Kit suitable for preparing biocompatible polymer hydrogel

Abstract

The present invention relates to a kit suitable for preparing a biocompatible polymer hydrogel, the kit comprising at least two containers, each container comprising an aqueous composition comprising at least one of tyramine functionalized dextran (Dex-TA), tyramine functionalized hyaluronic acid (HA-TA), a peroxidase and hydrogen peroxide, the present invention relates to a biocompatible polymer hydrogel comprising Dex-TA and HA-TA, which components when mixed are capable of forming said biocompatible polymer hydrogel wherein peroxidase and hydrogen peroxide are each present in different containers wherein the total weight ratio of Dex-TA to HA-TA is between 75: 25 and 25: 75, preferably between 60: 40 and 40: 60, more preferably between 55: 45 and 45: 55; wherein the concentration of Dex-TA is between 2 wt% and 7 wt% relative to the total weight of the aqueous composition in the kit; wherein the concentration of HA-TA varies between 2 wt% and 7 wt% relative to the total weight of the aqueous composition in the kit; wherein Dex-TA comprises unsubstituted dextran repeat units and TA substituted dextran repeat units, and the amount of TA substituted repeat units (DSDEX) in DEX-TA is between 5 mol% and 20 mol% relative to the total number of unsubstituted repeat units and TA substituted repeat units according to proton nuclear magnetic resonance; wherein the HA-TA comprises unsubstituted hyaluronic acid repeat units and TA-substituted hyaluronic acid repeat units, and the amount of TA-substituted repeat units (DSHA) in the HA-TA is between 5 mol% and 20 mol% according to proton nuclear magnetic resonance; wherein the Dex-TA has a weight average molecular weight (Mw) of between 15 kDa and 650 kDa; wherein the HA-TA has a weight average molecular weight (Mw) of between 15 kDa and 550 kDa; wherein the peroxidase is present at a concentration of between 0.25 unit / ml and 10 unit / ml and the hydrogen peroxide is present at a concentration of between 0.01 wt% and 0.10 wt% relative to the total weight of the aqueous composition in the kit.

Description

Technical Field

[0001] The present invention relates to a kit suitable for preparing a biocompatible polymer hydrogel, a method for preparing the biocompatible polymer hydrogel, a mixed polymer composition and the biocompatible polymer hydrogel.

[0002] The present invention also relates to the use of the biocompatible polymer hydrogel of the present invention in repairing damaged cartilage to prevent or inhibit joint pain in humans or animals. Background Art

[0003] Cartilage is a type of connective tissue found in various parts of the body of higher animals, including humans, such as joints between bones and bronchi. It is composed of specialized cells called chondrocytes, which produce a large amount of extracellular matrix composed of collagen fibers, proteoglycans, and elastic fibers. Because cartilage lacks blood vessels (avascular) or nerves (aneurotic), it has a limited ability to regenerate itself. Cartilage can be damaged by a variety of factors, including injury, accidents, and age-related degeneration caused by arthritis, particularly osteoarthritis. Patients with cartilage damage experience joint pain, stiffness, and reduced range of motion, which can affect their daily lives. Functional and physical limitations can also lead to anxiety and depression. In these cases, pain management and total or partial joint replacement are the most common treatments. However, pain management does not address the underlying cause and only provides temporary relief. Joint replacement, on the other hand, is typically reserved for elderly patients and only in the end stage of the disease. For younger patients, joint replacement is generally not recommended due to the limited lifespan of artificial joints. Young patients often develop osteoarthritis due to untreated cartilage damage caused by sports injuries or accidents. For these reasons, finding alternatives to joint replacement surgery and repairing cartilage damage early are crucial for preventing the onset of joint diseases such as osteoarthritis. Therefore, preventive treatments for damaged joints in humans and animals (aimed at preventing the development of osteoarthritis) are under investigation.

[0004] Although there is currently no definitive cure for cartilage defects, tissue engineering is a promising approach for regenerating degenerated or missing cartilage in humans or animals. Tissue engineering approaches typically involve the use of cells placed in a three-dimensional scaffold that acts as a temporary artificial extracellular matrix (ECM). Hydrogels are three-dimensional networks of water-insoluble polymer chains that can hold large amounts of water. They are among the biomaterials designed specifically for use in the human body and are therefore used in biomedical applications. Biocompatible polymer hydrogels can serve as temporary scaffolds to guide the growth of chondrocytes and the differentiation of chondrocytes and / or their progenitor cells, thereby forming new cartilage tissue. The use of biocompatible polymer hydrogels offers several advantages. They can be prepared in situ by mixing polymer compositions and applied locally via minimally invasive surgery. They can fill irregularly shaped defects and facilitate the integration of cells and bioactive molecules.

[0005] Therefore, biocompatible polymer hydrogels have received extensive attention in cartilage tissue engineering in recent years. Typically, a mixed polymer composition is prepared, in which a chemical cross-linking reaction occurs, thereby initiating a gelation process and ultimately forming a biocompatible polymer hydrogel.

[0006] Studies have disclosed the enzymatic cross-linking reaction of dextran-tyramine conjugate (Dex-TA) in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (Jin R. et al., Biomaterials. 2009(13):2544-51 and Jin R. et al., Biomaterials, 2007(18):2791-800).

[0007] WO 2011 / 059326 discloses a kit suitable for preparing a biocompatible polymer hydrogel, which comprises a heparin-tyramine (Hep-TA) conjugate, a dextran-tyramine (Dex-TA) conjugate, hydrogen peroxide and peroxidase.

[0008] The gelation process and the formation of a suitable polymer hydrogel are complex. During the polymerization (gelation) process of a mixed polymer composition, numerous networks are generated. The key to this process is bond formation, which in turn triggers cluster formation. The formation of more clusters ultimately leads to gelation. Methods known in the art for preparing biocompatible polymer hydrogels have several disadvantages. A mixed polymer composition with a gelation time that is too fast can make it difficult to handle the mixed polymer composition during application, as a biocompatible polymer hydrogel may form during injection rather than in situ. A gelation time that is too slow requires a long processing time, making it extremely impractical and cost-ineffective. Due to the formation of the polymer hydrogel network, the volume of the polymer hydrogel can vary significantly due to slight changes in external stimuli (such as temperature), which in turn causes the polymer to swell. If the polymer hydrogel swells excessively, it can be dragged out of the application area due to joint movement. The polymer hydrogel may have a low density in the cross-linked network, resulting in a low storage modulus and excessive degradation, making it impossible to achieve proper cartilage regeneration. In addition, polymer hydrogels may be susceptible to mechanical erosion between two moving joints.

[0009] The kits suitable for preparing biocompatible polymer hydrogels in the prior art and the biocompatible polymer hydrogels in the prior art are still far from being optimal.

[0010] The present invention aims to overcome at least some of these drawbacks. More specifically, the present invention aims to develop an efficient kit for preparing a biocompatible polymer hydrogel, and an efficient biocompatible polymer hydrogel that is advantageously used to repair damaged cartilage to prevent or inhibit joint pain in humans or animals.

[0011] After extensive research, the inventors have solved at least some of the above problems and developed an efficient kit for preparing biocompatible polymer hydrogels. The prepared biocompatible polymer hydrogels can be used to repair damaged cartilage. Summary of the Invention

[0012] The present invention relates to a kit suitable for preparing a biocompatible polymer hydrogel, the kit comprising at least two containers, each container containing an aqueous composition, the aqueous composition comprising at least one of tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide, which components, when mixed, are capable of forming the biocompatible polymer hydrogel.

[0013] Wherein, peroxidase and hydrogen peroxide are respectively present in different containers.

