WO2025226888A1 - Ecm-derived injectable embolic for permanent treatment of cerebral aneurysms - Google Patents

Abstract

Provided herein is a method of treating a defect in the vasculature of a patient, including delivering to the defect a gellable composition comprising a first modified extracellular matrix (ECM) component and a second modified ECM component.

Description

ECM-DERIVED INJECTABLE EMBOLIC FOR PERMANENT TREATMENT OF CEREBRAL ANEURYSMS

CROSS REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to United States Provisional Patent Application No. 63/639,203, filed April 26, 2024, the disclosure of which is hereby incorporated by reference in its entirety.

REFERENCE TO A SEQUENCING LISTING

[0002] The Sequence Listing associated with this application is filed in electronic format via Patent Center and is hereby incorporated by reference into the specification in its entirety. The name of the file containing the Sequence Listing is 25OO678.xmL The size of the file is 4,575 bytes, and the file was created on April 22, 2025.

BACKGROUND OF THE INVENTION

Field of the Invention

[0003] Provided herein are compositions, methods, and kits for treating vascular conditions.

Description of Related Art

[0004] Cerebral aneurysms pose a severe risk of rupture, leading to potentially fatal subarachnoid hemorrhage, often leading to mortality (25-50%) and permanent disability (nearly 50%) in the survivors. Non-bioabsorbable platinum coils are typical options for cerebral aneurysm embolization. Additionally, flow-diverting devices (FDD) stents with high metal coverage and low porosity have been introduced. While FDD has greater rates of aneurysm obliteration than intrasaccular therapy, careful patient selection is necessary for typical side-wall aneurysms, and extended anti-platelet therapy is required. Despite its advantages, endovascular therapy by coiling is not without limitations and complications. These include well-known issues such as coil migration, coil compaction, and recanalization. Recanalization has been observed in approximately 17% of patients who undergo bare coiling for aneurysms, necessitating retreatment in 10.3% of cases.

[0005] As an alternative to metallic coiling, researchers have investigated injectable embolic agents to fill vascular defects including arteriovenous malformations and aneurysms. These include Onyx™, n-butyl cyanoacrylate, and EmboGel™. In practice, the target site is filled by administering a controlled amount of the embolic agent to occlude the aneurysm. However, Onyx™ and n-butyl cyanoacrylate have limitations primarily because of uncontrolled reflux into the parent artery, creating a higher risk of stroke. Accordingly, there exists a significant need in the art to address aneurysms.

SUMMARY OF THE INVENTION

[0006] Provided herein is a method of treating a defect in the vasculature of a patient, including delivering to the defect a gellable composition including a first modified extracellular matrix (ECM) component and a second modified ECM component.

[0007] Also provided herein is a kit including a delivery device having a first reservoir including a first modified extracellular matrix (ECM) component and a second reservoir including a second modified ECM component, wherein the first ECM component and the second ECM component are configured to gel when mixed.

[0008] Also provided herein is a gellable composition including a first modified ECM component, a second modified ECM component, a polymer, a therapeutic composition, and a self-assembling peptide.

[0009] Also provided herein is a gellable composition for use in treating a defect in the vasculature of a patient, the composition including a first modified ECM component, a second modified ECM component, a polymer, a therapeutic composition, and a self-assembling peptide.

[0010] Also provided herein is use of a gellable composition including a first modified ECM component, a second modified ECM component, a polymer, a therapeutic composition, and a self-assembling peptide for treating a defect in the vasculature of a patient.

[0011] Further non-limiting embodiments are set forth in the following numbered clauses:

[0012] 1 . A method of treating a defect in the vasculature of a patient, comprising delivering to the defect a gellable composition comprising a first modified extracellular matrix (ECM) component and a second modified ECM component.

[0013] 2. The method of clause 1 , wherein the first modified ECM component and/or the second ECM component is one or more of gelatin, hyaluronic acid, fibronectin (FN), collagen, laminin (LM), vitronectin (VN), thrombospondin (TSP), tenascin (TN), a proteoglycan, and/or a glycosaminoglycan.

[0014] 3. The method of clause 1 or clause 2, wherein the first modified ECM component is gelatin and the second modified ECM component is hyaluronic acid.

[0015] 4. The method of any of clauses 1 -3, wherein the modification comprises one or more of a thiol group, a sulfone group, an azide group, an alkyne group, a succinimide group, and/or a malemide group.

[0016] 5. The method of any of clauses 1 -4, wherein the sulfone group is a vinyl sulfone group.

[0017] 6. The method of any of clauses 1 -5, wherein the first modified ECM component is thiolated gelatin and the second modified ECM component is vinyl sulfonated hyaluronic acid.

[0018] 7. The method of any of clauses 1 -6, further comprising a therapeutic composition.

[0019] 8. The method of any of clauses 1 -7, wherein the therapeutic composition is one or factors that promote coagulation and/or fibrosis.

[0020] 9. The method of any of clauses 1 -8, wherein the therapeutic composition comprises one or more of a transforming growth factor (TGF), substance P, a platelet- derived growth factor (PDGF), a pro-inflammatory cytokine, and/or a monocyte chemoattractant protein (MCP).

[0021] 10. The method of any of clauses 1 -9, wherein the gellable composition further comprises a self-assembling peptide.

[0022] 1 1 . The method of any of clauses 1 -10, wherein the self-assembling peptide has the following sequence: RADARADARADARADA (SEQ ID NO: 2).

[0023] 12. The method of any of clauses 1 -1 1 , wherein the self-assembling peptide further comprises a therapeutic composition conjugated thereto.

[0024] 13. The method of any of clauses 1 -12, wherein the therapeutic composition is substance P.

[0025] 14. The method of any of clauses 1 -13, further comprising a contrast agent.

[0026] 15. The method of any of clauses 1 -14, wherein the contrast agent is iohexol.

[0027] 16. The method of any of clauses 1 -15, wherein the gellable composition further comprises a polymer. [0028] 17. The method of any of clauses 1 -16, wherein the polymer is a natural polymer.

[0029] 18. The method of any of clauses 1 -17, wherein the natural polymer is one or more of a polysaccharide, a polypeptide, a nucleic acid-based polymer, silk, wool, and/or cellulose.

[0030] 19. The method of any of clauses 1 -18, wherein the polysaccharide is chitin, chitosan, and/or alginate.

[0031] 20. The method of any of clauses 1 -19, wherein the defect is an aneurysm.

[0032] 21 . The method of any of clauses 1 -20, wherein the aneurysm is a cerebral aneurysm.

[0033] 22. The method of any of clauses 1 -21 , wherein the first modified ECM component and the second modified ECM component are delivered to the defect from separate reservoirs.

[0034] 23. The method of any of clauses 1 -22, wherein the first modified ECM component and the second modified ECM component are delivered with a catheter.

[0035] 24. A kit comprising: a delivery device comprising a first reservoir including a first modified extracellular matrix (ECM) component and a second reservoir including a second modified ECM component, wherein the first modified ECM component and the second modified ECM component are configured to gel when mixed.

[0036] 25. The kit of clause 24, wherein the first modified ECM component and/or the second modified ECM component is one or more of gelatin, hyaluronic acid, fibronectin (FN), collagen, laminin (LM), vitronectin (VN), thrombospondins (TSPs) tenascin (TN), a proteoglycan, and/or a glycosaminoglycan.

[0037] 26. The kit of clause 24 or clause 25, wherein the first modified ECM component is gelatin and the second modified ECM component is hyaluronic acid.

[0038] 27. The kit of any of clauses 24-26, wherein the modification comprises one or more of a thiol group, a sulfone group, an azide group, an alkyne group, a succinimide group, and/or a malemide group.

[0039] 28. The kit of any of clauses 24-27, wherein the sulfone group is a vinyl sulfone group.

[0040] 29. The kit of any of clauses 24-28, wherein the first modified ECM component is thiolated gelatin and the second modified ECM component is vinyl sulfonated hyaluronic acid. [0041] 30. The kit of any of clauses 24-29, wherein the first reservoir and/or the second reservoir further includes a therapeutic composition.

[0042] 31 . The kit of any of clauses 24-30, wherein the therapeutic composition is one or factors that promote coagulation and/or fibrosis.

[0043] 32. The kit of any of clauses 24-31 , wherein the therapeutic composition comprises one or more of a transforming growth factor (TGF), substance P, a platelet- derived growth factor (PDGF), a pro-inflammatory cytokine, and/or a monocyte chemoattractant protein (MCP).

[0044] 33. The kit of any of clauses 24-32, wherein the first reservoir and/or the second reservoir further includes a self-assembling peptide.

[0045] 34. The kit of any of clauses 24-33, wherein the self-assembling peptide has the following sequence: RADARADARADARADA (SEQ ID NO: 2).

[0046] 35. The kit of any of clauses 24-34, wherein the self-assembling peptide further comprises a therapeutic composition conjugated thereto.

[0047] 36. The kit of any of clauses 24-35, wherein the therapeutic composition is substance P.

[0048] 37. The kit of any of clauses 24-36, wherein the first reservoir and/or the second reservoir further includes a contrast agent.

[0049] 38. The kit of any of clauses 24-37, wherein the contrast agent is iohexol.

[0050] 39. The kit of any of clauses 24-38, wherein the first reservoir and/or the second reservoir further includes a polymer.

[0051 ] 40. The kit of any of clauses 24-39, wherein the polymer is a natural polymer. [0052] 41 . The kit of any of clauses 24-40, wherein the natural polymer is one or more of a polysaccharide, a polypeptide, a nucleic acid-based polymer, silk, wool, and/or cellulose.

