Molecular linker

Abstract

This disclosure relates to crosslinking of endogenous proteins in tissues using molecular linkers, and therapeutic compositions comprising such linkers. This disclosure further relates to therapeutic methods and therapeutic compositions for the treatment of diseases and disorders.

Description

[Technical Field]

[0001] This disclosure relates to crosslinking of endogenous proteins in tissues using molecular linkers, and therapeutic compositions comprising molecular linkers. This disclosure further relates to therapeutic methods and therapeutic compositions for the treatment of diseases and disorders. [Background technology]

[0002] Corneal ectasia (ECD), or corneal ectasia, is a group of progressive eye disorders characterized by progressive corneal ablation and thinning. Corneal ectasia is associated with decreased uncorrected visual acuity (UCVA), increased ocular aberrations, and, often, loss of best-corrected distance visual acuity. Corneal ectasia can lead to serious eye diseases and may require surgical intervention.

[0003] Corneal ectasias, such as keratoconus (KC), pellucid corneal marginal degeneration, and post-refractory or post-traumatic corneal ectasia, are characterized by progressive corneal ablation, thinning, and refractive changes. Keratoconus is a bilateral, progressive degenerative corneal condition that results in degeneration, thinning, and weakening of the corneal extracellular matrix (ECM), leading to corneal bulging and progressive loss of vision. Keratoconus is characterized by progressive, non-inflammatory subcorneal thinning, ablation, and apical scarring, affecting approximately 2-3 people per 1000 in developed countries and being the second most common reason for corneal transplantation worldwide. Corneal ectasias can severely impair an individual's visual field and quality of life, potentially leading to functional impairment.

[0004] The pathogenesis of keratoconus is not fully understood, but an interaction between genetic factors and environmental stressors is suspected. If left untreated, approximately 20% of patients require a corneal transplant, and in some countries, keratoconus is reported as a leading indication, accounting for 40%–45% of annual corneal transplants. Several treatment options are available to patients with KC. These include refractive correction (with glasses or contact lenses), intracorneal ring segment grafting, and, in advanced stages, corneal transplantation (full-thickness keratografting or deep anterior keratografting). While such approaches may be effective, they can be expensive, and a good prognosis depends on the skill of the surgeon. Furthermore, corneal grafting requires patients to take immunosuppressants for a long period, and the procedure can expose patients to a high risk of lifelong ocular rupture due to weakening of corneal structure.

[0005] Corneal crosslinking (CXL) represents a relatively recent method that can avoid the more dramatic surgical step of corneal transplantation. It is FDA-approved for the treatment of keratoconus and corneal ectasia. The Dresden protocol, the first CXL technique described for the treatment of keratoconus, uses a 3.0 mW / cm² method. 2 This includes 30 minutes of ultraviolet-A (UVA, 370nm) irradiation at a radiant intensity of 5.4 J / cm². 2This results in a total surface dose (Wollensak et al., 2003). It is generally understood that exposure of riboflavin (Rv) to UV-A light, in an oxygen-containing aqueous environment, induces the formation of highly reactive singlet oxygen radicals, which interact with protein components in corneal tissue to produce additional crosslinking (Kamaev et al., 2012). Based on the Bunsen-Roscoe reciprocity rule, the same photochemical effect can be achieved with reduced light exposure time and correspondingly increased irradiation intensity. Several newly commercialized CXL devices offer high UVA irradiance, thereby enabling similar energy delivery with shorter treatment times (Dervenis et al., 2020). For example, currently available accelerated crosslinking (A-CXL) protocols involve 18 minutes of UVA (370 nm) (20 mW / cm²). 2 7.2 J / cm 2 The pulse profile includes a pulse (1 second active, 2 seconds pause). An alternative A-CXL protocol is 9 mW / cm² for 10 minutes without an optical pulse. 2 This includes UV-A irradiation. A standard CXL protocol (Wollensak et al., 2003; Spoerl et al., 1998) consists of applying riboflavin (vitamin B2) to the epithelialized cornea for 30 minutes to promote molecular diffusion into the stroma to a depth of approximately 200 μm. Riboflavin diffusion plays a crucial role in the CXL procedure, and the axial corneal diffusion in a typical epithelialized CXL is 6.5 × 10⁻⁶. -5 mm 2 It has been found to be equal to / second, and the typical diffusion time is 30 minutes (Seiler et al., 2014).

[0006] The principle of this approach is that UV-A-induced photopolymerization in the presence of the photosensitizer riboflavin induces the formation of covalent bonds between substrates within the corneal stroma, including between collagen, proteoglycans, and other stromal proteins (Wollensak et al., 2003; Brummer et al., 2011; Hayes et al., 2013). The effects of these induced crosslinks have been reported in various ways, including increased tissue stiffness, resistance to enzymatic digestion, changes in ultrastructure, and altered expansion behavior (Akhtar et al., 2013; Spoerl et al., 2004a; Spoerl et al., 2004b; Wollensak et al., 2004). The mechanical properties of biological tissues depend heavily on the entanglement of collagen fibrils, the bound lamina layers, and the spacing between fibrils. Corneal collagen crosslinking (CXL) can lead to increased corneal stiffness and potentially alter the natural course of corneal cornea (KC). This is a well-established method that can reduce the risk of KC progression over the long term (Raiskup-Wolf et al., 2008; O'Brart et al., 2015).

[0007] The CXL method, however, only works over short molecular distances (less than 5 Å) because this crosslinking protocol relies on the localized diffusion of highly reactive singlet oxygen species formed by UV irradiation of riboflavin. Hayes et al. (2013) concluded that riboflavin / UVA therapy does not result in widespread crosslinking of collagen molecules. Furthermore, evidence has been presented that riboflavin / UVA-induced crosslinking does not exist between or within collagen fibrils, but may occur on the surface of individual collagen molecules constituting the fibrils and within the proteoglycan-rich coating surrounding the collagen fibrils.

[0008] Despite FDA approval and clinical adoption for KC treatment, the current riboflavin / UV-A CXL approach has several practical and potential drawbacks. (i) Use of UV irradiation (light irradiation at a wavelength of 370 nm) that may damage DNA and have adverse and detrimental effects on endothelial cells. (ii) Production of highly reactive and nonspecific singlet oxygen free radicals. These free radicals can cause damage to sensitive endothelial cells and keratocytes. The potential for damage to endothelial cells is to prevent widespread diffusion of riboflavin into deeper layers of the corneal stroma (above 300 μm) and to keep the total dose of UV energy at a biologically safe level (5.4 J / cm²). 2 This is avoided by limiting it to less than . This method relies on the diffusion and substantial availability of high concentrations of molecular oxygen, which are the substrates for the photochemical production of reactive singlet oxygen species. Because the Dresden photocrosslinking technique consumes molecular oxygen, the treatment process is necessarily time-consuming (requiring approximately 9-18 minutes of irradiation per eye), even when using various pulsed laser methods that allow time to maintain sufficiently high levels of oxygen in the substantial matrix during the irradiation process. (iii) Complications of corneal crosslinking include corneal opacity, corneal scarring, infectious keratitis, sterile infiltrates, delayed epithelial healing, treatment failure, excessive corneal flattening with hyperopia, and endothelial dysfunction. (iv) Corneal endothelial damage may occur if safety limits regarding corneal thickness to prevent endothelial toxicity are not observed. Sharma et al. (2012) reported a 1.4% incidence of persistent endothelial dysfunction in 350 eyes treated with a standard epithelial removal protocol, even when safety limits for corneal thickness exceeding 400 μm (epithelial removal) were observed (Vimalin et al., 2012). This may be due to intraoperative thinning of the stroma, lack of homogeneity, and focusing / alignment problems with the UV device, resulting in stroma dehydration.

[0009] The contraindications and complications of the CXL procedure, including the following, are described: corneal infections; corneal sensitivity and tear function; effects on corneal limbal epithelial cells; transient and persistent corneal stromal opacities and sterile infiltrates; endothelial toxicity and corneal thinning; corneal drug penetration and IOP measurement; changes in corneal thickness; postoperative pain; infectious keratitis; development of persistent epithelial defects; development of persistent epithelial defects; stromal scarring, corneal melting; corneal endothelial decompensation and lens or retinal damage; and development of late-onset marginal ulcerative keratitis.

Summary of the Invention

Problems to be Solved by the Invention

[0010] Therefore, there remains a need for methods of crosslinking endogenous proteins in tissue, as well as methods of treating or preventing ectatic corneal diseases, such as keratoconus, and / or methods for strengthening the cornea.

Means for Solving the Problems

[0011] The present disclosure is based on the identification by the inventors of molecular linkers suitable for crosslinking endogenous proteins in tissue. The inventors have shown that by including a molecular linker polymer, having a suitable molecular weight and containing two or more crosslinking functional groups, such as, by way of example, tyramine or tyrosine, also referred to herein as a "molecular linker" or "molecular staple", protein crosslinking of more distally located endogenous protein molecules in tissue is promoted. The molecular linkers and crosslinking methods described herein have use in the treatment of diseases.

[0012] Thus, in one aspect, a method of crosslinking an endogenous protein in a tissue, comprising: (i) contacting the tissue with a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor, whereby the molecular linker contacts the endogenous protein in the tissue; and (ii) irradiating the tissue. A method is provided.

[0013] Cross-linking of the molecular linker and the endogenous protein occurs by irradiating the tissue in the presence of a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor.

[0014] In certain embodiments, the molecular linker is a polymer comprising two or more tyrosine and / or tyramine residues.

[0015] In other embodiments, the polymer comprising one or more tyrosine and / or tyramine residues may be selected from tyrosine and / or tyramine-derivatized gelatin, tyrosine and / or tyramine-derivatized dextran, and / or tyrosine and / or tyramine-derivatized xylan, and / or tyrosine and / or tyramine-derivatized low molecular weight nanocellulose.

[0016] In some embodiments, the molecular linker has a molecular weight of about 5 kDa to about 50 kDa, or about 5 kDa to 40 kDa. Alternatively, the molecular linker has a molecular weight of about 10 kDa to about 20 kDa, or about 12 kDa to about 20 kDa, or about 10 kDa to about 15 kDa.

[0017] In some embodiments of the cross-linking method, the photoactivatable metal ligand complex is ruthenium(III) bipyridyl chloride.

[0018] In some embodiments, the electron acceptor is sodium persulfate.

[0019] In certain embodiments, the tissue is corneal tissue.

[0020] In some embodiments, the endogenous protein is a corneal stromal protein. For example, the corneal stromal protein can be selected from collagen and / or stromal proteoglycan. In some embodiments, the corneal stromal proteins include collagen (types I, III, V, and VI), and proteoglycans (aggrecan, keratocan, lumican, mimecan, decorin, and biglycan).

[0021] In some embodiments, the step of contacting the corneal tissue with a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor includes administering a solution containing the molecular linker, the photoactivatable metal ligand complex, and the electron acceptor to the surface of the cornea, whereby the solution diffuses into the corneal stroma.

[0022] In some embodiments, the step of contacting the corneal tissue with a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor includes administering two or more solutions collectively containing the molecular linker, the photoactivatable metal ligand complex, and the electron acceptor to the surface of the cornea, whereby the solutions diffuse into the corneal stroma.

[0023] In some embodiments, the solution or the two or more solutions further contain high molecular weight gelatin.

[0024] In some embodiments, the tissue is irradiated with light having a wavelength of approximately 430 nm to 480 nm. In certain embodiments, the tissue is irradiated with blue light having a peak wavelength of about 455 nm.

[0025] In some embodiments, the step of irradiating the tissue includes delivering a light intensity of about 200 mW / cm 2 ~1,200 mW / cm 2 to the tissue surface.

[0026] In a further embodiment, the step of irradiating the tissue includes about 10 J / cm 2This includes delivering radiant energy less than 1 J / cm². For example, in some embodiments, the step of irradiating tissue is about 1 J / cm². 2 ~About 5J / cm 2 This includes irradiating the tissue with light from a 455 nm light source to deliver a maximum of 3.4 J / cm². In some embodiments, the step of irradiating the tissue is performed with a maximum of 3.4 J / cm². 2 This includes light irradiation of the tissue using a 455nm LED light source to deliver light.

