. In other embodiments, the method is a method of the repair of post- surgical reconstruction. In other embodiments, the method is a method of the repair of cardiovascular disease. In a particularly preferred embodiment, the method is a method for the repair of a hernia.

[0138] Typically, the method further comprises visualising the implanted scaffold using X-rays, e.g. a computed tomography (CT) scan or a planar X-ray scan. The radiopacity of the scaffold (as a consequence of the iodination of the protein molecules) ensures that the scaffold can be visualised against adjacent native tissue which contains non-iodinated protein.

[0139] The method may further comprise a surgical intervention following the visualisation of the implantable scaffold. Such a surgical intervention may comprise repositioning, reattachment, replacement or removal of the scaffold. Removal of the implant may be carried out in order to replace the implant with a new implant. A new implant may be similar in size, bigger, or smaller than the original implant. A new implant may be made of the same material as the original implant, or may be made of a different material. Repositioning of the implant may optionally include of re-attachment of an implant that has become dislocated from its original location.

[0140] The implantable scaffold also finds utility in methods of guiding resection or ablation a tumour. Such methods may involve resection of a tumour. Alternatively, such methods may involve ablation of a tumour. In such methods, the scaffold acts as a surgical marker that assists in locating identified tumours to guide resection or ablation procedures such as gamma-knife therapy, proton beam therapy, hyperthermia therapy, or cryo-ablation.

[0141] The present invention therefore also provides a method of using an implantable scaffold as described herein for resection or ablation of a tumour, wherein said method comprises positioning the implantable scaffold in or on a subject such that the scaffold extends across, or adjacent to, all or a proportion of tumour to be resected or ablated. Thus, in some embodiments, the scaffold extends across all or a proportion of the tumour to be resected or ablated. In other embodiments, the scaffold is adjacent to all or a proportion of the tumour to be resected or ablated. In some embodiments, the scaffold extends across, or is adjacent to, all of tumour to be resected or ablated. In other embodiments, the scaffold extends across, or is adjacent to, a proportion of tumour to be resected or ablated. Preferably, the scaffold extends across, or is adjacent to, a proportion of the tumour to be resected or ablated. Typically “a proportion of’ the tumour to be resected or ablated means that the scaffold extends across, or is adjacent to (as the case may be) at least 5% of the tissue defect or tissue to be reconstructed, preferably at least 10%, more preferably at least 20%. In some embodiments, the scaffold extends across, or is adjacent to, at least 30%, or at least 50%, or at least 75%, or at least 90%, of the tumour to be resected or ablated. The present invention further provides a protein comprising at least one aromatic amino acid residue that is substituted with iodine on its aromatic moiety, for use in a method of repairing a tissue defect or reconstructing tissue, or a method for resection or ablation of a tumour, comprising:

[0142] (i) incorporating said protein into an implantable scaffold;

[0143] (ii) positioning said implantable scaffold in or on a subject such that the scaffold extends across, or adjacent to, all or a proportion of the tissue defect or tissue to be reconstructed or the tumour to be resected or ablated; and

[0144] (iii) visualising the implanted scaffold using a computed tomography (CT) scan or a planar X-ray scan.

[0145] The protein for use in such a method may be any of the proteins described herein.

[0146] The at least one aromatic amino acid may be any of the aromatic acids as described herein.

[0147] The method of repairing a tissue defect or reconstructing tissue, or the method for resection or ablation of a tumour, may be any of the methods as described herein.

[0148] The method may in particular further comprise a surgical intervention following the visualisation of the implantable scaffold. Such a surgical intervention may comprise repositioning, reattachment, replacement or removal of the scaffold. Removal of the implant may be carried out in order to replace the implant with a new implant. A new implant may be similar in size, bigger, or smaller than the original implant. A new implant may be made of the same material as the original implant, or may be made of a different material. Repositioning of the implant may optionally include of re-attachment of an implant that has become dislocated from its original location.

[0149] The present invention further provides the use of a protein comprising at least one aromatic amino acid residue that is substituted with iodine on its aromatic moiety for the manufacture of a medicament for application in a method of repairing a tissue defect or reconstructing tissue, or a method for resection or ablation of a tumour, comprising:

[0150] (i) incorporating said protein into an implantable scaffold; (ii) positioning said implantable scaffold in or on a subject such that the scaffold extends across, or adjacent to, all or a proportion of the tissue defect or tissue to be reconstructed or the tumour to be resected or ablated; and

[0151] (iii) visualising the implanted scaffold using a computed tomography (CT) scan or a planar X-ray scan.

[0152] The protein for use in such a method may be any of the proteins described herein.

[0153] The at least one aromatic amino acid may be any of the aromatic acids as described herein.

[0154] The method of repairing a tissue defect or reconstructing tissue, or the method for resection or ablation of a tumour, may be any of the methods as described herein.

[0155] The method may in particular further comprise a surgical intervention following the visualisation of the implantable scaffold. Such a surgical intervention may comprise repositioning, reattachment, replacement or removal of the scaffold. Removal of the implant may be carried out in order to replace the implant with a new implant. A new implant may be similar in size, bigger, or smaller than the original implant. A new implant may be made of the same material as the original implant, or may be made of a different material. Repositioning of the implant may optionally include of re-attachment of an implant that has become dislocated from its original location.

[0156] Methods of preparing the implantable scaffolds

[0157] The implantable scaffolds of the present invention can be prepared either by carrying out an iodination reaction on a pre-formed scaffold comprising a plurality of protein molecules, or alternatively, the protein molecules may be iodinated prior to assembly of the protein molecules into the implantable scaffold.

[0158] The iodination reaction may be carried out under any conditions suitable for electrophilic substitution of iodine on aromatic amino acid residues, which do not lead to denaturation of the protein. However, the present inventors have also surprisingly found specific conditions that can be utilised to iodinate an implantable scaffold comprising a plurality of protein molecules, under mild conditions such that the proteins are not denatured but the extent of iodination sufficiently increases the radiopacity of the protein so that it is useful in the visualisation of protein scaffolds. In particular, the present inventors have found that an implantable scaffold comprising a plurality of protein molecules, or molecules of an insoluble protein in solution, can be effectively iodinated on their aromatic amino acid residues using either potassium triiodide (KI3; also referred to as Lugol’s solution) or iodine monochloride.

[0159] A: Methods for direct or indirect iodination of a protein scaffold

[0160] Thus, the present invention provides a method of iodinating a protein scaffold, wherein said protein scaffold comprises a plurality of protein molecules, comprising:

[0161] (i) contacting the scaffold with a buffer solution; and

[0162] (ii) incubating said solution with KI3 or IC1 under suitable conditions to result in iodination of the aromatic moiety of at least one aromatic amino acid residue in at least one of the plurality of protein molecules.

[0163] In some embodiments, the present invention provides a method of iodinating a protein scaffold, wherein said protein scaffold comprises a plurality of protein molecules, comprising:

[0164] (i) contacting the scaffold with a buffer solution; and

[0165] (ii) incubating said solution with KI3 under suitable conditions to result in iodination of the aromatic moiety of at least one aromatic amino acid residue in at least one of the plurality of protein molecules.

[0166] The scaffold can be any of the implantable scaffolds described herein.

[0167] The nature of the buffer solution is not particularly limited. The skilled person is able to select an appropriate buffer solution in which to perform the reaction. Preferably, though, the buffer solution has a pH of from 7 to 9, for example from 7 to 8 or from 8 to 9. More preferably, the buffer solution has a pH of about 8. Preferably the buffer is selected from phosphate-buffered saline (PBS) and 4-(2 -hydroxy ethyl)- 1 -piperazineethanesulfonic acid solution (HEPES).

[0168] In such methods, the at least one aromatic amino acid residue is any aromatic amino acid residue described herein. However, typically the amino acid residue in tyrosine. Thus, in some embodiments, such methods result in iodination of the aromatic moiety of tyrosine in at least one of the plurality of protein molecules. The tyrosine residues may be substituted with one or two iodine atoms on its aromatic moiety. Typically, such methods lead to diiodination of each tyrosine residue. Alternatively, such methods lead to mono-iodination of each tyrosine residue.

[0169] Therefore, in such methods, typically at least 10% of the tyrosine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Typically, at least 10% of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety. Preferably, at least 20% of the tyrosine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Typically, at least 20% of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety. More preferably, at least 30% of the tyrosine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Typically, at least 30% of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety. Still more preferably, at least 40% of the tyrosine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Typically, at least 40% of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety. Most preferably, at least 50% of the tyrosine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Typically, at least 50% of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety.

[0170] Typically, up to 100% (e.g. up to 90%, or up to 80%) of the tyrosine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, up to 100% (e.g. up to 90%, or up to 80%) of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety.

[0171] The KI3 reagent may comprise any isotopic form of iodine but preferably comprises a nonradioactive isotope of iodine. Thus, preferably, the KI3 reagent comprises127I. In other embodiments, the present invention provides a method of iodinating a protein scaffold, wherein said protein scaffold comprises a plurality of protein molecules, comprising:

[0172] (i) contacting the scaffold with a buffer solution; and

[0173] (ii) incubating said solution with IC1 under suitable conditions to result in iodination of the aromatic moiety of at least one aromatic amino acid residue in at least one of the plurality of protein molecules.

[0174] The scaffold can be any of the implantable scaffolds described herein.

[0175] The nature of the buffer solution is not particularly limited. The skilled person is able to select an appropriate buffer solution in which to perform the reaction. Preferably, though, the buffer solution has a pH of from 7 to 10, for example from 7 to 9 or from 8 to 10. More preferably, the buffer solution has a pH of from 8 to 9. Preferably the buffer is selected from glycine solution and tris(hydroxymethyl)aminomethane solution (TRIS).