[0053] 42. The kit of any of clauses 24-41 , wherein the polysaccharide is chitin, chitosan, and/or alginate.

[0054] 43. The kit of any of clauses 24-42, wherein the delivery device is a syringe.

[0055] 44. The kit of any of clauses 24-43, further comprising a catheter.

[0056] 45. The kit of any of clauses 24-44, wherein the catheter comprises a radiopaque material.

[0057] 46. A gellable composition comprising: a first modified ECM component; a second modified ECM component; a polymer; a therapeutic composition; and a selfassembling peptide. [0058] 47. The gellable composition of clause 46, wherein the first modified ECM component is a thiolated gelatin, the second modified ECM component is a vinyl sulfonated hyaluronic acid, and the therapeutic composition is substance P.

[0059] 48. A gellable composition for use in treating a defect in the vasculature of a patient, the composition comprising: a first modified ECM component; a second modified ECM component; a polymer; a therapeutic composition; and a selfassembling peptide.

[0060] 49. The gellable composition of clause 48, wherein the first modified ECM component is a thiolated gelatin, the second modified ECM component is a vinyl sulfonated hyaluronic acid, and the therapeutic composition is substance P.

[0061] 50. Use of a gellable composition for treating a defect in the vasculature of a patient, the composition comprising: a first modified ECM component; a second modified ECM component; a polymer; a therapeutic composition; and a selfassembling peptide.

[0062] 51 . The use of clause 50, wherein the first modified ECM component is a thiolated gelatin, the second modified ECM component is a vinyl sulfonated hyaluronic acid, and the therapeutic composition is substance P.

BRIEF DESCRIPTION OF THE DRAWINGS

[0063] FIG. 1 shows a non-limiting embodiment of a device suitable for delivering composition for use in non-limiting embodiments of methods as described herein;

[0064] FIGS. 2A-2C show (A) the synthesis of gelatin/hyaluronic acid-based injectable hydrogel; (B) 1 H-NMR of thiolated gelatin (Gel-SH) and vinyl sulfonated hyaluronic acid (HA-VS); (C) an image taken 30 sec after mixing and SEM of gelatin/hyaluronic acid-based injectable hydrogel;

[0065] FIG. 3 shows steps for in vitro injection study with fresh ovine blood; cross- sectional SEM images of injection of (B) 20% 0.2 M CaCI2, (C) 2% alginic acid + 20% 0.2 M CaCI2, (D) HA-VS/Gel-SH with 10% (w/v) iohexol, or (E) HA-VS/Gel-SH gel with 15% (w/v) iohexol into 50 pL fresh citrated ovine blood (n = 4). Arrows indicate blood cell adhesion and incorporation;

[0066] FIG. 4 shows in vitro blood-contacting test where formed HA-VS/Gel-SH gel was lyophilized and contacted ovine blood for 3 h with gentle rocking (n = 4): (A) image of freeze-dried HA-VS/Gel-SH with 10% iohexol; (B) freeze-dried HA-VS/Gel-SH with 10% iohexol after blood contact; (C) HA-VS/Gel-SH with 15% iohexol after blood contact; (D) SEM images of surfaces and cross sections from C and D;

[0067] FIGS. 5A-5D show in vivo embolization studies using the mice aneurysm model: (A) diagram of schedule of the animal study; (B) aneurysm creation and embolization steps: 1 ) murine common carotid artery is exposed and a latex cuff is placed underneath; 2) most distal portion of the artery is ligated using 8-0 nylon suture to create a stump; 3) the artery is incubated with elastase for 20 min; 4) at the end of the incubation period an aneurysm has formed, the latex cuff is removed and the neck incision is closed; 5) 3 weeks later the carotid aneurysm is re-exposed and a temporary clip is placed around the proximal part of the artery-aneurysm complex; 6) embolic is injected through a 30G needle; 7) waiting for 5 min; 8) the temporary clip is removed; and (C) intraoperative photographs of the injection. (D) Pro-inflammatory cytokine production (n = 3) after the injection of (a) HA-VS/Gel-SH with 10% iohexol and (b) HA-VS/Gel-SH with 10% iohexol and RADA-SP.

[0068] FIG. 6 shows H&E (upper), trichrome (bottom left), and picrosirrus-red (bottom right) images of 3 weeks after the embolization: (A) HA-VS/Gel-SH with 10% iohexol; and (B) HA-VS/Gel-SH with 10% iohexol and RADA-SP (Scale bar: 60 pm, *: middle of aneurysm sac);

[0069] FIGS. 7A-7C show Immunofluorescence (CD68, FSP-1 , MECA-32, aSMA, and DAPI) images of 3 weeks after the embolization (n = 4): HA-VS/Gel-SH with 10% iohexol, and HA-VS/Gel-SH with 10% iohexol and RADA-SP (scale bar: 100 pm, *: middle of aneurysm sac;

[0070] FIGS. 8A-8B show immunofluorescence (CD80, CD206, and DAPI) images of 3 weeks after the embolization (n = 4): HA-VS/Gel-SH with 10% iohexol, and HA- VS/Gel-SH with 10% iohexol and RADA-SP (scale bar: 50 pm, *: middle of aneurysm sac);

[0071] FIGS. 9A-9B show (A) Viscosity test and elasticity evaluation, and (B) modulus of HA-VS/Gel-SH and in vitro degradation profile of HA-VS/Gel-SH with 10% iohexol scaffolds in PBS. Weight change versus exposure time was determined for 4 weeks (n =5); and

[0072] FIG. 10 shows a photo image of HA-VS/Gel-SH gel with 10% (w/v) iohexol after reacted and lyophilized in 96-well plate; (B) X-ray (OEC 9800 Plus); and (C) SEM images of HA-VS/Gel-SH gel with different concentrations of iohexol (n=5). DESCRIPTION OF THE INVENTION

[0073] The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges are both preceded by the word "about". In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, unless indicated otherwise, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values. As used herein, "a" and "an" refer to one or more.

[0074] As used herein, the term "comprising" is open-ended and may be synonymous with 'including', 'containing', or 'characterized by'. The term "consisting essentially of" limits the scope of a claim to the specified materials or steps, and those that do not materially affect basic and novel characteristic(s). The term "consisting of" excludes any element, step, or ingredient not specified in the claim. As used herein, embodiments "comprising" one or more stated elements or steps also include but are not limited to embodiments "consisting essentially of" and "consisting of" these stated elements or steps.

[0075] As used herein, the term "patient" or "subject" refers to members of the animal kingdom including but not limited to human beings, and "mammal" refers to all mammals, including, but not limited to human beings.

[0076] As used herein, the "treatment" or "treating" of a condition means administration to a patient by any suitable dosage regimen, procedure and/or administration route an amount of a composition, device or structure effective to, and with the object of achieving a desirable clinical/medical end-point, including filling and/ro isolation of the aneurysm from the remaining vasculature, attracting progenitor cells, healing a wound, correcting a defect, etc. In the context of the present disclosure, wound-treating compositions, kits, and methods provided herein, "treatments" may include delivery of the composition described herein to a target site, such as an aneurysm, bioerosion or bioabsorption of one or more components of the composition, and cellular infiltration and/or remodeling at the site.

[0077] As used herein, the terms "extracellular matrix" and "ECM" refer to a natural scaffolding for cell growth obtained from the decellularization or devitalization of tissue or a synthetic scaffolding designed to mimic natural scaffolding. Extracellular matrix (ECM) is a complex mixture of structural and non-structural biomolecules, including, but not limited to, collagens, elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and growth factors. In mammals, ECM often comprises about 90% collagen, in its various forms, derivatives of which include gelatin. The composition and structure of ECM varies depending on the source of the tissue. For example, small intestine submucosa (SIS), urinary bladder matrix (UBM), liver stroma ECM, and dermal ECM each differ in their overall structure and composition due to the unique cellular niche needed for each tissue. Methods of isolating, and sources of, ECM are known, for example as described in U.S. Patent Nos. 4,902,508; 4,956,178; 5,281 ,422; 5,352,463; 5,372,821 ; 5,554,389; 5,573,784; 5,645,860; 5,711 ,969; 5,753,267; 5,762,966; 5,866,414; 6,099,567; 6,485,723; 6,576,265; 6,579,538; 6,696,270; 6,783,776; 6,793,939; 6,849,273; 6,852,339; 6,861 ,074; 6,887,495; 6,890,562; 6,890,563; 6,890,564; and 6,893,666, each of which is incorporated herein by reference in their entirety.

[0078] As used herein, the term "derive" and any other word forms or cognates thereof, such as, without limitation, "derived" and "derives", refers to a component or components obtained from any stated source by any useful method. For example and without limitation, an ECM-derived gel refers to a gel comprised of one or more components of ECM obtained from any tissue by any number of methods known in the art for isolating ECM. In another example, mammalian tissue-derived ECM refers to ECM comprised of components of a particular mammalian tissue obtained from a mammal by any useful method.

[0079] ECM components, whether derived from natural sources or produced by other means, may be modified, including by known methods and/or with known groups and/or moieties. ECM components may be modified to alter characteristics such as rheology (including crosslinking and gelation), controlled drug delivery (including release rates and protection of bound and unbound therapeutics), porosity (including pore size and morphology), cell-matrix interaction (including cell recruitment, migration, infiltration, penetration, attachment, and retention), blood-matrix interaction (including thrombogenicity and adhesion), stiffness and other rheological characteristics, imageability (including radiopacity), immunogenicity and cytocompatibility, degradability, and tissue ingrowth and remodeling. ECM and ECM components may be modified in a number of ways.