[0027] In some embodiments, the tissue is irradiated for about 1 to about 10 seconds or about 1 to 5 seconds.

[0028] In another embodiment, a method for treating weakening of the corneal stroma in a subject, (i) The step of administering a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor to the target cornea, (ii) The step of irradiating the cornea and A method is provided that includes this.

[0029] The step of irradiating the cornea in the presence of a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor initiates a crosslinking reaction between the molecular linker and endogenous proteins in the corneal stroma.

[0030] In another embodiment, a method for treating or preventing at least one symptom of keratoconus in a subject, (i) The step of administering a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor to the target cornea, (ii) The step of irradiating the cornea and A method is provided that includes this.

[0031] The step of irradiating the cornea in the presence of a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor initiates a crosslinking reaction between the molecular linker and endogenous proteins in the corneal stroma.

[0032] In some embodiments, the molecular linker is a polymer containing two or more tyrosine and / or tyramine residues.

[0033] In some embodiments, polymers comprising two or more tyrosine and / or tyramine residues are selected from tyrosine and / or tyramine-derivative gelatin, tyrosine and / or tyramine-derivative dextran, and / or tyrosine and / or tyramine-derivative xylan, and / or tyrosine and / or tyramine-derivative low molecular weight nanocellulose.

[0034] In some embodiments, the molecular linker has a molecular weight of approximately 5 kDa to approximately 40 kDa, approximately 10 kDa to approximately 20 kDa, or approximately 12 kDa to approximately 20 kDa, or approximately 10 kDa to approximately 15 kDa.

[0035] In some embodiments, the photoactivatable metal ligand complex is ruthenium(tris)bipyridyl chloride.

[0036] In some embodiments, the electron acceptor is sodium persulfate.

[0037] In some embodiments, the endogenous protein is a corneal stromal protein. For example, the corneal stromal protein may be selected from collagen and / or stromal proteoglycans. In some embodiments, the corneal stromal protein may be collagen (type I, type III, type V, and type VI), as well as proteoglycans (agrecan, keratocan, lumican, mimican, decorin, and biglycan).

[0038] In some embodiments of the methods described herein, the step of administering a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor includes administering a solution containing the molecular linker, the photoactivatable metal ligand complex, and the electron acceptor to the surface of the cornea, thereby allowing the solution to diffuse into the corneal stroma.

[0039] In some embodiments described herein, the step of administering a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor involves administering two or more solutions collectively comprising the molecular linker, the photoactivatable metal ligand complex, and the electron acceptor to the surface of the cornea, thereby allowing the solutions to diffuse into the corneal stroma.

[0040] In some embodiments, the solution diffuses into the corneal stroma for up to approximately 5 minutes.

[0041] In some embodiments, the cornea is irradiated with light having a wavelength of approximately 430 nm to 480 nm. For example, the cornea may be irradiated with blue light from an LED light source having a peak wavelength of approximately 455 nm. In some embodiments, the LED light emits blue light at a usable wavelength of 430 nm to 480 nm, with a central wavelength of 455 ± 10 nm. In some embodiments, the light intensity (400 to 515 nm) is approximately 1200 mW / cm². 2 The range can be -10% / +20% (independent of battery power level). In some embodiments, the step of irradiating tissue is up to 3.4 J / cm². 2 This includes light irradiation of the tissue using a 455nm LED light source to deliver light.

[0042] In one embodiment, the tissue is irradiated for about 1 to about 10 seconds or about 1 to about 5 seconds.

[0043] In some embodiments, crosslinking molecular linkers and endogenous proteins improves one or more mechanical properties of the corneal stroma. For example, one or more improvements in the mechanical properties of the corneal stroma may be an increase in the elastic modulus of the stroma.

[0044] In another embodiment, the use of a molecular linker is provided in the manufacture of a pharmaceutical product for the treatment or prevention of one or more symptoms of a disease in a subject.

[0045] In some embodiments, the disease is keratoconus and / or weakening of the corneal stroma.

[0046] In another embodiment, a composition for treating keratoconus in a subject is provided, comprising a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor.

[0047] In yet another embodiment, a composition is provided for intermolecular crosslinking between endogenous stromal collagen fibrils and other endogenous proteins in the cornea, comprising a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor.

[0048] In some embodiments, the endogenous stromal proteins in the cornea are selected from collagen and proteoglycans.

[0049] In some embodiments of the methods described herein, the method further includes the step of applying high molecular weight gelatin to the surface of the cornea.

[0050] For clarity, it is understood that certain characteristics described herein in the context of separate embodiments may also be provided in combination in a single embodiment. In contrast, for brevity, the various characteristics described in the context of a single embodiment may also be provided separately or in any partial combination.

[0051] Throughout this specification, variations of the word “comprise,” “comprises,” or “comprising” indicate the inclusion of the element, integer, or step, or group of elements, integers, or steps, mentioned, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0052] Throughout this specification, unless otherwise specifically indicated or required by the context, any reference to a single step, characteristic, composition in question, group of steps, or characteristic or group of compositions in question shall be interpreted as encompassing one or more (i.e., one or more) such steps, characteristics, composition in question, group of steps, or group of characteristics or compositions in question.

[0053] This disclosure can be better understood by referring to the following drawings. [Brief explanation of the drawing]

[0054] [Figure 1] This is a diagram of the tissue structure of collagen and proteoglycans in corneal fibrils. Source: Box 4.9 - Organization of tissue proteoglycans (In: Chapter 4; Biochemistry and cell biology. Forrester, JV, Dick, AD, McMenamin, PG, Roberts, F., & Pearlman, E. (2020). The Eye: basic sciences in practice (5th Edition Elsevier) (1A). Diagram of crosslinking scenario: tyr-dextran molecular linker crosslinking within proteoglycan core proteins in intramolecular and intermolecular spaces on the collagen fibril surface and in interfibril spaces (1B). [Figure 2] This is an SDS-PAGE image of autoclaved gelatin solutions. Each lane shows 75 mg / ml of BH-Nippi gelatin autoclaved for the time indicated in each lane, in PBS pH 7.4. 1: Standard, 2: Nippi gelatin, 3: BH-Nippi gelatin, 4: Autoclaved for 20 minutes, 5: Autoclaved for 40 minutes, 6: Autoclaved for 60 minutes, 7: Autoclaved for 80 minutes, 8: Autoclaved for 5 hours, 9: Autoclaved for 9 hours, 10: Autoclaved for 12 hours. [Figure 3]This figure shows dextran functionalized or derivatized with an amine group (3A). A 10 kDa tyramine-derivative dextran can form dityrosine crosslinks between endogenous proteins separated by intermolecular distances of up to 25 nm (3B). [Figure 4] This is a diagram of dextran polymer containing primary amines at each end. [Figure 5] Photograph of fresh, intact epithelium from a bovine eye (5A). Epithelium debridement using an 8mm diameter biopsy punch (5B). [Figure 6] Photograph (6A) showing the addition of 25 μL of 1 mM Ru(bpy)3 to the corneal debridement area. Frozen corneal button showing an 8 mm debridement area to which 25 μL of 1 mM Ru(bpy)3 was added (6B). Frozen cornea - 1 mm thick cross-sectional section cut after 6 minutes of Ru(bpy)3 diffusion into the cornea (6C). [Figure 7] This image shows the visualization of Ru(bpy)3 fluorescence after diffusion into the debridemented cornea (fluorescence image captured through a red filter). [Figure 8] Photograph of riboflavin diffused into a new bovine cornea (after blotting) (8A). Diffusion of riboflavin into the cornea - frozen section (8B). Visualization of riboflavin fluorescence after diffusion into the debridemented cornea (fluorescence image captured through a red filter) (8C). [Figure 9] This is a photograph of the entire epithelium debridement from the cornea. 1 mM Ru(bpy)3 + 300 μL of 50 mM SPS was added to the anterior corneal surface, allowing a 10-minute diffusion time (9A). A 450 nm blue LED was used at a distance of 1 cm using a piping nozzle (metal) as a mold for 20 seconds (9B). [Figure 10] Photograph of an unstructured cornea fixed to the shape of a mold after photocrosslinking in the presence of Ru(bpy)3+SPS (10A). Morphology of the photocrosslinked cornea held with forceps (10B). [Figure 11]This is a diagram of the cornea after crosslinking on a template without SPS - no template-like structure formation is observed (11A). Cornea after photochemical crosslinking without SPS - no template-like structure formation is observed (11B). Cornea after photochemical crosslinking without SPS held with forceps - no template-like structure formation is observed (11C). [Figure 12] This is a graph of the diffusion of 0.5 mM Ru(bpy)3 (Figure 12A). Diffusion of 1.0 mM Ru(bpy)3 (12B). Diffusion of 2.0 mM Ru(bpy)3 (12C). [Figure 13] This graph shows the diffusion of 0.5 mM Ru(bpy)3 + 50 mM SPS(13A), 1.0 mM Ru(bpy)3 + 50 mM SPS(13B), and 2.0 mM Ru(bpy)3 + 50 mM SPS(13C) into a 1 mm thick, 12% gelatin sheet. [Figure 14] This graph shows the diffusion of 2.6 mM (0.1%) riboflavin into a 12% gelatin gel over a maximum time of 14 minutes. [Figure 15]This is a photograph of a 12% gelatin strip (15A) crosslinked by a photochemical crosslinking reaction including 1 mM Ru(bpy)3 + 50 mM SPS in the presence of autoclaved BH-derivativeated Nippi gelatin (150 mg / ml). Gelatin strip with one end fixed: mass added 0 mg. This is a photograph of a 12% gelatin strip (15B) crosslinked by a photochemical crosslinking reaction including 1 mM Ru(bpy)3 + 50 mM SPS in the presence of BH-derivativeated Nippi gelatin (150 mg / ml) that has been autoclaved seven times. Gelatin strip with one end fixed: mass added 150 mg. This is a photograph of a 12% gelatin strip (15C) crosslinked by a photochemical crosslinking reaction including 1 mM Ru(bpy)3 + 50 mM SPS in the presence of autoclaved BH-derivativeated Nippi gelatin (150 mg / ml). Gelatin strip with one end fixed: 150 mg removed by mass. A 12% gelatin strip (15D) crosslinked by a photochemical crosslinking reaction including 1 mM Ru(bpy)3 + 50 mM SPS in the presence of BH derivatized Nippi gelatin (150 mg / ml) that has been autoclaved 7 times. A 12% gelatin strip (without addition of "molecular staples") (15E) crosslinked by a photochemical crosslinking reaction including 1 mM Ru(bpy)3 + 50 mM SPS. Gelatin strip with one end fixed: 0 mg added by mass. A 12% gelatin strip (without addition of "molecular staples") (15F) crosslinked by a photochemical crosslinking reaction including 1 mM Ru(bpy)3 + 50 mM SPS. Gelatin strip with one end fixed: 150 mg added by mass. 12% gelatin strips (without addition of "molecular staples") (15G) crosslinked by photochemical crosslinking reaction including 1 mM Ru(bpy)3 + 50 mM SPS. Gelatin strips with one end fixed: 150 mg mass removed. 12% gelatin strips (without addition of "molecular linker / staples") (15H) crosslinked by photochemical crosslinking reaction including 1 mM Ru(bpy)3 + 50 mM SPS. [Figure 16]These are photographs of new bovine corneal strips (10 mm × 5 mm) crosslinked in the presence (16A) or absence (16B) of low molecular weight BH-derivative gelatin (150 mg / ml). Bovine corneas (10 mm × 5 mm) were crosslinked by photochemical crosslinking using 1 mM Ru(bpy)3 + 50 mM SPS in the presence of BH-derivative Nippi gelatin (lane 10, see Figure 2) (150 mg / ml) that had been autoclaved seven times (total volume = 60 μL). Keys in an array list

[0055] This application is filed together with an electronic sequence listing. The entire contents of the sequence listing are incorporated herein by reference. Sequence ID 1 - Amino acid sequence of human collagen type 1A Amino acid sequence of type IA of tyrosine-derivativeized human collagen (gelatin) (SEQ ID NO: 2) SEQ ID NO: 3 - Amino acid sequence of human collagen type V Amino acid sequence of SEQ ID NO: 4 - Human vimentin SEQ ID NO: 5 - Amino acid sequence of human fibronectin Amino acid sequence of SEQ ID NO: 6 - human laminin SEQ ID NO: 7 - Amino acid sequence of human elastin Sequence ID 8 - Amino acid sequence of human aggrecan SEQ ID NO: 9 - Amino acid sequence of human keratocan Sequence ID 10 - Amino acid sequence of *Human Luminosa* Sequence ID 11 - Amino acid sequence of *Epipactis thunbergii* Amino acid sequence of SEQ ID NO: 12 - Starfish Choline Sequence ID 13 - Amino acid sequence of human biglycan [Modes for carrying out the invention]

[0056] overview Throughout this specification, unless otherwise specifically indicated or required by the context, any reference to a single step, composition in question, group of steps, or group of compositions in question shall be interpreted as encompassing one or more (i.e., one or more) of those steps, compositions in question, groups of steps, or groups of compositions in question.