[0176] In such methods, the at least one aromatic amino acid residue is any aromatic amino acid residue described herein. However, typically in such methods both tyrosine and histidine residues are iodinated. Thus, in some embodiments, such methods result in iodination of the aromatic moiety of tyrosine in at least one of the plurality of protein molecules and the aromatic moiety of histidine in at least one of the plurality of protein molecules. The tyrosine residues may be substituted with one or two iodine atoms on its aromatic moiety. Typically, such methods lead to di-iodination of each tyrosine residue. Alternatively, such methods lead to mono-iodination of each tyrosine residue. Typically, each histidine residue is substituted with a single iodine substituent.

[0177] Therefore, in such methods, typically at least 10% of the tyrosine residues and at least 10% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Typically, at least 10% of the tyrosine residues in the plurality of protein molecules are substituted with two iodine substituents on their aromatic moiety and at least 10% of the histidine residues in the plurality of protein molecules are substituted with a single iodine substituent on their aromatic moiety. Preferably, at least 20% of the tyrosine residues and at least 20% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Typically, at least 20% of the tyrosine residues in the plurality of protein molecules are substituted with two iodine substituents on their aromatic moiety and at least 20% of the histidine residues in the plurality of protein molecules are substituted with a single iodine substituent on their aromatic moiety. More preferably, at least 30% of the tyrosine residues and at least 30% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Typically, at least 30% of the tyrosine residues in the plurality of protein molecules are substituted with two iodine substituents on their aromatic moiety and at least 30% of the histidine residues in the plurality of protein molecules are substituted with a single iodine substituent on their aromatic moiety. Still more preferably, at least 40% of the tyrosine residues and at least 40% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Typically, at least 40% of the tyrosine residues in the plurality of protein molecules are substituted with two iodine substituents on their aromatic moiety and at least 40% of the histidine residues in the plurality of protein molecules are substituted with a single iodine substituent on their aromatic moiety. Most preferably, at least 50% of the tyrosine residues and at least 50% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Typically, at least 50% of the tyrosine residues in the plurality of protein molecules are substituted with two iodine substituents on their aromatic moiety and at least 50% of the histidine residues in the plurality of protein molecules are substituted with a single iodine substituent on their aromatic moiety.

[0178] Typically, up to 100% (e.g. up to 90%, or up to 80%) of the tyrosine residues and up to 100% (e.g. up to 90%, or up to 80%) of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, up to 100% (e.g. up to 90%, or up to 80%) of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety and up to 100% (e.g. up to 90%, or up to 80%) of the histidine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety.

[0179] The IC1 reagent may comprise any isotopic form of iodine but preferably comprises a nonradioactive isotope of iodine. Thus, preferably, the IC1 reagent comprises127I.

[0180] Alternative iodination reagents are also available and can be used in conjunction with the present invention. For instance, the chloramine-T, Bolton-Hunter, and lodo-bead-based methods of iodination are alternative ways of preparing the iodinated scaffold described herein. However, these methods are more complex and involve more expensive reagents than the KI3 and IC1 methods, which are accordingly preferred.

[0181] Notwithstanding the above, in yet further embodiments where the protein comprises at least one modified lysine residue (as described herein) substituted with iodine, an alternative method is required which makes use of Bolton-Hunter type and analogous reagents (see Biochemical Journal, 1973, 133, 529-538). In these embodiments, native lysine residues in the protein may be acylated with a Bolton-Hunter type reagent (i.e. an N- hydroxysuccinamide ester) and then subsequently iodinated, or alternatively, native lysine residues in the protein may be acylated with a pre-iodinated reagent (e.g. 7V-succinimidyl 3 -iodobenzoate).

[0182] Thus, the present invention provides a method of iodinating a protein scaffold, wherein said protein scaffold comprises a plurality of protein molecules, comprising:

[0183] (i) contacting the scaffold with a buffer solution; and

[0184] (ii) incubating said solution with an acylating reagent X-(C=O)-R under suitable conditions to result in acylation of the primary amino group of at least one lysine residue in at least one of the plurality of protein molecules, wherein:

[0185] - X is a leaving group under addition-elimination reaction conditions; and

[0186] - R is Ce-Cio aryl or Ce-Cio aralkyl, which is optionally substituted with a hydroxy group on the aromatic ring; and

[0187] (iii) incubating said solution with KI3 or IC1 under suitable conditions to result in iodination of the aromatic moiety of at least one acylated lysine residue in at least one of the plurality of protein molecules.

[0188] In this method, the leaving group X is not particularly limited but is typically selected from Cl, OH, O-alkyl, O-aryl, SH, S-alkyl, S-aryl, NH2, NH-alkyl, NH-aryl, N(alkyl)2, N(aryl)i, O-2-Cl-Trt, ODmb, O-2-PhiPr, O-EDOTn-Ph, O-NHS, OFm, ODmab and OCam. Preferably the leaving group X is O-NHS (i.e. O-A'-hydroxysuccinirnide).

[0189] In this method, typically R is as defined in formula (I) but without any iodine substituents, i.e.: wherein:

[0190] (i) X is H or OH;

[0191] (ii) m is an integer from 0 to 4; and

[0192] (iii) the dotted line represents the point of attachment to -(C=O)-.

[0193] More preferably, m is 0 or 2. Still more preferably, when m is 0, X is H. Still more preferably, when m is 2, X is OH.

[0194] Thus, in a preferred embodiment of this method, the acylating reagent is selected from N- succinimidyl-3-(4-hydroxyphenyl)propionate or A'-succinimidyl benzoate.

[0195] Typically in this method, the acylation reaction (i.e. step (ii)) is left to run for from 2 to 24 hours. Typically in this method, the acylation reaction is carried out at room temperature. Typically in this method, the acylation reaction comprises using a 0.5 to 1,000-fold molar excess of the acylating reagent compared to the lysine content in the plurality of protein molecules.

[0196] Typically in this method, the nature of the buffer solution is not particularly limited. The skilled person is able to select an appropriate buffer solution in which to perform the reaction. Preferably, though, the buffer solution has a pH of from 7 to 10. An example buffer solution that is appropriate is phosphate-buffered saline (PBS).

[0197] Typically in this method, step (iii) can be carried out using any of the preferred methods described herein relating to use of KI3 or IC1 as an iodinating reagent.

[0198] Alternatively, the present invention provides a method of iodinating a protein scaffold, wherein said protein scaffold comprises a plurality of protein molecules, comprising:

[0199] (i) contacting the scaffold with a buffer solution; and

[0200] (ii) incubating said solution with an acylating reagent X-(C=O)-R under suitable conditions to result in acylation of the primary amino group of at least one lysine residue in at least one of the plurality of protein molecules, wherein:

[0201] - X is a leaving group under addition-elimination reaction conditions; and - R is Ce-Cio aryl or Ce-Cio aralkyl, which is substituted with one or more iodine atoms (preferably, one or two iodine atoms) on the aromatic ring, and which is further optionally substituted with a hydroxy group on the aromatic ring.

[0202] In this method, the leaving group X is not particularly limited but is typically selected from Cl, OH, O-alkyl, O-aryl, SH, S-alkyl, S-aryl, NH2, NH-alkyl, NH-aryl, N(alkyl)2, N(aryl)i, O-2-Cl-Trt, ODmb, O-2-PhiPr, O-EDOTn-Ph, O-NHS, OFm, ODmab and OCam. Preferably the leaving group X is O-NHS (i.e. O-7V-hydroxysuccinimide).

[0203] In this method, typically R is as defined in formula (I), i.e.:

[0204] (i) X is H or OH;

[0205] (ii) n is 1 or 2;

[0206] (iii) m is an integer from 0 to 4; and

[0207] (iv) the dotted line represents the point of attachment to -(C=O)-.

[0208] Preferably R is as defined in formula (la) or formula (lb), i.e.:

[0209] More preferably, m is 0 or 2. Still more preferably, when m is 0, X is H. Still more preferably, when m is 2, X is OH.

[0210] Thus, in a preferred embodiment of this method, the acylating reagent is selected from N- succinimidyl-3-(4-hydroxy-3-iodophenyl)propionate, 7V-succinimidyl-3-(4-hydroxy-3,5-di- iodophenyl)propionate), 7V-succinimidyl 3 -iodobenzoate and 7V-succinimidyl-3,5-di- iodobenzoate. Typically in this method, the acylation reaction (i.e. step (ii)) is left to run for from 2 to 24 hours. Typically in this method, the acylation reaction is carried out at room temperature. Typically in this method, the acylation reaction comprises using a 0.5 to 10,000-fold molar excess of the acylating reagent compared to the lysine content in the plurality of protein molecules. Typically in this method, the product of the acylation reaction is washed with saline, optionally 3 times, optionally with an incubation period between each wash of from 1 to 24 hours, optionally about 8 hours.

[0211] Typically in this method, the nature of the buffer solution is not particularly limited. The skilled person is able to select an appropriate buffer solution in which to perform the reaction. Preferably, though, the buffer solution has a pH of from 7 to 10. An example buffer solution that is appropriate is phosphate-buffered saline (PBS).