[0080] Chemical modification may include but are not limited to the covalent and non-covalent binding of moieties, addition of chemical motifs that enable click chemistry and components like cell attachment molecules that promote cell adhesion and migration, and/or grafting of substances like iohexol that alter macroscopic characteristics like imageability and porosity. Chemical motifs that enable click chemistry include but are not limited to methacrylates, acrylates, thiols, alkenes, alkynes, maleimides, sulfones (e.g., vinyl sulfones), dienes, azides, oximes, hydrazones, and combinations thereof (see, for example, Nicolas, et al. Biomacromolecules 2020 21 (6): 1968-1994, incorporated herein by reference in its entirety). Non-chemical modifications may include mixing components into the ECM, including but not limited to therapeutic compositions that alter cell-matrix and bloodmatrix interactions. Additional components may also be mixed with ECM for purposes that may include, but are not limited to, altering gelation rates, altering rheology, and/or altering the release, delivery, and degradation of therapeutic compositions within the ECM.

[0081] Provided herein are compositions, including gellable compositions, including modified components of extracellular matrix (ECM), kits including such compositions, and methods, of using such compositions and/or kits for treating various conditions.

[0082] In non-limiting embodiments, a useful composition may include a first ECM component, for example a first modified ECM component, and a second ECM component, for example a second modified ECM component. Suitable ECM components may be obtained from any useful source, including, without limitation, urinary bladder matrix (UBM), intestines (including small intestinal submucosa, SIS), skin, fat, bone, muscle, heart, lung, esophagus, colon, and/or the like. Suitable ECM components may include one or more of a hydroxy apatite or a derivative thereof, a collagen or a derivative thereof (including, without limitation, a gelatin), an elastin or a derivative thereof, cell adhesion proteins or derivatives thereof (including, without limitation, a fibronectin (FN) and/or a laminin (LM)), a chondroitin or a derivative thereof (such as a chondroitin sulfate), a vitronectin (VN) or a derivative thereof, a thrombospondin (TSP) or a derivative thereof (including at least TSP1 and/or TSP2), a tenascin (TN) or a derivative thereof, a proteoglycan or a derivative thereof (including, without limitation, a heparan and/or a heparan sulfate), and/or a glycosaminoglycan or a derivative thereof (including, without limitation, a hyaluronic acid and/or a keratan, such as a keratan sulfate).

[0083] In non-limiting embodiments the first and second ECM components are the same component, and in non-limiting embodiments, the first and second ECM component are different components. In non-limiting embodiments, the first and/or second ECM component may be a mixture of components. In non-limiting embodiments, the first ECM component is a gelatin, and the second ECM component is a hyaluronic acid.

[0084] As referenced above, the composition may be a gellable composition. In non-limiting embodiments, the composition is configured to gel only after mixing of the first ECM component and the second ECM component. In non-limiting embodiments, the composition is a reverse-gelling composition, or can be said to exhibit reverse thermal gelation, in that it forms a gel (sol to gel transition) upon an increase in temperature. The lower critical solution temperature (LCST) in a reverse gel is a temperature below which a reverse-gelling polymer is soluble in its solvent (e.g. water or an aqueous solvent). As the temperature rises above the LCST in a reverse gel, a hydrogel is formed for example. In non-limiting embodiments, the composition may be a composition having a LCST of at least 37^QC.

[0085] As noted above, the ECM component may be modified. Such modifications may be covalent modifications. In non-limiting embodiments, the modification is one that includes a motif and/or group that allows for click chemistry-based binding between the ECM components, including, without limitation, a thiol group, a sulfone group, an azide group, an alkyne group, a succinimide group, and/or a malemide group. In non-limiting embodiments, the first ECM component is a thiolated gelatin (for example as shown in FIG. 2A) and the second ECM component is a vinyl sulfonated hyaluronic acid (for example as shown in FIG. 2A).

[0086] In non-limiting embodiments, the composition may further include a therapeutic composition. In non-limiting embodiments, for example where the first ECM component and the second ECM component are gellable following mixing, a therapeutic composition may be included with the first ECM component and/or the second ECM component. In non-limiting embodiments the therapeutic composition may be one that promotes clot formation (e.g., a clot activator, such as, without limitation, thrombin, ellagic acid, thromboplastin, glass, silica, kaolin, bentonite, and diatomaceous earth) and/or fibrosis, for example, and without limitation, a transforming growth factor (TGF) (including, without limitation, TGF-[3), substance P, a platelet- derived growth factor (PDGF), a fibroblast growth factor (FGF), a connective tissue growth factor (CTGF), a cytokine (including, without limitation, pro-inflammatory cytokines such as IL-1 , TNF-a, IL-6, IL-8, IL-12, IL-17, and/or INF-y), and/or a monocyte chemoattractant protein (MCP).

[0087] Other therapeutic compositions, for example and without limitation chemoattractants and/or chemokines (including, without limitation, CXCL2, CXCL9, and/or CXCL12), adhesion molecules (including, without limitation, integrins, selectins, and/or immunoglobulins), cytokines (including, without limitation, pro- inflammatory cytokines), growth factors (including, without limitation, PDGF and/or vascular endothelial growth factor (VEGF)), tissue factors, anti-microbials, von Willebrand factor (vWF), and/or platelet agonists (such as, without limitation, thrombin, thromboxane A2, and adenosine diphosphate (ADP)) may be included, with the caveat that the therapeutic composition, in non-limiting embodiments, does not include any fibrolytic or anti-coagulant compositions.

[0088] In non-limiting embodiments, the composition may further include a selfassembling peptide. In non-limiting embodiments, for example where the first ECM component and the second ECM component are gellable following mixing, a selfassembling peptide may be included with the first ECM component and/or the second ECM component. As used herein, the term “self-assembling peptide” means a plurality of monomers of short amino acids, which may be repeated sequences, that self-assemble to form a nano- or-microstructure. Self-assembling peptides are known to those of skill in the art (see, e.g., Lee et al., ‘Self-Assembling Peptides and Their Application in the Treatment of Diseases’ Int. J. Mol. Sci. 2019, 20(23): 5850, incorporated herein by reference in its entirety) and include, without limitation, RADA (SEQ ID NO: 1 ) peptides (e.g., peptides with a ‘RADA’ repeat). In non-limiting embodiments, the self-assembling peptide has a sequence of RADARADARADARADA (SEQ ID NO: 2), RARADADARARADADA (SEQ ID NO: 3), and/or RARADADARARADADAGGRPKPQQFFGLM (SEQ ID NO: 4). In non-limiting embodiments, the self-assembling peptide may include a therapeutic composition bound thereto. For example, and without limitation, the self-assembling peptide may be a RADA peptide with substance P bound thereto, for example as shown in SEQ ID NO: 4.

[0089] In non-limiting embodiments, the composition may further include a contrast agent. In non-limiting embodiments, for example where the first ECM component and the second ECM component are gellable following mixing, a contrast agent may be included with the first ECM component and/or the second ECM component. Suitable contrast agents may include radiopaque compositions, may be ionic or non-ionic, may be iodinated or non-iodinated, and/or may be barium-based, gadolinium-based, or may include microbubbles. In non-limiting embodiments, the contrast agent is iohexol. [0090] In non-limiting embodiments, the composition may further include a polymer. In non-limiting embodiments, for example where the first ECM component and the second ECM component are gellable following mixing, a polymer may be included with the first ECM component and/or the second ECM component. In non-limiting embodiments the polymer is a biocompatible polymer. In non-limiting embodiments, the polymer is bioerodible or is not bioerodible. In non-limiting embodiments, the polymer is a natural polymer, including, but not limited to, a polysaccharide, a polypeptide, a nucleic acid-based polymer, a silk, a wool, and/or a cellulose. Those of skill in the art will appreciate that the disclosure is not limited to the foregoing, and may include any natural polymer, including derivatives thereof. In non-limiting embodiments, the polymer is a chitin, a chitosan, and/or an alginate.

[0091] In non-limiting embodiments, the composition includes, when mixed, a plurality of ECM components, such as modified ECM components as described herein, a self-assembling peptide, a natural polymer, one or more therapeutic compositions that promote platelet adhesion and/or cell recruitment, and/or one or more therapeutic compositions that promote or cause fibrosis. In non-limiting embodiments, the composition is at least partially bioerodible and/or bioabsorbable, such that as fibrosis occurs and/or cells are recruited to the target site to which the composition is delivered, the exogenous factors that have been delivered are removed and replaced by the patient’s own cells and/or fibrotic tissue.

[0092] Also provided herein are kits including compositions, such as gellable compositions, described herein. In non-limiting embodiment a kit may include a delivery device and a first ECM component (such as a first modified ECM component) and a second ECM component (such as a second modified ECM component). The ECM components (and various therapeutic compositions, polymers, and/or selfassembling peptides as described herein) may be provided in vials or other containers, and/or within the delivery device. Any suitable delivery device may be included in such a kit. In non-limiting embodiments, the delivery device is one with a plurality of chambers and/or reservoirs, such that the first ECM component and the second ECM component are not in contact with one another during manufacture, shipping, and/or storage. In non-limiting embodiments, a suitable delivery device may include a mixing chamber, such that the first ECM component and the second ECM component, while stored separately, are mixed prior to delivery to a target site. In non-limiting embodiments, a suitable delivery device maintains isolation of the first ECM component from the second ECM component until the compositions have reached the target site. In non-limiting embodiments, the first ECM component and/or the second ECM component are provided in a kit as a lyophilized powder, and, in non-limiting embodiments, with a solvent or diluent for reconstitution. In non-limiting embodiments, the first ECM component and/or the second ECM component are provided in a kit as liquids for injection.