[0057] Those skilled in the art will recognize that this disclosure may be subject to variations and modifications other than those specifically described. It should be understood that this disclosure includes such variations and modifications. This disclosure also includes all of the steps, properties, compositions, and compounds referenced or indicated herein, as well as any combination or any two or more of such steps or properties, individually or collectively.

[0058] Unless otherwise specifically defined, all technical and scientific terms used herein shall be construed to have the same meaning as those commonly understood by those skilled in the art in that field (e.g., protein chemistry, chemistry, cell culture, molecular genetics, molecular biology, immunohistochemistry, and biochemistry).

[0059] The term "and / or," for example, "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be interpreted as providing explicit support for both meanings or either meaning.

[0060] Where used herein, the term “about” typically refers to a maximum of ±10% of the specified value, for example, ±5%, unless otherwise indicated.

[0061] As used herein, the term "subject" shall be interpreted as meaning any animal, including mammals, such as humans.

[0062] Throughout this specification, various aspects and components of the invention may be presented in range form. Range form is included for convenience and should not be construed as a firm limitation on the scope of the invention. Therefore, range descriptions should be considered to specifically disclose all possible subranges and the individual numerical values ​​within those ranges unless specifically indicated otherwise. For example, a range description such as 1–5 should be considered to specifically disclose subranges such as 1–3, 1–4, 1–5, 2–4, 2–5, 3–5, and the individual numerical values ​​and fractions within the enumerated range, e.g., 1, 2, 3, 4, 5, 5.5, and 6, unless integers are required or implied by context. This applies regardless of the breadth of the disclosed range. Where specific values ​​are required, they are provided herein.

[0063] Selective definition As used herein, the term “crosslinking” refers to the process of linking two amino acids or atoms to each other via a covalent bond. One example of a crosslinking process is the reaction of a protein linking group with a target protein, such as a side chain residue of an endogenous protein in a tissue, to result in a stable covalent linkage.

[0064] When used herein, the term "molecular linker" refers to a low molecular weight polymer comprising two or more residues having tyrosine or tyramine side chains capable of forming dityrosine crosslinks with tyrosine residues located in endogenous proteins in tissues.

[0065] The term "photoactivatable metal ligand complex," as used herein, refers to a metal ligand complex in which the metal can become an excited state upon irradiation, such that it can donate electrons to an electron acceptor to transition to a higher oxidation state, and subsequently extract electrons from an aromatic side chain, e.g., tyrosine, tyramine, or tyrosine-containing protein, or tyramine or tyrosine derivatized polymer, to produce a free radical without relying on the formation of singlet oxygen. Suitable metals include, but are not limited to, Ru(II), Pd(II), Cu(II), Ni(II), Mn(II), and Fe(III) in the form of complexes that can absorb light in the visible region, such as Ru(II) bipyridyl complexes, Pd(II) porphyrin complexes, sulfonatophenyl Mn(II) complexes, or Fe(III) protoporforin complexes.

[0066] As used herein, the term “electron acceptor” refers to a chemical entity that allows the transfer of electrons thereto, and therefore refers to an easily reducible molecule (or oxidizing agent) whose redox potential is sufficiently positive to facilitate a crosslinking reaction. Suitable electron acceptors for use in the methods of this disclosure can be readily determined by those skilled in the art using the techniques described herein. For example, in some embodiments, the electron acceptor may be a persulfate, periodate, perbromate, or perchlorate compound, or vitamin B12. In one embodiment, a persulfate anion is used as the electron acceptor.

[0067] Where used herein, the terms “contact” or “administer” refer to the application of the crosslinking reagents described herein to the surface of a tissue, for example, corneal tissue, for a sufficient amount of time for the reagent to diffuse into the tissue. One or more crosslinking reagents may be applied sequentially, in any order, or simultaneously to the surface of the tissue, for example, in a single solution comprising one or more molecular linkers, photoactivatable metal ligand complexes, and / or electron acceptors.

[0068] As used herein, the terms “polypeptide” and “protein” are interchangeable. The term “polypeptide” also includes post-translational modified polypeptides or proteins. The term “polypeptide” includes polypeptides in which a conventional backbone is not naturally present or is replaced by a synthetic backbone, and peptides in which one or more conventional amino acids are replaced by one or more naturally present or synthetic amino acids. Generally, polypeptides can be of any length, for example, more than two amino acids, more than four amino acids, more than about ten amino acids, more than about twenty amino acids, more than about fifty amino acids, more than about 100 amino acids, more than about 300 amino acids, usually up to about 500 or 1000, or more amino acids. “Peptides” generally have more than two amino acids, more than four amino acids, more than about ten amino acids, more than about twenty amino acids, usually up to about nine, ten, twenty, 30, or 50 amino acids. In some embodiments, peptides are 5 to 30 amino acids long. Peptides may also be produced by protease digestion of large polypeptides.

[0069] When used herein, the term "biocompatible" means, based on a clinical risk / benefit assessment, that it causes minimal or no clinically relevant tissue irritation, injury, toxic reaction, or immunological reaction to human tissue.

[0070] Molecular linker The inventors have developed molecular linkers useful for the rapid formation of intermolecular crosslinks between endogenous protein molecules within tissues. These molecular linkers (also referred to herein as “molecular staples”) effectively crosslink or link endogenous protein regions that are degraded and consequently weakened in one or more tissues. Molecular linkers, such as tyrosine-derivative low molecular weight linkers, can be selected from low molecular weight polymers containing side chains capable of forming π-π hydrophobic interactions with tyrosine residues located in endogenous parenchymal proteins.

[0071] The length of the molecular linker allows for the formation of covalent crosslinks between endogenous proteins that are separated in molecular space, exceeding what can be achieved by currently available methods, such as the riboflavin / UV-A based CXL method, which only function over short distances (less than 5 Å). Current CXL methods rely on the generation of diffusible singlet oxygen radicals with short lifetimes and short ranges, whereas the fast photochemical method described herein allows for the formation of crosslinks between the molecular linker polymer and two or more distally separated endogenous proteins. Thus, the molecular linkers described herein can covalently crosslink molecules of distally separated endogenous proteins, for example, as a non-limiting example, to separate collagen molecules located in separate collagen fibrils, thereby forming interfibril collagen crosslinks. Figure 1 shows a possible collagen fibril crosslinking scenario using a tyr-derivativeized dextran molecular linker. The inventors have shown that the molecular linkers described herein have sufficiently low molecular weights to allow the linkers to diffuse into tissues at clinically useful rates.

[0072] Diffusion of polymers into the corneal stroma One exemplary tissue from which molecular linkers can freely diffuse is the corneal stroma. The corneal stroma has a relatively open, "gel-like" structure that allows the diffusion of compounds with molecular weights less than 500,000 Da, although the actual molecular weights may be much lower (Prausnitz and Noonan, 1998). The negatively charged, hydrophilic corneal stroma forms a weak barrier against the penetration of lipophilic drugs.

[0073] Rajapaksha et al. (2015) investigated the diffusion transport rates in excised porcine corneal stroma using fluorescently labeled dextran molecules with hydrodynamic radii ranging from 1.3 nm to 34 nm (exhibiting molecular weights ranging from 3 kDa to 2,000 kDa). It was found that even dextran with a molecular weight nearly equal to 2,000 kDa could penetrate 30% of the stroma thickness in 30 minutes, compared to the diffusion distance recorded for the low molecular weight fluorophore molecule fluorescein (molecular weight = 332 Da). Low molecular weight fluorescein-labeled dextran (molecular weight ≈ 3,000 Da) diffused into the stroma at 70% the rate of free fluorescein (Cui et al., 2011). The corneal stroma is therefore a relatively permeable extracellular matrix, allowing for the free diffusion of low molecular weight proteins, including tyrosine (or tyramine) derivatized molecular linkers described herein.

[0074] The inventors have determined that any low molecular weight biocompatible polymer containing residues having side chains capable of photochemical crosslinking with tyrosine may be suitable for use in the crosslinking methods described herein. To ensure the free diffusion of molecular linkers into the corneal stroma matrix, the molecular weight of these polymers must be such that it allows free diffusion into and from the stroma within a practically useful short period of time, measured in minutes. Those skilled in the art can easily determine the time it takes for a polymer of a given molecular weight to diffuse into the corneal stroma using the methods described herein. Examples of suitable low molecular weight polymers include size-selected gelatin, dextran, xylan, or low molecular weight nanocellulose. Preferably, the polymer used as the molecular linker has a molecular weight of about 5 kDa to about 100 kDa, or about 10 kDa to about 60 kDa, or about 10 to about 50 kDa, or about 10 kDa to about 20 kDa, or about 12 kDa to about 20 kDa, or about 10 kDa to about 15 kDa, or alternatively about 5 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, or about 40, 50, 60, 70, 80, 90, or 100 kDa.

[0075] Synthesis of molecular linkers A molecular linker suitable for use in the crosslinking method described herein comprises at least two residues having side chains that can crosslink with tyrosine residues of endogenous proteins in tissues through the formation of covalent dityrosine bonds. For example, the molecular linker may be a peptide or protein polymer comprising two or more tyrosine or tyramine residues that can interact with and crosslink with amino acid residues in endogenous proteins using the method described herein. In one embodiment, the molecular linker is size-selected gelatin, e.g., tyramine-derivative gelatin. In other embodiments, the molecular linker may be, but is not limited to, another biocompatible polymer, e.g., dextran, xylan, or low molecular weight nanocellulose. Other tyramine-derivative carbohydrate molecules, e.g., chitosan and PVA, can also be photocrosslinked to polymer hydrogels through the formation of dityrosine covalent bonds.

[0076] Those skilled in the art will understand that, depending on the polymer selected, it may be necessary to perform a size selection method to obtain a polymer with a suitable length / molecular weight for use in the crosslinking method described herein.

[0077] In addition, the present inventors believe that the crosslinking method can be improved by increasing the number or density of residues that can crosslink with endogenous proteins, thereby resulting in a high covalent crosslink density in the tissue. Methods for chemical derivatization to increase the number of crosslinkable tyrosine or tyramine residues in polymers are known in the art.