[0014] wherein the total weight ratio of Dex-TA to HA-TA is between 75:25 and 25:75, preferably between 60:40 and 40:60, and more preferably between 55:45 and 45:55;

[0015] wherein the concentration of Dex-TA is between 2 wt % and 7 wt % relative to the total weight of the aqueous composition in the kit;

[0016] wherein the concentration of HA-TA is between 2 wt % and 7 wt % relative to the total weight of the aqueous composition in the kit;

[0017] wherein the DEX-TA comprises unsubstituted glucan repeating units and TA-substituted glucan repeating units, wherein the amount of TA-substituted repeating units in DEX-TA (DS) relative to the total amount of unsubstituted repeating units and TA-substituted repeating units according to proton nuclear magnetic resonance is DEX ) is between 5 mol% and 20 mol%;

[0018] wherein the HA-TA comprises unsubstituted hyaluronic acid repeating units and TA-substituted hyaluronic acid repeating units, wherein the amount of TA-substituted repeating units in HA-TA relative to the total amount of unsubstituted repeating units and TA-substituted repeating units according to proton nuclear magnetic resonance is (D SHA ) is 5 mol% to 20 mol%;

[0019] wherein the DEX-TA has a weight average molecular weight (Mw) between 15 kDa and 650 kDa;

[0020] wherein the HA-TA has a weight average molecular weight (Mw) between 15 kDa and 550 kDa;

[0021] Wherein, relative to the total weight of the aqueous composition in the kit, the peroxidase is present in a concentration between 0.25 units / ml and 10 units / ml, and the hydrogen peroxide is present in a concentration between 0.01 wt % and 0.10 wt %.

[0022] The kit suitable for preparing the biocompatible polymer hydrogel according to the present invention can regenerate cartilage and can be used to repair damaged cartilage to prevent or alleviate joint pain in humans or animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Shown are the gel times (in seconds) of the biocompatible polymer hydrogels of the present invention prepared using 0.03 wt% (dark black) or 0.05 wt% (light black) hydrogen peroxide. The biocompatible polymer hydrogels were prepared using 3 units / ml of peroxidase (HRP).

[0024] Figure 2 The storage modulus (in kilopascals) of the biocompatible polymer hydrogels of the present invention prepared using 0.03 wt% (dark black) or 0.05 wt% (light black) hydrogen peroxide is shown. The biocompatible polymer hydrogels were prepared using 3 units / ml of peroxidase (HRP).

[0025] Figure 3 The swelling behavior of the biocompatible polymer hydrogel of the present invention prepared using 0.03 wt% (dark black) or 0.05 wt% (light black) hydrogen peroxide is shown. The biocompatible polymer hydrogel was prepared using 3 units / ml of peroxidase (HRP).

[0026] Figure 4 The degradation of the biocompatible polymer hydrogel of the present invention prepared using 0.03 wt% (dark black) or 0.05 wt% (light black) hydrogen peroxide over time is shown. The biocompatible polymer hydrogel was prepared using 3 units / ml of peroxidase (HRP).

[0027] Figure 5 Shown are mold pieces used to evaluate the storage modulus and swelling behavior of the biocompatible polymer hydrogels of the present invention. DETAILED DESCRIPTION

[0028] The present invention relates to a kit suitable for preparing a biocompatible polymer hydrogel, the kit comprising at least two containers, each container containing an aqueous composition, the aqueous composition comprising at least one of tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide, which components, when mixed, are capable of forming the biocompatible polymer hydrogel.

[0029] Wherein, peroxidase and hydrogen peroxide are respectively present in different containers.

[0030] wherein the total weight ratio of Dex-TA to HA-TA is between 75:25 and 25:75, preferably between 60:40 and 40:60, and more preferably between 55:45 and 45:55;

[0031] wherein the concentration of Dex-TA is between 2 wt % and 7 wt % relative to the total weight of the aqueous composition in the kit;

[0032] wherein the concentration of HA-TA is between 2 wt % and 7 wt % relative to the total weight of the aqueous composition in the kit;

[0033] wherein the DEX-TA comprises unsubstituted glucan repeating units and TA-substituted glucan repeating units, wherein the amount of TA-substituted repeating units in DEX-TA (DS) relative to the total amount of unsubstituted repeating units and TA-substituted repeating units according to proton nuclear magnetic resonance is DEX ) between 5 mol % and 20 mol %;

[0034] wherein the HA-TA comprises unsubstituted hyaluronic acid repeating units and TA-substituted hyaluronic acid repeating units, wherein the amount of TA-substituted repeating units in HA-TA relative to the total amount of unsubstituted repeating units and TA-substituted repeating units according to proton nuclear magnetic resonance is (D SHA ) between 5 mol % and 20 mol %;

[0035] wherein the DEX-TA has a weight average molecular weight (Mw) between 15 kDa and 650 kDa;

[0036] wherein the HA-TA has a weight average molecular weight (Mw) between 15 kDa and 550 kDa;

[0037] Wherein, relative to the total weight of the aqueous composition in the kit, the peroxidase is present in a concentration between 0.25 units / ml and 10 units / ml, and the hydrogen peroxide is present in a concentration between 0.01% by weight and 0.10% by weight.

[0038] When the aforementioned tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide are mixed, a hybrid polymer composition is obtained in which the gelation process begins, thereby forming a biocompatible polymer hydrogel.

[0039] Feature Details

[0040] As used herein, the term "polymer hydrogel" refers to a three-dimensional hydrophilic polymer network. The polymer hydrogels of the present invention have a high water content and provide an environment similar to natural cartilage. Furthermore, the polymer hydrogels of the present invention effectively transport nutrients and metabolic waste, which is crucial for cell growth. Preferably, the polymer hydrogels of the present invention are formed in situ upon injection of the mixed polymer composition described above.

[0041] The kit of the invention and the mixed polymer composition of the invention comprise a polymer or functionalized polymer selected from a natural polymer, a polymer present in cartilage tissue, a modified natural polymer or a synthetic polymer such as a polyethylene glycol-based polymer.

[0042] The polymer used in the present invention may be gelatin (amide group), collagen, polysaccharides (such as dextran) and glycosaminoglycans (GAGs), including hyaluronic acid, chondroitin sulfate, keratin sulfate, heparin sulfate and chitosan.

[0043] Preferably, the polymers used in the present invention are based on dextran and hyaluronic acid, which are biocompatible and easy to modify, and therefore are excellent template materials for further functionalization.

[0044] As used herein, the term "biocompatible polymer hydrogel" refers to a polymer hydrogel that can come into contact with living systems without producing significant adverse effects. "Biocompatible" in this context means that the material does not induce a significant pathological response in the body to the biomaterial, or that the material is harmless to the patient. The biocompatibility of polymer hydrogels can be assessed using ISO standard tests, particularly with regard to cytotoxicity, irritation, sensitization, acute, subacute, or subchronic systemic toxicity, pyrogenicity, and genotoxicity.

[0045] The kit for preparing biocompatible polymer hydrogels of the present invention comprises at least two containers.

[0046] As used herein, the term "container" refers to an object used to hold or transport an aqueous composition. Any type of container suitable for holding or transporting a substance can be used. Preferably, the container is suitable for holding or transporting a medicinal substance or a substance such as tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide. More preferably, the container used in the kit of the present invention is assembled within an administration device.

[0047] Each container contains an aqueous composition comprising at least one of tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide, while the peroxidase and hydrogen peroxide are placed in different containers to avoid gelation and formation of polymer hydrogel within the application device.

[0048] Tyramine-functionalized dextran (Dex-TA) and tyramine-functionalized hyaluronic acid (HA-TA) are prepared by modifying dextran and hyaluronic acid with tyramine (TA) by coupling reaction, and the method follows steps known in the art (such as Dex-TA reported by R Jin et al. in Journal of Controlled Release 2008; 132: e24-e6, and Dex-TA conjugates and HA-TA conjugates disclosed in WO 2011 / 059326 and WO 2019 / 143247), all of which are incorporated herein by reference.