[0212] Typically in these methods for modifying a lysine residue, at least 10% of the lysine residues in the plurality of protein molecules are modified lysine residues which are modified as described herein and are substituted with iodine. Thus, at least 10% of the lysine residues in the plurality of protein molecules are modified as described herein and may comprise a single iodine substituent. Alternatively, at least 10% of the lysine residues in the plurality of protein molecules are modified as described herein and comprise two iodine substituents. Preferably, at least 20% of the lysine residues in the plurality of protein molecules are modified lysine residues which are modified as described herein and are substituted with iodine. Thus, at least 20% of the lysine residues in the plurality of protein molecules are modified as described herein and may comprise a single iodine substituent. Alternatively, at least 20% of the lysine residues in the plurality of protein molecules are modified as described herein and comprise two iodine substituents. More preferably, at least 30% of the lysine residues in the plurality of protein molecules are modified lysine residues which are modified as described herein and are substituted with iodine. Thus, at least 30% of the lysine residues in the plurality of protein molecules are modified as described herein and may comprise a single iodine substituent. Alternatively, at least 30% of the lysine residues in the plurality of protein molecules are modified as described herein and comprise two iodine substituents. Still more preferably, at least 40% of the lysine residues in the plurality of protein molecules are modified lysine residues which are modified as described herein and are substituted with iodine. Thus, at least 40% of the lysine residues in the plurality of protein molecules are modified as described herein and may comprise a single iodine substituent. Alternatively, at least 40% of the lysine residues in the plurality of protein molecules are modified as described herein and comprise two iodine substituents. Most preferably, at least 50% of the lysine residues in the plurality of protein molecules are modified lysine residues which are modified as described herein and are substituted with iodine. Thus, at least 50% of the lysine residues in the plurality of protein molecules are modified as described herein and may comprise a single iodine substituent. Alternatively, at least 50% of the lysine residues in the plurality of protein molecules are modified as described herein and comprise two iodine substituents.

[0213] Typically, up to 100% (e.g. up to 90%, or up to 80%) of the lysine residues in the plurality of protein molecules modified as described herein and are substituted with iodine. Thus, up to 100% (e.g. up to 90%, or up to 80%) of the lysine residues in the plurality of protein molecules are modified as described herein and may comprise a single iodine substituent. Alternatively, up to 100% (e.g. up to 90%, or up to 80%) of the lysine residues in the plurality of protein molecules are modified as described herein and comprise two iodine substituents.

[0214] The iodinating reagent (e.g. the Bolton-Hunter type reagent, KI3 and / or IC1) may comprise any isotopic form of iodine but preferably comprises a non-radioactive isotope of iodine.

[0215] B: Methods for direct or indirect iodination of a plurality of protein molecules, and subsequent assembly to a protein scaffold

[0216] In other embodiments, the present invention provides a method of preparing an iodinated protein scaffold, wherein said protein scaffold comprises a plurality of protein molecules, comprising:

[0217] (i) contacting the plurality of protein molecules with a buffer solution;

[0218] (ii) incubating said solution with KI3 under suitable conditions to result in iodination of the aromatic moiety of at least one aromatic amino acid residue in at least one of the plurality of protein molecules; and

[0219] (iii) assembling the plurality of protein molecules into a protein scaffold. The nature of the buffer solution is not particularly limited. The skilled person is able to select an appropriate buffer solution in which to perform the reaction. Preferably, though, the buffer solution has a pH of from 7 to 9, for example from 7 to 8 or from 8 to 9. More preferably, the buffer solution has a pH of about 8. Preferably the buffer is selected from phosphate-buffered saline (PBS) and 4-(2 -hydroxy ethyl)- 1 -piperazineethanesulfonic acid solution (HEPES).

[0220] Typically, the molecular weight of the protein molecules is at least 100 MDa. The total weight of protein molecules is typically at least 1 mg. The density of protein molecules in the buffer solution is typically 30% w / v or above. Typically such proteins are insoluble in water.

[0221] In such methods, the at least one aromatic amino acid residue is any aromatic amino acid residue described herein. However, typically the amino acid residue in tyrosine. Thus, in some embodiments, such methods result in iodination of the aromatic moiety of tyrosine in at least one of the plurality of protein molecules. The tyrosine residues may be substituted with one or two iodine atoms on its aromatic moiety. Typically, such methods lead to diiodination of each tyrosine residue. Alternatively, such methods lead to mono-iodination of each tyrosine residue.

[0222] Therefore, in such methods, typically at least 10% of the tyrosine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Typically, at least 10% of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety. Preferably, at least 20% of the tyrosine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Typically, at least 20% of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety. More preferably, at least 30% of the tyrosine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Typically, at least 30% of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety. Still more preferably, at least 40% of the tyrosine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Typically, at least 40% of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety. Most preferably, at least 50% of the tyrosine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Typically, at least 50% of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety.

[0223] Typically, up to 100% (e.g. up to 90%, or up to 80%) of the tyrosine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, up to 100% (e.g. up to 90%, or up to 80%) of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety.

[0224] The KI3 reagent may comprise any isotopic form of iodine but preferably comprises a nonradioactive isotope of iodine. Thus, preferably, the KI3 reagent comprises127I.

[0225] The method by which the plurality of protein molecules are assembled into the implantable scaffold are not particularly limited, and the skilled person is aware of appropriate methods for assembly of the scaffold. Such methods may include cryop alternation, 3D printing, light-induced polymerisation, 2-photon polymerisation, moulding, reverse moulding, templating, electrospinning, molecularly patterned assembly and lithography.

[0226] Cryopatternation is a method of self-assembly whereby a solution comprising the plurality of protein molecules is frozen, with the sites of crystal nucleation forming pores, and the proteins increasing in concentration in the remaining areas until they precipitate and join together into a 3D matrix around the ice crystals.

[0227] 3D printing (also known as “additive manufacturing”) refers to the construction of a three- dimensional object from a computer-aided design (CAD) model or a digital 3D model. It can refer to a variety of processes in which material is joined or solidified under computer control to create a three-dimensional object. Typically, in such processes, material is added in a layer by layer fashion, whereby successive layers of material are laid down or formed at precise positions. Alternatively, a 3D model can be printed simultaneously, such as in tomographic volumetric additive manufacturing. Examples of materials that can be layered by a 3D printing process include liquids, which solidify after being added to the growing object, or solids, which can be fused onto the growing object as they are applied. 3D printing can be carried out at room temperature or in combination with cryopattemation.

[0228] Light-induced polymerisation and 2-photon polymerisation are useful techniques for microscale 3D printing of scaffolds.

[0229] Moulding is a process of manufacturing by shaping liquid or pliable raw material using a rigid frame called a mould or matrix. In reverse moulding or templating, the mould itself is prepared using a pattern or model of the final scaffold.

[0230] Electrospinning is a fibre production method that uses electric force to draw charged threads of polymer solutions or polymer melts up to fibre diameters in the order of some hundred nanometers. This is a particularly useful method for preparation of scaffolds when the protein is a fibrous protein.

[0231] Molecularly patterned assembly is a process whereby proteins self-assemble in a designed shape due to their specific binding sites with other proteins. This process is carried out in a modular fashion with a number of different proteins.

[0232] Lithography is a planographic method of printing in which a design is printed onto a substrate and affixed via a chemical reaction. Types of lithography include stereolithography (SLA), micro-stereolithography, and low-force stereolithography (LFS).

[0233] In other embodiments, the present invention provides a method of preparing an iodinated protein scaffold, wherein said protein scaffold comprises a plurality of protein molecules, comprising:

[0234] (i) contacting the plurality of protein molecules with a buffer solution;

[0235] (ii) incubating said solution with KE under suitable conditions to result in iodination of the aromatic moiety of at least one aromatic amino acid residue in at least one of the plurality of protein molecules; and

[0236] (iii) assembling the plurality of protein molecules into a protein scaffold.

[0237] Typically, the molecular weight of the protein molecules is at least 100 MDa. The total weight of protein molecules is at least 1 mg. The density of protein molecules in the buffer solution is typically 30% w / v or above. Typically such proteins are insoluble in water. The nature of the buffer solution is not particularly limited. The skilled person is able to select an appropriate buffer solution in which to perform the reaction. Preferably, though, the buffer solution has a pH of from 7 to 10, for example from 7 to 9 or from 8 to 10. More preferably, the buffer solution has a pH of from 8 to 9. Preferably the buffer is selected from glycine solution and tris(hydroxymethyl)aminomethane solution (TRIS).

[0238] In such methods, the at least one aromatic amino acid residue is any aromatic amino acid residue described herein. However, typically in such methods both tyrosine and histidine residues are iodinated. Thus, in some embodiments, such methods result in iodination of the aromatic moiety of tyrosine in at least one of the plurality of protein molecules and the aromatic moiety of histidine in at least one of the plurality of protein molecules. The tyrosine residues may be substituted with one or two iodine atoms on its aromatic moiety. Typically, such methods lead to di-iodination of each tyrosine residue. Alternatively, such methods lead to mono-iodination of each tyrosine residue. Typically, each histidine residue is substituted with a single iodine substituent.

[0239] Therefore, in such methods, typically at least 10% of the tyrosine residues and at least 10% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Typically, at least 10% of the tyrosine residues in the plurality of protein molecules are substituted with two iodine substituents on their aromatic moiety and at least 10% of the histidine residues in the plurality of protein molecules are substituted with a single iodine substituent on their aromatic moiety. Preferably, at least 20% of the tyrosine residues and at least 20% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Typically, at least 20% of the tyrosine residues in the plurality of protein molecules are substituted with two iodine substituents on their aromatic moiety and at least 20% of the histidine residues in the plurality of protein molecules are substituted with a single iodine substituent on their aromatic moiety. More preferably, at least 30% of the tyrosine residues and at least 30% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Typically, at least 30% of the tyrosine residues in the plurality of protein molecules are substituted with two iodine substituents on their aromatic moiety and at least 30% of the histidine residues in the plurality of protein molecules are substituted with a single iodine substituent on their aromatic moiety. Still more preferably, at least 40% of the tyrosine residues and at least 40% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Typically, at least 40% of the tyrosine residues in the plurality of protein molecules are substituted with two iodine substituents on their aromatic moiety and at least 40% of the histidine residues in the plurality of protein molecules are substituted with a single iodine substituent on their aromatic moiety. Most preferably, at least 50% of the tyrosine residues and at least 50% of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Typically, at least 50% of the tyrosine residues in the plurality of protein molecules are substituted with two iodine substituents on their aromatic moiety and at least 50% of the histidine residues in the plurality of protein molecules are substituted with a single iodine substituent on their aromatic moiety.