[0078] Size-selected tyrosine-derivative gelatin In some embodiments, the molecular linker comprises size-selected tyrosine-derivative gelatin. For example, pharmaceutical-grade Bolton-Hunter derivatized gelatin can be used as a molecular linker to enhance substantial protein crosslinking. Type B porcine gelatin (type I gelatin) can be chemically derivatized (using Bolton-Hunter reagent) to replace up to 38 lysine (K) amino acid residues with tyrosine residues, as described (Elvin et al., 2010). In this regard, gelatin can be derivatized using Bolton-Hunter reagent (N-succinimidyl-3-[4-(hydroxy)propionate) to increase its Tyr content with slight modifications, as described (Bolton and Hunter, 1973; Miller, 1996). A 10% solution of Nippi Inc. MediGelatin can be dissolved in sodium borate / saline buffer pH 8.6 and reacted with Bolton-Hunter reagent (Sigma) in a ratio of 25:1 (w:w) at 37°C for 1 hour. This reaction mixture is thoroughly dialyzed against PBS at 37°C and subsequently freeze-dried. This derivatization process increases the tyrosine content of the gelatin from 4 / 1,000 residues (0.4 mol%) in natural gelatin to 38 / 1,000 residues (3.8 mol%) in Bolton Hunter derivatized gelatin. The average molecular weight (MW) of BH derivatized gelatin is approximately 100 kDa, and strong bands are observed at 100 kDa (α band), 200 kDa (β band), and 300 kDa (γ band) on SDS-PAGE. For the production of molecular linkers based on gelatin, the average molecular weight of the derivatized gelatin should preferably be reduced to a size range of 10 kDa to 20 kDa. Several approaches exist to reduce the average molecular weight of gelatin, including repeated cycles of either autoclaving or sonication.

[0079] As described by Vanhoecke and Olijve, 2018, the molecular weight of gelatin can be reduced by autoclaving a gelatin solution. For example, a 10% solution of type A gelatin (G1890) from Sigma-Aldrich can be autoclaved at 121°C for 30 minutes, resulting in a reduction in molecular weight from 150 kDa to approximately 70 kDa. Furthermore, increasing the autoclaving time induces a higher level of degradation of the gelatin solution, resulting in the production of gelatin fragments with lower molecular weight. The inventors have found that a 150 mg / ml (15% w / v) solution of Bolton Hunter derivatized Nippi Medigelatin can be autoclaved to MW after up to 14 hours of autoclaving (121°C). ave This indicates that the polymer was broken down into fragments of 15 kDa (SDS-PAGE analysis, see Figure 2). Polymers in this molecular weight range can diffuse into the corneal stroma with little interference.

[0080] Another approach to preparing low molecular weight gelatin fractions is sonication (Kanegae et al., 1992). The starting material can be a high molecular weight gelatin sample (e.g., bone-derived type B gelatin) and may contain gelatin fractions of a certain molecular weight range, including γ (approximately 300 kDa), β (approximately 200 kDa), and α (100 kDa). Sonication results in the rapid decomposition of the high molecular weight gelatin fractions (γ, β), while the low molecular weight fractions (α and smaller fragments) are decomposed at a slower rate. For example, sonication at an intensity of 6 V and a frequency of 19.5 kHz for 4 minutes decomposes a 7.5% gelatin solution of the gamma fraction (γ=300 kDa) into low molecular weight (M ave (β = 40 kDa) is necessary to break it down into gelatin molecules, and 8 minutes of sonication is required to break down the beta fraction (β = 200 kDa) into low molecular weight (M ave (α=40kDa) is necessary to break it down into gelatin molecules, and 16 minutes of sonication is required to break down the alpha fraction (α=100kDa) into low molecular weight (M aveIt was necessary to decompose it into gelatin molecules (=40kDa). Based on this, to prepare a 10kDa-20kDa sample of decomposed Nippi medicelatin (including γ:β:α relative size fraction abundances of 1:1:2), a person skilled in the art would sonicate a 7.5% solution of high molecular weight gelatin dissolved in deionized water in a 20°C water bath for approximately 30 minutes.

[0081] Those skilled in the art will understand, based on the teachings herein, that a size-selected derivatized gelatin sample may contain molecular linkers within a range of molecular weights, for example, approximately 5 kDa to approximately 60 kDa or approximately 10 kDa to approximately 50 kDa, depending on the method used to prepare the size-selected molecular linkers. Preferably, a portion of the molecular linkers is within the molecular weight range of approximately 5 kDa to approximately 20 kDa or approximately 10 kDa to approximately 15 kDa.

[0082] An exemplary amino acid sequence of tyrosine-derivativeized human type I collagen (gelatin) is provided in Sequence ID No. 2.

[0083] Non-degradable (protease-insensitive) molecular linkers Several approaches exist for preparing suitable non-degradable molecular linkers ("molecular staples") that are not susceptible to proteolysis for crosslinking endogenous proteins in tissues. These include carbohydrate-based polymers, such as dextran, xylan, and nanocellulose. Dextran is a biopolymer approved by the FDA for use in eye drops and for the treatment of dry eye. They consist of α-1,6-linked D-glucopyranose residues with several percent of α-1,2, α-1,3, or α-1,4-linked side chains.

[0084] Xylan is a polysaccharide made from β-1,4-linked xylose residues having α-arabinofuranose and / or α-glucuronic acid side chains. Xylan (molecular weight = 12.7 kDa, 20 kDa) is a naturally occurring hemicellulose polysaccharide composed of arabinose, 4-O-methylglucuronic acid (10% by weight), and xylose in a ratio of 1:2:11, respectively (Escalante et al., 2012).

[0085] Cellulose is a plant-derived polysaccharide polymer composed of repeating units of β-(1-4)-linked D-glucose assembled into nano-sized thread-like aggregates called microfibrils.

[0086] Non-limiting examples of different classes of biocompatible, non-degradable, protease-insensitive polymers as starting materials for the synthesis of molecular linkers suitable for photochemical crosslinking of endogenous corneal stromal proteins include: (i) Low molecular weight tyrosine derivatized dextran (10 kDa and 20 kDa), (ii) Low molecular weight bis-tyrosine derivatized dextran (10 kDa and 20 kDa), (iii) Low molecular weight tyramine-derivative xylan (12.7 kDa and 20 kDa), and (iv) Low molecular weight tyramine-derivative nanocellulose (10kDa~20kDa) These are some examples.

[0087] Dextran Dextran polymers with a molecular weight of less than 40 kDa can be eliminated through renal clearance and have a half-life of 8 hours, while molecules with a molecular weight greater than 40 kDa have a longer half-life and are sequestered in the liver and spleen, where they are then hydrolyzed by endodextranase and exodextranase. Biodegradation occurs through the innate enzymatic cleavage of sugar bonds by dextran-1,6-glucosidase found in the spleen, liver, lungs, kidneys, brain, and muscle tissue, as well as by dextranase expressed by bacteria in the colon.

[0088] (i) Tyrosine-derived aminodextran Those skilled in the art can readily obtain amino-functionalized dextrans. For example, amino-functionalized dextrans are commercially available from FinaBioSolutions. 10 kDa aminodextrans with 10 primary amine groups per polymer and 20 kDa aminodextrans with 20 primary amine groups per polymer are preferred candidates, their low molecular weight allowing for free diffusion into the corneal stroma. Tyrosine groups can be added to reactive primary amine groups using the -NH2 reactive Bolton-Hunter method, as described by Elvin et al. (2010). Tyrosine-derivativeized dextrans and BIS-dextrans can be purified by methods known in the art, for example, by ethanol precipitation using the method described by Xu et al. (2014). Figure 3A shows dextran polymers derivatized with amine groups. Figure 3B shows a 10 kDa tyramine-derivative dextran that can form dityrosine crosslinks between endogenous proteins separated by an intermolecular distance of up to 25 nm. Considering that a single glucose molecule is approximately 1 nm long, a 20 kDa tyramine-derivative dextran molecular linker can form dityrosine crosslinks between endogenous proteins separated by an intermolecular distance of up to 50 nm.

[0089] (ii) Tyrosine-derived bisaminodextran Those skilled in the art will understand that di-aminodextran is also suitable for use as a molecular linker in the methods described herein. Di-aminodextran is commercially available from FinaBioSolutions. Dextran contains a primary amine at each end of the dextran polymer (Figure 4). Both 10 kDa and 20 kDa bis-aminodextran can be derivatized using the Bolton-Hunter method described by Elvin et al. (2010). Tyramine-derivativeized dextran can then be purified by ethanol precipitation to remove non-dextran reagents, as described by Xu et al. (2014). For dextran polymers of approximately 20 kDa, an ethanol concentration of about 90% is required for quantitative precipitation of derivatized dextran.

[0090] (iii) Tyramine-derived xylan Xylan is a polysaccharide made from β-1,4-linked xylose residues with α-arabinofuranose and / or α-glucuronic acid side chains. Xylan (molecular weight = 12.7 kDa, 20 kDa) is a naturally occurring hemicellulose polysaccharide composed of arabinose, 4-O-methylglucuronic acid (10 wt%), and xylose in a ratio of 1:2:11, respectively (Escalante et al., 2012). Xylan remains undigested in human tissues and can only be degraded by anaerobic colon microbiota. Xylan is biocompatible and non-cytotoxic—hydrogels based on xylan have been shown to be non-cytotoxic (Khaire et al., 2022). Tyr-derivativeized xylan is soluble in water up to a concentration of 40% (w / v) (Kuzmenko et al., 2014).

[0091] It has been previously shown that hemicellulose extracted from spruce, such as xylan and galactoglucomannan, can be modified with tyramine, a molecule similar to lignin, which enables crosslinking of aromatic residues using the subsequent enzyme horseradish peroxidase (Kuzmenko et al., 2014; Markstedt et al., 2017).

[0092] We successfully conjugated xylan isolated from spruce with tyramine using EDAC / NHS activation of glucuronic acid in spruce arabinoglucuronoxylan. This conjugate was enzymatically crosslinked with HRP, resulting in gelation within 20±5 seconds at room temperature. The hydrogel exhibited mechanical integrity, an interconnected porous structure, and a high degree of expansion. When this system was used with in-situ immobilized mesenchymal stem cells, the cells were able to differentiate into adipocytes without evidence of cytotoxicity. Previous in vivo studies have shown that hydrogels crosslinked in 100 mM H2O2 are biocompatible and non-toxic.

[0093] (iv) Tyramine-derivative nanocellulose Cellulose and its derivatives are widely used in textile fibers, chemical precursors, and papermaking and food additives. Cellulose whiskers (nanocrystals) obtained by acid hydrolysis of cellulose represent a more recent application area for nanocomposite materials. Acid hydrolysis yields crystalline rod or whisker-shaped nanoparticles with diameters ranging from 8 to 20 nm and lengths from 100 nm to several micrometers, depending on the cellulose source (Habibi et al., 2010; Lima et al., 2004). Such whiskers possess high elastic modulus and high aspect ratios, and their surface chemistry can be modified to expand their use in high-value applications.

[0094] Chemical modifications of cellulose nanocrystals and nanofibers are known in the art and include esterification, cationization, carboxylation, silylation, and polymer grafting. Most of these techniques utilize the abundance of hydroxyl groups on the surface to facilitate the easy conjugation of desired molecules.

[0095] Nanocellulose fibers of a desired molecular weight (e.g., approximately 10 kDa to approximately 20 kDa) and length (e.g., nanofibers of approximately 50 nm to approximately 100 nm in length) can be produced by using high-temperature and high-pressure water (supercritical water) to decompose and extract short, low-molecular-weight cellulose nanofibers (Buffiere et al., 2016).

[0096] Nanocellulose fibers and crystals of desired molecular weight and size are biocompatible and safe in vitro for cells and in vivo for animals. Due to their favorable biocompatibility and relatively low toxicity, nanocellulose represents a promising biomaterial for tissue regeneration. Several independent studies have explained the low toxicity and biocompatibility of cellulose nanoparticles and fibers.

[0097] Amino-functionalized nanocellulose fibers can be prepared using several chemical synthesis methods known to those skilled in the art. Such amine-derivativeized nanocellulose whiskers can be prepared as a 20% (w / v) aqueous suspension. Synthetic chemical approaches for introducing amino-reactive groups into cellulose polymers include the methods described by Shaghaleh et al. (2021), Heinze et al. (2016), and Jardine (2022).