[0093] A non-limiting embodiment of a suitable delivery device is shown in FIG. 1 , where device 100 includes a first reservoir 1 10 and a second reservoir 120, each configured to hold, or holding, a composition including an ECM component as described herein. While the illustrated embodiment resembles a dual-chamber syringe with a side-by-side arrangement or chambers, those of skill will appreciate that syringes with coaxial chambers (allowing for sequential delivery of compositions) and automatic delivery devices, such as pumps, are included within the scope of the present disclosure. In the illustrated embodiment, fluid conduit 140 maintains separation of the composition held in the first reservoir 1 10 and the composition held in the second reservoir 120 until the compositions are delivered to a target site. Suitable devices for guiding the composition to a target site are known and include, without limitation, catheters and like devices configured to enter and pass through a patient’s vasculature. In non-limiting embodiments, a distal end of fluid conduit 140 and/or a catheter may include a radiopaque component, to allow for visualization during delivery of the composition. In non-limiting embodiments, the kit is included in a blister pack, and may be sterilized.

[0094] Also provided herein are methods of using the compositions and/or kits described herein in the delivery of a composition, such as a gellable composition, to a target site within a patient. In non-limiting embodiments, the method includes delivering a first ECM component and a second ECM component as described herein to a target site and allowing the first ECM component and the second ECM component to come into contact, thus forming a gel. Compositions, kits, and methods described herein are suitable for treatment of defects and/or abnormalities, for example a defect and/or abnormality associated with the vasculature of a patient. In non-limiting embodiments, the defect and/or abnormality is an aneurysm. In non-limiting embodiments, the defect and/or abnormality is a cerebral aneurysm. In non-limiting embodiments, the defect and/or abnormality is arteriovenous malformation (AVM). In non-limiting embodiments, the composition (e.g., a first ECM component, which may be modified, and a second ECM component, which may be modified), are delivered into the aneurysm sac, where a gel is formed. In non-limiting embodiments, a gel is formed from the composition at a sufficient rate such that leakage of the composition out of the aneurysm sac is reduced and/or eliminated as compared to other embolic treatments.

Example

MATERIALS & METHODS

[0095] 2.1 . Materials

[0096] Gelatin (type A, from porcine skin), 1 ,4-dithiothreitol (DTT), hydrazine hydrate solution (78-82%, iodometric), N-(3-(dimethylamino)propyl)-N^!- ethylcarbodiimide hydrochloride (EDC, 98%), and alginic acid sodium salt from Macrocystis pyrifera (kelp) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 0.2 M calcium chloride (CaCI₂) was purchased from Hemonetics (Clinton, PA, USA). Sodium hyaluronate was purchased from Fisher Scientific (Hampton, NH, USA). Divinyl sulfone (stabilized with HQ) was purchased from TCI Chemicals (Tokyo, Japan). Dimethyl 3,3-dithiodipropionate was purchased from Ambeed, Inc. (Arlington Heights, IL, USA). Peptides RADA16 (Ac-RARADADARARADADA-NH₂) (SEQ ID NO: 3) and RADA-SP (Ac-RARADADARARADADAGGRPKPQQFFGLM-NH₂) (SEQ ID NO: 4) were purchased from Peptron (Daejeon, South Korea).

[0097] 2.2. Synthesis and Characterization of Thiolated Gelatin (Gel-SH) and Vinyl

Sulfonated Hyaluronic Acid (HA-VS)

[0098] Thiolated gelatin (Gel-SH) and vinyl sulfonated hyaluronic acid (HA-VS) were prepared using the methodology described previously. Briefly, Gel-SH was synthesized from gelatin and 3,3'-dithiobis(propionic hydrazide) (DTPH) which is a reaction product of 3,3'-dithiopropionic acid dimethyl ester with hydrazine hydrate. A solution of 1.0 g of gelatin in 100 mL of distilled water was prepared, and 0.5 g of DTPH was added while stirring. The pH of the solution was adjusted to 4.75 by adding 1 M HCI, followed by the addition of 0.3 g of EDC. After 2 h, the reaction was stopped by adding 1 M NaOH solution to raise the pH to 7.0, then, 2.2 g of DTT was slowly added to the reactor, and the pH was increased to 8.5 using 1 M NaOH. The reaction proceeded for 24 h at room temperature. The pH was adjusted to 3.5 using 1 M HCI, and the acidified product was purified using dialysis membrane tubing (MWCO 12000) in 0.03 M HCL The resulting fine powder was obtained through lyophilization.

[0099] Hyaluronic acid (HA) and divinyl sulfone (DVS) were used to synthesize HA- VS. A solution containing 0.5 g of HA in 100 mL of 0.02 M NaOH was prepared. Then 1 .3215 g of DVS was added to the reactor in an ice bath. The reaction was allowed to proceed for 30 min and then terminated by adjusting the pH to 5.0 using 1 M HCI solution. The product was purified using dialysis membrane tubing (MWCO 12,000) in distilled water at pH 5.3. The fine powder was obtained after lyophilization. The synthesized Gel-SH and HA-VS chemical structures were confirmed by 1 H-nuclear magnetic resonance (1 H NMR).

[00100] Viscosity and elasticity of 2% alginic acid, 1 % HA HA-VS, 2% Gel-SH, 2% Gel-SH + 20% iohexol, and 2% Gel-SH + 20% iohexol in 20 mM HEPES were evaluated at 37 ^QC using a Vilastic-3 viscoelasticity analyzer (Vilastic Scientific, Austin, TX, USA). For the evaluation, shear rate sweep, measurement sequence, measurement frequency, and integration time were preset as 5 to 80 (1/s), 50 increasing shear rate, 2.0 Hz, and 5 s, respectively.

[00101] For the inverted vial gelation test, solutions of HA-VS and Gel-SH were prepared in 20 mM HEPES at concentrations of 1 % (w/v) and 2% (w/v), respectively. A 0.5 mL HA-VS solution and a 0.5 mL Gel-SH solution were preheated at 37 °C and injected into a 3.7 mL glass vial using a 25 G needle. The vial containing the mixture was then incubated at 37 °C for 30 s, and the sol-gel phase transition was evaluated using the vial inverting method. The internal structure of the formed gel was examined by cross-sectional images of lyophilized scaffolds using a scanning electron microscope (SEM, JSM 6335F, JEOL, Tokyo, Japan).

[00102] A rheometer (AR2000EX, TA Instruments, New Castle, DE, USA) was used for evaluation of the rheological change at 37 °C during the gelation. A 0.5 mL portion of 1 % (w/v) HA-VS solution and 0.5 mL of 2% (w/v) Gel-SH solution were injected into the center of the Peltier plate at the same time using separated nozzles. The shear storage modulus (G') and loss modulus (G") were measured at a strain of 5%.

[00103] The swelling ratio and degradation profiles of HA-VS/Gel-SH were evaluated in vitro. Solutions of 1 % (w/v) HA-VS and 2% (w/v) Gel-SH were prepared in 20 mM HEPES, and 75 pL of each solution was injected into each well of a 96 well plate and then mixed and kept at 37 °C for 1 h. The HA-VS/Gel-SH gels were freeze- dried, washed with distilled (DI) water, and freeze-dried before use. For the swelling ratio, the mass of dried samples was measured and then immersed in Dulbecco’s phosphate-buffered saline (DPBS) at 37 °C for 3 h. The mass of swollen samples was measured for the calculation of the swelling ratio (S) using the equation , where Ms and Md are the mass of swollen and dried samples, respectively (n = 5).

[00104] The degradation profile was evaluated by a change in mass for 4 weeks. The mass of samples (WO) was measured and then immersed in DPBS for 4 weeks. At time points of 1 , 2, 3, and 4 weeks, the samples were pulled out, washed with DPBS and DI water three times for each, and freeze-dried before checking the mass (Wi). The degradation of samples was evaluated by an equation change of mass (%) = ((Wi - Wo)/Wo) x 100 (n = 5).

[00105] 2.3. HA-VS/Gel-SH Gelation with lohexol

[00106] HA-VS was dissolved at 1 % (w/v), and Gel-SH was dissolved at 2% (w/v) in 20 mM HEPES separately after being sterilized under UV for 10 min. lohexol was then dissolved in the Gel-SH solution with various concentrations at 5%, 10%, or 15% (w/v, mass of iohexol/a total volume of HA-VS and Gel-SH mixture solution). 75 pL of each solution HA-VS and Gel-SH+iohexol were injected into each 96 well at 37 °C and then kept in an incubator for 30 min followed by lyophilization. X-ray images of the resulting freeze-dried hydrogels were captured by using an OEC 9800 Plus X-ray machine. In addition, cross-sectional images of the freeze-dried hydrogels were obtained using SEM. Each sample was immersed in liquid nitrogen and sliced using a surgical-grade blade to expose the cross section. Before SEM imaging, the sliced samples were mounted on a sample holder and coated with a 5 nm layer of Pt. The average diameters of the pores were measured from the SEM images using a free photo editing software program Fiji (https://imagej.net/software/fiji/).

[00107] 2.4. Blood-Contacting Test of HA-VS/Gel-SH with lohexol

[00108] Fresh whole ovine blood was obtained for this study through jugular venipuncture, using a sodium citrate-containing tube. The collection and handling of the blood samples followed the guidelines set by the National Institute of Health (NIH) for the care and use of laboratory animals. All animal procedures conducted as part of this study were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pittsburgh.

