Hydrogel compositions for vascular associated treatment and methods

Abstract

Hydrogel compositions for vascular associated treatments or promotion of desirable vascular characteristics are disclosed. The hydrogel compositions can be carbohydrate based self-assembled natural bio-polymers, composed of a protein and / or polypeptide component, a polysaccharide component, and an active agent, configured for direct and localized administration to a target tissue, such as a vascular tissue, and configured for gradual, sustained release of the active agent.

Description

[0001] HYDROGEL COMPOSITIONS FOR VASCULAR ASSOCIATED TREATMENT AND METHODS

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims priority under 35 U.S.C. 119(e) to U.S. Provisional Application Nos. 63 / 640,606 filed April 30, 2024; 63 / 682,661 filed August 13, 2024; 63 / 695,175 filed on September 16, 2024; and 63 / 711 ,243 filed October 24, 2024 all of which are hereby incorporated by reference herein in their entirety.

[0004] TECHNICAL FIELD

[0005] Hydrogels used for vascular associated treatments and medical procedures requiring vascular access particularly, for example, hydrogels promoting vascular maturation and healing.

[0006] DISCUSSION OF RELATED ART

[0007] In the United States, over half a million individuals with end-stage renal disease (ESRD) are in need of treatment. Approximate 90% of ESRD patients opt for hemodialysis as the preferred renal replacement therapy. Worldwide, hemodialysis remains a need for around 2 million patients who require a well-functioning vascular access for this procedure. An exemplary and common vascular access procedure involves formation of an arteriovenous fistula (AVF), which is formed by direct anastomosis between a native artery and vein.

[0008] Although formation of AVFs is a common practice for vascular access, and the favored method for dialysis access, such procedures are susceptible to complications. Complications with vascular access significantly contribute to the health complications and deaths of those on hemodialysis, incurring costs exceeding a billion dollars annually. For example, a 60% failure rate in AVF maturation has been observed, and is primarily due to insufficient widening of blood vessels (vasodilation), poor outward remodeling, and the rapid development of thickened vein walls (early venous neointimal hyperplasia development). It is estimated that approximately 44% of patients with AVFs ultimately need another procedure to establish new vascular access, due to failure of an AVF. Currently, there are no sufficiently effective treatments to enhance AVF maturation. Further, there are no known reports describing localized application of a hydrogel comprising an active agent, such as a PDE5 inhibitor (e.g., sildenafil citrate; an FDA-approved drug commonly recognized as Viagra), directly to AVFs. Effective, localized treatment would improve outward remodeling of AVF vessels and support the maturation and development of AVFs.

[0009] There are a few periadventitial hydrogel treatments being investigated for AVF applications. One method employs thermo-responsive polymers, specifically a PLGA- PEG-PLGA triblock copolymer, to administer Sirolimus, aiming to prevent the development of intimal hyperplasia. This polymer, however, exhibits weak adhesion needed for secure wrapping, low mechanical stability, and has not shown sustained drug delivery (referenced in J Control Release 2012; 160(3): 459-467). Moreover, its stability is significantly compromised when exposed to any liquid, such as body fluids or blood.

[0010] Another conventional approach involves a nitric oxide (NO) delivery system using a peptide amphiphile-based hydrogel. See Patrick Hwang (Biomaterials 2022; 280: 121254). This approach has been shown to dilate vessels and reduce intimal hyperplasia in the AVF vein. However, NO has stability and delivery challenges and hydrogels show relatively low mechanical stability.

[0011] Additionally, various scaffold applications have been explored to support AVF maturation, but these often trigger foreign body responses and present administration challenges, including the need for specialized surgical skills (as discussed in J Control Release 2012; 161 (1 ): 81-89). While particle-based methods have been attempted, they lack mechanical support and pose integration difficulties with AVF vessels (highlighted in J Am Heart Assoc 2020; 9(24): e018418).

[0012] What is needed is a novel approach to vascular access that enhances maturation, promotes healing and avoids problems associated with insufficient widening of blood vessels and the rapid development of thickened vein walls.

[0013] BRIEF SUMMARY OF EMBODIMENTS

[0014] In accordance with the present invention, various embodiments of the hydrogel compositions for vascular treatment or therapy (hereinafter “hydrogel compositions”), methods of preparation thereof, and methods of use thereof are herein disclosed. Generally, the hydrogel compositions are hydrogels as described below and the methods generally relate to administration of the hydrogel composition directly to a target tissue requiring treatment.

[0015] In some embodiments, the hydrogel compositions are configured for direct and localized administration to a target site or target tissue, such as a bodily tissue. In one embodiment, the bodily tissue is a vascular tissue needing treatment or conditioning / maturation. In another embodiment, the target tissue is a diseased or damaged vascular tissue. In one embodiment, the vascular tissue is an arteriovenous fistula (AVF). In yet another embodiment, the hydrogel is configured to be administered by injection.

[0016] In some embodiments, the hydrogel compositions can be composed of biomolecules, including but not limited to 1) a protein, polypeptide or molecule containing amino acid residues catalyzed by enzymes component, and 2) a polysaccharide based component composed of a polysaccharide molecule, in some embodiments, crosslinked by divalent ions. In some embodiments, the content of protein or polypeptide based component is the dry weight of hydrogel 1 %-99% of the total mass, and the content of the polysaccharide based component is 1 %-99% of the total dry weight of the hydrogel.

[0017] In one embodiment, the protein or polypeptide based component can be one or more of gelatin, collagen, silk, fibroin, zein, elastin, fibrinogen, glycoprotein, and laminin fibronectin. In another embodiment, the protein or polypeptide based component can be gelatin. The protein or polypeptide based component can be a protein or polypeptide or a molecule containing amino acid residues that is catalyzed by an enzyme through peptide bond cross-linking or through Schiff base (Schiff base) crosslinking or phenolic hydroxyl oxidation cross-linking or phosphate groups of coenzyme A. It is formed by binding and cross-linking with serine residues of proteins, or is physically cross-linked by self-assembly caused by changes in the hydrophilicity of molecules caused by proteins or polypeptides through enzyme-catalyzed reactions. The catalytic enzymes can be transglutaminase, transglutaminase isozyme, lysyl oxidase, tyrosinase, peroxidase, horseradish peroxidase, thermolysin, phosphatase, [3- lactamase, plasma amine oxidase.

[0018] In some embodiments, the polysaccharide based component can be guar gum, alginate, gellan gum, and the like. In one embodiment, the polysaccharide component is be guar gum.

[0019] In some embodiments, the hydrogel compositions can contain additives that are one or more mixtures of enzymes, cell growth factors, bone morphogenic proteins, vitamins, insulin, and dexamethasone.

[0020] In some embodiments, the hydrogel compositions are carbohydrate based selfassembled natural bio-polymers. In one embodiment, the hydrogel compositions can comprise a gelatin, a guar gum, and an active agent, for example, a PDE5 inhibitor, such as sildenafil citrate. In alternative embodiments, when a PDE5 inhibitor is used as the active agent, the PDE5 inhibitor can be selected from compounds including, but not limited to vardenafil, tadalafil, and avanafil. PDE3 inhibitors can also be used as active agents, including but not limited to cliostazol and inamrinone (aka amrinone). In other embodiments, active agents can be vasodilator compounds, such as, for example, nitrates (e.g., isorbide dinitrite), minoxidil, hydralazine; calcium channel blockers, such as amlodipine and flodipine; and ACE inhibitors, such as captopril. Anti-proliferative agents, such as sirolimus, everolimus, and paclitaxel; anti-platelet agents, such as aspirin and clopidogrel; and statins, such as simvastatin and atrovastatin may also be used.

[0021] The polysaccharide component (e.g., guar gum) used in the composition can be oxidized in some embodiments. Oxidation can be performed by sodium periodate oxidation through Schiff base formation in some embodiments. In one embodiment, the sodium periodate is metaperiodate. In yet another embodiment, sodium periodate can be added to a concentration of about 1 mg / ml, 2 mg / ml, or 3 mg / ml.

[0022] In some embodiments, the hydrogel compositions are crosslinked with a crosslinking agent. In one embodiment, the compositions are crosslinked with a natural crosslinking agent, such as an enzyme. In one embodiment, the crosslinking agent is microbial transglutaminase. In other embodiments, the hydrogel compositions can be configured to gradually release a therapeutic amount of the active agent (e.g., PDE5 inhibitor) at a desired release rate over a sustained period of time, for example, at least one month or at least two months.