[0240] Typically, up to 100% (e.g. up to 90%, or up to 80%) of the tyrosine residues and up to 100% (e.g. up to 90%, or up to 80%) of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety. Thus, up to 100% (e.g. up to 90%, or up to 80%) of the tyrosine residues in the plurality of protein molecules may be substituted with two iodine substituents on their aromatic moiety and up to 100% (e.g. up to 90%, or up to 80%) of the histidine residues in the plurality of protein molecules may be substituted with a single iodine substituent on their aromatic moiety.

[0241] The IC1 reagent may comprise any isotopic form of iodine but preferably comprises a nonradioactive isotope of iodine. Thus, preferably, the IC1 reagent comprises127I.

[0242] The method by which the plurality of protein molecules are assembled into the implantable scaffold are not particularly limited, and the skilled person is aware of appropriate methods for assembly of the scaffold. Such methods may include cryopatternation, 3D printing, light-induced polymerisation, 2-photon polymerisation, moulding, reverse moulding, templating, electrospinning, molecularly patterned assembly and lithography.

[0243] Alternative iodination reagents are also available and can be used in conjunction with the present invention. For instance, the chloramine-T, Bolton-Hunter, and lodo-bead-based methods of iodination are alternative ways of preparing the iodinated scaffold described herein. However, these methods are more complex and involve more expensive reagents than the KF and IC1 methods, which are accordingly preferred. Notwithstanding the above, in yet further embodiments where the protein comprises at least one modified lysine residue (as described herein) substituted with iodine, an alternative method is required which makes use of Bolton-Hunter type and analogous reagents. In these embodiments, native lysine residues in the protein may be acylated with a Bolton-Hunter type reagent (i.e. an A'-hydroxysuccinamide ester) and then subsequently iodinated, or alternatively, native lysine residues in the protein may be acylated with a preiodinated reagent (e.g. 7V-succinimidyl 3 -iodobenzoate).

[0244] Thus, the present invention provides a method of iodinating a protein scaffold, wherein said protein scaffold comprises a plurality of protein molecules, comprising:

[0245] (i) contacting the plurality of protein molecules with a buffer solution;

[0246] (ii) incubating said solution with an acylating reagent X-(C=O)-R under suitable conditions to result in acylation of the primary amino group of at least one lysine residue in at least one of the plurality of protein molecules, wherein:

[0247] - X is a leaving group under addition-elimination reaction conditions; and

[0248] - R is Ce-Cio aryl or Ce-Cio aralkyl, which is optionally substituted with a hydroxy group on the aromatic ring;

[0249] (iii) incubating said solution with KI3 or IC1 under suitable conditions to result in iodination of the aromatic moiety of at least one acylated lysine residue in at least one of the plurality of protein molecules; and

[0250] (iv) assembling the plurality of protein molecules into a protein scaffold.

[0251] In this method, the leaving group X is not particularly limited but is typically selected from Cl, OH, O-alkyl, O-aryl, SH, S-alkyl, S-aryl, NH2, NH-alkyl, NH-aryl, N(alkyl)2, N(aryl)i, O-2-Cl-Trt, ODmb, O-2-PhiPr, O-EDOTn-Ph, O-NHS, OFm, ODmab and OCam. Preferably the leaving group X is O-NHS (i.e. O- -hydroxysuccinimide).

[0252] In this method, typically R is as defined in formula (I) but without any iodine substituents, i.e.: wherein: (iv) X is H or OH;

[0253] (v) m is an integer from 0 to 4; and

[0254] (vi) the dotted line represents the point of attachment to -(C=O)-.

[0255] More preferably, m is 0 or 2. Still more preferably, when m is 0, X is H. Still more preferably, when m is 2, X is OH.

[0256] Thus, in a preferred embodiment of this method, the acylating reagent is selected from N- succinimidyl-3-(4-hydroxyphenyl)propionate or A-succinimidyl benzoate.

[0257] Typically in this method, the acylation reaction (i.e. step (ii)) is left to run for from 2 to 24 hours. Typically in this method, the acylation reaction is carried out at room temperature. Typically in this method, the acylation reaction comprises using a 0.5 to 1,000-fold molar excess of the acylating reagent compared to the lysine content in the plurality of protein molecules.

[0258] Typically in this method, the nature of the buffer solution is not particularly limited. The skilled person is able to select an appropriate buffer solution in which to perform the reaction. Preferably, though, the buffer solution has a pH of from 7 to 10. An example buffer solution that is appropriate is phosphate-buffered saline (PBS).

[0259] Typically in this method, step (iii) can be carried out using any of the preferred methods described herein relating to use of KI3 or IC1 as an iodinating reagent.

[0260] Alternatively, the present invention provides a method of iodinating a protein scaffold, wherein said protein scaffold comprises a plurality of protein molecules, comprising:

[0261] (i) contacting the plurality of protein molecules with a buffer solution; and

[0262] (ii) incubating said solution with an acylating reagent X-(C=O)-R under suitable conditions to result in acylation of the primary amino group of at least one lysine residue in at least one of the plurality of protein molecules, wherein:

[0263] - X is a leaving group under addition-elimination reaction conditions; and

[0264] - R is Ce-Cio aryl or Ce-Cio aralkyl, which is substituted with one or more iodine atoms (preferably, one or two iodine atoms) on the aromatic ring, and which is further optionally substituted with a hydroxy group on the aromatic ring; and (iii) assembling the plurality of protein molecules into a protein scaffold.

[0265] In this method, the leaving group X is not particularly limited but is typically selected from Cl, OH, O-alkyl, O-aryl, SH, S-alkyl, S-aryl, NH2, NH-alkyl, NH-aryl, N(alkyl)2, N(aryl)2, O-2-Cl-Trt, ODmb, O-2-PhiPr, O-EDOTn-Ph, O-NHS, OFm, ODmab and OCam.

[0266] Preferably the leaving group X is O-NHS (i.e. O-7V-hydroxysuccinimide).

[0267] In this method, typically R is as defined in formula (I), i.e.:

[0268] (v) X is H or OH;

[0269] (vi) n is 1 or 2;

[0270] (vii) m is an integer from 0 to 4; and

[0271] (viii) the dotted line represents the point of attachment to -(C=O)-.

[0272] Preferably R is as defined in formula (la) or formula (lb), i.e.:

[0273] More preferably, m is 0 or 2. Still more preferably, when m is 0, X is H. Still more preferably, when m is 2, X is OH.

[0274] Thus, in a preferred embodiment of this method, the acylating reagent is selected from N- succinimidyl-3-(4-hydroxy-3-iodophenyl)propionate, 7V-succinimidyl-3-(4-hydroxy-3,5-di- iodophenyl)propionate), 7V-succinimidyl 3 -iodobenzoate and 7V-succinimidyl-3,5-di- iodobenzoate.

[0275] Typically in these methods for modifying a lysine residue, the molecular weight of the protein molecules is at least 100 MDa. The total weight of protein molecules is at least 1 mg. The density of protein molecules in the buffer solution is typically 30% w / v or above. Typically such proteins are insoluble in water.

[0276] Typically in this method, the acylation reaction (i.e. step (ii)) is left to run for from 2 to 24 hours. Typically in this method, the acylation reaction is carried out at room temperature. Typically in this method, the acylation reaction comprises using a 0.5 to 10,000-fold molar excess of the acylating reagent compared to the lysine content in the plurality of protein molecules. Typically in this method, the product of the acylation reaction is washed with saline, optionally 3 times, optionally with an incubation period between each wash of from 1 to 24 hours, optionally about 8 hours.

[0277] Typically in this method, the nature of the buffer solution is not particularly limited. The skilled person is able to select an appropriate buffer solution in which to perform the reaction. Preferably, though, the buffer solution has a pH of from 7 to 10. An example buffer solution that is appropriate is phosphate-buffered saline (PBS).

[0278] Typically in these methods for modifying a lysine residue, at least 10% of the lysine residues in the plurality of protein molecules are modified lysine residues which are modified as described herein and are substituted with iodine. Thus, at least 10% of the lysine residues in the plurality of protein molecules are modified as described herein and may comprise a single iodine substituent. Alternatively, at least 10% of the lysine residues in the plurality of protein molecules are modified as described herein and comprise two iodine substituents. Preferably, at least 20% of the lysine residues in the plurality of protein molecules are modified lysine residues which are modified as described herein and are substituted with iodine. Thus, at least 20% of the lysine residues in the plurality of protein molecules are modified as described herein and may comprise a single iodine substituent. Alternatively, at least 20% of the lysine residues in the plurality of protein molecules are modified as described herein and comprise two iodine substituents. More preferably, at least 30% of the lysine residues in the plurality of protein molecules are modified lysine residues which are modified as described herein and are substituted with iodine. Thus, at least 30% of the lysine residues in the plurality of protein molecules are modified as described herein and may comprise a single iodine substituent. Alternatively, at least 30% of the lysine residues in the plurality of protein molecules are modified as described herein and comprise two iodine substituents. Still more preferably, at least 40% of the lysine residues in the plurality of protein molecules are modified lysine residues which are modified as described herein and are substituted with iodine. Thus, at least 40% of the lysine residues in the plurality of protein molecules are modified as described herein and may comprise a single iodine substituent. Alternatively, at least 40% of the lysine residues in the plurality of protein molecules are modified as described herein and comprise two iodine substituents. Most preferably, at least 50% of the lysine residues in the plurality of protein molecules are modified lysine residues which are modified as described herein and are substituted with iodine. Thus, at least 50% of the lysine residues in the plurality of protein molecules are modified as described herein and may comprise a single iodine substituent. Alternatively, at least 50% of the lysine residues in the plurality of protein molecules are modified as described herein and comprise two iodine substituents.