[00109] To investigate the gelation behavior of HA-VS/Gel-SH+iohexol in blood, solutions of 1 % HA-VS and 2% Gel-SH+iohexol (10%) were prepared following the procedure described in section 2.3. Subsequently, 75 pL of each solution was injected into 50 pL of fresh ovine blood in individual wells of a 96-well plate, maintained at 37 °C. As a comparison, 0.2 M CaCl2 (20v/v%, relative to the total blood volume) or 2% (w/v) alginic acid in PBS + 0.2 M CaCh (20v/v%, relative to the total blood volume) were injected into 50 pL of fresh blood. After injection, the plate was placed in an incubator for 30 min. The samples were then washed with DPBS five times to remove any unattached blood cells, followed by fixation with 2.5% glutaraldehyde at room temperature for 2 h. The samples were subjected to lyophilization after being washed with distilled water. The internal structure of the freeze-dried hydrogels was examined by using cross-sectional SEM images. The number of entrapped or encapsulated red blood cells was counted from the SEM images.

[00110] To explore the interaction between blood cells and the scaffolds following gelation, lyophilized HA-VS/Gel-SH+iohexol (10% or 15% of the total mixture volume) and 2% alginic acid+0.2 M CaCI2 scaffolds were exposed to fresh ovine blood, and the results were observed by SEM imaging. The lyophilized hydrogels were prepared as described previously and immersed in 5 mL of fresh ovine blood. The bloodcontaining tubes with the samples added were maintained at 37 °C with gentle rocking for 3 h. Following blood contact, the samples were washed with DPBS five times to remove any unattached blood cells. Subsequently, the samples were fixed with 2.5% glutaraldehyde at room temperature for 2 h, followed by washing with distilled water. The samples were then lyophilized and sectioned by using a surgical blade in liquid nitrogen to examine their surface and cross-sectional morphologies through SEM imaging. The number of entrapped or encapsulated red blood cells was counted from the SEM images.

[00111] 2.5. rSMC Penetration and Proliferation in HA-VS/Gel-SH with lohexol

[00112] The penetration and proliferation of rat aortic smooth muscle cells (rSMCs) within the HA-VS/Gel-SH+iohexol hydrogel were assessed by the in vitro seeding of rSMCs followed by DAPI imaging. Lyophilized HA-VS/Gel-SH+iohexol (10% or 15% of the total mixture volume) and 2% alginic acid+0.2 M CaCh scaffolds were prepared following the aforementioned method. The samples were sterilized under UV light for 10 min and placed in separate wells of a 96-well plate. rSMCs were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (HI FBS) and 1 % penicillin/streptomycin at 37 'C and 5% CO2. A volume of 150 pL of rSMCs at a concentration of 5 x 106 cells/mL was added to each well containing the sample. The culture medium was refreshed daily. After 1 and 7 days, the samples were transferred to fresh wells and washed with DPBS. Following fixation with 2.5% glutaraldehyde, the samples were embedded in an optimal cutting temperature (OCT) compound and then sectioned using a microtome-cryostat (CryoStar NX50, Thermo Scientific). The mounted slices were washed with PBS and stained with 4',6-diamidino-2-phenylindole (DAPI). The images were captured using a Zeiss fluorescence microscope.

[00113] 2.6. In Vitro Cell Response to HA-VS/Gel-SH with lohexol and RADA-SP [00114] The gelation of HA-VS/Gel-SH with RADA-SP was tested in vitro, and their morphology was observed by SEM imaging. To prepare the gel incorporating RADA- SP, the following steps were taken: first, HA-VS was dissolved at a concentration of 1 % (w/v) and Gel-SH was dissolved at a concentration of 2% (w/v) in 1 mL of 20 mM HEPES solution. Both solutions were sterilized under UV for 10 min. Then, iohexol was added to the Gel-SH solution at a concentration of 20% (w/v), and 2 mg of either RADA or RADA-SP was dissolved in 1 mL of the Gel-SH+iohexol solution. Next, 75 pL of each solution (HA-VS and Gel-SH+iohexol+RADA or RADA-SP) were separately injected into individual 96-well plates at a temperature of 37 ^QC. The plates were incubated for 30 min in an incubator and subsequently subjected to lyophilization. Finally, the cross-sectional morphologies of the resulting gels were observed by using SEM imaging. The average diameters of the pores were measured from the SEM images using Fiji.

[00115] The cell responses to the RADA-SP-incorporated injectable embolic were evaluated and compared with those of RADA and RADA-SP free embolic and RADA- incorporated embolic in vitro. For the evaluation of the effect of RADA-SP, rat vascular endothelial cells (rECs, from aortic tissue) or rSMCs (150 pL of 1 x 107 cells/mL) were seeded on the lyophilized gels in each well of a 96-well plate and incubated for 7 d. The cell medium was changed daily. After incubation, samples were washed in PBS and fixed in 2% paraformaldehyde for 1 h at room temperature, followed by permeabilization and blocking in PBS containing 0.2% Triton X-100, 3% bovine serum albumin, and 10% goat serum for 2 h at room temperature. After blocking, samples were incubated with primary antibody (ab6994, rabbit polyclonal to Von Willebrand Factor, Abeam, Cambridge, UK, 1 :300) or (ab5694, rabbit polyclonal to alpha-smooth muscle actin, Abeam, 1 :100) overnight at 4 °C, then incubated with the solution containing secondary antibody (A21442, Alexa Fluor 594 chicken antirabbit IgG H&L, Invitrogen, Eugene, Oregon, USA, 1 :500) or (A1 1034, Alexa Fluor 488 goat antirabbit IgG H&L, Invitrogen, 1 :250) and DAPI for 2 h at room temperature. Primary and secondary antibodies were diluted in PBS containing 5% goat serum. Stained samples were kept in PBS until imaging. Fluorescent images were acquired using either a Zeiss fluorescence microscope (for VWF) or a Nikon A1 confocal laser scanning microscope (for aSMA). Image analysis was performed using either Zen lite (version 2.3) or Fiji.

[00116] 2.7. In Vivo Studies Using a Mouse Aneurysm Model

[00117] All animal experimentation conducted in this study adhered to a protocol approved by the IACUC at the University of Pittsburgh, in accordance with the guidelines set by the NIH. Given that cerebral aneurysm formation and rupture affect women at a 60% higher rate than men, we used C57BL/6 female mice (Charles River) to establish murine carotid aneurysm. Briefly, mice were anesthetized with an isoflurane and oxygen gas mixture, and then microsurgical exposure of the right common carotid artery (RCCA) was performed under sterile conditions using an operating microscope. The RCCA was exposed, and then a latex cuff was placed around the vessel. The RCCA was bathed with porcine pancreatic elastase solution (10 U/1 mL of PBS) for 20 min, and the vessel was occluded distally. A saccular aneurysm formed over the next 3 weeks. For control purposes, sham-operated animals underwent microsurgical exposure to the RCCA, which was then bathed with phosphate-buffered saline rather than elastase solution and no distal occlusion of the artery. A second control group underwent the same procedure but received elastase but no follow-up treatment with an embolic.

[00118] Two different components HA-VS/Gel-SH with 10% (w/v) iohexol and HA- VS/Gel-SH with 10% iohexol and 2 mg of RADA-SP were tested in vivo using the murine model (n = 7 for each group). Here, a temporary aneurysm clip with a relatively gentle closing force of 69 gf (0.68N; MIZUHO Sugita Titanium temporary miniclip II) was placed at the neck of the aneurysm to prevent retrograde embolization. A 30G needle was then used to inject ~20 pL of the embolic agent into the aneurysm. The gel mixture was allowed to cross-link for 5 min. The temporary clip was then removed, and the aneurysm was allowed to heal for 3 weeks, after which the carotid arteries that had been embolized or underwent a sham procedure were collected for proinflammatory cytokine assay, histologic evaluation, and immunofluorescence analysis.

[00119] 2.8. Proinflammatory Cytokine Assay [00120] For proinflammatory cytokine expression, to extract proteins from these arteries, a Cell Lysis Buffer from RayBiotech was utilized for their lysis. Subsequently, the isolated protein was applied onto standard mouse cytokine antibody array membranes (RayBiotech’s Mouse Cytokine Array C1000), which enabled the analysis of 96 different cytokines. The membranes underwent steps involving incubating them with the protein sample, removing any unbound proteins through washing and treating them with a detection reagent. The emitted signals resulting from the bound cytokines were visualized by capturing film images. To assess the intensity of these signals, densitometry analysis was performed using Imaged. Morpheus, software for matrix visualization and analysis (https://software.broadinstitute.org/morpheus) was employed to visualize the data. Additionally, data clustering was carried out by applying the hierarchical clustering algorithm within Morpheus, using the Spearman rank correlation similarity metric (n = 3 animals in each group).

[00121] 2.9. Histologic Evaluation

[00122] Aneurysm tissues treated with the injectable embolic were collected and preserved in a 30% sucrose solution at 4 °C for 3 days before use. The samples were embedded in O.C.T. compound and cut into 5 pm serial sections. Subsequently, the slides were subjected to hematoxylin and eosin (H&E), trichrome, and picrosirius red staining according to standard procedures to visualize general morphology, connective tissue formation, and cellular infiltration. The images were taken by a slide scanner (MoticEasyScan Pro 6, Motic, Kowloon City, Kowloon, Hong Kong) and processed with Aperio ImageScope (Leica Biosystems, Wetzlar, Germany).