[0049] Tyramine (TA) has been selected as a reactive side group for enzymatic cross-linking, but other reactive side groups containing hydroxyphenyl groups (such as those present in tyrosine) or dihydroxyphenyl groups (such as those present in dopamine) may also be used. Thanks to the formation of tyramine-tyramine bonds in the presence of catalysts such as horseradish peroxidase (HRP) and oxidants such as hydrogen peroxide (H2O2), tyramine-functionalized dextran (Dex-TA) and tyramine-functionalized hyaluronic acid (HA-TA) are cross-linked in situ in biocompatible polymer hydrogels. Hyaluronic acid is preferred because it is naturally present in joint tissue and synovial fluid, acting as a lubricant and shock absorber in joints. In addition, considering that the degradation characteristics of hydrogels can affect cell morphogenesis and tissue regeneration after implantation, hyaluronic acid is preferred because it is easily degraded by the hydrolytic enzymes (hyaluronidase) in the human body. Therefore, the tyramine-functionalized hyaluronic acid (HA-TA) used in the present invention can not only simulate the natural environment of the joint, but also control the degradation characteristics of the hydrogel.

[0050] As used herein, the term "tyramine functionalized dextran (Dex-TA)" refers to a dextran molecule coupled to a tyramine molecule, wherein the dextran molecule is linked to the tyramine via a carbamate bond, or via any bond that allows coupling of the dextran molecule to tyramine.

[0051] As used herein, the term "tyramine functionalized hyaluronic acid (HA-TA)" refers to a hyaluronic acid molecule coupled to a tyramine molecule, wherein the hyaluronic acid molecule and the tyramine are linked via an amide bond, or via any bond that allows coupling of the hyaluronic acid molecule to the tyramine.

[0052] The functional properties of the biocompatible polymer hydrogels of the present invention are influenced by a variety of factors. Important properties include shear storage modulus, swelling or shrinkage (related to the volume change of the biocompatible polymer hydrogel after in situ formation), glucose permeability, gelation time (related to the time required for the cross-linked polymer to transform from a free-flowing solution to a gel or semi-gel state), viscosity, and the degradability of the biocompatible polymer hydrogel.

[0053] The term "shear storage modulus" as used herein refers to a parameter calculated from the strain and stress measurements obtained when the biocompatible polymer hydrogel is tested in a shear plate-plate geometry. The storage modulus is related to the ability of the biocompatible polymer hydrogel to elastically store energy (energy stored in the elastic structure of the biocompatible polymer hydrogel). Preferably, the storage modulus is high so that the biocompatible polymer hydrogel becomes a strong gel that can withstand all forces that may be encountered in the joint; preferably, the storage modulus should not be too high to allow cells to penetrate and proliferate in the biocompatible polymer hydrogel.

[0054] As used herein, the term "swelling" refers to the increase in volume of a biocompatible polymer hydrogel after in situ formation. As a porous, fluid-saturated material, swelling of the biocompatible polymer hydrogel can be triggered by a variety of stimuli. Swelling can cause the biocompatible polymer hydrogel to deform, potentially squeezing out of the injection area (e.g., a joint) during exercise, leading to displacement of the hydrogel from the defect.

[0055] As used herein, the term "shrinkage" refers to the reduction in volume of the mixed polymer composition converted into a biocompatible polymer hydrogel. Preferably, the shrinkage of the biocompatible polymer hydrogel is small to prevent the biocompatible hydrogel from bulging out of the injection area and causing it to move out of the defect during exercise.

[0056] As used herein, the term "gel time" refers to the time required for a cross-linked polymer to transition from a free-flowing solution to a gel or semi-gel state.

[0057] As used herein, the term "degradation" refers to the mass loss of a biocompatible polymer hydrogel sample when exposed to a 5 U / mL hyaluronidase solution at 37°C. Preferably, the degradation rate should not be too rapid to allow sufficient time for chondrocytes to migrate through the biocompatible polymer hydrogel and produce extracellular matrix. In a preferred embodiment, degradation is less than 50% by weight within the first 30 days after hydrogel formation.

[0058] The inventors have been searching for a hydrogel whose composition balances several unique properties: mechanical stability against intra-articular forces, optimal degradation time to support joint defect healing, and an excellent gelation time that is long enough for injection without requiring an excessively long wait for the gel to solidify within the joint. The kit of claim 1 defines several essential features that must be combined to produce a biocompatible polymer hydrogel useful for treating cartilage defects. Each of these essential features is discussed in detail below, along with preferred ranges for each feature.

[0059] The first factor affecting the functional properties of the biocompatible polymer hydrogel of the present invention is the weight ratio of Dex-TA to HA-TA.

[0060] The inventors have discovered that using only HA-TA to prepare biocompatible polymer hydrogels results in poor mechanical properties. Using only Dex-TA results in no in situ degradation or very slow degradation. Conversely, the inventors have discovered that when the total weight ratio of Dex-TA to HA-TA in the kit of the present invention is between 75:25 and 25:75, preferably between 60:40 and 40:60, and more preferably between 55:45 and 45:55, the biocompatible polymer hydrogels of the present invention can achieve acceptable functional properties.

[0061] The inventors of the present invention have found that when the weight ratio of Dex-TA / HA-TA is between 75:25 and 25:75, preferably between 60:40 and 40:60, and more preferably when the weight ratio of Dex-TA / HA-TA is between 55:45 and 45:55, the storage modulus and swelling / shrinkage of the biocompatible polymer hydrogel are improved. The obtained biocompatible polymer hydrogel is able to withstand forces within the injection area (such as a joint) and provide a suitable environment for cell migration and proliferation. The inventors have found that if the weight ratio of Dex-TA / HA-TA exceeds the above range, the biocompatible polymer hydrogel is either too soft to withstand the forces or too hard to hinder cell migration into the hydrogel.

[0062] Regarding the effect of the Dex-TA / HA-TA weight ratio on the degradation of the biocompatible polymer hydrogel of the present invention, the inventors noted that degradation was slower when the Dex-TA / HA-TA weight ratio was between 75:25 and 25:75, preferably between 60:40 and 40:60, and more preferably between 55:45 and 45:55. This slow degradation provides ample time for cells from surrounding tissue to migrate into the hydrogel, proliferate, and generate new extracellular matrix while repairing the defect.

[0063] Regarding the effect of the weight ratio of Dex-TA / HA-TA on the gelation time of the biocompatible polymer hydrogel of the present invention, the inventors noted that when the weight ratio of Dex-TA / HA-TA is between 75:25 and 25:75, preferably between 60:40 and 40:60, and more preferably when the weight ratio of Dex-TA / HA-TA is between 55:45 and 45:55, the surgeon has enough time to inject the hydrogel into the defect site without having to wait too long for its gel to solidify.

[0064] The second factor is the concentration of Dex-TA and HA-TA relative to the total weight of the aqueous composition in the kit. The inventors have found that acceptable functional properties can be achieved for the biocompatible polymer hydrogels of the present invention when the concentration of Dex-TA is between 2% and 7% by weight, preferably between 3% and 6% by weight, and more preferably between 3% and 5% by weight, relative to the total weight of the aqueous composition in the kit, and the concentration of HA-TA is between 2% and 7% by weight, preferably between 3% and 6% by weight, and more preferably between 3% and 5% by weight, relative to the total weight of the aqueous composition in the kit. The inventors studied the storage modulus and swelling or shrinkage properties of the biocompatible polymer hydrogels. Furthermore, the degradation of the biocompatible polymer hydrogels within the first 30 days after hydrogel formation, gelation time, and viscosity were also studied.

[0065] Table 1 reproduces the data and observed effects related to the concentrations of Dex-TA and HA-TA on the functional properties of the biocompatible polymer hydrogels.