[0098] After preparing amine-derivatized nanocellulose fibers, the conversion of such nanofibers to the desired tyramine-derivatized form is carried out using synthetic methods described in the literature. These include the use of Bolton-Hunter reagents to convert primary amine groups to tyramine derivatives, as described (Bolton, AE; Hunter, WM., 1973).

[0099] Other approaches for the synthesis of tyramine-derivativeized cellulose, including the synthesis of cellulose acetacetate (CAA) as a precursor for preparing tyramine-cellulose derivatives, are also described.

[0100] An alternative method for the synthesis of tyramine-derivativeized cellulose was described by Simon et al. (2023), in which the authors used reductive amination of dialdehyde cellulose (DAC) with 2-picoline borane to produce a range of bio-derived thermoplastics and thereby introduced the primary amine tyramine into the cellulose backbone.

[0101] Photochemical crosslinking using molecular linkers In contrast to current CXL methods, the inventors hereby describe the design and synthesis of several different molecular linkers useful for improving tissue strength by photochemical crosslinking, taking into account the composition and structure of corneal stroma, the molecular dimensions of this specialized extracellular matrix (ECM), and the properties of its component protein species. The proposed short (low molecular weight) polymer reagents enable the formation of crosslinks by covalent bonds between distally separated endogenous proteins. In corneal stroma tissue, such crosslinks cannot be produced using the conventional riboflavin / UV-A Dresden protocol. The inventors have determined that the crosslinking procedure described herein brings about crosslinks between stroma proteins that were previously impossible to crosslink because the proteins are physically separated beyond the distance at which covalent crosslinking can be formed between proteins by conventional CXL methods. Covalent crosslinking of stroma collagen and proteoglycans mediated by molecular linkers results in a mechanically stiffer (higher modulus of elasticity) ECM material compared to conventional CXL methods, as described herein.

[0102] The photochemical crosslinking described herein avoids one or more problems associated with current CXL methods, due to both the chemical components of the visible light photocrosslinking method and the wavelength and energy of the blue light irradiation. The method described herein involves direct photochemical crosslinking of tyrosine residues and, to a lesser extent, histidine residues located in various endogenous parenchymal ECM proteins. Exemplary amino acid sequences of major endogenous parenchymal ECM proteins are provided in SEQ ID NOs: 1 and 3-13. These proteins include type I and V collagen, vimentin, fibronectin, laminin, elastin, aggrecan, keratocan, lumican, mimican, decorin, and biglycan, all of which contain multiple tyrosine residues suitable for photochemical crosslinking in the method described herein.

[0103] The crosslinking method described herein avoids the production of damaging singlet oxygen free radicals. Furthermore, the efficiency of this process is high, requiring only a few seconds of blue light irradiation to activate quantitative crosslinking of hydrophobic associated proteins in the corneal stroma. Diffusion of low molecular weight "molecular linker" polymers into the corneal stroma allows crosslinking of stroma collagen protein chains located up to 55 nm away from adjacent protein chains. In this manner, degraded interfibrillary collagen stroma protein chains can be crosslinked by intact "molecular linkers" that are covalently crosslinked. Refer to Figure 1A, which shows the arrangement of collagen molecules within a fibril and the spacing between fibrils. Note that a "molecular linker" based on 16 kDa tyramine-derivativeized dextran (molecular length = 42 nm) can span the distance between two adjacent collagen fibrils (approximately 40 nm) and thus can form crosslinks between collagen molecules located in adjacent collagen fibrils.

[0104] While we do not wish to be limited by the molecular dimensions of molecular linkers or by theory, collagen molecules located in individual but adjacent collagen fibril bundles can be physically crosslinked by molecular linkers that function as crosslinkers. In contrast, the current riboflavin / UV-A-induced CXL method is a crosslinking method that can only induce crosslinking between individual collagen chains located within a collagen fibril. This limitation applies because the mechanism of this singlet oxygen-mediated free radical crosslinking method functions only over very short molecular distances (less than 5 Å) and allows for crosslinking of hydrophobic associated tyrosine residues located in adjacent triple-chain collagen molecules within the same collagen fibril.

[0105] The inventors have shown that photochemical crosslinking of endogenous proteins via treatment with a solution containing a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor, and that this photochemical crosslinking can cause stable readjustment of the 3D structure of stromal matrix proteins in the cornea. This suggests that collagen and / or other stromal ECM proteins become covalently crosslinked via hydrophobic molecular interactions involving tyrosine residues located in both the endogenous stromal proteins (collagen and / or proteoglycans) and the tyramine-derivative molecular linker. The fact that the method described herein can form novel molecular associations induced by external physical stress / strain stresses imparted during corneal stromal template formation suggests that, as described in this embodiment, endogenous physical interactions must occur between tyrosine / histidine / tryptophan residues located in the various stromal matrix proteins constituting the ECM.

[0106] During the 3D structuring / templated processes described in the examples, these novel molecular associations are permanently "fixed" through the formation of new covalent bonds that occur during photochemical crosslinking reactions over short periods, e.g., 1–5 seconds. Importantly, given the molecular linker's ability to span intermolecular distances of up to 55 nm, new intermolecular covalent bonds can be formed between collagen proteins located on separate collagen fibrils.

[0107] The photochemical crosslinking method disclosed herein involves a 3-minute period to allow the diffusion of photochemical components, such as molecular linkers, into the corneal stromal matrix, followed by the application of a light source (e.g., a 455 nm blue LED dental curing lamp, 1,200 mW / cm²). 2 This provides short-duration (e.g., less than approximately 5 seconds) light irradiation of tissue, such as the cornea, using a light source.

[0108] Therefore, the steps of the crosslinking method are: (i) A step of applying a photochemical component containing a molecular linker to the surface of the tissue, (ii) A step that allows the photochemical components to diffuse into the tissue, (iii) The step of irradiating the tissue with light of a wavelength and energy suitable for promoting crosslinking of molecular linkers to endogenous proteins. Includes.

[0109] The use of short exposure times with visible blue light represents a safer alternative to the potentially harmful use of UV light in conventional CXL procedures.

[0110] Those skilled in the art will understand that, in some embodiments, it is desirable for the crosslinking reagent to diffuse into the cornea without exceeding approximately half the thickness of the cornea, or exceeding approximately 300 μm, or exceeding approximately 250 μm, or exceeding approximately 200 μm. Those skilled in the art can determine a suitable diffusion time based on the information provided herein.

[0111] Safety of photochemical curing using blue light (455nm) The inventors of this invention have found that the requirement for blue light irradiation in the molecular linker crosslinking method is 3M ESPE S10 LED curing light (430nm~480nm, peak wavelength 455nm±10nm, 1,200mW / cm² at light source) 2 ) is transmitted from the target at a distance of 2 cm (≡ 2 J / cm 2 ) at 400mW / cm² 2 It is estimated to be delivered within 5 seconds*.

[0112] Regarding the safety of blue light exposure, the methods described herein are in line with the current ISO standard (ISO 15004-2) and have a maximum exposure of 10 J / cm². 2 It is possible to deliver visible light irradiation to the retina (Sliney et al., 2005).

[0113] The current riboflavin-based CXL method is 5.4 J / cm². 2 It delivers UV-A light (light irradiation at 370 nm). A recent study by Ziolkowska et al. (2023) showed that in rats exposed to LED light (463 ± 10 nm), up to 3.4 J / cm² was delivered to retinal ganglion cells. 2 The safe ophthalmic level was indicated. The proposed blue light exposure described herein (1-5 seconds with a 455±10nm blue LED light source at a distance of 50mm from the cornea using a 3M ESPE S10 LED curing lamp) is 0.4 J / cm². 2 ~2.0J / cm 2 This is equivalent to delivery.

[0114] Blue light does not cause the type of DNA damage caused by UV light. One cause of cytotoxicity associated with photocuring with blue light is related to the formation of singlet oxygen free radicals. The photochemical crosslinking method described herein, however, does not produce singlet oxygen formation during the photochemical crosslinking reaction in the presence of persulfates. In addition, light irradiation of the cornea from a distance of 10 mm for 10 seconds using this LED source yields 2.55 J / cm². 2 Energy (defined by ISO 15004-2 standard as 10 J / cm²) 2It will deliver (well below) (Sliney et al., 2005).

[0115] The inventors have further demonstrated that, in some embodiments, by reducing the light irradiation time to approximately 2.5 seconds, a low dose of energy (1 J / cm²) can be achieved. 2 (Less than) Use a 3M dental curing light at a distance of 20 mm from the light guide tip at 400 mW / cm². 2 It is determined that the light can be delivered at a given illuminance. In some embodiments, crosslinking can be achieved with light irradiation for 1 second at a distance of 50 mm.

[0116] Treatment method and composition The photochemical crosslinking method described by the present inventors can be used for preventive and therapeutic purposes when improvement of the mechanical properties and / or strength of tissues is required.

[0117] Therefore, this disclosure relates to a method for crosslinking endogenous proteins in tissue, (i) The tissue is brought into contact with a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor, thereby allowing the molecular linker to come into contact with endogenous proteins in the tissue. (ii) The step of irradiating the tissue to crosslink it with molecular linkers. This provides a method that includes [something].

[0118] Irradiation of tissue in the presence of these reagents results in covalent crosslinking of molecular linkers and endogenous proteins.

[0119] In some embodiments, this method may be used in the treatment or prevention of ectodialytic corneal disease (ECD) or corneal ectasia, which is a group of progressive eye disorders characterized by progressive corneal ablation and thinning. In some embodiments, this method may be used in the treatment or prevention of at least one symptom of keratoconus in a subject.

[0120] Therefore, the present disclosure further relates to a method for treating weakening of the corneal stroma in a subject, (i) The step of administering a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor to the target cornea, (ii) The step of irradiating the cornea and This provides a method that includes [something].

[0121] The step of irradiating the cornea may be performed using a blue LED light curing lamp. The proposed blue light exposure described herein (1-5 seconds with a 455±10nm blue LED light source at a distance of 50 mm from the cornea using a 3M ESPE S10 LED curing lamp) is 0.4 J / cm². 2 ~2.0J / cm 2 This is equivalent to delivery.

[0122] The step of irradiating the cornea in the presence of a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor initiates a covalent crosslinking reaction between the molecular linker and endogenous proteins in the cornea.

[0123] This disclosure further relates to a method for treating or preventing at least one symptom of keratoconus in a subject, (i) The step of administering a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor to the target cornea, (ii) The step of irradiating the cornea and This provides a method that includes [something].

[0124] The step of irradiating the cornea in the presence of a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor initiates a crosslinking reaction between the molecular linker and endogenous proteins in the cornea.

[0125] The reagents used in treatment may be applied separately to the tissue, for example, the surface of the cornea, in any order, or one or more reagents may be contained in a single solution for application to the tissue.

[0126] This disclosure further provides therapeutic compositions and / or pharmaceutical compositions comprising molecular linkers as defined herein. Furthermore, therapeutic compositions or pharmaceutical compositions comprising one or more molecular linkers, as well as photoactivatable metal ligand complexes and / or electron acceptors are provided.

[0127] This disclosure further provides a kit comprising a molecular linker and one or more of the following: a photoactivatable metal ligand complex and / or an electron acceptor.

[0128] Those skilled in the art can easily determine pharmaceutically acceptable buffers, excipients, and carriers suitable for preparing crosslinking reagents. A wide range of pharmaceutically acceptable excipients and carriers are known in the art. Such pharmaceutical carriers and excipients, as well as suitable pharmaceutical formulations, are well described in various publications (see, for example, "Pharmaceutical Formulation Development of Peptides and Proteins," Frokjaer et al., Taylor & Francis (2000) or "Handbook of Pharmaceutical Excipients," 3rd edition, Kibbe et al., Pharmaceutical Press (2000), A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wlkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999), edited by H. Ansel et al., 7th edition, Lippincott, Williams, & Wlkins, and Handbook of Pharmaceutical Excipients (2000), edited by A. Kibbe et al., 3rd edition, A. Pharmaceutical Assoc).