[0023] In some embodiments, the concentration of the active agent (e.g., sildenafil citrate) is between about .5 and 3.5 mg / ml. In other embodiments, the concentration is about 1.5 mg / ml.

[0024] In another embodiment, the hydrogel compositions are configured for sustained release of the active agent. In one embodiment, the release rate of the active agent is about 70% over the course of 63 days.

[0025] In some embodiments, the hydrogel compositions can be configured to perform at least one of the following vascular associated therapies or treatments: promoting vascular maturation, promoting vascular healing, promoting blood vessel dilation, promoting vascular outward remodeling, limiting smooth muscle cell proliferation, limiting intimal hyperplasia, reducing inflammation, and / or generally promoting vascular access. In one embodiment, the hydrogel compositions are configured to promote arteriovenous fistula (AVF) maturation, thus improving vascular access during dialysis.

[0026] In yet another embodiment, a method of preparation of the hydrogel compositions is provided. In some embodiments, the preparation method can comprise the following steps including, but not limited to: 1 ) dissolve the polysaccharide component in water to obtain an aqueous solution, dissolve proteins or polypeptides in the aqueous solution; 2) add active agent to obtain a mixed solution; 3) add an enzyme catalyst, mix evenly, and catalyze the cross-linking reaction at 0°C-50°C for 0.5-24 hours, so that the protein or polypeptide can be cross-linked.

[0027] In another embodiment, the method of preparing a hydrogel vascular therapeutic composition can comprise the following steps: dissolving a quantity of a polysaccharide component in solvent to obtain a first aqueous solution; dissolving a quantity of an oxidizing agent in solvent to obtain a second aqueous solution; combining a volume of said second aqueous solution to said first aqueous solution to oxidize said polysaccharide component and obtain a third aqueous solution; dissolving a quantity of an enzyme catalyst in solvent to obtain a fourth aqueous solution; dissolving a quantity of a protein or polypeptide component to obtain a fifth aqueous solution; adding a quantity of an active agent, a volume of said fourth solution, and a volume of said fifth aqueous solution to said third aqueous solution to obtain a sixth solution; and stirring the sixth solution for 5 minutes and incubating the sixth solution at the 37°C for a predetermined time.

[0028] In yet another embodiment, the method of preparation of the hydrogel compositions can comprise the following steps: dissolving gelatin in water at approximately 60°C to obtain a homogeneous aqueous solution; adding an enzyme catalyst, such as microbial transglutaminase (mTG), to the gelatin solution to initiate protein crosslinking; cooling the mixture to approximately 37°C; and adding an oxidized polysaccharide component, such as oxidized guar gum, to the cooled mixture to further facilitate cross-linking and hydrogel network formation.

[0029] In some embodiments of the preparation methods described above, the hydrogel composition may be further cross-linked by divalent ions, for example, calcium or magnesium ions, to obtain a high-strength interpenetrating hydrogel. In another embodiment, an additive can be added to the mixed solution. In some embodiments, the protein component can be one or more of gelatin, collagen, silk fibroin, zein, elastin, and fibronectin; the polypeptide or molecule containing amino acid residues can be a small molecule peptide containing lysine and glutamine; the polysaccharide component can be one or more of guar gum, alginate, and gellan gum; enzymes can be transglutaminase, lysyl oxidase, tyrosine enzymes, peroxidase, horseradish peroxidase, including enzymes of plant, animal or microbial origin; the active agent can be one or more of 1) a PDE5 inhibitor selected from compounds including, but not limited to sildenafil citrate, vardenafil, tadalafil, and avanafil; 2) a PDE3 inhibitor selected from compounds including, but not limited to cliostazol and inamrinone (aka amrinone); 3) a charged vasodilator molecule, such as, for example, nitrates, minoxidil sulfate, and hydralazine hydrochloride; and / or 4) an uncharged drug molecule, such as, for example, iloprost and ambrisentan; and the additive can be one or more mixtures of cell growth factors, bone morphogenic proteins, vitamins, insulin, and dexamethasone. In one embodiment, the hydrogel vascular therapeutic composition can comprise a gelatin, an oxidized guar gum, and a PDE5 inhibitor, such as sildenafil citrate. In another embodiment, the guar gum can be oxidized with a sodium periodate and the crosslinking can be enzymatically catalyzed using microbial transglutaminase.

[0030] In another embodiment, a method for treating vascular tissue is provided. In some embodiments, the method for treating vascular tissues can comprise steps including, but not limited to 1 ) providing a hydrogel vascular therapeutic composition comprising a protein / polypeptide component as described above, a polysaccharide component as described above, an active agent as described above and, in some embodiments, an additive can be added as described above; 2) applying the hydrogel vascular therapeutic composition directly to a vascular tissue with a vascular condition requiring treatment. In some embodiments, the hydrogel vascular therapeutic composition is a carbohydrate based self-assembled natural bio-polymer. In other embodiments, the hydrogel vascular therapeutic composition is configured to release a therapeutic amount of the active agent gradually over a sustained period of time. In one embodiment, the hydrogel vascular therapeutic composition can comprise a gelatin, an oxidized guar gum, and a PDE5 inhibitor, such as sildenafil citrate.

[0031] The hydrogel vascular therapeutic composition of the present invention can be administered or applied by injection; topical applications like gel sheets or wraps, sprays, implants, or direct placement; in situ gelling by, for example, injecting a liquid precursor that gels upon reaching body temperature or pH at the site. Micro-bead hydrogels may also be used.

[0032] Certain aspects of the presently disclosed subject matter having been stated hereinabove, are addressed in whole or in part by the presently disclosed subject matter. Other aspects will become evident as the description proceeds when taken in conjunction with the accompanying Examples and Figures as best described herein below.

[0033] BRIEF DESCRIPTION OF THE FIGURES

[0034] For the purposes of illustrating the invention, depicted in the drawings are certain embodiments. However, the invention is not limited to the precise arrangements and instrumentalities depicted in the embodiments. FIG. 1A-D are illustrations exhibiting molecular group interactions between sildenafil citrate, oxidized guar gum, and gelatin in the hydrogel compositions.

[0035] FIGS. 2A-C are images showing the results of the gel injectability assay of the hydrogel compositions.

[0036] FIG. 3 is an image showing the results of the gel stability test of the hydrogel compositions.

[0037] FIG. 4A-B show the results of the gel adhesion test of the hydrogel compositions.

[0038] FIG. 5 is a graph showing the results of the rheology assay of the hydrogel compositions - oscillatory time sweep.

[0039] FIG. 6 is a graph showing the results of the rheology assay analyzing shear thinning of the hydrogel compositions.

[0040] FIG. 7A-B is a graph exhibiting the results of the FTIR assay confirming Schiff base bonding in the hydrogel compositions.

[0041] FIG. 8A-B are graphs exhibiting the results of the XPS assay and molecular groups indicating Schiff base bonding in the hydrogel compositions.

[0042] FIG. 9A-B are graphs exhibiting the results of the XPS assay and molecular groups indicating Schiff base bonding in the hydrogel compositions.

[0043] FIG. 10A-B is a graph exhibiting the results of the TNBS assay confirming oxidation of the hydrogel compositions.

[0044] FIG. 11 are images generated via SEM showing porosity of the hydrogel compositions.

[0045] FIG. 12A-B is a graph exhibiting the results of the porosimetry assay of the hydrogel compositions.

[0046] FIG. 13 is a graph exhibiting the results of the cell viability assay on raw 264.7 cells over 1 day and 7 days.

[0047] FIG. 14 is a graph exhibiting the results of the cell viability assay on raw 264.7 cells over 1 day and 7 days. FIG. 15 is a graph exhibiting the results of the cell viability test of human umbilical derived vein endothelial cells (HLIVEC) over 1 day and 7 days.

[0048] FIG. 16 is a graph exhibiting the results of the cell viability test of human umbilical derived vein endothelial cells (HLIVEC) over 1 day and 7 days.

[0049] FIG. 17A-B are illustrations depicting how hydrogel compositions effect inflammatory response in cell culture.