[0066] Table 1

[0067]

[0068]

[0069] The inventors discovered that if the concentration of Dex-TA and HA-TA falls below 2 wt%, the storage modulus of the biocompatible polymer hydrogel decreases to an unmeasurable level because the gel disintegrates. In effect, the gel is too soft to withstand the forces applied to the injection site, such as a joint.

[0070] When the concentrations of Dex-TA and HA-TA were both higher than 2 wt % or lower than 7 wt %, the storage modulus was measurable, and the biocompatible polymer hydrogel was able to withstand some forces within the injection area (e.g., joints) and provide a suitable environment for cell migration and proliferation.

[0071] When the concentration of Dex-TA and HA-TA was higher than 7 wt%, the hydrogel network structure could not form properly. The viscosity of the polymer solution increased, hindering the cross-linking process of the hydrogel.

[0072] When the concentration of Dex-TA and HA-TA was between 3 wt% and 5 wt%, the storage modulus was optimal, and the biocompatible polymer hydrogel was able to withstand forces within the injection area (such as the joint) and provide an excellent environment for cell migration and proliferation.

[0073] The second important factor is the shrinkage properties of the hydrogel during use. The inventors found that if the concentration of Dex-TA and HA-TA is below 2% by weight, the shrinkage of the hydrogel is too high to be measurable because the gel disc used for measurement is damaged by the hydrogel, and the deformation of the hydrogel causes it to fall away from the injection area.

[0074] When the concentrations of Dex-TA and HA-TA were both higher than 2 wt % or lower than 7 wt %, swelling / shrinkage was measurable and the deformation of the biocompatible polymer hydrogel was within an acceptable range.

[0075] When the concentration of Dex-TA and HA-TA was higher than 7 wt %, the hydrogel swelled too much, causing it to deform and bulge out from the injection area during exercise.

[0076] When the concentration of Dex-TA and HA-TA was between 3 wt% and 5 wt%, the hydrogel neither swelled nor bulged excessively, nor did it shrink excessively. The biocompatible polymer hydrogel did not dislodge or detach from the injection area.

[0077] Another important factor is the degradation of the hydrogel during use, allowing for the regeneration of healthy cartilage tissue within the joint. The inventors anticipate that if the concentration of Dex-TA and HA-TA is below 2% by weight, the biocompatible polymer hydrogel will degrade too rapidly within the first 30 days after formation, resulting in the gel degrading before cartilage regeneration occurs. This product would be ineffective for cartilage repair.

[0078] When the concentrations of Dex-TA and HA-TA are both above 2% by weight or below 7% by weight, the degradation rate is acceptable, providing sufficient time for chondrocytes to migrate within the biocompatible polymer hydrogel and produce extracellular matrix. Therefore, this product is effective for cartilage repair.

[0079] When the concentration of Dex-TA and HA-TA is higher than 7% by weight, the degradation rate is very slow. Due to the slow degradation of the polymer network, the healing time of the defect will be prolonged. Therefore, the effect of this product on cartilage repair will be poor.

[0080] When the concentration of Dex-TA and HA-TA is between 3% and 5% by weight, the degradation rate is moderate, providing ample time for cells from surrounding tissue to migrate into the hydrogel, proliferate, and produce new extracellular matrix while repairing the defect. This product has a significant effect on cartilage repair.

[0081] Another important factor is the gelation of the liquid composition into a hydrogel after injection into the joint. The inventors found that when the concentration of Dex-TA and HA-TA was less than 2% by weight, the gelation time was 25 seconds. They also noted that the transition from liquid to solid was very abrupt, which was a problem for surgeons applying the hydrogel.

[0082] Ideally, the hydrogel should gel quickly enough for surgeons to administer, but also slowly enough so that treatment can be completed within a reasonable timeframe. This ideal situation was observed when the concentrations of both Dex-TA and HA-TA were above 2 wt% or below 7 wt%, and particularly when the concentrations of Dex-TA and HA-TA were between 3 and 5 wt%, providing surgeons with ample time to inject the gel into the defect without having to wait too long for the gel to solidify.

[0083] The inventors of the present invention have found that if the concentration of Dex-TA and HA-TA is lower than 2 wt %, the viscosity of the hydrogel is very low and the hydrogel flows out from the injection site.

[0084] When the concentrations of Dex-TA and HA-TA were both higher than 2 wt % or 7 wt %, the viscosity of the hydrogel was within an acceptable range and the hydrogel did not flow out from the injection site.

[0085] When the concentration of Dex-TA and HA-TA was higher than 7 wt %, the viscosity was too high to prevent gel formation and mixing problems occurred.

[0086] When the concentration of Dex-TA and HA-TA is between 3 and 5 weight percent, the viscosity avoids mixing problems while allowing the gel to be easily ejected from a syringe as a liquid without applying excessive force. This makes it easier for surgeons to administer the hydrogel by injection. Furthermore, the gel has proven to be secure at the defect site, with no risk of leakage from the injection area.

[0087] When DEX-TA and / or HA-TA are present in the container, the concentration of each of DEX-TA and HA-TA in the container is preferably between 2-10 wt%.

[0088] The third factor is the degree of substitution (DS) of tyramine on dextran and hyaluronic acid. The term "degree of substitution (DS)" with respect to dextran and hyaluronic acid refers to the (average) number of substituent (TA) groups attached per repeating unit, taking into account that the repeating units of dextran are monosaccharides and those of hyaluronic acid are disaccharides.

[0089] The inventors have found that when DEX-TA comprises unsubstituted glucan repeating units and TA-substituted glucan repeating units, wherein the amount of TA-substituted repeating units in DEX-TA (DS DEX ) is between 5 mol % and 20 mol %, preferably between 6 mol % and 17 mol %, more preferably between 7 mol % and 15 mol %, even more preferably between 7.5 mol % and 12 mol %, and even further more preferably between 7.5 mol % and 10.5 mol %, the biocompatible polymer hydrogel of the present invention can be obtained with acceptable functional properties.

[0090] When the degree of substitution (DS DEX ) is between 5 mol % and 20 mol %, the storage modulus of the hydrogel enables the hydrogel to withstand forces within the injection site (such as a joint) and provide a favorable environment for cell migration and proliferation.

[0091] The inventors anticipate that if the DEX-TA (DS DEX ) Beyond the above range, the biocompatible polymer hydrogel may be too soft to withstand the force and be squeezed out from the injection site; or too hard to hinder cell migration into the hydrogel. DEXIf the concentration is too high, the product will become insoluble in water.

[0092] Discover when DS DEX When the content is less than 5 mol%, the degradation of biocompatible polymer hydrogel is slightly slowed down; while DS DEX Above 12 mol %, the formation of biocompatible polymer hydrogels is very slow. Ideally, degradation should not be too rapid to allow sufficient time for cells from the surrounding tissue to migrate into the hydrogel and proliferate and produce new extracellular matrix while repairing the defect.

[0093] The inventors also found that when HA-TA comprises unsubstituted hyaluronic acid repeating units and TA-substituted hyaluronic acid repeating units, wherein the amount of TA-substituted repeating units in HA-TA (DS 1 ) relative to the total amount of unsubstituted repeating units and TA-substituted repeating units according to proton nuclear magnetic resonance is 1 . HA ) between 5 mol % and 20 mol %, preferably between 6 mol % and 17 mol %, more preferably between 7 mol % and 15 mol %, even more preferably between 10 mol % and 14.5 mol %, may provide acceptable functional properties.

[0094] The fourth factor is the weight-average molecular weight (Mw) of DEX-TA and HA-TA. The inventors have found that acceptable functional properties can be achieved for the biocompatible polymer hydrogel of the present invention when DEX-TA has a weight-average molecular weight (Mw) between 15 kDa and 650 kDa, preferably between 20 kDa and 125 kDa, and more preferably between 30 kDa and 60 kDa, and HA-TA has a weight-average molecular weight (Mw) between 15 kDa and 550 kDa, preferably between 15 kDa and 300 kDa, and more preferably between 15 kDa and 60 kDa.