[0129] In some embodiments, the addition of a high molecular weight gelatin component (e.g., Nippi Medigelatin) to the formulations described herein will seal the exposed corneal surface after epithelial debridement. This photocurable hydrogel enhances epithelial regrowth and healing, and also provides delivery of antibiotics, anti-inflammatory drugs, and / or analgesics to the wounded corneal surface. The use of this high molecular weight hydrogel treatment will improve patient comfort and reduce the incidence of keratitis and corneal perforation, which may rarely occur after “epithelial removal” surgical methods. [Examples]

[0130] This disclosure is not limited in scope by the specific examples described herein, which are intended solely for illustrative purposes. Functionally equivalent products, compositions, and methods are clearly within the scope of the present invention as described herein.

[0131] Example 1. Study on diffusion in bovine eyes Ten fresh bovine eyes were obtained from the slaughterhouse (stored at 4°C for less than 10 hours). Corneal thickness was determined using a digital caliper and was found to be in the range of 740 μm to 820 μm. The eye epithelium was debridemented with an 8 mm biopsy punch and then scraped off with a scalpel. The fresh bovine eyes are shown in Figure 5A, and the bovine eyes from which the epithelium has been debridemented are shown in Figure 5B.

[0132] 1 mM Ru(bpy)3 25 μL was added to the corneal debridement area (Figure 6A) and allowed to diffuse into the cornea for 6 minutes. Excess Ru(bpy)3 solution was removed by blotting using a fresh Kimwipe tissue.

[0133] The cornea was frozen on the surface of an aluminum block at -20°C for 2 minutes (Figure 7B). Transverse (bisection) sections of the cornea were prepared by cutting 1 mm thick sections using a razor blade. The corneal cross-sectional sections were placed on a glass slide to allow visualization of the cross-sectional area of ​​the corneal stroma (Figure 7C). Ru(bpy)3 was visualized through a red filter by irradiation with 450 nm LED light (Figure 7).

[0134] Example 2. Diffusion of riboflavin (0.1% [2.6 mM] in PBS at pH 8.5) Debridemented corneas were prepared from fresh bovine eyes as described in Example 1. 25 μL of 0.1% riboflavin was diffused into the debridemented cornea for 5 minutes, and then the excess liquid was blotted (Figure 8A).

[0135] The cornea was frozen as described in Example 1 and cut into 1 mm thick transverse sections (Figure 8B). Riboflavin fluorescence was visualized under a red light filter (illumination using a 450 nm blue LED) (Figure 8C).

[0136] Example 3. Photochemical crosslinking of the corneal stroma using a 3D template mold. A. Corneal crosslinking using 1 mM Ru(bpy)3 + 50 mM sodium persulfate (SPS). The entire epithelium was debridemented from the cornea. 300 μL of 1 mM Ru(bpy)3 + 50 mM SPS was applied to the anterior corneal surface with a diffusion time of 10 minutes (Figure 9A). The cornea was wrapped around a metal piping nozzle that functioned as a mold, and irradiated for 20 seconds using a 450 nm blue LED dental lamp at a distance of 1 cm from the light guide tip (Figure 9B). After photocrosslinking, the cornea was fixed to the shape of the mold and could support its weight when held with forceps (Figures 10A and 10B).

[0137] B. Corneal crosslinking using only 1 mM Ru(bpy)3 (without SPS). Except for the omission of sodium persulfate (SPS), the corneas were subjected to the crosslinking protocol described in Example 3A. After irradiation with 450 nm blue LED light, no mold template-like structures were formed, and the corneas remained unstructured and deformable (Figures 11A-11C). This experiment demonstrates that electron acceptors, such as SPS, play a crucial role in the photochemical formation of dityrosine covalent bonds between endogenous corneal proteins. In its absence, short-term (20-second) irradiation of Ru(bpy)3 in aqueous solution with blue light to produce singlet oxygen radicals is insufficient to induce crosslinking of endogenous parenchymal proteins by any measurable covalent bonds. In contrast, in its presence, crosslinking of endogenous corneal proteins and "molecular linkers" occurs rapidly and quantitatively.

[0138] Example 4. Model gelatin hydrogel and [Ru(bpy)] applied to cadaveric corneal stroma 3 ] 2+ spread The diffusion rate of Ru(bpy)3 into both new bovine cadaveric corneal stroma and a heat-gelled 12% gelatin hydrogel (as a model corneal stroma matrix) was studied. The results showed that a PBS solution containing 0.5 mM Ru(bpy)3 allowed free diffusion of Ru(bpy)3 to a depth of 400 μm into an 800 μm thick bovine cadaveric corneal stroma within 6 minutes. For comparison, the Dresden CXL method, a standard protocol for crosslinking corneal proteins conventionally used, consists of applying a 0.1% riboflavin solution (vitamin B2) to a deepithelialized cornea for 30 minutes to promote diffusion into the stroma to a depth of approximately 200 μm.

[0139] Diffusion rate of Ru(bpy)3 in the corneal stroma The observed rate of diffusion of Ru(bpy)3 into the parenchyma should require no more than 3 minutes of diffusion, followed by 1 second of blue light exposure, to induce photochemical crosslinking of the endogenous parenchyma proteins. Thus, the entire crosslinking procedure is reduced from 30 minutes per eye (current procedure) to less than 3 minutes per eye (procedure described herein). This allows surgeons to perform the procedure 10 times faster in the operating room, subjecting patients to a shorter and less painful surgical procedure.

[0140] [Ru(bpy)3] bound from the corneal stroma after photochemical crosslinking 2+ Removal of. Literature reports indicate that Ru(bpy)3 does not bind to protein hydrogels (Kurimura et al., 1982). In any case, the interaction between Ru(bpy)3 and corneal stromal proteins can lead to residual non-covalent bonds of Ru(bpy)3 within the crosslinked stromal hydrogel, potentially causing temporary discoloration of the stroma. This temporary discoloration can be addressed by rinsing the crosslinked cornea with a solution containing low molecular weight (PSS)-poly(styrenesulfonic acid) sodium salt (e.g., [molecular weight approximately 4,600 Da] poly(p-styrenesulfonate), available from Polysciences catalog number 16248-250, https: / / www.polysciences.com / german / polystyrenesulfonic-acid-sodium-salt-mw-4600), which is delivered as eye drops. Poly(p-styrenesulfonic acid) is a polystyrene polymer used as a cation exchange resin. The strong charge and hydrophobic interactions between the bipyridyl ligand of the Ru(bpy)3 complex and the PSS effectively extract residual Ru(bpy)3 from the cured hydrogel, thereby removing the retained Ru(bpy)3 (Tuite et al., 2012).

[0141] Example 5. Ru(bpy) in 12% gelatin gel 3 The spread of [this]. A 12% gelatin (Sigma G1890) type A, 300 Bloom solution was prepared in PBS (pH 7.4) by melting at 50°C. 60 μL was added to a circular Teflon mold measuring 8.5 mm (diameter) × 1 mm (depth). The gelatin was heated and solidified at 18.5°C for 10 minutes. At 10 minutes, 60 μL of 0.5 mM Ru(bpy)3 (0.5 mM solution in PBS) was added onto the 12% gelatin disc. The gelatin disc was covered with foil for the diffusion time. Excess solution was blotted from the top of the gel using a Kimwipe. The gelled disc was removed using a spatula and added to 940 μL of PBS at 55°C. The gel was melted (for 30 seconds), and the solution was added to a cuvette. 452 Read it.

[0142] If diffusion is expressed as a percentage of the total disk volume in equilibrium with the applied 0.5 mM Ru(bpy)3 solution, then assuming complete mixing of the applied 60 μL solution with the 60 μL gelatin disk, 100% diffusion is 0.205 A 452 This would correspond to [0.221 - 0.016 = gelatin-PBS blank]. Therefore, diffusion to 30% of the hydrogel depth / thickness (corresponding to approximately 300 μm in this case) would be achieved in approximately 2.3 minutes. This is the desired penetration depth of the crosslinking agent in the human cornea (compared to the riboflavin-based CXL method, which requires 30 minutes of diffusion).

[0143] Example 6. Ru / SPS diffusion model: Diffusion rate to a 12% gelatin / PBS hydrogel that has been heat-solidified (at room temperature for 10 minutes). Objective: The objective of these experiments was to describe the diffusion rate of solution (i) or (ii) (see below) into a thermogelatin hydrogel representing an in vitro model of the corneal stroma: (i) Ru(bpy)3 (0.5mM, 1.0mM, 2.0mM - proposed concentrations), and (ii) Riboflavin (2.6 mM) 0.1% (the concentration currently used in FDA-approved CXL crosslinking treatments).

[0144] Method: 12% gelatin (Sigma G1890) Type A 300 Bloom was melted in PBS (pH 7.4) at 50°C, and 60 μL was added to an 8.5 mm (diameter) x 1 mm (depth) Teflon mold. The gelatin was heated to solidify at 18.5°C for 10 minutes. 60 μL of Ru(bpy)3 / SPS solution (either 0.5 mM Ru(bpy)3 + 50 mM SPS, 1.0 mM Ru(bpy)3 + 50 mM SPS, or 2.0 mM Ru(bpy)3 + 50 mM SPS) was added and diffused into the gel disc at various times (0, 2, 4, 6, 8, 10, 14 minutes). Excess liquid was blotted from the top surface of the mold. At the end of the diffusion period, a 3M ESPE blue LED lamp (1,200 mW / cm²) is used to heat-solidify the gelatin hydrogel. 2 The gelatin hydrogel was irradiated with light for 5 seconds at a distance of 1 cm from the optical guide tip using a 0.5 mm meter. The crosslinked gelatin hydrogel was weighed using a microbalance and then placed in a 65°C water bath for 30 seconds. Excess liquid was removed from the crosslinked hydrogel, and it was weighed again to measure the residual crosslinked gelatin hydrogel. Plots showing the percentage of crosslinked gelatin against diffusion time (0 to 14 minutes) were prepared. The diffusion of 0.5 mM, 1.0 mM, and 2.0 mM Ru(bpy)3 into the gelatin disk is shown in Figures 12A-12C.

[0145] Example 7. Ru(bpy) 3 +SPS diffusion: Photochemical crosslinking of gelatin hydrogels Objective: To determine the time required for a solution containing Ru(bpy)3+SPS to diffuse into a 1 mm thick heat-gelling gelatin sheet, subsequently forming covalent crosslinks between gelatin molecules, thereby making the hydrogel resistant to subsequent melting at high temperatures. This is a test to indicate the time required for photochemical crosslinking by covalent bonding of gelatin molecules in a hydrogel.

[0146] Method: 60 μL of Ru(bpy)3+ SPS solution was diffused into a 12% gelatin gel (60 μL in PBS buffer) that had been heat-solidified (at room temperature for 10 minutes) with diffusion times of 2 to 14 minutes. The gel was irradiated with a 450 nm blue LED lamp for 5 seconds. After crosslinking, the gel was weighed and then immersed in a 65°C water bath for 30 seconds. Excess liquid was blotted from the gel, and then the gel was weighed.

[0147] Any uncrosslinked gelatin chains are dissolved in hot water, and the covalently crosslinked chains will become part of a crosslinked polymer gelatin hydrogel that is resistant to dissolution by heat. If all gelatin molecules are crosslinked, no gelatin will melt from the hydrogel, and the weight of the gelatin hydrogel disk after heat treatment will be equal to the weight before heat treatment (this situation represents 100% crosslinking). Diffusion of 0.5 mM Ru(bpy)3 + 50 mM SPS, 1.0 mM Ru(bpy)3 + 50 mM SPS, and 2.0 mM Ru(bpy)3 + 50 mM SPS into a 12% gelatin disk (Figures 13A, 13B, and 13C, respectively).

[0148] Results: These studies demonstrate that Ru(bpy)3 can diffuse throughout a 1.0 mm thick 12% (w / v) gelatin hydrogel within 6 minutes. Following the diffusion step, the entire gelatin hydrogel can be covalently crosslinked by 5 seconds of light irradiation with a 450 nm blue LED lamp. The thickness of the human cornea is approximately 450 μm, and therefore, a diffusion time of approximately 3 minutes followed by 5 seconds of light irradiation is sufficient to form a fully crosslinked corneal stroma.