[00123] 2.10. Immunofluorescence Analysis

[00124] For evaluation of cell infiltration, tissue sections were stained with markers for CD68 (macrophages), FSP-1 (fibroblast), MECA32 (endothelial cell), aSMA (smooth muscle cell), CD80 (M1 -macrophage), and CD206 (M2-macrophage). The samples were double stained with CD68 (ab53444, rat antimouse recombinant antibody, Abeam, 1 :100) and FSP-1 (ab197896, rabbit antimouse recombinant antibody, Abeam, 1 :125) and also MECA32 (ab27853, rat antimouse recombinant antibody, Abeam, 1 :500) and aSMC (ab5694, rabbit antimouse recombinant antibody, Abeam, 1 :100). Also, samples were single stained with CD80 (ab254579, rabbit antimouse recombinant antibody, Abeam, 1 :50) and CD206 (ab300621 , rabbit antimouse antibody, Abeam, 1 :50). Alexa Fluor 488 donkey antimouse IgG H&L (A1 1006, Invitrogen, 1 :500), Alexa Fluor 594 donkey antirabbit IgG H&L (A21442, Invitrogen, 1 :500), Alexa Fluor 647 donkey antirat IgG H&L (A78947, Invitrogen, 1 :500), and Alexa Fluor 647 donkey antirabbit IgG H&L (A31573, Invitrogen, 1 :500) were used for secondary antibodies.

[00125] Antisubstance P antibody (NC1/34HL, sc-21715, rat monoclonal IgG Substance P antibody, Santa Cruz Biotechnology, Dallas, TX, 1 :125) was used for staining the tissue sections to estimate remaining RADA-SP in the treated aneurysm. Alexa Fluor 647 donkey antirat IgG H&L (A78947, Invitrogen, 1 :125) was used as a secondary antibody. DAPI Fluoromount-G (0100-20, SouthernBiotech, Birmingham, AL, USA) was used for DAPI staining and mounting. Image analysis was performed using Fiji (n = 4 animals in each group).

[00126] 2.1 1. Statistical Analysis

[00127] The n-value refers to the number of replicates for each test. Data are presented as mean ± standard deviation (SD). One-way ANOVA along with Tukey’s test was performed, and p < 0.05 was considered statistically significant.

RESULTS

[00128] 3.1. Synthesis and Characterization of HA-VS and Gel-SH and their Gelation with lohexol

[00129] HA-VS and Gel-SH were successfully synthesized with the expectation of rapid gelation by the Michael Addition reaction when they were mixed at physiological temperature (37 ^QC) and pH (~7.4) (FIG. 2A). The chemical structure of synthesized HA-VS and Gel-SH were confirmed by 1 H NMR spectra, which showed new methylene sulfone (-CH2CH2SO2) peaks at 6.25, 6.35, and 6.80 for HA-VS and new side chain methylene (-CH2CH2SH) peaks at 2.54 and 2.68 ppm for Gel-SH (FIG. 2B). The conversion rate of the repeat unit of HA to HA-VS is 106.5% and that of Gel-SH is 48.1 %; therefore, the substitution degree of VS and SH is 106.5% and 48.1 %, respectively. The ratio between VS and SH was 1 :0.45.

[00130] Viscosity and elasticity of 2% alginic acid, 1 % HA-VS, 2% Gel-SH, 2% Gel- SH + 10% iohexol, and 2% Gel-SH + 10% iohexol + 2 mg of RADA-SP were evaluated. Their average viscosity with shear rate ranged from 5 to 80 (1 /s) were 0.0414 ± 0.0006, 0.033 ± 0.007, 0.0004 ± 0.0002, 0.0024 ± 0.0002, and 0.0028 ± 0.0001 Pa-s, respectively (FIGS. 9A-9B). These viscosities are close to a typical blood viscosity 0.03 Pa-s or lower; therefore, all the prepared solutions can be injected through 5-Fr or smaller catheters. The mixture of 1 % HA-VS and 2% Gel-SH became a chemically cross-linked hydrogel at 37 °C and pH 7.4 (FIG. 2C), and its lyophilized scaffold showed a porous structure observed by SEM (FIG. 2C).

[00131] The results of the rheological change and reaction time of the mixture of 1 % HA-VS and 2% Gel-SH showed a fast storage modulus increase for 5 min to 88 ± 1 Pa from 49 ± 1 Pa, and then reached 100 Pa within 10 min. The initial storage modulus may be influenced by the premixing of 1 % HA-VS and 2% Gel-SH solutions on the Peltier plate before measurement.

[00132] The swelling ratio of HA-VS/Gel-SH hydrogel is 271 ± 35%. The mass of HA-VS/Gel-SH scaffolds decreased -24 ± 13% at 1 week and reached -37 ± 16% at 4 weeks (Supplementary Figure 1 D).

[00133] In consideration of the future potential for angiography-guided transcatheter injection, the FDA-approved water-soluble contrast agent iohexol (Omnipaque) was dissolved in a 2% Gel-SH solution at concentrations of 5%, 10%, or 15% (w/v, mass of iohexol/a total volume of HA-VS and Gel-SH mixture solution). Following the lyophilization process, the resulting chemically cross-linked scaffolds containing iohexol were examined using X-ray and SEM imaging (FIG. 10). It was observed that all scaffolds with different concentrations of iohexol exhibited radiopacity, with higher concentrations of iohexol yielding greater radiopacity. The SEM images showed that all scaffolds, irrespective of iohexol concentration, displayed porous cross sections. The average pore size (diameter, pm) measured for the iohexol concentration of 5, 10, and 15% was 43 ± 14, 49 ± 12, and 47 ± 1 1 , respectively. However, the scaffolds incorporating 10% and 15% iohexol were deemed better than scaffolds with 5% iohexol considering clear visualization under an X-ray machine.

[00134] 3.2. In vitro blood-contacting of HA-VS/Gel-SH with iohexol

[00135] SEM images of the cross sections of gels injected into ovine blood are depicted in FIG. 3. The HA-VS/Gel-SH gels revealed evident entrapment and encapsulation of blood cells (including red blood cells and platelets) on the porous walls of the gel. In contrast, SEM images of gels formed using 0.2 M CaCI2 and 2% alginic acid + 0.2 M CaCh displayed a less obvious blood cell entrapment in the cross section compared to HA-VS/Gel-SH gels. The number of encapsulated red blood cells was 0, 8 ± 3, 100, and 63 ± 10% for 20% 0.2 M CaCl2, 2% alginic acid + 20% 0.2 M CaCh, HA-VS/Gel-SH gel with 10% (w/v) iohexol, and HA-VS/Gel-SH gel with 15% (w/v) iohexol, respectively, when normalized to 20% 0.2 M CaCI2 as 0% and HA- VS/Gel-SH gel with 10% (w/v) iohexol as 100%. Overall, the HA-VS/Gel-SH gel incorporating 10% iohexol exhibited the most pronounced attachment of blood cells in the cross section. Distinguishing the formed fibrin from the scaffold was not possible from the images.

[00136] From the rocking test with fresh ovine blood, it becomes evident that blood cells were deposited on the surface and penetrated the inner pores of the lyophilized scaffolds (which were formed by the chemically cross-linked HA-VS/Gel-SH+iohexol gels) (FIG. 4). The lyophilized scaffold allowed the penetration of blood cells and supported the formation of blood clots within the scaffold. The number of encapsulated red blood cells was 100, 79 ± 6, 67 ± 8, and 72 ± 5% for surface and cross section of HA-VS/Gel-SH gel with 10% (w/v) iohexol, and surface and cross section of HA- VS/Gel-SH gel with 15% (w/v) iohexol, respectively, when normalized to the surface of HA-VS/Gel-SH gel with 10% (w/v) iohexol as 100%. On the other hand, the scaffolds created using 2% alginic acid and 0.2 M CaCl2 underwent the same procedure of being in contact with excess blood with gentle rocking for 3 h. However, these scaffolds were fragile and dispersed in the blood, leading to an inability to collect SEM images depicting the blood-contacted 2% alginic acid + 0.2 M CaCl2 scaffolds.

[00137] 3.3. In vitro cell response to the HA-VS/Gel-SH

[00138] The infiltration and proliferation of cells within the HA-VS/Gel-SH+iohexol gels were assessed for 7 days using rSMCs. The number of cells in the cross section of the gels was estimated by fluorescent images with DAPI staining and the intensity of the DAPI (FIG. 4). It was observed that the HA-VS/Gel-SH gel containing 10% iohexol exhibited a significantly higher DAPI intensity on both day 1 and day 7 compared to the HA-VS/Gel-SH gel with 15% iohexol. Furthermore, the DAPI intensity on day 7 was significantly greater than on day 1 for both the 10% and 15% iohexol- containing HA-VS/Gel-SH gels. 2% alginic acid + 20% 0.2 M CaCh were tested as well, but data could not be collected because of degradation and the fragility of fragments.

[00139] The HA-VS/Gel-SH+iohexol gels containing RADA or RADA-SP exhibited porous cross-sectional images (FIG. 4). The average pore size (diameter, pm) measured for the peptide-free, with RADA, or with RADA-SP was 43 ± 14, 35 ± 9, and 29 ± 7, respectively. Among the different types of prepared scaffolds (peptide-free, with RADA, or with RADA-SP) seeded with rECs or rSMCs, the gel incorporating RADA-SP demonstrated significantly higher expression of both von Willebrand factor (vWF) and alpha-smooth muscle actin (aSMA) compared to the peptide-free and with RADA gels. In the case of rEC seeding, the DAPI intensity in the cross section of the RADA-SP added gel was significantly lower, but the ratio of vWF/DAPI was significantly higher compared to other gel compositions (FIGS., 13A-13D). When rSMCs were seeded, the gel incorporating RADA-SP displayed significantly higher DAPI intensity, aSMA expression, and aSMA/DAPI ratio compared to the other gel compositions (FIGS. 7A-7C).