[0050] FIG. 17C is a graph exhibiting the results of the inflammatory cell test on the hydrogel compositions.

[0051] FIG. 18A-B is an illustration (FIG. 18A) depicting sildenafil citrate’s effect on cGMP expression smooth cell relaxation and a graph (FIG. 18B) showing the results of a cGMP expression test on the hydrogel compositions.

[0052] FIG. 19A-B is a graph showing the results of the in vitro drug release kinetics assay of the hydrogel compositions.

[0053] FIG. 20A-B is a graph showing the results of the in vitro drug release kinetics assay of the hydrogel compositions.

[0054] FIG. 21 is a graph showing the results of an in vitro cell proliferation assay of human umbilical vein smooth muscle cells (HLIVSMC) using the hydrogel compositions.

[0055] FIG. 22 is a graph showing the results of an in vitro test of human umbilical vein endothelial cells (HLIVEC) and tube formation using the hydrogel compositions.

[0056] FIG. 23 is an image exhibiting junction formation in HllVECs using the hydrogel compositions.

[0057] FIG. 24 is an illustration depicting an in vivo assay design and effect on arteriovenous fistula (AVF) maturation in a rabbit model.

[0058] FIG. 25 is an image showing the results of an in vivo histological analysis and AVF maturation in a rabbit model.

[0059] FIG. 26 is a graph showing of an in vivo analysis of AVF maturation in a rabbit model and quantitative data of neointimal area and intima-to-media ratio. DETAILED DESCRIPTION

[0060] Provided herein are novel hydrogel compositions and novel methods of preparation and use of the compositions. The compositions can be used to treat vascular associated conditions or promote desired vascular characteristics, such as promoting vascular maturation, promoting vascular healing, promoting blood vessel dilation, promoting vascular outward remodeling, limiting smooth muscle cell proliferation, limiting intimal hyperplasia, reducing inflammation, and / or generally promoting vascular access.

[0061] As an example, the hydrogel compositions can be configured to promote arteriovenous fistula (AVF) maturation and avoid problems associated with insufficient widening of blood vessels and the rapid development of thickened vein walls. The hydrogel compositions can decrease the failure rate of AVF maturation in dialysis and / or other medical procedures and significantly decrease the need for creating new vascular access points, a common issue stemming from unsuccessful AVF maturation. Consequently, the hydrogel compositions provided herein greatly improve treatments aimed at efficient AVF maturation.

[0062] The hydrogel compositions described herein are carbohydrate based selfassembled natural bio-polymers comprising 1 ) a protein, polypeptide or molecule containing amino acid residues catalyzed by enzymes component, and 2) a polysaccharide based component composed of a polysaccharide molecule, in some embodiments, crosslinked by divalent ions. In some embodiments, the content of protein or polypeptide based component is the dry weight of hydrogel 1 %-99% of the total mass, and the content of the polysaccharide based component is 1 %-99% of the total dry weight of the hydrogel.

[0063] The protein or polypeptide based component can be one or more of gelatin, collagen, silk, fibroin, zein, elastin, fibrinogen, glycoprotein, and laminin fibronectin. Gelatin was used to prepare the hydrogel compositions OGG1 , OGG2, and OGG3 in the Examples. The polysaccharide based component can be guar gum, alginate, gellan gum, and the like. Guar gum was used to prepare the hydrogel compositions OGG1, OGG2, and OGG3. In some embodiments, the hydrogel compositions can contain additives that are one or more mixtures of enzymes, cell growth factors, bone morphogenic proteins, vitamins, insulin, and dexamethasone.

[0064] The hydrogel compositions disclosed herein can comprise an active agent. Suitable active agents include but are not necessarily limited to PDE5 inhibitors, PDE3 inhibitors, charged vasodilator compounds, and uncharged vasodilator compounds. PDE5 inhibitors can be selected from compounds including, but not limited to vardenafil, tadalafil, and avanafil. Suitable PDE3 inhibitors include but not limited to cliostazol and inamrinone (aka amrinone). Charged vasodilator molecules include, but are not limited to nitrates, minoxidil sulfate, and hydralazine hydrochloride and uncharged drug molecules include, but are not limited to iloprost and ambrisentan. The PDE5 inhibitor compound, sildenafil citrate, was used to prepare the hydrogel compositions in the Examples at a concentration of 1.5 mg / ml; however, in some embodiments, active agent concentration can be between about .5 and 3.5 mg / ml. Delivery of sildenafil via inhalation at a concentration of approximately 1 .25 mg / mL has been shown. See Journal of Controlled Release, 250(2017): 96-106. 0.5 to 3.5 mg / mL concentrations for hydrogels (vs. aerosols) encompass effective therapeutic levels while allowing formulation flexibility.

[0065] The polysaccharide component used in the composition can be oxidized, in some embodiments, using various methodologies. To prepare the hydrogel compositions described in the Example section, guar gum was oxidized using a sodium periodate oxidation through Schiff base formation, namely metaperiodate. Guar gum is an FDA approved natural polysaccharide and its oxidized structure can form self-assembled hydrogel by cyclic acetal reaction. Also, oxidized guar gum interacts with gelatin by Schiff’s base reaction allowing better control of gelation property. As described in the Example section, three levels of oxidized guar gum were used to prepare the hydrogel compositions OCG1 (1 mg / ml metaperiodate), OGG2 (2 mg / ml meta periodate), and OGG3 (3 mg / ml metaperiodate).

[0066] In some embodiments, the hydrogel compositions are prepared using a crosslinking agent to improve gel stability. In one embodiment, the compositions are enzymatically crosslinked. Enzyme catalyzed crosslinking possesses advantages, such as mild reaction condition, structurally stable products, superior selectivity and substrate specificity, simple operation, sustainable enzyme sources, safe and large- scale industrial production. For example, the common commercial enzyme, transglutaminase, can selectively crosslink peptide bonds in proteins between the s- amino groups of lysine residues and the y-carboxyamide groups of glutamine residues to form £-(y-glutamyl)-lysine peptide bonds. Transglutaminase crosslinking has been shown to improve the gelling, thermal stability, rheological properties, and emulsifying properties of gelatin. Microbial transglutaminase (mTG) has been used commercially as an emulsifying, stabilizing, gelation, viscosifier, foaming, and water holding agent since approximately 1998 and has been shown to facilitate stable integration between native host tissue and hydrogels. mTG is considered as GRAS (Generally Recognized As Safe) by Food and Drug Administration (FDA). According to European Parliament Directive 2000 / 13 EC, mTG is not an ingredient, but a processing aid, therefore, it is not listed in the ingredients of finished products. Other potentially suitable catalytic enzymes include lysyl oxidase, tyrosinase, peroxidase, horseradish peroxidase, thermolysin, phosphatase, p-lactamase, plasma amine oxidase. For the hydrogel compositions described in the Examples, microbial transglutaminase was added to crosslink gelatin.

[0067] Other commonly used crosslinking methods include physical crosslinking, such as hydrophobic interaction, hydrogen bond interaction, etc.; chemical crosslinking often uses crosslinking agents to polymerize, such as glutaraldehyde, epichlorohydrin, diisocyanate, carbodiimide, etc. Chemical crosslinking agents include, but are not limited to formaldehyde, glutaraldehyde, carbodiimide, and polyepoxide, owing to its numerous functionalized side groups. Such chemical crosslinkers, while potentially useful for the purposes described herein, have drawbacks, such as lower biocompatibility, higher cytotoxicity, uncontrollable reactivity, and high cost. Natural crosslinking agents are environmentally friendly alternatives to synthetic agents due to their abundance in nature, biocompatibility, and green methods for extraction, purification, and processing. Moreover, natural crosslinking agents contain various readily modifiable structures and functional groups. For example, polysaccharides that are oxidized by periodate to dialdehyde polysaccharides (DPs) can be useful and prospective environmentally friendly crosslinking agents for hydrogels. The aldehyde group of DP is crosslinked to the amino group of proteins or chitosan derivatives via Schiff base bonding to enhance the structure and properties of the hydrogel. Dialdehyde sodium alginate (DSA), (dialdehyde guar gum (DGG), dialdehyde dextran (DDE), dialdehyde starch, dialdehyde hyaluronic acid, dialdehyde Pullula), and dialdehyde succinoglycan have been reported to be nontoxic green crosslinking agents that improve the mechanical properties of hydrogels.