[0149] Diffusion of riboflavin into a 12% gelatin gel For comparison, the inventors also investigated the diffusion of riboflavin (0.1%) into a 12% gelatin hydrogel model. The diffusion rate of riboflavin is slightly slower than that of a 0.5 mM Ru(bpy)3 solution. The current riboflavin / UV-A CXL method requires a 30-minute riboflavin diffusion period, followed by a 10-minute (accelerated) or 30-minute (conventional Dresden protocol) UV-A irradiation period. Riboflavin was used at 0.1% for CXL. ≡0.1g / 100ml ≡1g / L ≡1 mg / ml = 1 / 376.37 mol = 2.6 mM in PBS (pH 7.4) (not completely soluble in PBS*) *1 μL of 10N NaOH was added to 2 ml of a 0.1% solution in PBS - the pH was adjusted to approximately 8.5 (pH paper test). The riboflavin solution became completely soluble at 0.1% (2.6 mM) at room temperature.

[0150] Figure 14 shows the diffusion of 2.6 mM (0.1%) riboflavin into a 12% gelatin gel over a maximum of 14 minutes.

[0151] Example 8. Gelatin "Molecular Linker" - Enhancement of Mechanical Properties The intrinsic crosslinking of a thermogelling 12% gelatin (Sigma G-1890 - high molecular weight type A gelatin) hydrogel, A. 1 mM Ru(bpy)3 + 50 mM SPS alone solution B. A solution containing autoclaved (size-selected) BH-derivativeated Nippi medigelatin (150 mg / ml) + 1 mM Ru(bpy)3 + 50 mM SPS. The comparison was made using one of the following methods.

[0152] material and method. A highly tyrosine-derivatized low molecular weight gelatin sample (BH Nippi Medigelatin sample containing 3.8 mol% tyrosine) was resuspended in water at 100 mg / ml and heated at 50°C until dissolved. The solution was autoclaved in seven consecutive cycles of 120 minutes each. This procedure describes the effect of autoclaving on the molecular weight of gelatin and was based on the study by Vanhoecke and Olijve (2018) in which the molecular weight of G1890 Sigma gelatin was reduced from 150 kDa to 75 kDa in a single autoclave cycle. Based on this, the inventors performed seven consecutive autoclave cycles of a G1890 gelatin solution (in 1×PBS) at 100 mg / ml. After four autoclave cycles, the G1890 gelatin sample (100 mg / ml) did not gel at 4°C. A study by Mrak and Stewart (1957) showed that "the molecular weight of decomposed gelatin that did not gel at 0°C is approximately 10 kDa to 15 kDa."

[0153] After autoclaving, approximately 2 μL to 5 μL of sample (from a 100 mg / ml solution) was removed and frozen. SDS-PAGE was performed to analyze the success of autoclaving in reducing the gelatin molecular weight to approximately 10 kDa to 15 kDa. Please refer to the SDS-PAGE image (Figure 2) showing the molecular weight range of autoclaved gelatin against autoclaving time. The objective was to produce samples of BH-derivativeized gelatin exhibiting an average molecular weight range of 10 kDa to 15 kDa.

[0154] Example 9. Effect of autoclaved gelatin molecular linker on the elastic modulus of crosslinked gelatin hydrogel. Methods. A 12% (w / v) solution of Sigma G1890 (Type A) gelatin was prepared by dissolving solid gelatin in PBS buffer at 50°C. The solution was maintained at 40°C until it was ready for use in forming a mold for the heat-solidified hydrogel. The molten solution was pipetteed into a Teflon mold to prepare a 1 mm thick gelatin hydrogel sheet with dimensions of 10 mm × 5 mm × 1 mm (volume of 50 μL).

[0155] Two experiments were conducted to evaluate the effect of including a tyrosine derivatized molecular linker on the mechanical properties of crosslinked hydrogels. A thermally solidified gelatin strip was immersed for 10 minutes in a solution (60 μL) containing either of the following two solutions (A) or (B). After a 10-minute diffusion period, excess liquid was blotted from the crosslinked gelatin strip using Kimwipe tissue, and the gelatin strip was exposed to light from a 450 nm blue LED dental lamp (3M ESPE blue LED lamp (1,200 mW / cm²)). 2 Photocrosslinking was performed using a light guide tip at a distance of 5 cm for a 5-second irradiation period.

[0156] (A) A 12% gelatin strip measuring 1 mm thick, 10 mm x 5 mm, was crosslinked by photochemical crosslinking using 1 mM Ru(bpy)3 + 50 mM SPS in the presence of BH-derivativeized Nippi gelatin (150 mg / ml) that had been autoclaved seven times (total volume = 60 μL), or (B) 1 mm thick, 10 mm x 5 mm, 12% gelatin strips were crosslinked by a photochemical crosslinking reaction in the presence of only 1 mM Ru(bpy)3 + 50 mM SPS (total volume = 60 μL). No tyrosine derivatized molecular linker solution was added during the crosslinking reaction.

[0157] A photochemically crosslinked gelatin strip was positioned so that a 5mm length of the strip was fixed to the end of a PTFE block. A 150mg weight was attached to the protruding strip. A series of photographs were taken to determine the load-bearing capacity of the gelatin strip fixed at one end.

[0158] A. 12% gelatin strips measuring 1 mm thick, 10 mm x 5 mm, were crosslinked by photochemical crosslinking using 1 mM Ru(bpy)3 + 50 mM SPS in the presence of BH-derivativeized Nippi gelatin (150 mg / ml) that had been autoclaved seven times (total volume = 60 μL).

[0159] B. 12% gelatin strips measuring 1 mm thick and measuring 10 mm x 5 mm were crosslinked by a photochemical crosslinking reaction in the presence of only 1 mM Ru(bpy)3 + 50 mM SPS (total volume = 60 μL). No tyrosine derivatized molecular linker solution was added during the crosslinking reaction.

[0160] Results: The inventors demonstrated that a thin (1 mm) gelatin hydrogel sheet photochemically crosslinked in the presence of low molecular weight (LMW - decomposed gelatin with a molecular weight of approximately 10 kDa to 15 kDa) tyrosine-derivativeized gelatin molecules could support a larger mass with less displacement compared to the same gelatin gel crosslinked using Ru(bpy)3+SPS alone (Figures 16A-16H).

[0161] These results indicate that the addition of a solution of decomposed (tyrosine-derivativeized) gelatin (150 mg / ml) to a Ru(bpy)3+SPS photochemical reagent solution improves the mechanical properties of the crosslinked gelatin hydrogel compared to the mechanical properties of a gelatin hydrogel photochemically crosslinked using a Ru(bpy)3+SPS solution alone. The gelatin hydrogel model exhibits a higher elastic modulus when photochemically crosslinked in the presence of a low molecular weight tyrosine-derivativeized gelatin molecular linker.

[0162] Example 10. Effect of autoclaved BH-derivativeized gelatin molecular linker on the elastic modulus of bovine cornea. method Bovine corneas, specifically "buttons," were prepared by excising corneas from fresh slaughterhouse specimens (collected on ice within 4 hours of slaughter). The corneal epithelium was removed from the entire corneal surface before corneal excision. The newly prepared corneas were rinsed in saline solution and blot-dried using Kimwipe tissue.

[0163] Two experiments were conducted to evaluate the effect of including an autoclaved low molecular weight tyrosine derivatized molecular linker on the mechanical properties of crosslinked hydrogels. Bovine corneas were immersed for 10 minutes in a solution (60 μL) containing either of the following two solutions (A) or (B). After a 10-minute diffusion period, excess liquid was blotted from the crosslinked gelatin strip using Kimwipe tissue, and the gelatin strip was cured using a 450 nm blue LED dental lamp (The Elipar® S10 LED curing light (1,200 mW / cm²)). 2 Photocrosslinking was performed using a light guide tip at a distance of 5 cm for a 5-second irradiation period.

[0164] A photochemically crosslinked corneal strip was positioned so that one end of the 10 mm length of the strip was fixed to the end of a PTFE block. A weight of 111 mg was attached to the protruding strip. A series of photographs were taken to determine the load-bearing capacity of the bovine corneal strip with one end fixed.

[0165] A. (Figure 16A) Bovine corneal strips (10 mm × 5 mm) were crosslinked by photochemical crosslinking using 1 mM Ru(bpy)3 + 50 mM SPS in the presence of BH-derivativeized Nippi gelatin (150 mg / ml) that had been autoclaved seven times (total volume = 60 μL).

[0166] B. (Figure 16B) Bovine corneal strips (10 mm × 5 mm) were crosslinked by a photochemical crosslinking reaction in the presence of only 1 mM Ru(bpy)3 + 50 mM SPS (total volume = 60 μL). No tyrosine derivatized molecular linker solution was added during the crosslinking reaction.

[0167] result The inventors have shown that a bovine corneal strip (5 mm × 10 mm) photochemically crosslinked in the presence of low molecular weight (LMW - decomposed gelatin with a molecular weight of approximately 12 kDa to 20 kDa) tyrosine-derivative gelatin molecules can support the same mass with less vertical displacement compared to the same corneal strip crosslinked using Ru(bpy)3 + SPS only (Figure 16B) (Figure 16A). Note that the deflection angle of a strip with one end fixed and crosslinked in the presence of a low molecular weight BH-derivative gelatin "molecular linker" is lower than the deflection recorded for a strip crosslinked in the absence of the low molecular weight BH-derivative gelatin molecular linker (Figure 16B - 31°) (Figure 16A - 12°).

[0168] These results indicate that the addition of a solution (150 mg / ml) of decomposed (tyrosine-derivativeized) gelatin (molecular weight 12 kDa to 20 kDa) to a Ru(bpy)3+SPS photochemical reagent solution improves the mechanical properties of the crosslinked gelatin hydrogel compared to the mechanical properties of bovine cornea photocrosslinked using a Ru(bpy)3+SPS solution alone. The photocrosslinked corneal model exhibits a higher elastic modulus when photocrosslinked in the presence of a low molecular weight tyrosine-derivativeized gelatin molecular linker ("molecular staple") compared to a corneal strip photocrosslinked in the absence of the low molecular weight tyrosine-derivativeized gelatin molecular linker.

[0169] The inventors have shown that the elastic modulus of bovine cornea crosslinked in the presence of decomposed (BH-derivativeized) low molecular weight gelatin (molecular weight 12kDa~20kDa) molecular linker (150 mg / ml solution) was 5.3 times higher than that of bovine cornea crosslinked in the absence of the molecular linker.

[0170] For both experiments, the calculation of the elastic modulus (E) was as follows: E (crosslinking without molecular linker) ≈ 266.23 kPa E_(with molecular linker and cross-linking) ≈ 1.41 MPa The elastic modulus (E) from the "beam fixed at one end" experiment can be calculated using the following elastic modulus calculation notes: Beam dimensions = 5mm (w) × 0.8mm (h) × 10mm (l) The density of the beam material is assumed to be approximately 1.05 g / cm³. If we model the mass of the beam and the mass of the aluminum foil as point masses at the center of the beam, we obtain 153 mg at 5 mm from the support point of the beam. Convert this mass to Newtons (=0.0014994), and convert mm to m to obtain the moment in Nm: The total moment of equilibrium of the beam at the support is 0.0014994N * 0.005m = 0.000007497Nm. The second moment of area is a multiple integral of the square of the distance from the bending axis of the beam. Since the beam is approximately rectangular in cross-section, the formula bh^3 / 12 (considering the square of both the distance above and below the bending axis) is appropriate. Therefore, the second moment of area of ​​this beam is (0.0008^3)0.005 / 12 = 2.13e10^-13m^4. B_untreated:=beam before linker treatment B_treated:=beam after linker treatment The curvature at the support point of B_untreated can be obtained by overlapping the contact circles and determining their radii. It is found that such curvature is 0.132(mm / mm) / mm = 0.132mm^-1 = 132m^-1. Similarly, it can be found that the curvature at the support point of B_treated is 0.0249(mm / mm) / mm = 0.0249mm^-1 = 24.9m^-1. E_untreated:=elastic modulus before linker treatment E_treated:=elastic modulus after linker treatment Using the formula M=EIk (where M is the moment at the support, E is the modulus of elasticity, I is the second moment of area, and k is the curvature), therefore, E_(without molecular linker) ≈ 266.23 kPa E_(with molecular linker) ≈ 1.41 MPa.