[0279] Typically, up to 100% (e.g. up to 90%, or up to 80%) of the lysine residues in the plurality of protein molecules modified as described herein and are substituted with iodine. Thus, up to 100% (e.g. up to 90%, or up to 80%) of the lysine residues in the plurality of protein molecules are modified as described herein and may comprise a single iodine substituent. Alternatively, up to 100% (e.g. up to 90%, or up to 80%) of the lysine residues in the plurality of protein molecules are modified as described herein and comprise two iodine substituents.

[0280] The iodinating reagent (e.g. the Bolton-Hunter type reagent, KI3 and / or IC1) may comprise any isotopic form of iodine but preferably comprises a non-radioactive isotope of iodine.

[0281] The method by which the plurality of protein molecules is assembled into the implantable scaffold are not particularly limited, and the skilled person is aware of appropriate methods for assembly of the scaffold. Such methods may include cryop alternation, 3D printing, light-induced polymerisation, 2-photon polymerisation, moulding, reverse moulding, templating, electrospinning, molecularly patterned assembly and lithography. Examples

[0282] The present invention is illustrated by the following examples. However, these examples do not limit the scope of the invention. All statistical analysis discussed below was performed in Graph Pad Prism 6.

[0283] Example 1: Enhanced visibility of protein scaffolds

[0284] The efficacy of (a) iodination with Potassium Triiodide (KE / Lugol’s solution), and (b) iodination with Iodine Monochloride (IC1), was evaluated on samples of a representative clinically approved collagen-based scaffold produced from decellularised porcine dermis (XenMatrix™ hernia mesh, Bard). Two buffers were compared per reaction, chosen for their suitable pH range and established use in protein biochemistry: phosphate-buffered saline (PBS) and HEPES for the KI3 method (at pH 7.4); and Glycine and Tris buffers for the IC1 method (at pH 8.5). Both reactions were expected to result in iodination of tyrosine residues within the collagen scaffold (see Fig. 1 A).

[0285] For the triiodide labelling method, 5x10mm hernia mesh samples (Xenmatrix, Bard) were added to either ImL of HEPES buffer (200 mM, pH 7.4), or PBS (phosphate buffered saline, pH 7.4, Gibco) with 400 pL Lugol’s iodine solution (SLS, CHE2380, 38 mM), and incubated for 24 hours, before being transferred into 0.9% w / v NaCl solution for washing.

[0286] For the iodine monochloride (McFarlane’s) labelling method, mesh samples (5* 10mm) were added to either 1 mL of glycine buffer (200 mM, pH 8.6) or Tris-HCl buffer (200 mM, pH 8.6), containing 10 pL of iodine monochloride (Alfa Aesar, 39104), and incubated for 24 hours, before being transferred into 0.9% w / v NaCl solution. Washing was done with 3 changes of fresh saline with 1 hour incubation each on a tube rotator (20 rpm), with a final 24 hour incubation also with rotation.

[0287] Each reaction / buffer combination increased radiopacity of labelled materials following 24- hour incubation and removal of unbound iodine, with significantly increased Hounsfield Units (HU) measured with X-ray CT versus unlabelled control samples (Fig IB). For the KI3 / Lugol’s method, PBS showed superior labelling to HEPES buffer.

[0288] Assuming an iodine radiopacity of 14-16 HU per mg Iodine / mL (see Jeong et al., Biomaterials Research, 2022, 26(1):27), a tyrosine weight fraction of 1.96% for porcine collagen type 1 (UniProt ref: A0A287BLD2), and a protein weight / volume fraction for decellularised porcine dermis of 40% (see Zhang et al., Heliyon, 2018, 4(4):e00600), then total di-iodination of tyrosine residues would be expected to increase radi opacity by 154- 175 HU. This was comparable to the increase in HU measured for KE with HEPES buffer (112 HU) and PBS (160 HU); see Fig. IB. Fluorescence spectroscopy gave a characteristic 300 nm peak for tyrosine in control (unmodified) samples, which decreased significantly in intensity by 98% following labelling with either KE / PBS or ICl / Glycine reactions (see Fig. 6; n=3, p<0.005 2-tailed t-test).

[0289] Interestingly the iodine monochloride reaction showed significantly higher levels of CT contrast (p<0.05 versus KE / PBS, Student’s 2-tailed t-test; 210 HU for Glycine buffer, 226 HU for Tris buffer), suggesting additional modification sites. Without wishing to be bound by any particular theory, it is believed that the higher pH conditions of the iodine monochloride reaction lead to iodination of histidine residues in collagen in addition to diiodination of tyrosine residues; complete iodination of both histidine and tyrosine residues would give a predicted increase in radiopacity of 220-251 HU, which is similar to the observed results under both the Glycine and Tris conditions.

[0290] Homogeneity of labelling, and iodine incorporation throughout the scaffold, was confirmed by plotting line profiles of signal intensity for samples labelled with each of the four reaction combinations (Fig 1C). To assess labelling stability, samples were incubated at 37°C in 1.5 mL human serum for 16 days, with radiopacity measured at 0, 1, 2, 5, 7, 9, 12 and 16 days post-labelling using X-ray CT (Figs. ID and IE). Retention of contrast was assessed using a PET-CT system (Mediso nanoScan®) with a semi-circular scan at 50kVp with 170ms exposure, 720 projections, 1 :4 binning, and medium FOV setting. Region of interest analysis was performed using VivoQuant software (Invicro) to quantify X-ray absorbance. No contrast reduction was identified over this period, suggesting stable iodine retention within the material under biol ogically-relevant conditions.

[0291] For longer term stability, samples (Neuragen, ESM, and Xenmatrix hernia mesh) were incubated at room temperature in saline (0.9% w / v NaCl), and imaged up to 17 months post labelling. To ensure greater selectivity of modification and to minimise alterations to biological function, the milder conditions of the potassium tri-iodide method were chosen for use throughout the subsequent examples, unless otherwise indicated. Potassium triiodide, in the form of Lugol’s Iodine as used here, also has the advantage of being clinically established as an antiseptic agent.

[0292] Investigation of the kinetics of iodination with KI3 / PBS showed 80% of the peak labelling was achieved in 45 minutes (see Fig. 7), and was saturated after 3 hours. The stability of contrast during storage of the labelled Xenmatrix in saline at room temperature was monitored over 17 months, with the material found to retain its contrast over this period (see Fig. 7).

[0293] To demonstrate versatility, four additional protein-based materials of interest were also labelled using the KI3 method (Figs. IF- 1 J).

[0294] Neuragen nerve guide (Integra Life Sciences) was labelled as above with Lugol’s / PBS in 1.5 cm lengths, before washing as above. Egg shell membrane was supplied in 40x40mm pieces*, but otherwise the Lugol’s / PBS method was as described above. Silk sutures (Permahand, Ethicon, 3-0) were de-waxed for 5 minutes in xylenes, washed twice with 70% ethanol, and twice with PBS, and labelled with the above Lugol’s / PBS or IC1 Glycine method, before washing as above. Purified collagen (Research Grade Jellyfish Collagen, Jellagen), 100 pL at 4mg / mL, was labelled as above with the Lugol’s / PBS method, and precipitated with ethanol, before washing as above.

[0295] [*Egg shell membranes (ESM) were extracted by a method adapted from Mensah et al. (Journal of Biomaterials Applications, 2021, 36(5), 912-929). Briefly, after washing with deionised water (DI), fresh chicken eggs were immersed in 0.5 M acetic acid for 44 hours at room temperature to dissolve the calcium carbonated (CaCCL) shell. After CaCCL shell dissolution was complete, the eggs were washed again using DI and the membrane was perforated to empty the albumen and yolk. A final washing step was done before storage in PBS to prevent dehydration.]

[0296] NeuraGen (Fig. 1G) is an FDA-approved hollow tube formed of porous bovine collagen, implanted to repair peripheral nerve damage, and of interest in combination with autologous stem cell therapy. However, surgical implantation is challenging, implants must be located and removed when unsuccessful. Labelling with KE / PBS increased radi opacity by around 200 HU, and labelling stability was confirmed up to 15 months at room temperature in saline (see Fig. 9). Egg-shell membrane (ESM) was also successfully labelled (Fig. 1H), which contains a number of proteins alongside type I and type IV collagen. ESM is typically discarded as a waste product by the food industry, but due to its favourable biocompatibility and material properties is attracting attention as a new scaffold for tissue engineering and cell therapy. Again, an increase of -200 HU was achieved with KI3 / PBS, and labelling stability was preserved over 15 months (see Fig. 10). A commercially-available hydrogel of “Type 0” jellyfish-derived collagen could also be labelled and imaged (Fig. 11), a material that has been suggested as more sustainable than established porcine and bovine collagen that are typically used clinically. Finally, a commercially-available silk-suture (Ethicon, 3-0), was also labelled and showed an increase in HU of over -1200 (Fig. 1 J), consistent with the unusually high tyrosine content of silk worm (Bombyx mori) silk, at 5 to 6-fold that of collagen (UniProt: P05790 • FIBH BOMMO).

[0297] Example 2: Preservation of surface morphology and material strength

[0298] To demonstrate preservation of native surface structure, collagen scaffolds (XenmatrixTM hernia mesh) labelled with potassium tri-iodide (PBS buffer), and iodine monochloride (glycine buffer) were analysed using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS), and compared to unmodified samples.