[0068] The hydrogel compositions can be configured to gradually release a therapeutic amount of the active agent at a desired release rate over a sustained period of time to treat vascular associated conditions, for example, at least one month or at least two months. This is advantageous when sustained exposure to the active agent is required to promote or induce a desired characteristic (e.g., AVF maturation) that takes time to develop. For example, the initial weeks following the creation of an AVF are crucial for determining its clinical success, with the first 1 -2 weeks being particularly vital. Within this timeframe, blood flow necessary for the AVF to become suitable for dialysis is established, typically within the first week, and the peak of cell proliferation leading to neointimal hyperplasia occurs around the fifth day. The hydrogel compositions prepared in the Example exhibited sustained release of sildenafil citrate over a duration of about 2 months and a release rate of about 70% during that time frame and thus exhibit enhanced treatment effectiveness during these early weeks improving blood vessel dilation and inflammatory response reduction. This helps ensure successful promotion of desired vascular conditions, such as AVF maturation.

[0069] The hydrogel compositions can be configured to perform at least one of the following vascular associated therapies or treatments: promoting vascular maturation, promoting vascular healing, promoting blood vessel dilation, promoting vascular outward remodeling, limiting smooth muscle cell proliferation, limiting intimal hyperplasia, reducing inflammation, and / or generally promoting vascular access. In one embodiment, the hydrogel compositions are configured to promote arteriovenous fistula (AVF) maturation, thus improving vascular access during dialysis.

[0070] The hydrogel compositions described herein are capable of local and direct application to a target site, such as a bodily or vascular tissue by injection to optimize the drug's effectiveness while minimizing systemic side effects. Other methods include topical applications like gel sheets or wraps, sprays, implants, or direct placement; in situ gelling by, for example, injecting a liquid precursor that gels upon reaching body temperature or pH at the site. Micro-bead hydrogels may also be used.

[0071] In some embodiments, the hydrogel compositions are synthesized using oxidized guar gum (OGG) and gelatin, both of which are biocompatible, biodegradable, and FDA-approved natural polymers. In one embodiment, oxidized guar gum (OGG) and gelatin are used to create the hydrogel by cross-linking through Schiff base bonding, as described in detail below. The synergy of these components allows for the modulation of gelation properties and drug release dynamics, while also eliminating the need for toxic chemical crosslinking agents if desired. The combination of these polymers allows for tunable gelation properties and drug release kinetics. In certain embodiments, gelatin crosslinking is achieved through the action of microbial transglutaminase, which catalyzes the conjugation of glutamine and lysine residues. mTG, an FDA-approved substance, is extensively used in food processing and ensures a stable bond between the native host tissue and the hydrogel.

[0072] The exemplary hydrogel compositions described in the Examples below comprising guar gum (OGG) oxidized using a sodium periodate, gelatin, sildenafil citrate, and enzymatically crosslinked using microbial transglutaminase, exhibit vastly improved properties including, but not limited to improved mechanical properties, stability in fluid, injectability, gel adhesiveness, and sustained active agent release. Three different oxidation levels were prepared by adding different amounts of sodium periodate - 1 mg / ml (OGG1), 2 mg / ml (OGG2), or 3 mg / ml (OGG3). Various properties were examined including, but not limited to rheology, injectability, gel stability, adhesion property, chemical composition, and porosity, in vitro biocompatibility, inflammatory responses, drug or active agent release kinetics, as well as in vivo treatment efficacy.

[0073] Definitions

[0074] As used herein, each of the following terms has the meaning associated with it in this section.

[0075] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.

[0076] Generally, the nomenclature used herein and the laboratory procedures in tissue engineering and biomaterial science are those well-known and commonly employed in the art. As used herein, the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0077] As used herein, the term “about” is understood by persons of ordinary skill in the art and varies to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods.

[0078] As used herein, the term “hydrogel” or “gel” refers to a three-dimensional polymeric structure that itself is insoluble in a particular liquid but which is capable of absorbing and retaining large quantities of the liquid to form a stable, often soft and pliable, but always to one degree or another shape-retentive, structure. When the liquid is water, the gel is referred to as a hydrogel. Unless expressly stated otherwise, the term “gel” is used throughout this application to refer both to polymeric structures that have absorbed a liquid other than water and to polymeric structures that have absorbed water, it being readily apparent to those skilled in the art from the context whether the polymeric structure is simply a “gel” or a “hydrogel.”

[0079] As used herein, the term “protein or polypeptide component” refers to protein and polypeptide molecules substances commonly used in the synthesis of biocompatible hydrogels, including but not limited to gelatin, collagen, fibroin, fibrin, zein, elastin, fibrinogen, glycoprotein, and laminin fibronectin. In another embodiment, the protein or polypeptide based component can be gelatin. The protein or polypeptide based component can be a protein or polypeptide or a molecule containing amino acid residues that is catalyzed by an enzyme through peptide bond cross-linking or through Schiff base (Schiff base) cross-linking or phenolic hydroxyl oxidation cross-linking or phosphate groups of coenzyme A. It is formed by binding and cross-linking with serine residues of proteins, or is physically cross-linked by self-assembly caused by changes in the hydrophilicity of molecules caused by proteins or polypeptides through enzyme- catalyzed reactions. The catalytic enzymes can be transglutaminase, transglutaminase isozyme, lysyl oxidase, tyrosinase, peroxidase, horseradish peroxidase, thermolysin, phosphatase, fMactamase, plasma amine oxidase.

[0080] As used herein the term, “gelatin” refers to a common collagen derived polymer component of hydrogels with physicochemical features that can be altered with application requirements. Gelatin is derived from collagen through hydrolysis and its chemical structure is primarily composed of amino acid chains forming a three- dimensional network through hydrogen bonding and hydrophobic interactions, which give gelatin its gelling properties. Gelatin dissolves well in water and can form reversible gel-like substances. When cooled, water is trapped within its network structure, resulting in what is known as a hydrogel. Gelatin based hydrogels often require crosslinking to improve mechanical characteristics and structural stability since the network structure of gelatin is formed via non-covalent bonds.

[0081] As used herein the term “polysaccharide component” refers to a polysaccharide based or derived molecule used in the synthesis of biocompatible hydrogels, including but not limited to guar gum, alginate, alginate salts, pectinate, pectinate salts, carrageenan, cellulose derivatives, gellan gum, agarose, dextran, xanthan gum, and the like, a derivative of any thereof, and a mixture of any two or more thereof. In one embodiment, the polysaccharide component is guar gum.

[0082] As used herein, the term “guar gum” refers to a polysaccharide composed of galactose and mannose sugars with a linear chain skeleton of p 1 ,4 linked mannose residues to which galactose residues are 1 ,6 linked at every second mannose, forming short lateral branches. Guar gum is extracted from guar beans and has binding, thickening, and stabilizing properties. Examples of commercially available non-ionic guar gums include products marketed under the name Vidogum® GH174 by Unipectine and Meypro-Guar 50 and Jaguar® C by Solvay.

[0083] As used herein, the term “crosslinking” refers to the process of bonding and entanglement between hydrogel molecular constituents to enhance mechanical strength and slow degradation. Commonly used cross-linking methods include physical cross-linking, such as hydrophobic interaction, hydrogen bond interaction, etc.; chemical cross-linking often uses cross-linking agents to polymerize, such as glutaraldehyde, epichlorohydrin, diisocyanate, carbodiimide, etc.

[0084] As used herein, the term “crosslinking agent” refers to synthetic or natural chemicals, enzymes, or substances that are used to crosslink hydrogel components. Chemical crosslinking agents, including formaldehyde, glutaraldehyde, carbodiimide, and polyepoxide, owing to its numerous functionalized side groups. Such chemical crosslinkers, while potentially useful for the purposes described herein, have drawbacks, such as lower biocompatibility, higher cytotoxicity, uncontrollable reactivity, and high cost. Natural crosslinking agents are environmentally friendly alternatives to synthetic agents due to their abundance in nature, biocompatibility, and green methods for extraction, purification, and processing. Moreover, natural crosslinking agents contain various readily modifiable structures and functional groups. For example, polysaccharides that are oxidized by periodate to dialdehyde polysaccharides (DPs) can be useful and prospective environmentally friendly crosslinking agents for hydrogels. The aldehyde group of DP is crosslinked to the amino group of proteins or chitosan derivatives via Schiff base bonding to enhance the structure and properties of the hydrogel. Dialdehyde sodium alginate (DSA), (dialdehyde guar gum (DGG), dialdehyde dextran (DDE), dialdehyde starch, dialdehyde hyaluronic acid, dialdehyde Pullula), and dialdehyde succinoglycan have been reported to be nontoxic green crosslinking agents that improve the mechanical properties of hydrogels.