[0171] A comparison of the photochemical crosslinking method presented by the inventors (using blue LED light at 455 nm in the presence of Ru(bpy)3+SPS) with the conventional CXL method can be made by referring to the study published by Alenezi et al. (2022). These researchers measured the elastic modulus (E) of various segments of human cornea using a nanoindentation method. It was reported that the elastic modulus (E) of the anterior segment of human cornea crosslinked by CXL was 178% higher than that of the anterior segment of uncrosslinked cornea. The actual elastic modulus (E) reported for the anterior segment of younger cornea treated with riboflavin-UV-A based CXL was 285 kPa. In other words, the photochemical crosslinking method using Ru(bpy)3+SPS alone yielded an elastic modulus nearly equivalent to that achieved after a total treatment of 39 minutes per cornea (30 minutes of riboflavin application, followed by 9 minutes of UV-A (10 mW / cm2) irradiation, totaling 5.4 J / cm2), including 5 seconds of irradiation with 455 nm blue LED light (in 3 minutes). Significantly, the addition of a solution containing 150 mg / ml of decomposed (12 kDa~20 kDa) BH-derivative gelatin resulted in a 5.3-fold increase in the elastic modulus (E) up to 1.41 MPa.

[0172] To achieve the desired increase in elastic modulus after a Ru(bpy)3+SPS-mediated crosslinking method, those skilled in the art can adjust the desired degree of photochemical crosslinking (and therefore the level of crosslinking that causes an increase in elastic modulus) by changing the amount of each component of the photochemical crosslinking method. For example, the amount of crosslinking induced by the photochemical crosslinking method described herein can be varied by (i) changing the concentration of Ru(bpy)3 from about 0.25 mM to about 2 mM, (ii) changing the SPS concentration from about 10 mM to about 100 mM, and (iii) changing the concentration of the BH derivatized molecular linker from about 10 mg / ml to about 200 mg / ml. In addition, the light irradiation time can be varied from about 0.5 seconds to about 10 seconds, which has the effect of changing the blue light energy transmitted to the cornea. This can also be achieved by varying the distance of the light source from the corneal surface from about 20 mm to about 200 mm.

[0173] It was concluded that short tyrosine-derivatized gelatin chains can form crosslinks between distally separated gelatin chains in a thermally gelled hydrogel (described in Experiment 9) or between distally separated collagen chains in bovine cornea (described in Experiment 10). Such crosslinks contribute to enhanced mechanical properties when gelatin molecular linkers are present during the photochemical crosslinking reaction. In both Experiments 9 and 10, the inclusion of low molecular weight tyrosine-derivatized gelatin molecular linkers resulted in a clear increase in the elastic modulus of both thermally gelled gelatin strips (Experiment 9) and newly prepared corneal tissue strips (Experiment 10) during the diffusion of the crosslinking reagent (Ru(bpy)3+SPS). In the absence of molecular linkers, dityrosine crosslinks can only be formed through short-range hydrophobic π interactions between closely related tyrosine residues located proximal (less than 5 Å) to adjacent gelatin chains (in Experiment 9) or between closely related tyrosine residues located in proteoglycan and collagen chains (in Experiment 10).

[0174] In both Experiments 9 and 10, the inclusion of tyrosine-derivatized low molecular weight linkers was shown to increase the elastic modulus of the test material, whether it was a thermally gelled gelatin strip (in Experiment 9) or bovine cornea (in Experiment 10). This increase in material stiffness resulted from the introduction of novel intermolecular crosslinks induced by the presence of tyrosine-derivatized low molecular weight linkers.

[0175] This application claims priority to Australian Provisional Application No. 2023900571.

[0176] Any discussion of documents, laws (acts), materials, devices, articles, etc., included herein is solely for the purpose of providing context for the present invention. Nothing or all of these matters shall be construed as forming part of the prior art foundation or being common general knowledge in the art relating to the present invention, if they existed prior to the priority date of each claim of this application.

[0177] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0178] References Akhtar et al., (2013), Acta Ophthalmol 86: 764-772 Alenezi et al., (2022) Experimental Eye Research 214: 108839 Bolton and Hunter, (1973), Biochem J. 133: 529-539 Brummer et al., (2011), Invest Ophthalmol Vis Sci. 52: 6363-6369 Buffiere et al., (2016) Green Chem. 18: 6516-6525 Cui et al., (2011), Invest Ophthalmol Vis Sci. 52: 2556-2564 Dervenis et al., (2020), Med Princ Pract 29: 332-337 Elvin et al., (2010), Biomaterials 31: 8323-8331 Escalante et al. (2012) Carbohydrate Polymers 87: 42381-2387 Forrester et al., (2020) The Eye: basic sciences in practice (5th Edition Elsevier) Habibi et al., (2010) Chemical Reviews, 110(6), 3479-3500 Hayes et al., (2013), PLoS ONE 8: e52860 Heinze et al., (2016) Macromol Biosci 16: 10-42 Jardine (2022) Current Research in Green and Sustainable Chemistry 5: 100309 Kamaev et al. (2012) Invest Ophthalmol. Vis. Sci. 53: 2360-2367 Khaire et al., (2022) Industrial Crops and Products 187: 115545 Kurimuraet al., (1982), Bull Chem Soc Jpn 55: 55-58 Kuzmenko et al., (2014) Carbohydr Polym. 102: 862-868 Lima et al., (2004) Macromolecular Rapid Communications, 25, 771-787 Markstedt et al., (2017) Carbohydr Polym. 157: 1349-1357) Miller, (1996), Biochem Biophys Res Commun. 218: 377-382 Mrak and Stewart (1957), Advances in Food Research (Academic Press) p 307 O’Brart et al., (2015), Am J Ophthalmol. 160: 1154-1163 Prausnitz and Noonan, (1998), J Pharm Sci 87: 1479. Raiskup-Wolf et al., (2008), J Cataract Refract Surg. 34: 796-801 Rajapaksha et al., (2015), Molecular and Cellular Biomechanics. 12: 215-230 Seiler et al., (2014), Invest Ophthalmol Vis Sci. 56: 6740-6746 Shaghaleh et al., (2021) Journal of Cleaner Production 297: 126664 Sharma et al., (2012), Am J Ophthalmol. 154: 922-926 Simon et al., (2023) Biomacromolecules 24: 166-177 Sliney, D. et al., (2005) Applied Optics 44: 2162-2176. Spoerl et al., (1998), Ex Eye Res. 66: 97-103 Spoerl et al., (2004a), Curr Eye Res. 29: 35-40 Spoerl et al., (2004b), Ophthalmo-logica 218: 136-140 Tuite et al., (2012), Phys. Chem. Chem. Phys. 14:3681–3692 van Dijk et al., (2018), Graefes Arch. Clin. Exp. Ophthalmol. 256:1151–1158 Vanhoecke and Olijve (2018), Rousselot Report, 2018 Vimalin et al., (2012), Cornea 31: 1052-1059 Wollensak et al., (2003), Ophthalmologist. 100:44–49 Wollensak et al., (2004), Cornea. 23:503–507 Xu et al., (2014), International Journal of Biological Macromolecules 67: 205-209 Ziolkowska, et al., (2023) Cells 12: 1014

Claims

1. A method for cross-linking endogenous proteins in tissues, (i) The tissue is brought into contact with a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor, thereby bringing the molecular linker into contact with the endogenous protein in the tissue; (ii) The step of irradiating the tissue and Methods that include...

2. The method according to claim 1, wherein the molecular linker is a polymer containing two or more tyrosine and / or tyramine residues.

3. The method according to claim 2, wherein the polymer comprising one or more tyrosine and / or tyramine residues is selected from tyrosine and / or tyramine-derivative gelatin, tyrosine and / or tyramine-derivative dextran, and / or tyrosine and / or tyramine-derivative xylan, and / or tyrosine and / or tyramine-derivative low molecular weight nanocellulose.

4. The method according to any one of claims 1 to 3, wherein the molecular linker has a molecular weight of about 5 kDa to about 50 kDa.

5. The method according to any one of claims 1 to 4, wherein the photoactivatable metal ligand complex is ruthenium (tris)bipyridyl chloride.

6. The method according to any one of claims 1 to 5, wherein the electron acceptor is sodium persulfate.

7. The method according to any one of claims 1 to 6, wherein the tissue is corneal tissue.

8. The method according to claim 7, wherein the step of contacting the corneal tissue with a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor comprises administering a solution comprising the molecular linker, the photoactivatable metal ligand complex, and the electron acceptor to the surface of the cornea, thereby allowing the solution to diffuse into the corneal stroma.

9. The method according to any one of claims 1 to 8, wherein the tissue is irradiated with light having a wavelength of approximately 430 nm to 480 nm.

10. The step of irradiating the aforementioned tissue is approximately 200 mW / cm². ２ ~1,200mW / cm ２ The method according to any one of claims 1 to 9, comprising delivering the light intensity of the tissue to the tissue surface.

11. The method according to any one of claims 9 to 10, wherein the tissue is irradiated for about 1 to about 10 seconds.

12. A method for treating weakening of the corneal stroma in a subject, (i) The step of administering a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor to the cornea of ​​the subject, (ii) The step of irradiating the cornea and Methods that include...

13. A method for treating or preventing at least one symptom of keratoconus in a subject, (i) The step of administering a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor to the cornea of ​​the subject, (ii) The step of irradiating the cornea and Methods that include...

14. The method according to claim 12 or claim 13, wherein the molecular linker is a polymer containing two or more tyrosine and / or tyramine residues.

15. The method according to claim 14, wherein the polymer comprising two or more tyrosine and / or tyramine residues is selected from tyrosine and / or tyramine-derivative gelatin, tyrosine and / or tyramine-derivative dextran, and / or tyrosine and / or tyramine-derivative xylan, and / or tyrosine and / or tyramine-derivative low molecular weight nanocellulose.

16. The method according to any one of claims 12 to 15, wherein the molecular linker has a molecular weight of about 5 kDa to about 50 kDa.

17. The method according to any one of claims 12 to 16, wherein the photoactivatable metal ligand complex is ruthenium (tris)bipyridyl chloride.

18. The method according to any one of claims 12 to 17, wherein the electron acceptor is sodium persulfate.

19. The method according to any one of claims 12 to 18, wherein the step of administering the molecular linker, the photoactivatable metal ligand complex, and the electron acceptor comprises administering a solution comprising the molecular linker, the photoactivatable metal ligand complex, and the electron acceptor to the surface of the cornea, thereby allowing the solution to diffuse into the corneal stroma.

20. The method according to claim 19, wherein the solution diffuses into the corneal stroma for a maximum of about 5 minutes.

21. The method according to any one of claims 12 to 20, wherein the cornea is irradiated with light having a wavelength of approximately 430 nm to 480 nm.

22. The method according to claim 21, wherein the tissue is irradiated for about 1 to about 10 seconds.

23. The method according to any one of claims 12 to 22, wherein crosslinking the molecular linker and endogenous proteins brings about an improvement in one or more mechanical properties of the corneal stroma.

24. The use of molecular linkers in the manufacture of pharmaceuticals for the treatment or prevention of one or more symptoms of a disease in a target area.

25. A composition for the treatment of keratoconus in a subject, comprising a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor.

26. A composition for intermolecular crosslinking between endogenous stromal collagen fibrils and other endogenous proteins in the cornea, comprising a molecular linker, a photoactivatable metal ligand complex, and an electron acceptor.

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