[0299] For SEM analysis, collagen scaffold samples were mounted on specimen stubs fitted with adhesive carbon pads, sputter-coated with carbon and examined using a Zeiss Evo50 (Oxford Instruments, Cambridge, UK) scanning electron microscope, with micrographs obtained at an acceleration voltage of 20 kV. Point EDS spectra were acquired using and Oxford Instruments x-act EDS detector running INCA software. Silk samples were fixed to adhesive 12 mm carbon tabs (Agar Scientific, Stansted, UK) pre-mounted onto 0.5 aluminium spectrum stubs (Agar Scientific, UK), and imaged using a field emission scanning electron microscope, (FE)SEM, (Zeiss, EVO HD, Jena, Germany) with operation voltage of 5 kV. Samples on the stubs were sputter-coated with 95% gold and 5% palladium (Polaron E500, Quorum Technology, UK) and imaged at magnifications of x500 and X1K. No change in large- or small-scale structure was identified via SEM after labelling with either iodination reaction (see Fig. 2A), consistent with the mild reaction conditions. EDS confirmed iodine was only present in labelled samples (see Figs. 11 A-l ID).

[0300] Dynamic mechanical analysis testing of control and KE (PBS) labelled collagen scaffolds (XenmatrixTM hernia mesh) showed comparable stress-strain curves (see Fig. 2B), and percent elongation at fracture (see Fig. 2C).

[0301] Tensile strength of labelled and unlabelled hernia mesh samples (n=5) was evaluated at room temperature using a uniaxial testing device (AGS-X 200kN, Shimadzu), with the following measurement of dimensions prior to testing: 8.298±0.50 mm wide, 14.9±0.24 mm long and 2.16±0.08 mm thick. Each specimen was clamped an average of 3.12±0.44 mm on each end of the long axis before starting the test. During the test the samples were pulled apart at 5mm / min in the Y-axis while recording force and displacement until failure, which was defined as a drop by 50% in the force needed to continue pulling the sample apart. After the initial ‘fresh’ test, the samples were trimmed at the fracture point and rehydrated in PBS for 20 minutes prior to clamping and retesting under the same conditions (labelled ‘Re-test’ in the results). Silk sutures (80mm long and 0.2mm diameter) were double knotted at each of a metal 75mm mending plate and the space from knot to knot was measured as the gap Gauge length (19.46±3.16 mm). Both mending plates were clamped on each end of the long axis and the gap increased enough to slightly tense the suture with minimal force before starting the tensile test. During the test the sutures were pulled apart at a rate of 5mm / min in the Y-axis while recording force and displacement until failure, which was defined as a 50% drop in the force.

[0302] Ultimate tensile strength (UTS) was however increased significantly in labelled meshes (see Fig. 2D; 8.46 ± 0.24 (SD) MPa vs 6.32 ± 1.19 MPa, p<0.005 2-tailed t-test), though remained within the range of 2.53 ± 0.25 MPa to 28.54 ± 1.99 MPa reported for commercially available bovine and porcine-derived biological hernia meshes (see Deeken et al.. Annals of Surgery, 2012, 255(3), 595-604). This difference was not significant upon re-testing the material (7.72 ± 1.31 MPa vs 6.84 ± 1.33 MPa, p=0.32), suggesting that the effect of iodination on material behaviour was at least partially reversible with stretching. Similarly, a small but significant increase in Young’s modulus was measured upon labelling from 15.3 to 18.1 MPa (p<0.01, Student’s 2-tailed t-test), which again was reversible post-stretching. FT-IR and Differential Scanning Calorimetry showed comparable spectra for labelled and unlabelled materials, albeit with minor peak shifts presumably due to the incorporation of iodine (see Figs. 12 and 13).

[0303] As silk is typically one to two orders of magnitude stronger than collagen, and with much higher tyrosine content, the preservation of its material properties were also investigated post-iodination. Consistent with the collagen samples, surgical silk sutures (as shown in Fig. 1 J) showed no morphological changes via SEM (see Fig. 14) when labelled with either the KE or IC1 methods. Mechanical properties were also preserved, with both the UTS and Young’s modulus of labelled samples showing no significant difference when compared to unlabelled controls (see Figs. 15 and 16, and Table 1 below).

[0304] Sample Ultimate tensile Young Modulus strength UTS (MPa)

[0305] Control 390.66±17.13 1781.09±147.40

[0306] KE / PBS (2 hours) 389.45±39.25 1984.76±414.78

[0307] KE / PBS (2 days) 335.61±44.06 1891.55±127.54

[0308] ICI / Glycine (2 days) 341.3±38.91 1556.28±1156.99

[0309] Table 1. No significant change was found in the UTS and Youngs modulus of silk sutures (Ethicon Permahand, 3-0) after labeling with KI3 or IC1 reactions, as compared to control (unmodified) samples. Values are presented as means with SD, p>0.05 ANOVA.

[0310] Example 3: Trackability of iodinated hernia meshes in vivo

[0311] To demonstrate clinical utility, immunocompetent mice were subcutaneously implanted with labelled (KE / PBS) and unlabelled samples of the collagen -based hernia mesh (XenmatrixTM). Female C57BL / 6 mice were purchased from Charles River and were 3 weeks old at point of implantation. Mice were housed 5 per individually ventilated cage, at 21 °C with normal day / night cycles. All animal studies were licensed under UK Home Office regulations and approved by the UCL Biological Services Ethical Review Committee, with all regulated in compliance with UCL experimentation guidelines and regulations. Mice were anesthetised with 2% isoflurane in 100% O2 and surgically implanted with 4x4mm squares of Xenmatrix mesh (BD Bard) subcutaneously under the scruff (n=10 each for labelled and unlabelled mesh). For imaging, mice were anesthetised with 2% isoflurane in 100% O2, and maintained under anaesthesia during imaging with a preclinical micro-CT device (Quantum GX2, PerkinElmer). Mice were scanned at 0,7,14,21,28,35,56, and 84-day timepoints with 4- minute high resolution scans (Voxel size 72 pm, 90 kVp, filters Cu 0.06+ Al 0.5). On the 56- and 84-day timepoints mice were also imaged with 18-second (Standard), 8-second (High speed), and 3.9-second (High speed) scans for comparison, also at 90 kVp with Cu 0.06+ Al 0.5 filters. 3D ROI analysis was done using Analyze 14.0 software (AnalyzeDirect, Overland Park, KS) using manually-drawn volumes of interest on labelled and unlabelled meshes, and corresponding upper thigh muscles for each animal. Intensitybased semi-automatic segmentation was used to analyse mesh volume over time, which was only possible on the labelled mesh due to its greater radiopacity versus surrounding tissue.

[0312] Subsequent X-ray CT imaging extending to 3 months (12 weeks) post-implantation showed significant differences in radiopacity (HU) between labelled and unlabelled meshes (p<0.0001), and between labelled meshes and muscle within the same animal (p<0.0001), confirming radiopacity was sustained over time (see Figs. 3A-3C). No difference in radiopacity between unlabelled meshes and muscle was found (p>0.05), illustrating the existing challenge when attempting to discriminate commercially-available meshes from surrounding tissue. Highlighting utility to detect material migration from implantation site, one labelled mesh had moved laterally between implantation and subsequent imaging 1 week post-implantation (Fig. 3D), a phenomenon that can occur clinically when the mesh detaches from surrounding tissues.

[0313] Example 4: Preserved visibility using clinically relevant X-ray doses

[0314] To assess feasibility of imaging the labelled material using clinically relevant X-ray doses, implanted meshes were imaged with a range of scan times at 2 and 3 months postimplantation. At all scan times between 4 minutes and 4 seconds, labelled meshes showed good visibility on CT sections (see Fig. 4A) and a significantly higher HU than both muscle and unlabelled meshes following region of interest analysis (see Fig. 4B). To put these results into perspective, average clinical volumetric CT doses of 25 and 17 mGy (with 1 mGy equalling 1 Joule of energy absorbed per kg) were reported for abdominal imaging in Europe and the US respectively (see Atli et a!.. Diagn Interv Radiol. , 2021, 27(1), 147-151), which corresponded most closely to the 18 second / 16.4 mGy scan in the present experiment. This X-ray dose was sufficient to produce comparable quantification of intensity (HU) to the 4-minute scan for mesh and muscle, and to visualise the migrated mesh at 3 months post-implantation in 3D following intensity-based 3D rendering (see Fig. 4D).

[0315] Example 5: Biocompatibility of visible mesh and preservation of mesh integrity

[0316] To assess the effect of iodination on the immune response to implanted meshes, samples were explanted at 2, 4, and 12 weeks, and H and E staining performed on histological sections (see Fig. 5A).

[0317] Implanted material was dissected out at the relevant time point post-implantation, and freeze-embedded in OCT media (optimal cutting temperature). Samples were cryosectioned (Leica, Bright 5040) at 15 pm and 5 pm onto glass slides (Thermo-Fisher, Superfrost Plus), and left to air dry at room temperature. Tissue was then fixed on the slides in 4% buffered formaldehyde solution for 5 minutes, before washing in phosphate buffered saline (x3). Staining was done using an automated H and E protocol using a Tissue-TEK DRS autostainer (Sakura). Neutrophils and Leukocytes were counted manually at 40* magnification from representative slides at each timepoint using a AE2000 microscope, with five random fields per animal. Digital images were taken at 5 mega-pixel with an eyepiece camera (Dino-Eye-Lite). Collagen staining was done using a Piero Sirius Red kit (Abeam, abl50681) according to the manufacturer’s instructions. Representative images of collagen matrixes were taken at 10x magnification, and threshold analysis was done manually using ImageJ’s (NIH) measure function to measure % of area coverage.