[0085] Enzyme catalyzed crosslinking possesses advantages, such as mild reaction condition, structurally stable products, superior selectivity and substrate specificity, simple operation, sustainable enzyme sources, safe and large-scale industrial production. For example, the common commercial enzyme, transglutaminase, can selectively crosslink peptide bonds in proteins between the s-amino groups of lysine residues and the y-carboxyamide groups of glutamine residues to form E-(y-glutamyl)- lysine peptide bonds. Transglutaminase crosslinking has been shown to improve the gelling, thermal stability, rheological properties, and emulsifying properties of gelatin. Microbial transglutaminase (mTG) has been used commercially as an emulsifying, stabilizing, gelation, viscosifier, foaming, and water holding agent since approximately 1998. mTG is considered as GRAS (Generally Recognized As Safe) by Food and Drug Administration (FDA). According to European Parliament Directive 2000 / 13 EC, mTG is not an ingredient, but a processing aid. Therefore, it is not listed in the ingredients of finished products.

[0086] The term “oxidation” as used herein refers to the chemical modification of a polysaccharide (e.g., guar gum) to induce a crosslinking interaction with the amino groups of polymers, such as gelatin. In some uses, through oxidation, self-crosslinking can be induced in the absence of external crosslinking agent. For example, periodate oxidation of polysaccharides form dialdehydes that interact with amino groups of gelatin through Schiff’s base.

[0087] The term “active agent” as used herein refers to a molecule or compound that exhibits a desired therapeutic effect on the target tissue (e.g., vascular tissue). In certain embodiments, the active agent in the hydrogel composition is phosphodiesterase-5 (PDE5) inhibitor, which is known to promote vasodilation and smooth muscle cell (SMC) relaxation by limiting enzyme activity of PDE5, which catalyzes cGMP hydrolysis. In other embodiments, the PDE5 inhibitor can be sildenafil, tadalafil, vardenafil, avanafil, mirodenafil, udenafil, gisadenafil, yonkenafil, lodenafil, fenspiride, MBCQ, zaprinast, and / or icariin. In yet another embodiment, the PDE5 inhibitor is sildenafil citrate. PDE3 inhibitors can also be used as active agents including, but not limited to cliostazol and inamrinone (aka amrinone). Charged vasodilator molecules, such as, for example, nitrates, minoxidil sulfate, and hydralazine hydrochloride and / or uncharged drug molecules, such as iloprost and ambrisentan can also be used.

[0088] The terms “patient,” “subject” or “individual” are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In a non-limiting embodiment, the patient, subject or individual is a human.

[0089] As used herein, the term “treatment” or “treating” is defined as the application or administration of a therapeutic agent, i.e., a compound of the invention (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a condition contemplated herein, a symptom of a condition contemplated herein or the potential to develop a condition contemplated herein, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect a condition contemplated herein, the symptoms of a condition contemplated herein or the potential to develop a condition contemplated herein. Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.

[0090] Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range and, when appropriate, partial integers of the numerical values within ranges. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0091] Every formulation or combination of components described or exemplified can be used to practice the invention, unless otherwise stated. Specific names of compounds are intended to be exemplary, as it is known that one of ordinary skill in the art can name the same compounds differently. When a compound is described herein such that a particular isomer or enantiomer of the compound is not specified, for example, in a formula or in a chemical name, that description is intended to include each isomers and enantiomer of the compound described individual or in any combination. Although the description herein contains many embodiments, these should not be construed as limiting the scope of the invention but as merely providing illustrations of some of the presently preferred embodiments of the invention.

[0092] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this invention and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size / volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing / oxidizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application. In general the terms and phrases used herein have their art- recognized meaning, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art. Any preceding definitions are provided to clarify their specific use in the context of the invention.

[0093] EXAMPLES

[0094] The following Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.

[0095] Materials and Methods: Generally, guar gum was modified using sodium metaperiodate to produce guar gum with three levels of oxidation (OGG1 , OGG2, and OGG3) and evaluated by TNBS assay. A sildenafil-encapsulated hydrogel was fabricated using the oxidized guar gum, gelatin, and microbial transglutaminase as the gelatin crosslinking agent. Gel properties were characterized as described below.

[0096] Results Summary: Developed hydrogels were injectable, with those containing higher levels of oxidized guar gum exhibiting a more viscous appearance. Based on rheology tests, increased oxidization level correlated with a higher storage modulus and showing a shear-thinning behavior. Structural evaluation shows Schiff’s base formation between gelatin and oxidized guar gum as well as ester bond formation between oxidized guar gum and citrate salt of sildenafil. Drug release kinetics test demonstrated sustained release over 1 month. Where p values were calculated and shown in a graph - *p<0.05, **p<0.01 , ***p<0.001 , and ****p<0.0001.

[0097] Unlike a prior approach involving use of a peptide amphiphile-based hydrogel for nitric oxide (NO) delivery as the active agent, the natural polymer based hydrogel compositions prepared in the Examples and described herein avoid undesirable characteristics exhibited by nitric oxide based hydrogels, such as less stability, less active agent delivery control, and poorer mechanical properties. The present hydrogel technology is able to employ more stable and proven active agents; offers enhanced control over gelation properties, demonstrates superior mechanical stability, injectability, tissue adhesiveness, and maintains its structural integrity in aqueous environments, alongside sustained active agent release properties. Moreover, it incorporates components that are not only FDA approved but also widely utilized. Consequently, this hydrogel product leverages natural, safe, and proven components, along with exceptional gel characteristics.

[0098] Example 1 - Hydrogel Preparation

[0099] The PDE5 inhibitor, e.g. sildenafil, was selected as the active agent. PDE5 inhibitor is known to promote vasodilation and SMC relaxation by limiting enzyme activity of PDE5 which catalyzes cGMP hydrolysis. PDE5 inhibitor also showed a relaxant effect on radial arteries and cephalic veins harvested from organ donors which are commonly used to create AVFs in patients on chronic hemodialysis. In addition, PDE5 inhibitor has been shown to inhibit the migration of SMC and platelet aggregation by cGMP-dependent protein kinase activation thereby reducing the formation of neointimal hyperplasia. As illustrated in FIGS. 1A-D, the carboxyl groups of sildenafil citrate and the hydroxyl groups of oxidized guar gum and gelatin can form ester bonds. The ester bond is formed by the reaction between the carboxyl group of sildenafil citrate and the hydroxyl groups of oxidized guar gum and gelatin.

[0100] A highly biocompatible guar gum and gelatin based hydrogel was prepared. Guar gum is an FDA approved natural polysaccharide and its oxidized structure can form self-assembled hydrogel by cyclic acetal reaction. Also, gelatin was added to the oxidized guar gum, which can interact together by Schiff’s base reaction allowing better control of gelation property. Furthermore, mTG was also added to crosslink gelatin by formation of covalent bonds between the y-carboxamide group of glutamine residues and the s-amino group of lysine residues in gelatin, which improved gel stability. mTG is FDA-approved material and widely applied for food processing, and it facilitates stable integration between native host tissue and the hydrogel. a. Preparation of Gelation Materials

[0101] 1. Preparation of oxidized guar gum (OGG)

[0102] Step 1 ■ OGG production (500ml base)

[0103] 1) 500 ml of Guar gum solution (0.5-10%, w / v) was prepared in DW by continuous stirring and heating at 70 °C for 1 h.

[0104] 2) Divide the guar gum solution in to 3 solutions (160ml / 160ml / 160ml).

[0105] 3) Then, add an aqueous solution of sodium periodate, specifically metaperiodate (NalO4) dropwise into GG aqueous solution and stir the whole reaction mixture with a speed of 800 rpm for 24 h in the dark at 25 °C. See Table 1 .