[0095] Furthermore, the preparation process of biocompatible polymer hydrogels is affected by a variety of factors. In order to obtain an efficient process, the viscosity, injectability, and gelation time of the mixed polymer composition need to be considered in particular.

[0096] As used herein, the term "injectable" means that the mixed polymer composition can be introduced into the body using an injection administration device that may have a needle.

[0097] The first factor to consider when preparing the biocompatible polymer hydrogels of the present invention is the amount of peroxidase. The inventors have discovered that an efficient process for producing the biocompatible polymer hydrogels of the present invention can be achieved when the peroxidase is used at a concentration between 0.25 units / ml and 10 units / ml, relative to the total aqueous composition in the kit. Preferably, the peroxidase is used at a concentration between 1 unit / ml and 5 units / ml, more preferably between 2 units / ml and 4 units / ml, and even more preferably between 2 units / ml and 3.5 units / ml, relative to the total aqueous composition in the kit.

[0098] The term "units / ml" (also known as "pyrogallol units / ml"), as used with respect to peroxidase, refers to the concentration of peroxidase expressed in "activity units," which correlates to enzyme activity, a factor controlling the formation of biocompatible polymer hydrogels. Specifically, it refers to the amount of enzyme required to form 1.0 mg of purpurogallin from pyrogallol in 20 seconds, as measured spectrophotometrically at pH 6.0 and 20°C.

[0099] Regarding the effect of the amount of peroxidase on the gelation time of the mixed polymer composition of the present invention, the inventors monitored the gelation process of the mixed polymer composition and observed that reducing the amount of peroxidase would prolong the gelation time, thereby improving the operability and practicality of the kit of the present invention. Figure 1 As shown, when a peroxidase concentration of 3 units / ml is used relative to the total aqueous composition in the kit, an optimal gelation time ranging from 35 seconds to 60 seconds can be obtained. This gelation time improves the operability and practicality of the kit of the present invention.

[0100] The amount of peroxidase also affects the storage modulus (e.g. Figure 2 ) and the swelling / shrinkage properties of the biocompatible polymer hydrogel of the present invention (as shown in Figure 3 The results show that the peroxidase concentration of 3 units / ml relative to the total aqueous composition in the kit has an effect on the hydrogel. When a peroxidase concentration of 3 units / ml is used, an optimal storage modulus between 30 kPa and 60 kPa is achieved. Furthermore, a peroxidase concentration of 3 units / ml relative to the total aqueous composition in the kit prevents hydrogel swelling and achieves an optimal shrinkage between -10% and 10%.

[0101] Any peroxidase known to the skilled person and suitable for preparing biocompatible polymer hydrogels can be used. Preferably, the peroxidase used in the present invention is selected from horseradish peroxidase (HRP), soybean peroxidase, myeloperoxidase, lactoperoxidase, Nitrospira sp. recombinant peroxidase and Lyngbya sp. recombinant lysyloxidase. Preferably, the peroxidase used in the present invention is horseradish peroxidase (HRP).

[0102] The second factor is the amount of hydrogen peroxide. The inventors have discovered that an efficient process for obtaining the biocompatible polymer hydrogel of the present invention is achieved when hydrogen peroxide is used at a concentration between 0.01% and 0.10% by weight, relative to the total weight of the aqueous composition in the kit. Preferably, the hydrogen peroxide is used at a concentration between 0.02% and 0.07% by weight, more preferably between 0.025% and 0.06%, and even more preferably between 0.03% and 0.055% by weight, relative to the total weight of the aqueous composition in the kit.

[0103] Regarding the effect of the amount of hydrogen peroxide on the gelation time of the mixed polymer composition, the inventors found that when a concentration of 0.17% by weight of hydrogen peroxide was used relative to the total amount of the aqueous composition in the kit, the mixed polymer composition would gel in 13 minutes. Reducing the hydrogen peroxide concentration to 0.1% by weight relative to the total amount of the aqueous composition in the kit resulted in a gelation time of 197 seconds. Further reducing the hydrogen peroxide concentration to 0.01% by weight relative to the total amount of the aqueous composition in the kit resulted in a gelation time of only 14 seconds. When a concentration of 0.05% by weight of hydrogen peroxide was used relative to the total amount of the aqueous composition in the kit, an optimal gelation time between 35 seconds and 60 seconds was obtained (e.g., Figure 1 The optimal gelation time improves the operability and practicality of the kit of the present invention.

[0104] In addition, if Figure 2 and Figure 3 As shown, the biocompatible polymer hydrogel of the present invention exhibits optimal storage modulus and swelling / contraction properties when hydrogen peroxide is used at a concentration of 0.05 wt%. The optimal storage modulus, between 30 kPa and 60 kPa, allows the biocompatible polymer hydrogel to withstand the forces of an injection site (e.g., a joint), while remaining flexible enough to allow cells to migrate and proliferate within the hydrogel. Furthermore, when hydrogen peroxide is used at a concentration of 0.05 wt%, relative to the total aqueous composition in the kit, the hydrogel avoids swelling and achieves an optimal shrinkage between -10% and 10%.

[0105] Therefore, when using a concentration of 0.05 wt% hydrogen peroxide, the biocompatible polymer hydrogel has an appropriate stiffness, making it less susceptible to mechanical wear between two active joints. At the same time, the hydrogel exhibits an optimal density, allowing chondrocytes to migrate into and grow within the biocompatible polymer hydrogel, thereby achieving cartilage regeneration.

[0106] Finally, the best biocompatible polymer hydrogels were stably obtained when using a concentration of 0.05 wt % hydrogen peroxide, e.g. Figure 4 It is shown to be characterized by slow degradation, which provides time for chondrocytes to migrate and grow within the biocompatible polymer hydrogel.

[0107] In summary, the inventors found that the amount of peroxidase and hydrogen peroxide affects the gelation time of the mixed polymer composition, as well as the storage modulus and swelling / shrinkage (measured by gap size) of the biocompatible polymer hydrogel. Therefore, as shown in Table 2, the effects of both were evaluated at a fixed polymer concentration of 8.9 wt% (4.45 wt% Dex-TA + 4.45 wt% HA-TA). The gelation time was measured at a temperature of 18-22°C, and the storage modulus and gap size were measured at a temperature of 20±0.1°C, while considering various combinations of peroxidase (HRP) and hydrogen peroxide concentrations.

[0108]

[0109]

[0110] Table 2 shows that the combination of a high hydrogen peroxide concentration (maximum) and a low peroxidase (HRP) concentration (minimum) results in very slow gelation, which requires a long operation time and is therefore extremely impractical and cost-effective. The combination of a low hydrogen peroxide concentration (minimum) and a high peroxidase (HRP) concentration (maximum) results in very rapid gelation, reducing the operability of the kit of the present invention and causing inconvenience in its use.

[0111] Furthermore, low hydrogen peroxide (minimum) concentrations lead to the formation of a less dense cross-linked network, as evidenced by a lower storage modulus and a higher degree of swelling, which can lead to the gel being carried away from the injection area (eg, a joint) by movement.

[0112] In addition, the biocompatible polymer hydrogel prepared by the present invention exhibits a relatively low storage modulus compared to the biomaterials and biocompatible polymer hydrogels of the prior art, and because it does not swell, it is not easily mechanically taken out or removed between two movable joints, while having a good structural support density. In addition, the degradation time of the polymer hydrogel is slower, enabling sufficient structural support to be provided at the defect site, thereby providing more time for chondrocyte migration and growth to regenerate new cartilage. Therefore, relative to the total amount of the aqueous composition in the kit, when the concentration of peroxidase is between 0.25 units / ml and 10 units / ml and the concentration of hydrogen peroxide is between 0.01% by weight and 0.10% by weight, the kit of the present invention obtains an advantageous biocompatible hydrogel / cell construct. Preferably, relative to the total amount of the aqueous composition in the kit, when the concentration of the peroxidase is between 1 and 5 units / ml, the concentration of hydrogen peroxide is between 0.02 wt % and 0.07 wt %; more preferably, when the concentration of the peroxidase is between 2 units / ml and 4 units / ml, the concentration of hydrogen peroxide is between 0.025 wt % and 0.06 wt %; even more preferably, when the concentration of the peroxidase is between 2 units / ml and 3.5 units / ml, the concentration of hydrogen peroxide is between 0.03 wt % and 0.055 wt %.