[0318] Neutrophil and lymphocyte numbers were counted at 40* magnification by a trained histopathologist on n>5 sections per time-point and sample (see Fig. 5B), showing comparable numbers between labelled and unlabelled meshes at each time point (p>0.05 2-tailed unpaired T-tests). Infiltration of native tissue was comparable in labelled and unlabelled meshes, with representative sections at 12 weeks post-implantation shown in Figs. 5C and 5D. To investigate the biological response to labelled meshes further, samples were explanted at 2, 4, and 12 weeks, and stained using Piero Sirius Red stain for collagen (see Figs. 5E and 5F). Labelled and unlabelled meshes appeared similar at each time point, with quantification of stained area not significantly different in coverage between mesh types at any time point. No change in overall mesh volume for labelled meshes was measured based on semi-automatic intensity-based segmentation of the labelled implants (see Fig. 17), consistent with the overall preservation of collagen coverage seen histologically.

[0319] Example 6: Modification of lysine residues with iodine-containing moieties

[0320] To demonstrate the utility of embodiments of the invention in which native lysine residues are modified with iodine-containing substituents, the following protocol was carried out.

[0321] 10mm hernia mesh samples (Xenmatrix, Bard) were added to Bolton-Hunter reagent precursor (i.e. 2,5-dioxopyrrolidin-l-yl 3-(4-hydroxyphenyl)propanoate; 3-(4- hydroxyphenyl)propionic acid N-hydroxysuccinimide ester) in ImL of 0.25 M PBS (phosphate buffered saline, pH 7.5, Gibco), and incubated for 24 hours. The amount of Bolton-Hunter precursor reagent was calculated as being a 100-fold excess over the total lysine content of the hernia mesh samples.

[0322] Subsequently, the product of this reaction step was added to PBS (phosphate buffered saline, pH 7.4, Gibco) containing one third by volume of Lugol’s iodine solution (SLS, CHE2380, 38 mM), and incubated for 8 hours. The sample was then transferred into 0.9% w / v NaCl solution for washing; the sample was washed for 72 hours with three changes of the saline solution.

[0323] To assess label uptake, samples were incubated at 37°C in 1.5 mL human serum, with radiopacity measured using X-ray CT (PET-CT system (Mediso nanoScan®) with a semicircular scan at 50kVp with 170ms exposure, 720 projections, 1 :4 binning, and medium FOV setting). As a control, hernia mesh samples which were not exposed to acylation / iodination conditions were also incubated in human serum and imaged. The resulting CT scan is shown in Fig. 18. There is a high contrast between the hernia mesh that was subjected to acylation / iodination conditions and the hernia mesh that was not. Accordingly, this demonstrates successful labelling of the mesh with iodine using this method. As an alternative to the second step of the procedure, the acylated intermediate mesh could be transferred to glycine buffer (200 mM, pH 8.6) or Tris-HCl buffer (200 mM, pH 8.6), containing 1 / 1000 by volume of iodine monochloride (Alfa Aesar, 39104, 1 M), and incubated for 24 hours, before washing as described above.

[0324] Conclusions

[0325] Iodine labelling of implantable protein scaffolds has the benefit of amino-acid selectivity, providing a well-characterised and homogenously labelled product, but (unlike with other selective labelling agents for amino acids) does not require a bulkier, higher molecular weight carrier molecule or linker. The synthetic manipulation of the protein scaffold is straightforward, cost-effective and can be carried out under mild conditions, and does not require complex method steps, as is the case with many bioconjugate or click chemistry reagents.

[0326] The products synthesised in this study had an enhanced radiopacity such that the materials were visible using CT imaging, compared to the native protein background. Imaging using CT scanning is advantageous: in terms of spatial resolution, CT imaging is comparable to MRI in the clinic, and greatly superior to PET or SPECT, as well as typically being cheaper and more widely-available. Furthermore, due to the differences in peak x-ray absorption energy of iodine and water, acquisition of two images with different energies (one nearer the iodine peak at 33 KeV, one further away), allows a background-subtraction approach to increase iodine sensitivity. The in vivo results show clear visibility of labelled hernia meshes above both endogenous muscle and control unlabelled mesh samples. This was maintained over a biologically relevant time frame of three months, and under X-ray doses at and below those typically measured in the clinic. Both labelled and unlabelled materials showed otherwise similar behaviour in vivo, indicating the retention of biocompatibility post-labelling.

[0327] The results also demonstrate that this labelling method was compatible with a range of biomaterials including collagen, silk, hydrogels, and egg shell membranes (ESM), providing scope for implementation in a wide range of implantable medical devices.

[0328] The publications, patent publications and other patent documents cited herein are entirely incorporated by reference. Herein, any reference to a term in the singular also encompasses its plural. Where the term “comprising”, “comprise” or “comprises” is used, said term may substituted by “consisting of’, “consist of’ or “consists of’ respectively, or by “consisting essentially of’, “consist essentially of’ or “consists essentially of’ respectively. Any reference to a numerical range or single numerical value also includes values that are about that range or single value. Any reference to alginate encompasses any physiologically acceptable salt thereof unless otherwise indicated. Unless otherwise indicated, any % value is based on the relative weight of the component or components in question.

Claims

CLAIMS1. An implantable scaffold, wherein:(a) the scaffold comprises a plurality of protein molecules; and(b) at least one of said plurality of protein molecules comprises at least one aromatic amino acid residue that is substituted with iodine on its aromatic moiety.

2. The implantable scaffold of claim 1, wherein the aromatic amino acid residue is selected from tyrosine, phenylalanine, histidine, tryptophan and combinations thereof, and preferably wherein the aromatic amino acid residue is selected from tyrosine, histidine and a combination thereof.

3. The implantable scaffold of claim 1 or claim 2, wherein at least 10%, preferably at least 20%, and more preferably at least 30%, of the tyrosine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety, preferably wherein each of the said tyrosine residues are substituted with one or two iodine substituents on their aromatic moiety.

4. The implantable scaffold of any one of claims 1 to 3, wherein at least 10%, preferably at least 20%, and more preferably at least 30%, of the histidine residues in the plurality of protein molecules are substituted with iodine on their aromatic moiety, preferably wherein each of the said histidine residues are substituted with one iodine substituent on their aromatic moiety.

5. The implantable scaffold of any one of claims 1 to 4, wherein the iodine substituent is a non-radioactive isotope of iodine.

6. The implantable scaffold of any one of claims 1 to 5, wherein (i) the scaffold has a radi opacity of from 100 HU to 10,000 HU and / or (ii) the iodine substituents on the scaffold can be visualised with X-rays having a peak kilovoltage from 30 kVp to 200 kVp.

7. The implantable scaffold of any one of claims 1 to 6, wherein the protein molecules are molecules of a fibrous protein.

8. The implantable scaffold of claim 7, wherein the fibrous protein is selected from collagen, keratin, silk, eggshell membrane, elastin, fibrin, extracellular matrix proteins, and the protein components of decellularized tissue or organs, and is preferably collagen.

9. The implantable scaffold of any one of claims 1 to 8, wherein the scaffold comprises at least 30% (w / w) protein molecules, preferably at least 50% (w / w) protein molecules, and more preferably at least 75% (w / w) protein molecules.

10. The implantable scaffold of any one of claims 1 to 9, wherein:(a) the plurality of protein molecules are overlapping; and / or(b) the plurality of protein molecules are assembled into a scaffold via non-covalent and / or covalent interactions between adjacent protein molecules; and / or(c) the scaffold is a porous 3-dimensional matrix; and / or(d) the scaffold is a sheet, a tube, a mesh, a cardiac patch, a vascular graft, or an injectable hydrogel optionally further comprising a small molecule drug, cells or nanoparticles; and / or(e) the scaffold is a micropattemed or a nanopattemed structure; and / or(f) the scaffold is a 3D-printed structure; and / or(g) the scaffold is a decellularized tissue or organ.

11. A method of using an implantable scaffold according to any one of claims 1 to 10 for repairing a tissue defect or reconstructing tissue, or for resection or ablation of a tumour, wherein said method comprises positioning the implantable scaffold in or on a subject such that the scaffold extends across, or adjacent to, all or a proportion of the tissue defect or tissue to be reconstructed, or the tumour to be resected or ablated.

12. The method of claim 11, wherein said method further comprises:(i) visualising the implanted scaffold using a computed tomography (CT) scan or a planar X-ray scan; and(ii) optionally, a surgical intervention.

13. The method of claim 11 or claim 12, wherein the said method of repairing a tissue defect or reconstructing tissue is a method for the repair of urogenital damage, skin wounds, bone defects, hernia, nerve damage, vascular damage, post-surgical reconstruction and cardiovascular disease.

14. A protein comprising at least one aromatic amino acid residue that is substituted with iodine on its aromatic moiety, for use in a method of repairing a tissue defect or reconstructing tissue, or a method for resection or ablation of a tumour, comprising:(i) incorporating said protein into an implantable scaffold;(ii) positioning said implantable scaffold in or on a subject such that the scaffold extends across, or adjacent to, all or a proportion of the tissue defect or tissue to be reconstructed or the tumour to be resected or ablated; and(iii) visualising the implanted scaffold using a computed tomography (CT) scan or a planar X-ray scan.

15. The protein for use of claim 14, wherein the method further comprises a surgical intervention after visualisation of the implanted scaffold, optionally wherein said surgical intervention comprises repositioning, reattachment, replacement or removal of the scaffold.

16. The protein for use of claim 14 or claim 15, wherein the said method of repairing a tissue defect or reconstructing tissue is a method for the repair of urogenital damage, skin wounds, bone defects, hernia, nerve damage, vascular damage, post- surgical reconstruction or cardiovascular disease.

17. The protein for use of any one of claims 14 to 16, wherein the aromatic amino acid residue is selected from tyrosine, phenylalanine, histidine, tryptophan andcombinations thereof, and preferably wherein the aromatic amino acid residue is selected from tyrosine, histidine and a combination thereof.