[0106] Table 1

[0107] Step 2. 1-5 wt% OGG production (10ml base)

[0108] 1) Weigh the freeze dried OGG 0.1 -0.5 g and add in to a 20ml vial

[0109] 2) Put a stirring bar in to the 20ml vial and add 10 ml of 3dw

[0110] 3) Stir the solution with 300 rpm at 50°C overnight

[0111] 4) Centrifuge the solution with 6,000 rpm for 5 mins and use the supernatant (to remove some portion which is not dissolved)

[0112] Production of different degrees of (aldehyde group) oxidized Guar gum: OGG 1 < OGG 2< OGG 3 b. Preparation of 5-50wt% gelatin (20ml base)

[0113] 1) Add gelatin 1-10 g into an 100 ml vial

[0114] 2) Put the stirring bar and add the 3dw 20 ml in to the 100 ml vial

[0115] 3) Place the vial on the hot plate and stir the solution with 300rpm, 60 °C overnight cf). If the temperature is low, the solution quickly becomes thick gel and cannot be stirred anymore. c. Preparation of mTG 2U Concentration (10ml base) 1 ) Weigh 390 mg of mTG

[0116] 2) Add 10ml of 3DW in to the 50 ml conical tube

[0117] 3) Add a small amount of mTG in to the 3 dw and dissolve the mTG by tapping the 50 ml conical tube

[0118] 4) After completely dissolving, add more small amount of mTG in to the tube and dissolve it by tapping the tube (repeat the process until dissolving whole mTG). cf). Do not add a large amount of mTG together, it may cause aggregation and incomplete dissolving of mTG d. Gelation protocol (6ml base)

[0119] 1) w / o sildenafil citrate i) Take out the stored solutions in the refrigerator and warm them up at room temperature ii) These solutions will be mixed and stirred at 37°C iii) Prepare three 20ml vials iv) Add 3 ml of 1wt% OGG 1 ,2,3 solutions in to each 20ml vial v) Add 60-240 pl of mTG (2U) vi) Add 3 ml of 5-50wt% gelatin vii) Stir the solution for 5 mins, then place the vial at the 37°C incubator overnight

[0120] 2) w / sildenafil citrate i) Take out the stored solutions in the refrigerator and warm them up at room temperature ii) These solutions will be mixed and stirred at 37°C iii) Prepare three 20ml vials iv) Dissolve 0.3-27 mg of Sildenafil citrate in to the OGG 1 ,2,3 solutions (9mg / 3ml) v) Add 60-240 pl of mTG (2U) vi) Add 3 ml of 5-50wt% gelatin vii) Stir the solution for 5 mins, then place the vial at the 37°C incubator overnight

[0121] Example 2 - Hydrogel Characterization

[0122] The hydrogels (0CG1 -3) prepared according to the procedure described above in Example 1 was subjected to various tests to ascertain gel characteristics in order to demonstrate suitability for therapeutic administration and efficacy. a. Gel injectability test

[0123] The hydrogels prepared according to the procedure described above in Example 1 was subjected to an injectability test using a syringe. FIGS. 2A-C. All OGG gel conditions (OGG1 , 0GG2, and 0GG3) were injectable. Greater effort was required to inject the gel as oxidation level increased due to increased Schiff base bonding. FIG. 2B. b. Gel stability test

[0124] The hydrogels prepared according to the procedure described above in Example 1 and 0GG2 was subjected to a gel stability test by injecting the gel into 1X phosphate-buffered saline (PBS). As illustrated in FIG. 3, the hydrogel retained its stability, indicating that the gel is likely to maintain its physical integrity when exposed to body fluids after application. c. Gel adhesion test

[0125] Porcine skin was used to evaluate injectability and adhesiveness an important characteristic for the localized application of hydrogels to targeted tissues within the body. See FIG.4A. Effective adhesiveness and bonding ensures that the hydrogel does not prematurely detach, facilitating the sustained delivery of therapeutic agents directly to the desired sites. A lap shear test was conducted using overhead projector (OHP) film as the substrate to further test adhesiveness. The hydrogels were placed between two OHP films, and adhesion was quantified by interfacial toughness.

[0126] As shown in FIG. 4A, the injected hydrogel adheres very well to the porcine skin. Moreover, the hydrogels demonstrate improved interfacial toughness and tensile strength - OGG2-3 being the best performing gels. See FIG. 4B. Results showed a positive correlation between hydrogel oxidation rate and interfacial toughness: OGG3 reached 262kPa, OGG2 reached 17 kPa, and OGG1 24 kPa. d. Rheology Testing

[0127] 1) Oscillatory Time Sweep

[0128] The viscoelastic properties of the hydrogel were assessed by measuring the shear storage modulus (G') and loss modulus (G") using a rheometer (MCR 302, Anton Paar) equipped with a 25 mm diameter parallel plate at room temperature (RT). Oscillatory time sweep was conducted at a fixed strain amplitude (0.5%) and frequency (1 Hz) over a period of 400s to assess the temporal stability of the hydrogel network. The storage modulus (G') consistently exceeded the loss modulus (G"), confirming the long-term structural integrity of the hydrogel.

[0129] As illustrated in FIG. 5, G’ (Storage modulus) is greater than G” (Loss modulus) indicating that a solid like behavior predominates. G’ and G” plots in parallel as shown in FIG. 5 confirms that the hydrogels are in a gel state. G’ is greatest in the higher oxidation level i.e., OGG3. Higher oxidized OGG has more aldehyde groups which react with more amine groups in gelatin. Oxidation of OGG has more aldehyde group, it reacted with more amine group of gelatin. More Schiff base bonding contributes to increased storage and loss modulus.

[0130] 2) Shear viscosity (mPa*s) vs. shear rate (Pa)

[0131] The viscosity properties of the hydrogel compositions were assessed by measuring the shear viscosity with increase shear rate using a rheometer (MCR 302, Anton Paar) equipped with a 25 mm diameter parallel plate at room temperature (RT). Shear rate sweep was performed over a shear rate range of 0.1-1000 s-1to assess shear-thinning behavior.

[0132] Shear thinning is the result of micro-structural rearrangements occurring in the plane of applied shear and is commonly observed for dispersions, including emulsions and suspensions, as well as polymer solutions and melts. At low shear rates, materials tend to maintain an irregular order with a high zero shear viscosity resulting from particle / molecular interactions and the restorative effects of Brownian motion. Shear thinning is a material property indicative of a fluid like flow under pressure and suitability for administration via injection. As illustrated in FIG. 6, the hydrogel compositions exhibited a continuous decrease in viscosity with increasing shear rate, indicative of shear-thinning properties indicating their suitability for injection purposes. e. Chemical & Structural Analysis

[0133] 1) Fourier-transform Infrared Spectroscopy (FTIR)

[0134] FTIR is a technique used to obtain an infrared spectrum of absorption or emission of a solid, liquid, or gas. FTIR analyzes the chemical structure of gels and confirms the formation of Schiff base bonding (C=N) combined by OGG (C=O) and gelatin (N-H). As illustrated in FIG. 7A-B, as OGG oxidation increases, the aldehyde (C=O) and amine (N-H) peaks decrease, while the Schiff base bond (C=N) peak increases. In other words, higher oxidation rate of OGG makes more Schiff base bonding (C=N), while reducing amine bond (N-H) of gelatin and aldehyde bond (C=O) of Oxidized Guar gum.