[0113] A preferred embodiment of the present invention relates to a kit suitable for preparing a biocompatible polymer hydrogel, the kit comprising at least two containers, each container containing an aqueous composition, the aqueous composition comprising at least one of tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide, which components, when mixed, are capable of forming the biocompatible polymer hydrogel.

[0114] wherein peroxidase and hydrogen peroxide are each present in a different container;

[0115] Among them, the total weight ratio of Dex-TA to HA-TA is between 55:45 and 45:55;

[0116] wherein the concentration of Dex-TA is between 3 wt % and 5 wt % relative to the total amount of the aqueous composition in the kit;

[0117] wherein the concentration of HA-TA is between 3 wt % and 5 wt % relative to the total amount of the aqueous composition in the kit;

[0118] The Dex-TA comprises unsubstituted glucan repeating units and TA-substituted glucan repeating units, wherein the amount of TA-substituted repeating units in DEX-TA (DS) relative to the total amount of unsubstituted repeating units and TA-substituted repeating units according to proton nuclear magnetic resonance is DEX ) between 7.5 mol % and 10.5 mol %;

[0119] Wherein, the HA-TA comprises unsubstituted hyaluronic acid repeating units and TA-substituted hyaluronic acid repeating units, wherein the amount of TA-substituted repeating units in HA-TA (DS) relative to the total amount of unsubstituted repeating units and TA-substituted repeating units according to proton nuclear magnetic resonance is HA ) between 10 mol % and 14.5 mol %;

[0120] wherein the Dex-TA has a weight average molecular weight (Mw) between 30 kDa and 60 kDa;

[0121] wherein the HA-TA has a weight average molecular weight (Mw) between 15 kDa and 60 kDa;

[0122] The peroxidase is present in a concentration between 2 units / ml and 3.5 units / ml, and the hydrogen peroxide is present in a concentration between 0.03% by weight and 0.055% by weight relative to the total amount of the aqueous composition in the kit.

[0123] The inventors of the present invention have observed that the kit described above provides an ideal biocompatible polymer hydrogel with a storage modulus of about 30-60 kPa, which can withstand the forces in the injection area (such as a joint) and provide an ideal environment for cell migration and proliferation. Its swelling / contraction is about -10% to 10%, so there will be no excessive swelling or contraction, and the hydrogel will not fall off or separate from the injection area during joint movement. The biocompatible polymer hydrogel also exhibits excellent degradation properties within the first 30 days after formation. The degradation rate is not too fast, thereby providing sufficient time for cells to migrate from the surrounding tissue to the inside of the hydrogel and proliferate and produce new extracellular matrix while repairing the defect.

[0124] The gelation time (i.e., the time required for the cross-linked polymer to transform from a free-flowing solution to a gel or semi-gel state) is between 40 and 50 seconds, which provides ample time for surgeons to inject the hydrogel into the defect without having to wait for the hydrogel to solidify. Finally, the hydrogel has a viscosity of approximately 18-20 mPa·s, so it can be pushed out of the syringe as a liquid without mixing issues or the need to apply excessive force during injection / application. Furthermore, the hydrogel remains firmly in place at the defect site without the risk of it flowing out.

[0125] In the kit of the present invention as described above, the aqueous composition in each container contains between 80% and 99.9% by weight, preferably between 83% and 99.8% by weight, more preferably between 86% and 99.8% by weight, and most preferably between 89% and 99.8% by weight of water, water saline solution, phosphate buffer solution or phosphate buffered saline solution.

[0126] As used herein, the term "aqueous saline solution" refers to a salt solution (also known as normal saline) made by mixing sodium chloride (salt) and water, commonly used in medicine.

[0127] As used herein, the term "phosphate buffered saline" (also known as PBS) refers to a buffered solution having a pH of approximately 7.4.

[0128] As used herein, the term "phosphate-buffered saline solution" refers to a water-based saline solution with a pH of approximately 7.4, containing salts such as disodium hydrogen phosphate, sodium chloride, potassium chloride, or potassium dihydrogen phosphate. This buffer helps maintain a constant pH, and the osmotic pressure and ion concentration of the solution match those of the human body (isotonic).

[0129] Water or aqueous saline solutions or phosphate buffered solutions or phosphate buffered saline solutions are commonly used in medicine.

[0130] Typically, Dex-TA and HA-TA can be added to water, an aqueous saline solution, a phosphate buffer solution, or a phosphate buffered saline solution (preferably a phosphate buffered saline solution having a pH of approximately 7.4) to obtain a polymer solution. Peroxidase can also be dissolved in water, an aqueous saline solution, a phosphate buffer solution, or a phosphate buffered saline solution to obtain a peroxidase solution of the desired concentration. A hydrogen peroxide solution can be prepared, preferably freshly, in water, an aqueous saline solution, a phosphate buffer solution, or a phosphate buffered saline solution to obtain a hydrogen peroxide solution of the desired concentration.

[0131] A preferred embodiment of the present invention relates to a kit comprising a first container, wherein the first container comprises an aqueous composition A, wherein the aqueous composition A comprises

[0132] a. Tyramine functionalized dextran (Dex-TA),

[0133] b. Tyramine functionalized hyaluronic acid (HA-TA),

[0134] c. Peroxidase;

[0135] And, wherein the second container contains an aqueous composition B, which comprises

[0136] d. hydrogen peroxide, and

[0137] e. optionally at least one of tyramine-functionalized dextran (Dex-TA) and tyramine-functionalized hyaluronic acid (HA-TA), preferably comprising both Dex-TA and HA-TA,

[0138] wherein the total weight ratio of Dex-TA to HA-TA is between 75:25 and 25:75, preferably between 60:40 and 40:60, and more preferably between 55:45 and 45:55;

[0139] wherein the concentration of Dex-TA is between 2 wt % and 7 wt % relative to the total weight of the aqueous composition in the kit;

[0140] wherein the concentration of HA-TA is between 2 wt % and 7 wt % relative to the total weight of the aqueous composition in the kit;

[0141] Wherein, the DEX-TA comprises unsubstituted glucan repeating units and TA-substituted glucan repeating units. According to proton nuclear magnetic resonance, the amount of TA-substituted repeating units in DEX-TA (DS DEX ) between 5 mol % and 20 mol %;

[0142] Wherein, the HA-TA comprises unsubstituted hyaluronic acid repeating units and TA-substituted hyaluronic acid repeating units. According to proton nuclear magnetic resonance, the amount of TA-substituted repeating units in HA-TA (DS HA ) is between 5 mol% and 20 mol%;

[0143] wherein the Dex-TA has a weight average molecular weight (Mw) between 15 kDa and 650 kDa;

[0144] wherein the HA-TA has a weight average molecular weight (Mw) between 15 kDa and 550 kDa;

[0145] Wherein, relative to the total weight of the aqueous composition in the kit, the peroxidase is present at a concentration of 0.25 units / ml to 10 units / ml, and the hydrogen peroxide is present at a concentration of 0.01 weight % to 0.10 weight %.

[0146] Unless otherwise stated below, the detailed description of the features of the invention provided directly under the heading "Detailed description of features" also applies to this preferred embodiment.

[0147] In this embodiment, the kit of the present invention comprises two containers, the first container comprising the aqueous composition A as described above, and the second container comprising the aqueous composition B as described above.