18. The protein for use of any one of claims 14 to 17, wherein:(a) at least 10%, preferably at least 20%, and more preferably at least 30%, of the tyrosine residues in the protein molecule are substituted with iodine on their aromatic moiety, preferably wherein each of the said tyrosine residues are substituted with one or two iodine substituents on their aromatic moiety; and / or(b) at least 10%, preferably at least 20%, and more preferably at least 30%, of the histidine residues in the protein molecule are substituted with iodine on their aromatic moiety, preferably wherein each of the said histidine residues are substituted with one iodine substituent on their aromatic moiety.

19. The protein for use of any one of claims 14 to 18, wherein the protein is a fibrous protein, preferably wherein the fibrous protein is selected from collagen, keratin, silk, elastin, fibrin, extracellular matrix proteins, and the protein components of decellularized tissue or organs, and is more preferably collagen.

20. The protein for use of any one of claims 14 to 19, wherein the implantable scaffold is an implantable scaffold according to any one of claims 1 to 10.

21. A method of iodinating a protein scaffold, wherein said protein scaffold comprises a plurality of protein molecules, comprising:(i) contacting the scaffold with a buffer solution; and(ii) incubating said solution with KI3 or IC1 under suitable conditions to result in iodination of the aromatic moiety of at least one aromatic amino acid residue in at least one of the plurality of protein molecules, optionally wherein the KI3 or IC1 reagent comprises a non-radioactive isotope of iodine.

22. A method of preparing an iodinated protein scaffold, wherein said protein scaffold comprises a plurality of protein molecules, comprising:(i) contacting the plurality of protein molecules with a buffer solution;(ii) incubating said solution with KI3 or IC1 under suitable conditions to result in iodination of the aromatic moiety of at least one aromatic amino acid residue in at least one of the plurality of protein molecules; and(iii) assembling the plurality of protein molecules into a protein scaffold.

23. The method of claim 21 or claim 22, wherein the buffer solution is incubated with KI3, and wherein preferably:(a) the buffer solution has a pH from 7 to 9; and / or(b) the buffer solution is phosphate-buffered saline (PBS) or 4-(2-hy droxy ethyl)- 1- piperazineethanesulfonic acid solution (HEPES).

24. The method of claim 21 or claim 22, wherein the buffer solution is incubated with IC1, and wherein preferably:(a) the buffer solution has a pH from 7 to 10, preferably from 8 to 9; and / or(b) the buffer solution is glycine solution or tri s(hydroxymethyl)aminom ethane solution (TRIS).

25. The method of any one of claims 21 to 24, wherein the method is a method for preparing an implantable scaffold according to any of claims 1 to 10.

26. The implantable scaffold of any one of claims 1 or 5 to 10, wherein the aromatic amino acid residue is a non-natural amino acid residue.

27. The implantable scaffold of claim 26, wherein the aromatic amino acid residue is a modified lysine residue, the primary amino group of which is covalently bonded, via an amide bond, to a Ce-Cio aryl or Ce-Cio aralkyl group, which is substituted with one or more iodine atoms on the aromatic ring and which may be further optionally substituted with a hydroxy group on the aromatic ring.

28. The implantable scaffold of claim 26 or claim 27, wherein the aromatic amino acid residue has formula (I):wherein:(i) X is H or OH;(ii) n is 1 or 2;(iii) m is an integer from 0 to 4; and(iv) * andArepresent points of attachment of the aromatic amino acid residue to adjacent amino acid residues in the protein.

29. The implantable scaffold of claim 28, wherein the aromatic amino acid residue has formula (la) or formula (lb):

30. The implantable scaffold of claim 28 or claim 29, wherein:(a) m is 0, preferably wherein X is H; or(b) m is 2, preferably wherein X is OH.

31. The implantable scaffold of any one of claims 26 to 30, wherein at least 10%, preferably at least 20%, and more preferably at least 30%, of the lysine residues in the plurality of protein molecules are modified lysine residues, the primary amino group of which is covalently bonded, via an amide bond, to a Ce-Cio aryl or Ce-Cio aralkyl group, which is substituted with one or more iodine atoms on the aromaticring and which may be further optionally substituted with a hydroxy group on the aromatic ring.

32. A method of using an implantable scaffold according to any one of claims 26 to 31 for repairing a tissue defect or reconstructing tissue, or for resection or ablation of a tumour, wherein said method comprises positioning the implantable scaffold in or on a subject such that the scaffold extends across, or adjacent to, all or a proportion of the tissue defect or tissue to be reconstructed, or the tumour to be resected or ablated; optionally wherein said method further comprises:(i) visualising the implanted scaffold using a computed tomography (CT) scan or a planar X-ray scan; and(ii) optionally, a surgical intervention; further optionally wherein the said method of repairing a tissue defect or reconstructing tissue is a method for the repair of urogenital damage, skin wounds, bone defects, hernia, nerve damage, vascular damage, post-surgical reconstruction and cardiovascular disease.

33. The protein for use of any one of claims 14 to 16, 19 or 20, wherein the aromatic amino acid residue is a modified lysine residue, the primary amino group of which is covalently bonded, via an amide bond, to a Ce-Cio aryl or Ce-Cio aralkyl group, which is substituted with one or more iodine atoms on the aromatic ring and which may be further optionally substituted with a hydroxy group on the aromatic ring, preferably wherein the aromatic amino acid residue has formula (I), formula (la) or formula (lb) as defined in any one of claims 28 to 30.

34. The protein for use of any one of claims 14 to 16, 19, 20 or 33, wherein at least 10%, preferably at least 20%, and more preferably at least 30%, of the lysine residues in the plurality of protein molecules are modified lysine residues, the primary amino group of which is covalently bonded, via an amide bond, to a Ce-Cioaryl or Ce-Cio aralkyl group, which is substituted with one or more iodine atoms on the aromatic ring and which may be further optionally substituted with a hydroxy group on the aromatic ring.

35. A method of iodinating a protein scaffold, wherein said protein scaffold comprises a plurality of protein molecules, comprising:(i) contacting the scaffold with a buffer solution; and(ii) incubating said solution with an acylating reagent X-(C=O)-R under suitable conditions to result in acylation of the primary amino group of at least one lysine residue in at least one of the plurality of protein molecules, wherein:- X is a leaving group under addition-elimination reaction conditions; and- R is Ce-Cio aryl or Ce-Cio aralkyl group, which is optionally substituted with a hydroxy group on the aromatic ring; and(iii) incubating said solution with KI3 or IC1 under suitable conditions to result in iodination of the aromatic moiety of at least one acylated lysine residue in at least one of the plurality of protein molecules; optionally wherein the iodinating reagent comprises a non-radioactive isotope of iodine.

36. A method of iodinating a protein scaffold, wherein said protein scaffold comprises a plurality of protein molecules, comprising:(i) contacting the scaffold with a buffer solution; and(ii) incubating said solution with an acylating reagent X-(C=O)-R under suitable conditions to result in acylation of the primary amino group of at least one lysine residue in at least one of the plurality of protein molecules, wherein:- X is a leaving group under addition-elimination reaction conditions; and- R is Ce-Cio aryl or Ce-Cio aralkyl group, which is substituted with one or more iodine atoms (preferably, one or two iodine atoms) on the aromatic ring, and which is further optionally substituted with a hydroxy group on the aromatic ring;optionally wherein the iodinating reagent comprises a non-radioactive isotope of iodine.

37. A method of preparing an iodinated protein scaffold, wherein said protein scaffold comprises a plurality of protein molecules, comprising:(i) contacting the plurality of protein molecules with a buffer solution;(ii) incubating said solution with an acylating reagent X-(C=O)-R under suitable conditions to result in acylation of the primary amino group of at least one lysine residue in at least one of the plurality of protein molecules, wherein:- X is a leaving group under addition-elimination reaction conditions; and- R is Ce-Cio aryl or Ce-Cio aralkyl, which is optionally substituted with a hydroxy group on the aromatic ring;(iii) incubating said solution with KI3 or IC1 under suitable conditions to result in iodination of the aromatic moiety of at least one acylated lysine residue in at least one of the plurality of protein molecules; and(iv) assembling the plurality of protein molecules into a protein scaffold; optionally wherein the iodinating reagent comprises a non-radioactive isotope of iodine.

38. A method of iodinating a protein scaffold, wherein said protein scaffold comprises a plurality of protein molecules, comprising:(i) contacting the plurality of protein molecules with a buffer solution; and(ii) incubating said solution with an acylating reagent X-(C=O)-R under suitable conditions to result in acylation of the primary amino group of at least one lysine residue in at least one of the plurality of protein molecules, wherein:- X is a leaving group under addition-elimination reaction conditions; and- R is Ce-Cio aryl or Ce-Cio aralkyl group, which is substituted with one or more iodine atoms (preferably, one or two iodine atoms) on the aromatic ring, and which is further optionally substituted with a hydroxy group on the aromatic ring; and(iii) assembling the plurality of protein molecules into a protein scaffold; optionally wherein the iodinating reagent comprises a non-radioactive isotope of iodine.

39. The method of claim 35 or claim 37, wherein the acylating reagent is selected from7V-succinimidyl-3-(4-hydroxyphenyl)propionate or A'-succinimidyl benzoate.

40. The method of claim 36 or claim 38, wherein the acylating reagent is selected from 7V-succinimidyl-3-(4-hydroxy-3 -iodophenyl )propionate, 7V-succinimidyl-3-(4- hydroxy-3, 5-di-iodophenyl)propionate), 7V-succinimidyl 3 -iodobenzoate and N- succinimidyl-3,5-di-iodobenzoate.

41. The method of any one of claims 35 to 40, wherein the method is a method for preparing an implantable scaffold according to any of claims 1, 5 to 10 or 26 to 31.

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