[0135] 2) X-ray photoelectron spectroscopy (XPS) (C 1s and N 1s))

[0136] XPS is a surface sensitive quantitative spectroscopic technique that measures the very topmost 50-60 atoms, 0.01 urn, 5-10 nm of any surface and is a powerful measurement technique because it shows what elements are present and what they are bonded to. As shown in FIGS. 8A-B and 9A-B, the formation of Schiff base bonding (C=N) in the hydrogels was confirmed. Again, as the OGG oxidation rate increased, the aldehyde (C=O) and amine (N-H) peaks decreased, while the Schiff base bond (C=N) peak increased. Higher oxidation rate of OGG makes more Schiff base bonding (C=N), while reducing amine bond (N-H) of gelatin and aldehyde bond (C=O) of oxidized guar gum. f. 2,4,6-Trinitrobenzenesulfonic acid (TNBS) test

[0137] The percentage degree of oxidation of oxidized gar guam (OGG) was measured using TNBS assay method. The formation of carbazones by oxidized sugar after reacting with polyaldehyde sugar. This reaction allows the quantification of the aldehyde content in oxidized guar gum as the amine group bearing monomolecular reactant, tert-butyl carbazate (t-BC) reacts with aldehyde group of OGG. See FIG. 10A. As shown in FIG. 10B, the higher oxidized OGG reacts with the t-BC more, and the amount of remaining t-BC is relatively small. Thus, higher oxidation has lower t-BC concentration confirming that the guar gum has been properly oxidized. g. SEM analysis / Pore size / Theoretical porosity

[0138] The morphology of the hydrogels were investigated using a Scanning Electron Microscope (SEM, 7610F-Plus JEOL). Freeze-dried (lyophilized) hydrogel samples after swelling in PBS were mounted on a double sided tape before sputter coating with platinum (approximately 5 nm), then micrographs were captured using an accelerating voltage of 2-5kV. As shown in FIG. 11, the hydrogels showed porous structure formation. Porous structure (modified by crosslinking) impacts hydrogel characteristics including 1) controlled drug release kinetics (the pore size and porosity affects diffusion path of drugs); 2) swelling behavior (allowing hydrogel absorb water, promoting swelling and providing mechanical cushioning); and 3) degradation rate of a hydrogel.

[0139] Pore size and theoretical porosity were also measured using the analytical technique known as porosimetry. The technique involves intrusion of a non-wetting liquid at high pressure into the material being tested via porosimeter. In this case a PM33GT, Quantachrome porosimeter was used. Pore size can be determined based on the external pressure needed to force the liquid into a pore against the opposing force of the liquid’s surface tension. As shown in FIGS. 12A-B, higher oxidized OGG hydrogels exhibited smaller pore size and lower theoretical porosity resulting from greater degree of Schiff base bonding. For example, OGG1 showing the highest porosity (96%) and OGG3 the lowest (93%) reflecting the impact of crosslink density. h. In vitro test

[0140] 1 ) Cell viability test (Raw 264.7 Cells): 1 day test and 7 day test

[0141] RAW264.7 macrophages were cultured under standard conditions (37 °C, 5% CO2). After the cells were thawed, they were seeded in 24-well plates at a density of 50,000 cells / well (n = 4). After 24h of incubation, hydrogel samples were placed in transwell inserts (n = 3) and co-incubated with the cells for 1 day and 7 days. The 1- day assay was used to assess acute cytotoxicity, while the 7-day assay evaluates potential delayed or cumulative effects of the hydrogel and drug release over a longer period. Both assays consistently showed maintained cell viability, supporting the biocompatibility of the formulation over time.

[0142] Following treatment, the inserts were removed, and cells were washed with PBS. Subsequently, 1 mL of fresh growth medium and 1OOpL of Cell Counting Kit-8 (CCK-8) reagent (Dongin Biotech Co., Korea) were added to each well. After 2h of incubation, the colorimetric change in the medium was measured at 450 nm using a microplate reader (n = 3). As shown in FIGS. 13-14, cell viability was maintained posttreatment indicating the absence of hydrogel-related toxicity, which refers to both the bulk hydrogel material and the incorporated active agent, sildenafil citrate. The transwell setup allows for the evaluation of any leachable or diffusible components, including potential cytotoxicity from the hydrogel matrix itself as well as the sustained release of the active agent.

[0143] After treatment for 7 days cell viability was tested. There was no difference in cell viability, and thus no toxicity due to the active agent, sildenafil citrate, as between the drug-treated (w / drug) and untreated (w / o drug).

[0144] 2) Cell viability test (HUVEC): 1 day and 7 days

[0145] HLIVECs were cultured under standard conditions (37 °C, 5% CO2). Cells were seeded in 24-well plates at a density of 50,000 cells / well. After 24 h of incubation, hydrogel samples were placed in transwell inserts (n = 3) and co-incubated with the cells for 1 day and 7 days. Following treatment, the inserts were removed, and cells were washed with PBS. Subsequently, 1 mL of fresh growth medium and 100 pL of Cell Counting Kit-8 (CCK-8) reagent (Dongin Biotech Co., Korea) were added to each well. After 2 h of incubation, the colorimetric change in the medium was measured at 450 nm using a microplate reader (n = 3).

[0146] Cell viability of human umbilical derived vein endothelial cells was tested after 1 day and 7 days treatment with the hydrogel compositions. Endothelial cells are critical for maintaining vessel patency and overall vascular health. Cell viability was maintained post-treatment with the hydrogel, indicating the absence of hydrogel- related toxicity. No difference in cell viability was observed between the samples with the drug (w / drug) and without the drug (w / o drug), suggesting no drug-induced toxicity. See FIGS. 15-16. Further, hydrogel components released from the transwell promoted the proliferation of human umbilical vein endothelial cells (HUVECs). i. Inflammatory Cell Test (macrophage polarization)

[0147] Macrophage polarization refers to whether and how macrophages have been activated in response to signal. Macrophage polarization markers (M1 & M2 macrophage) were evaluated. Cell culture was incubated for 24 hours in hydrogel following LPS treatment. As illustrated in FIG.17A-C, the hydrogel reduced pro- inflammatory marker (M1 or CD80) and increased anti-inflammatory marker (M2 or CD206). The presence or absence of PDE5 inhibitor (sildenafil) had no significant effect. As shown in FIG. 17C, the p value was p<0.0001 (OGG1 , OGG2, and OGG3) for M1 and M2 assays. j. cGMP expression test

[0148] Smooth muscle cells (SMCs) were seeded in 6-well plates at a density of 120,000 cells per well and allowed to adhere overnight. The cells were then treated with either a sildenafil citrate solution or hydrogel formulations using transwell inserts (n = 3) for 24 h. After treatment, cells were lysed by repeated freeze-thaw cycles to release intracellular cGMP. The cGMP levels were quantified using a human cGMP ELISA kit (MyBioSource, CA, USA), according to the manufacturer’s instructions.

[0149] Referring now to FIG. 18A-B, sildenafil citrate (PDE5 inhibitor) was incorporated to promote smooth muscle relaxation via the cGMP-PKG signaling pathway. cGMP levels were measured in SMCs treated with hydrogel compositions (1 ml) loaded with 1.5 mg / ml sildenafil citrate. OGG1 and OGG3 hydrogels (w / sildenafil citrate) significantly increased cGMP production compared to the untreated control group. No significant difference was observed between OGG1 (w / o sildenafil citrate) and the untreated group. These results suggest that SC-loaded hydrogel facilitates vasodilation through upregulation of cGMP signaling. k. Drug release kinetics

[0150] To evaluate the drug release profile, 1 mL of OGG hydrogel was loaded into a dialysis membrane tube (molecular weight cutoff: 3.5-5 kDa). The dialysis tube was immersed in 15 mL of DI water contained in a conical tube, which was incubated in a shaking incubator at 37 °C. At predetermined time intervals, the DI water was collected and replaced with fresh DI water to maintain sink conditions. The concentration of the released drug was quantified using IIV-V spectroscopy (Model Evolution 300, Thermo Fisher Scientific).

[0151] Release kinetics were analyzed through day 69. The hydrogels included a total 22.275 mg sildenafil citrate at the start of the test. As shown in FIG. 19A-B and FIG. 20A-B, all hydrogel formulations exhibited a consistent and prolonged drug delivery for a duration of 67 days. Notably, the exhibited sustained release aligns with the standard time it takes for complete maturation of arteriovenous fistulas (AVFs).

[0152] I. In vitro drug effect

[0153] 1) Proliferation of human umbilical vein smooth muscle cells (HUVSMCs)

[0154] HUVSMCs were seeded in 6-well plates at a density of 120,000 cells per well and allowed to adhere overnight. The cells were then treated with sildenafil citrate and untreated. After 48hours, 1 mL of fresh growth medium and 100 pL of Cell Counting Kit-8 (CCK-8) reagent were added to each well. After 2 h of incubation, the colorimetric change in the medium was measured at 450 nm using a microplate reader (n = 3). HUVSMCs treated with 10 pM of sildenafil citrate showed a significant reduction in cell proliferation (p<0.05) compared to the untreated HUVSMCs after 48 hours. See FIG. 21.