[0148] The volume to volume ratio of aqueous composition A to aqueous composition B is in the range of 0.1:1 to 10:1, preferably in the range of 0.5:1 to 7:1, more preferably in the range of 0.8:1 to 5:1.

[0149] Composition A and composition B in the kit as described above are both fluid in the temperature range of 0°C to 37°C, and have a viscosity between 1 mPa·s and 50 Pa·s, preferably between 1 mPa·s and 100 mPa·s, more preferably less than 50 mPa·s, and even more preferably less than 35 mPa·s, as measured in a rheometer with a double gap device (low viscosity) or a cone-plate device (high viscosity) at 25±0.1°C and a shear rate of 10 Hz as required.

[0150] As used herein, the term "fluid" refers to a substance (eg, a liquid) whose molecules are free to move relative to each other, which is fluid, has no fixed shape, conforms to the shape of its container, and has little resistance to external stresses.

[0151] In the kit of this embodiment of the present invention as described above, composition A or composition B each contains between 80% and 99.9% by weight, preferably between 83% and 99.8% by weight, more preferably between 86% and 99.8% by weight, and most preferably between 89% and 99.8% by weight of water, saline solution, phosphate buffer solution or phosphate-buffered saline solution.

[0152] The terms "saline solution," "phosphate buffer solution," and "phosphate-buffered saline solution" have the same definitions as described above. Methods for preparing Dex-TA, HA-TA, hydrogen peroxide, and peroxidase (e.g., HRP) solutions using water, saline solution, phosphate buffer solution, or phosphate-buffered saline solution are also the same as described above.

[0153] In one embodiment, composition A is prepared by mixing Dex-TA, HA-TA, and peroxidase solution in phosphate-buffered saline solution in a first container; and composition B is prepared by preparing a phosphate-buffered saline solution containing only hydrogen peroxide solution in a second container, or by mixing a phosphate-buffered saline solution of hydrogen peroxide solution with a phosphate-buffered saline solution of Dex-TA and HA-TA solution.

[0154] The present invention can use any type of assembly kit known to those skilled in the art, which is suitable for preparing biocompatible polymer hydrogels and comprises at least two containers. Preferably, the outlets of the containers are connected to a mixing element (such as a static mixer) to effectively mix the contents of the different containers.

[0155] Preferably, the kit of the present invention is an injection kit. Common commercially available injection kits can be used, such as Nordson Medical Applicator Assembly device or Sulzer Medmix K-system.

[0156] Preferably, the kit has an independent outlet for each container to reduce the risk of cross-contamination during use. Preferably, the contents of each container are injected simultaneously, preferably mixed before leaving the kit. In a preferred embodiment, the kit for preparing a biocompatible polymer hydrogel is a syringe. The kit for preparing a biocompatible polymer hydrogel according to the present invention can also be marketed as a prefilled syringe.

[0157] The kit of the present invention as described above is applied to a surgical site or an incision site to repair damaged cartilage, thereby preventing or suppressing joint pain in humans or animals.

[0158] Administration by injection allows for the formation of microgels and macrogels in situ at the injection site (e.g., joint space). For example, surgery on areas such as joint spaces can be performed through open or arthroscopic incisions; the incisions need to be made based on the size and location of the defect. Retractors are usually used to visualize the defect. All rough and loose defect cartilage is removed, and the size and depth of the defect are determined. The defect is carefully debrided to remove any cracks or degenerated cartilage. The defect is then placed horizontally and allowed to dry. The biocompatible polymer hydrogel is then carefully injected until the edges of the defect are filled. After a few minutes, the stability of the biocompatible polymer hydrogel is checked by gently moving the application area, for example, if it is applied to the knee joint, this can be checked by bending the knee. The incision is then sutured according to standard procedures.

[0159] The present invention also relates to a method for preparing a biocompatible polymer hydrogel, comprising the following steps:

[0160] a. mixing an aqueous composition comprising tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide to obtain a mixed polymer composition;

[0161] b. allowing the mixed polymer composition to form a biocompatible polymer hydrogel at a temperature between 15 and 38° C. for a period of time between 10 and 200 seconds,

[0162] wherein the total weight ratio of Dex-TA to HA-TA is between 75:25 and 25:75, preferably between 60:40 and 40:60, and more preferably between 55:45 and 45:55;

[0163] wherein the concentration of Dex-TA is between 2 wt % and 7 wt % relative to the mixed polymer composition;

[0164] wherein the concentration of HA-TA is between 2 wt % and 7 wt % relative to the mixed polymer composition;

[0165] Dex-TA comprises unsubstituted dextran repeating units and TA-substituted dextran repeating units, wherein the amount of TA-substituted repeating units in DEX-TA (DS DEX ) is between 5 mol% and 20 mol%;

[0166] HA-TA comprises unsubstituted hyaluronic acid repeating units and TA-substituted hyaluronic acid repeating units, wherein the amount of TA-substituted repeating units in HA-TA (DS HA ) is between 5 mol% and 20 mol%;

[0167] wherein Dex-TA has a weight average molecular weight (Mw) between 15 kDa and 650 kDa;

[0168] wherein HA-TA has a weight average molecular weight (Mw) between 15 kDa and 550 kDa;

[0169] The peroxidase is present in a concentration of 0.25 to 10 units / ml, and the hydrogen peroxide is present in a concentration of 0.01 to 0.10 wt % relative to the mixed polymer composition.

[0170] Unless otherwise stated below, the detailed description of features of the present invention provided directly under the heading "Detailed Description of Features" also applies to the present embodiment.

[0171] The method for preparing a biocompatible polymer hydrogel comprises mixing an aqueous composition comprising tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide to obtain a mixed polymer composition. Mixing can be performed by procedures known in the art. Preferably, mixing is performed by a mixing connector that can be connected to an administration device or a cannula with a static mixer.

[0172] The inventors found that using the above concentrations of Dex-TA and HA-TA, combined with a specific weight ratio of Dex-TA to HA-TA, a specific DS DEX and DS HA, Dex-TA and HA-TA of specific weight-average molecular weight, and the above-mentioned specific concentrations of peroxidase and hydrogen peroxide, at a temperature between 15 and 38°C, can form a biocompatible polymer hydrogel within a time period between 10 and 200 seconds, preferably between 20 and 100 seconds, and more preferably between 30 and 60 seconds. Specifically, when the process is performed in vivo, the temperature is between 35 and 38°C; when the process is performed in vitro, the temperature is between 18 and 22°C, preferably between 17 and 20°C.

[0173] In a preferred embodiment, the present invention relates to a method for preparing a biocompatible polymer hydrogel, the method comprising the following steps:

[0174] a. The aqueous composition A and the aqueous composition B are mixed to obtain a mixed polymer composition;

[0175] b. forming a biocompatible polymer hydrogel at a temperature of 15 to 38 ° C. over a period of time between 10 and 200 seconds;

[0176] The aqueous composition A comprises tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and

[0177] wherein the aqueous composition B comprises hydrogen peroxide, and optionally at least one of tyramine-functionalized dextran (Dex-TA) and tyramine-functionalized hyaluronic acid (HA-TA), preferably comprising both Dex-TA and HA-TA;

[0178] wherein the total weight ratio of Dex-TA to HA-TA is between 75:25 and 25:75, preferably between 60:40 and 40:60, and more preferably between 55:45 and 45:55;

[0179] wherein the concentration of Dex-TA is between 2 wt % and 7 wt % relative to the sum of composition A and composition B;

[0180] wherein the concentration of HA-TA is between 2 wt % and 7 wt % relative to the sum of composition A and composition B;

[0181] Dex-TA comprises unsubstituted dextran repeating units and TA-substituted dextran repeating units, wherein the amount of TA-substituted repeating units in DEX-TA (DS DEX ) is between 5 mol% and 20 mol%;

[0182] HA-TA comprises unsubstituted hyaluronic acid repeating units and TA-substituted hyaluronic acid repeating units, wherein the amount of TA-substituted repeating units in HA-TA (DS HA ) is between 5 mol% and 20 mol%;

[0183] wherein Dex-TA has a weight average molecular weight (Mw) between 15 kDa and 650 kDa;

[0184] wherein HA-TA has a weight average molecular weight (Mw) between 15 kDa and 550 kDa;

[0185] The peroxidase is present at a concentration between 0.25 units / ml and 10 units / ml relative to the total of composition A and composition B; and the hydrogen peroxide is present at a concentration between 0.01% by weight and 0.10% by weight.