[00140] 3.4. In vivo injection of HA-VS/Gel-SH with iohexol and RADA-SP using murine carotid aneurysm model

[00141] The murine carotid aneurysm model was used for 3 weeks of in vivo assessment of the injectable embolics (FIGS. 5A-5C). From the proinflammatory cytokine array (FIG. 5D), there were no significant differences between the two embolic groups in the expression of major cytokines IL-1 [3, IL-6, IL-17A, TNF-a, MMP- 2, and MMP-3, which are mediators implicated in cerebral aneurysm formation and rupture.

[00142] Histologic evaluation of H&E, Trichrome, and PicroSirrus-Red showed successful embolization of the aneurysms by the injectable embolic (FIG. 6). All of the ECM-mimicking injectable embolic with and without RADA-SP embolized the sac of the aneurysm and allowed cell infiltration.

[00143] To evaluate the infiltration of cells (macrophages, fibroblasts, ECs, SMC, and M1 and M2 macrophages), immunofluorescence staining and imaging were executed with CD68, FSP-1 , MECA-32, aSMA, CD80, and CD206 (FIGS. 7A-8B). The quantification by fluorescence intensity/pm2 at the inside of the aneurysm sac showed that HA-VS/Gel-SH with 10% iohexol showed a significantly higher intensity of FSP-1 (15 ± 6) to the embolic with RADA-SP (2.3 ± 1 .8). On the other hand, HA-VS/Gel-SH with 10% iohexol and RADA-SP showed a significantly higher intensity of aSMA (32 ± 7) compared to the embolic without RADA-SP (10 ± 4). Both ECM-mimicking embolics showed a higher intensity of M1 macrophage CD80 rather than CD206 in 3 weeks.

[00144] The delivered RADA-SP was released within 3 weeks after the injection, and there was no remaining SP in the tissue.

DISCUSSION

[00145] Desirable properties for aneurysm embolization materials would include the promotion of clot formation to effectively exclude the aneurysm sac from the parental artery after embolization and subsequently support the growth of new tissue. Ultimately, the material should be remodeled by the infiltrating cells, allowing for permanent embolization without the presence of a foreign body. This would provide an optimal solution for treating intracranial aneurysms while minimizing potential complications and allowing for easier retreatment, if necessary.

[00146] HA and collagen are abundant ECM components broadly found in various tissues. Their cytocompatibility, support for cell adhesion, physical properties, and amenability to purification and modification have made derivatives of HA and collagen common building blocks for biomaterial design in the fields of tissue engineering and drug delivery systems. The use of HA and collagen derivatives (e.g., gelatin) in tissue engineering has shown promising results, as they can mimic critical aspects of the natural ECM and provide structural support. Moreover, their biodegradability ensures that these materials are gradually broken down in the body over time in native remodeling processes without leaving behind permanent foreign substances. Their versatility allows for various modifications and functionalization to tailor their properties for specific applications, such as controlled drug delivery systems.

[00147] In this report, we applied a chemically cross-linked hydrogel by synthesizing vinyl sulfonated hyaluronic acid and thiolated gelatin (collagen hydrolyzate functionalized with thiol groups). These components form a hydrogel through the Michael Addition reaction when mixed under physiological conditions. The HA-VS and Gel-SH can be individually delivered to an intracranial saccular aneurysm through two separate channels using a microcatheter connected to a dual-barrel syringe. The delivered two solutions would mix in the sac of the aneurysm, forming a cross-linked ECM-derived matrix while initiating and propagating local thrombus formation. Following in situ gelation, blood can infiltrate the porous embolic material and adhere to its surfaces. This phenomenon was verified through in vitro blood contacting tests conducted using the cross-linked scaffolds. For the test, we chose the alginic acid-based injectable embolic (2% alginic acid +0.2 M CaCte) as a control since the alginic acid is a reasonably close macromolecule to gelatin and hyaluronic acid, as well as that alginic acid-based injectable gel is an FDA-approved embolic and commercialized as Embogel. During 3 h of blood contact, lyophilized hydrogels consisting of 2% alginic acid + 0.2 M CaCh were observed to degrade in the blood. In contrast, the lyophilized HA-VS/Gel-SH + iohexol gels retained their shape while clot formation occurred. This different behavior between the 2% alginic acid + 0.2 M CaCl2 hydrogels and the HA-VS/Gel-SH+iohexol hydrogels can be attributed to differences in the cross-linking mechanism (ionic complexation for alginic acid with calcium ions versus chemical cross-linking for HA-VS/Gel-SH) and the extent of clot formation.

[00148] Based on the in vitro data regarding the penetration and proliferation of rSMCs, it was observed that HA-VS/Gel-SH with 10% iohexol had a higher number of cells within the cross section on both days 1 and 7, compared to HA-VS/Gel-SH with 15% iohexol. Iohexol exhibits only low systemic toxicity due to its low chemotoxicity and osmolality. However, a higher iohexol concentration might directly impact cell infiltration and proliferation within the hydrogel by changing the surface chemistry. HA- VS/Gel-SH with 10% iohexol was chosen for the next stage of the experiments.

[00149] In endovascular transcatheter embolization, both controlled localized thrombus formation and tissue ingrowth are key factors for successful aneurysm occlusion and healing. In addition to the thrombotic and cell support activity of gelatin, RADA-SP was loaded into the ECM-mimicking injectable embolic with the potential for stimulating improved healing. RADA-SP is a self-assembled peptide conjugated with SP. SP is a neuropeptide released from the peripheral terminals of sensory nerve fibers, and SP functions as both a neurotransmitter and a hormone. Studies have reported that RADA-SP has the ability to enhance the recruitment of endogenous cells into implanted scaffolds and expedite the process of wound healing. (22) While the effects of RADA-SP on wound healing, including ischemia, bone tissue engineering, skin regeneration, and vascular regeneration, have been extensively studied and established, its impact on aneurysm healing has not been investigated to our knowledge.

[00150] Self-assembling peptides, such as RADA, possess the ability to form stable [3-sheet structures and undergo self-assembly into nanofibers (ranging from 5 to 10 nm) through van der Waals and ionic interactions. RADA-SP has previously exhibited a sustained effect in contrast to free SP by impeding the rapid release of water-soluble SP from the injection site and protecting it from enzymatic digestion and deactivation. In an example utilizing a mouse hind limb ischemia model, self-assembled RADA-SP maintained a higher concentration of SP at the target site compared to free SP for 28 days. Resulting in an accelerated wound healing process, attributed to enhanced recruitment of mesenchymal stem cells into the ischemic region. The sustained release of SP is thus believed to play a vital role in promoting the continuous recruitment of cells and creating a favorable microenvironment for healing. In this regard, it was hypothesized that delivering RADA-SP would aid in healing by facilitating cell recruitment and tissue in-growth into the target aneurysm sac.

[00151] For the ECM-mimicking injectable embolic incorporating RADA-SP, given the intricate nature of the physical (e.g., self-assembly of RADA-SP) and chemical (e.g., Michael Addition of HA-VS with Gel-SH) gelation mechanisms involved, it was considered whether HA-VS/Gel-SH with 10% iohexol, in combination with RADA or RADA-SP, displayed a porous interconnected morphology that facilitates cell penetration and metabolite transport. The results demonstrated that the prepared gels containing RADA or RADA-SP exhibited a porous structure. The inclusion of RADA- SP likely introduced variations in surface chemistry, pore size, and structure, which may account for patterns of different cell infiltration into the ECM-mimicking gel. Furthermore, the gel system incorporating RADA-SP demonstrated notably elevated expression of VWF and aSMA in comparison to other formulations confirmed in vitro. Consequently, the HA-VS/Gel-SH gel with RADA-SP exhibited the potential ability to recruit vSMCs and stimulate angiogenesis. This activity may hold potential for enhancing the healing process of an embolized aneurysm.

[00152] IL-1 [3, TNF-a, and MMPs are mediators that play significant roles in the formation and rupture of cerebral aneurysms. IL-1 p is associated with endothelial dysfunction, while TNF-a is involved in phenotypic modulation and loss of SMCs. MMPs contribute to phenotypic modulation and SMC loss, macrophage activity, M1/M2 imbalance, leukocyte infiltration, vascular remodeling, and cell death. The expression levels of these mediators were evaluated using a cytokine expression array 3 weeks after implantation, and no significant differences were observed on both of the tested compositions of the injectable embolic. Based on the observation, it appears that integrating RADA-SP into the injectable embolic does not adversely impact cytokine expression associated with aneurysm development and rupture; however, a beneficial effect is also not apparent.