[0155] 2) Tube formation of Human umbilical vein endothelial cells (HUVECs)

[0156] Angiogenic potential of hydrogel formulations was evaluated by quantifying junction formation in HUVEC tube formation assay. To maintain a cold environment, 24-well plates were pre-chilled on ice. Matrigel (300 pL) was added to each well and evenly spread across the bottom surface by gentle shaking. HUVECs were seeded at a density of 50,000 cells per well and exposed to hydrogels placed in transwell inserts (n = 3) for 24 h. Tube formation was monitored at hourly intervals under an optical microscope. Images were acquired and analyzed using the Angiogenesis Analyzer plugin in Imaged to quantify the number of HUVEC junctions.

[0157] HUVECs were treated with 0.6 mg of sildenafil citrate in hydrogel for 12 hours. As demonstrated in FIGS. 22-23, hydrogels containing sildenafil citrate (OGG1 , OGG2, 0GG3 w / sildenafil citrate) significantly increased the number of junctions compared to OGG1 w / o sildenafil citrate with p values (p<0.0001 for OGG1), (p<0.01 for 0GG2), and (p<0.05 for 0GG3). 0GG1 (w / sildenafil citrate) showed the highest junction formation, including master junction, master segment and mesh. Notably, OGG1 also exhibits a relatively fast release of sildenafil citrate indicating enhanced endothelial network formation and superior pro-angiogenic capacity or effect. m. In-vivo tests

[0158] To evaluate the effects of OGG hydrogel on AVF maturation, an end-to-side anastomosis was created between the external jugular vein and the common carotid artery in rabbits. OGG2 (3 mg / ml sildenafil citrate) was applied around the grafted external jugular vein adjacent to the anastomosis site, with the aim of preventing neointimal hyperplasia and reinforcing the medial layer to promote vascular remodeling and maintain stable blood flow. See FIG. 24. Histological analysis at 1 month post-surgery revealed that OGG hydrogel treatment resulted in reduced neointimal thickening and preservation of the medial layer compared to the control group. See FIG. 25. Quantitative assessment confirmed a significant reduction in neointimal area (p<0.0001 ) and intima-to-media ratio (p<0.01) in the OGG hydrogel group, suggesting suppressed pathological remodeling. See FIG. 26. Additionally, the tunica media area was significantly increased in the OGG hydrogel group (p<0.001), indicating improved vessel wall stability and AVF maturation. Id. The quantitative histological analysis compares the neointimal area, intima-to-media ratio, and tunica media area between treatment groups. These measurements support the conclusion that the OGG hydrogel promotes favorable vascular remodeling and AVF maturation.

[0159] While there have been described herein the principles of the invention, it is to be understood by those skilled in the art that this description is made only by way of example and not as a limitation to the scope of the invention. Accordingly, it is intended by the appended claims, to cover all modifications of the invention which fall within the true spirit and scope of the invention.

Claims

What is claimed is:1 . A hydrogel composition for vascular associated treatments comprising: a protein or polypeptide component, a polysaccharide component, and an active agent; wherein said protein or polypeptide component comprises gelatin and said polysaccharide component comprises oxidized guar gum; and wherein said hydrogel is a carbohydrate based self-assembled natural biopolymer.

2. The composition of claim 1 wherein said active agent comprises a PDE5 inhibitor.

3. The composition of claim 2 wherein said PDE5 inhibitor is sildenafil citrate.

4. The composition of one or more of claims 1 through 3 wherein said oxidized guar gum is oxidized by sodium periodate through Schiff base formation.

5. The composition of claim 4 wherein said sodium periodate is metaperiodate.

6. The composition of one or more of claims 1 through 5 wherein said hydrogel is crosslinked with a biocompatible crosslinking agent.

7. The composition of claim 6 wherein said biocompatible crosslinking agent comprises an enzyme.

8. The composition of claim 7 wherein said enzyme comprises microbial transglutaminase.

9. The composition of one or more of claims 2 through 8 configured to release a therapeutic amount of said PDE5 inhibitor over a sustained period of time.

10. The composition of claim 9, wherein the sustained period of time is at least one month.1 1. The composition of claim 9, wherein the sustained period of time is at least two months.

12. The composition of one or more of claims 1 through 11 wherein said hydrogel is configured for direct and localized application to a vascular tissue.

13. The composition of claim 12 wherein said vascular tissue is an arteriovenous fistula.

14. The composition of claim 12 wherein said hydrogel is injectable.

15. The composition of one or more of claims 4 through 14 wherein said oxidized guar gum comprises about 3 mg / ml sodium periodate.

16. The composition of one or more of claims 4 through 14 wherein said oxidized guar gum comprise about 2 mg / ml sodium periodate.

17. The composition of one or more of claims 4 through 14 wherein said oxidized guar gum comprises about 1 mg / ml sodium periodate.

18. The composition of one or more of claims 2 through 17 comprising 1.5 mg / ml PDE5 inhibitor.

19. The composition of one or more of claims 1 through 18 wherein said hydrogel has a release rate of 70% over 63 days.

20. A method for treating vascular associated conditions comprising the steps of: providing a hydrogel composition comprising a gelatin, an oxidized guar gum, and an active agent; applying the hydrogel directly to a vascular tissue; wherein said hydrogel is a carbohydrate based self-assembled natural biopolymer; wherein said hydrogel is configured to release a therapeutic amount of the active agent gradually over a sustained period of time.

21. The method of claim 20 wherein said active agent is a PDE5 inhibitor.

22. The method of claim 21 wherein said PDE5 inhibitor is sildenafil citrate.

23. The method of claim 20 wherein said oxidized guar gum is oxidized by sodium periodate through Schiff base.

24. The method of claim 23 wherein said sodium periodate is metaperiodate.

25. The method of claim 20 wherein said hydrogel is enzymatically crosslinked with microbial transglutaminase.

26. The method of claim 20, wherein the sustained period of time is at least one month.

27. The method of claim 20, wherein the sustained period of time is at least two months.

28. The method of claim 20 wherein said hydrogel is applied by injection.

29. The method of claim 20 wherein said oxidized guar gum comprises about 3 mg / ml sodium periodate.

30. The method of claim 20 wherein said oxidized guar gum comprise about 2 mg / ml sodium periodate.

31. The method of claim 20 wherein said oxidized guar gum comprises about 1 mg / ml sodium periodate.

32. The method of claim 20 wherein said hydrogel comprises 1.5 mg / ml PDE5 inhibitor.

33. The method of claim 20 wherein said hydrogel has a release rate of about 70% over 63 days.

34. The method of claim 20 wherein said hydrogel is configured to perform at least one of promoting blood vessel dilation, promoting vascular outward remodeling, limiting smooth muscle cell proliferation, limiting intimal hyperplasia, and reducing inflammation.

35. The method of claim 20, wherein said bodily tissue is an arteriovenous fistula (AVF).

36. The method of claim 20, wherein said bodily tissue is a diseased vascular tissue.

37. A method of preparing a hydrogel composition for vascular associate treatment comprising the steps of: dissolving a quantity of a polysaccharide component in solvent to obtain a first aqueous solution; dissolving a quantity of an oxidizing agent in solvent to obtain a second aqueous solution; combining a volume of said second aqueous solution to said first aqueous solution to oxidize said polysaccharide component and obtain a third aqueous solution; dissolving a quantity of an enzyme catalyst in solvent to obtain a fourth aqueous solution; dissolving a quantity of a protein or polypeptide component to obtain a fifth aqueous solution; adding a quantity of an active agent, a volume of said fourth solution, and a volume of said fifth aqueous solution to said third aqueous solution to obtain a sixth solution;stirring the sixth solution for 5 minutes and incubating the sixth solution at the 37°C for a predetermined time.

38. The method of claim 37 wherein said polysaccharide component is guar gum.

39. The method of claim 37 wherein said oxidizing agent is sodium periodate.

40. The method of claim 37 wherein said enzyme catalyst is microbial transglutaminase.

41. The method of claim 37 wherein said active agent is selected from a PDE5 inhibitor, a PDE inhibitor, a charged vasodilator compound, and an uncharged vasodilator compound.

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