[00153] Histological analysis by H&E, trichrome, and picrosirius-red staining revealed that both injectable embolic with different components effectively filled the aneurysm sac and stimulated tissue ingrowth within 3 weeks. When RADA-SP was incorporated into the injectable embolic, potential capillary in-growth was observed. This angiogenesis effect was intended as intracranial aneurysm therapy but their effect on recanalization needs to be evaluated in longer-term animal studies. Immunofluorescent staining with biomarkers revealed that the RADA-SP incorporated injectable embolic showed significantly higher SMC infiltration reflected by aSMA. This result is along the lines of the in vitro aSMA expression data where the RADA-SP showed an effect on SMC proliferation. Nevertheless, given the successful embolization involving macrophages and fibroblast infiltration with the ECM-derived injectable embolic lacking RADA-SP, it is plausible that the injectable embolic without RADA-SP could exhibit superior efficacy in saccular aneurysm embolization and subsequent healing. However, it is worth noting that the hydrogel we developed is capable of incorporating other potential pro-healing molecules (e.g., TGF-[3, MCP-1 ) for cerebral saccular aneurysm treatment. Also, the infiltration of SMCs may induce better elasticity of new tissue in the sac than fibroblast infiltration. Both more extended studies temporally and broader investigation of bioactive factor loading would be logical next steps for this effort.

[00154] Several clinical limitations exist with the described approach including: 1 ) the experimental method used for embolic delivery in vivo, 2) the potential for downstream embolization resulting in cerebral ischemia, and 3) the rapid formation of a low-oxygen environment resulting in local aneurysm wall ischemia and rupture. In this work, we used an open approach and a temporary clip to prevent accidental downstream embolization. This was done to minimize unnecessary animal suffering. However, in clinical practice, such embolization would have to be performed via endovascular balloon-assisted methods. The potential for downstream embolization due to leakage around the balloon and causing a stroke cannot be overstated. In fact, in the CAMEO trial, a significant percentage of patients treated with Onyx had an intraprocedural complication resulting in permanent neurologic deficit. Another point of concern is that embolic use could result in local aneurysm wall ischemia and early rupture. Hoh et al. described the presence of endothelial cells and capillaries within both human and murine aneurysms. The function of these capillaries is unknown. However, rapid ischemia within the aneurysm wall could result in cellular apoptosis, a loss of structural integrity, and early rupture.

CONCLUSION

[00155] Thiolated gelatin (Gel-SH) and vinyl sulfonated hyaluronic acid (HA-VS) were successfully synthesized, and their composition was confirmed using ¹H NMR. By the rapid reaction of Gel-SH and HA-VS, upon mixing, ECM-derived, chemically cross-linked porous hydrogels were formed, incorporating the contrast agent iohexol. Furthermore, the bioactive, self-assembling peptide RADA-SP could be incorporated without compromising gel formation and structure. The injectable embolic system demonstrated promising potential for treating cerebral saccular aneurysms. In both in vitro and in vivo (murine) models, the embolic material exhibited rapid gelation, forming an in situ scaffold that enhanced thrombus formation and facilitated tissue ingrowth into the aneurysmal sac. Overall, the ECM-mimicking injectable embolic showed the potential for aneurysm treatment; although, further longer-term (over 3 months) in vivo studies may be necessary for a better understanding of their efficacy.

[00156] The present invention has been described with reference to certain exemplary embodiments, dispersible compositions and uses thereof. However, it will be recognized by those of ordinary skill in the art that various substitutions, modifications or combinations of any of the exemplary embodiments may be made without departing from the spirit and scope of the invention. Thus, the invention is not limited by the description of the exemplary embodiments.

Claims

THE INVENTION CLAIMED IS

1. A method of treating a defect in the vasculature of a patient, comprising delivering to the defect a gellable composition comprising a first modified extracellular matrix (ECM) component and a second modified ECM component.

2. The method of claim 1 , wherein the first modified ECM component and/or the second ECM component is one or more of gelatin, hyaluronic acid, fibronectin (FN), collagen, laminin (LM), vitronectin (VN), thrombospondin (TSP), tenascin (TN), a proteoglycan, and/or a glycosaminoglycan.

3. The method of claim 1 or claim 2, wherein the first modified ECM component is gelatin and the second modified ECM component is hyaluronic acid.

4. The method of claim 1 , wherein the modification comprises one or more of a thiol group, a sulfone group, an azide group, an alkyne group, a succinimide group, and/or a malemide group.

5. The method of claim 4, wherein the sulfone group is a vinyl sulfone group.

6. The method of claim 1 , wherein the first modified ECM component is thiolated gelatin and the second modified ECM component is vinyl sulfonated hyaluronic acid.

7. The method of claim 1 , further comprising a therapeutic composition.

8. The method of claim 7, wherein the therapeutic composition is one or factors that promote coagulation and/or fibrosis.

9. The method of claim 8, wherein the therapeutic composition comprises one or more of a transforming growth factor (TGF), substance P, a platelet- derived growth factor (PDGF), a pro-inflammatory cytokine, and/or a monocyte chemoattractant protein (MCP).

10. The method of claim 1 , wherein the gellable composition further comprises a self-assembling peptide.

11 . The method of claim 10, wherein the self-assembling peptide has the following sequence: RADARADARADARADA (SEQ ID NO: 2).

12. The method of claim 10 or claim 11 , wherein the self-assembling peptide further comprises a therapeutic composition conjugated thereto.

13. The method of claim 12, wherein the therapeutic composition is substance P.

14. The method of claim 1 , further comprising a contrast agent.

15. The method of claim 14, wherein the contrast agent is iohexol.

16. The method of claim 1 , wherein the gellable composition further comprises a polymer.

17. The method of claim 16, wherein the polymer is a natural polymer.

18. The method of claim 1 , wherein the natural polymer is one or more of a polysaccharide, a polypeptide, a nucleic acid-based polymer, silk, wool, and/or cellulose.

19. The method of claim 18, wherein the polysaccharide is chitin, chitosan, and/or alginate.

20. The method of claim 1 , wherein the defect is an aneurysm.

21. The method of claim 20, wherein the aneurysm is a cerebral aneurysm.

22. The method of claim 1 , wherein the first modified ECM component and the second modified ECM component are delivered to the defect from separate reservoirs.

23. The method of claim 1 , wherein the first modified ECM component and the second modified ECM component are delivered with a catheter.

24. A kit comprising: a delivery device comprising a first reservoir including a first modified extracellular matrix (ECM) component and a second reservoir including a second modified ECM component, wherein the first modified ECM component and the second modified ECM component are configured to gel when mixed.

25. The kit of claim 24, wherein the first modified ECM component and/or the second modified ECM component is one or more of gelatin, hyaluronic acid, fibronectin (FN), collagen, laminin (LM), vitronectin (VN), thrombospondins (TSPs) tenascin (TN), a proteoglycan, and/or a glycosaminoglycan.

26. The kit of claim 24 or claim 25, wherein the first modified ECM component is gelatin and the second modified ECM component is hyaluronic acid.

27. The kit of claim 24, wherein the modification comprises one or more of a thiol group, a sulfone group, an azide group, an alkyne group, a succinimide group, and/or a malemide group.

28. The kit of claim 27, wherein the sulfone group is a vinyl sulfone group.

29. The kit of claim 24, wherein the first modified ECM component is thiolated gelatin and the second modified ECM component is vinyl sulfonated hyaluronic acid.

30. The kit of claim 24, wherein the first reservoir and/or the second reservoir further includes a therapeutic composition.

31 . The kit of claim 30, wherein the therapeutic composition is one or factors that promote coagulation and/or fibrosis.

32. The kit of claim 31 , wherein the therapeutic composition comprises one or more of a transforming growth factor (TGF), substance P, a platelet- derived growth factor (PDGF), a pro-inflammatory cytokine, and/or a monocyte chemoattractant protein (MCP).

33. The kit of claim 24, wherein the first reservoir and/or the second reservoir further includes a self-assembling peptide.

34. The kit of claim 33, wherein the self-assembling peptide has the following sequence: RADARADARADARADA (SEQ ID NO: 2).

35. The kit of claim 33 or claim 34, wherein the self-assembling peptide further comprises a therapeutic composition conjugated thereto.

36. The kit of claim 35, wherein the therapeutic composition is substance P.

37. The kit of claim 24, wherein the first reservoir and/or the second reservoir further includes a contrast agent.

38. The kit of claim 37, wherein the contrast agent is iohexol.

39. The kit of claim 24, wherein the first reservoir and/or the second reservoir further includes a polymer.

40. The kit of claim 39, wherein the polymer is a natural polymer.

41 . The kit of claim 24, wherein the natural polymer is one or more of a polysaccharide, a polypeptide, a nucleic acid-based polymer, silk, wool, and/or cellulose.

42. The kit of claim 41 , wherein the polysaccharide is chitin, chitosan, and/or alginate.

43. The kit of claim 24, wherein the delivery device is a syringe.

44. The kit of claim 24, further comprising a catheter.

45. The kit of claim 44, wherein the catheter comprises a radiopaque material.

46. A gellable composition comprising: a first modified ECM component; a second modified ECM component; a polymer; a therapeutic composition; and a self-assembling peptide.

47. The gellable composition of claim 46, wherein the first modified ECM component is a thiolated gelatin, the second modified ECM component is a vinyl sulfonated hyaluronic acid, and the therapeutic composition is substance P.

48. A gellable composition for use in treating a defect in the vasculature of a patient, the composition comprising: a first modified ECM component; a second modified ECM component; a polymer; a therapeutic composition; and a self-assembling peptide.

49. The gellable composition of claim 48, wherein the first modified ECM component is a thiolated gelatin, the second modified ECM component is a vinyl sulfonated hyaluronic acid, and the therapeutic composition is substance P.

50. Use of a gellable composition for treating a defect in the vasculature of a patient, the composition comprising: a first modified ECM component; a second modified ECM component; a polymer; a therapeutic composition; and a self-assembling peptide.

51 . The use of claim 50, wherein the first modified ECM component is a thiolated gelatin, the second modified ECM component is a vinyl sulfonated hyaluronic acid, and the therapeutic composition is substance P.