[0186] Unless otherwise stated below, the detailed description of the features of the invention provided directly under the heading "Detailed description of features" also applies to this preferred embodiment.

[0187] The present invention also relates to a hybrid polymer composition obtained by mixing an aqueous composition comprising tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide.

[0188] wherein the total weight ratio of Dex-TA to HA-TA is between 75:25 and 25:75, preferably between 60:40 and 40:60, and more preferably between 55:45 and 45:55;

[0189] wherein the concentration of Dex-TA is between 2 wt % and 7 wt % relative to the mixed polymer composition;

[0190] wherein the concentration of HA-TA is between 2 wt % and 7 wt % relative to the mixed polymer composition;

[0191] Dex-TA comprises unsubstituted dextran repeating units and TA-substituted dextran repeating units, wherein the amount of TA-substituted repeating units in DEX-TA (DS DEX ) is between 5 mol% and 20 mol%;

[0192] HA-TA comprises unsubstituted hyaluronic acid repeating units and TA-substituted hyaluronic acid repeating units, wherein the amount of TA-substituted repeating units in HA-TA (DS HA ) is between 5 mol% and 20 mol%;

[0193] wherein Dex-TA has a weight average molecular weight (Mw) between 15 kDa and 650 kDa;

[0194] wherein HA-TA has a weight average molecular weight (Mw) between 15 kDa and 550 kDa;

[0195] wherein the peroxidase is present at a concentration between 0.25 units / ml and 10 units / ml, and the hydrogen peroxide is present at a concentration between 0.01% and 0.10% by weight relative to the mixed polymer composition.

[0196] Unless otherwise stated below, the detailed description of the features of the invention provided directly under the heading "Detailed description of features" also applies to this preferred embodiment.

[0197] The inventors have found that if the mixed polymer composition gels too quickly, it will be difficult to apply easily to the surgical site or incision site. The inventors have found that when the above concentrations of Dex-TA and HA-TA are combined with a specific weight ratio of Dex-TA to HA-TA, a specific DS DEX and DS HA , Dex-TA and HA-TA with a specific weight-average molecular weight, and the above-mentioned specific concentrations of peroxidase and hydrogen peroxide, the mixed polymer composition of the present invention can obtain an optimal gelation time; this enables the mixed polymer composition to complete gelation at a temperature between 15 and 38°C within a time between 10 and 200 seconds, preferably within a time between 20 and 100 seconds, and more preferably within a time between 30 and 60 seconds, thereby forming a biocompatible polymer hydrogel.

[0198] The optimal gelation time discovered by the inventors allows the mixed polymer composition to be an "injectable composition".

[0199] The term "injectable composition" refers to a solution that can form a polymer hydrogel after injection. Therefore, the mixed polymer composition has sufficient fluidity to enable the mixed polymer composition to be injected. The advantage of the injectable composition is that it can form microgels and macrogels in situ at the injection site (such as the joint gap), can fit irregularly shaped defects well, and allow chondrocytes to easily migrate and grow into the biocompatible polymer hydrogel, thereby achieving cartilage regeneration. Alternatively, the mixed polymer composition can be used to generate a microgel first, and then the microgel can be directly injected into the joint gap.

[0200] Injecting the mixed polymer composition into the surgical site or incision takes only seconds. After injection, several minutes are required for the biocompatible polymer hydrogel to form and stabilize. Furthermore, the stability of the biocompatible polymer hydrogel can be checked by gently moving the application area. For example, if the biocompatible polymer hydrogel is applied to the knee joint, this can be checked by bending the knee, followed by suturing the incision. This minimally invasive and simplified injection procedure offers significant advantages over traditional implants, which are often used for severe cartilage injuries.

[0201] The mixed polymer composition can be injected into the surgical site or incision site by methods known in the art, preferably using a needle or cannula. When a needle is used, the needle gauge is preferably between 12G and 23G, more preferably between 16G and 21G.

[0202] In order to facilitate the application of the mixed polymer composition of the present invention, the mixed polymer composition must have a suitable viscosity.

[0203] The inventors found that at a fixed polymer concentration of 8.9 wt% (4.45 wt% Dex-TA + 4.45 wt% HA-TA), at a shear rate of 10 Hz, the initial viscosity of the mixed polymer composition at T0 is between 15 and 25 mPa.s, preferably between 18 and 20 mPa.s, which is measured as required at 25±0.1°C in a double-gap or cone-plate apparatus rheometer.

[0204] The term "T0" refers to the initial time between the initial contact of tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide and the onset of the gelation process. Therefore, viscosity measurements at T0 must be performed in the absence of hydrogen peroxide to prevent the onset of the gelation process and the formation of the biocompatible polymer hydrogel of the present invention. Once the gelation process begins, the viscosity of the mixed polymer composition will gradually increase until a biocompatible polymer hydrogel is formed.

[0205] The present application also relates to a biocompatible polymer hydrogel, which is obtained by reacting tyramine-functionalized dextran (Dex-TA) with tyramine-functionalized hyaluronic acid (HA-TA) in the presence of peroxidase and hydrogen peroxide.

[0206] wherein the total weight ratio of Dex-TA to HA-TA is between 75:25 and 25:75, preferably between 60:40 and 40:60, and more preferably between 55:45 and 45:55;

[0207] Among them, DEX-TA contains unsubstituted glucan repeating units and TA-substituted glucan repeating units. According to proton nuclear magnetic resonance, the amount of TA-substituted repeating units in DEX-TA (DS DEX ) is between 5 mol% and 20 mol%;

[0208] HA-TA contains unsubstituted hyaluronic acid repeating units and TA-substituted hyaluronic acid repeating units. According to proton nuclear magnetic resonance, the amount of TA-substituted repeating units in HA-TA (DS HA ) is between 5 mol% and 20 mol%;

[0209] The biocompatible polymer hydrogel has a shear storage modulus between 10 and 100 kPa as measured in an oscillatory rheology setup and exhibits a shrinkage α between -20% and 20%, wherein α is determined according to formula (I):

[0210]

[0211] Where α is the shrinkage of the gel disc, expressed in %,

[0212] Among them, h mold is the height of the mold, which is 1.6 mm, and

[0213] Among them, h disc is the height of the gel disc in millimeters, measured in an oscillatory rheometer at a normal force of 0.05 N.

[0214] Unless otherwise stated below, the detailed description of the features of the invention described directly under the heading "Detailed description of features" also applies to this preferred embodiment.

[0215] The shear storage modulus of the biocompatible polymer hydrogel of the present invention has been evaluated in an oscillatory rheology apparatus. Samples for oscillatory rheology measurements were prepared in a mold 9 mm wide and 1.6 mm high to obtain cylindrical samples. After preparation, the cylindrical samples (gel discs) of the biocompatible polymer hydrogel were placed in phosphate-buffered saline and incubated overnight to reach a state of equilibrium swelling. The gel discs were then removed from the PBS, patted dry with a paper towel to remove excess PBS, and placed in an oscillatory rheology apparatus equipped with an 8 mm diameter shaft. The shaft was lowered onto the aligned gel discs until a normal force of 0.05 ± 0.005 N was obtained. The measurements were then performed at 20 ± 0.1°C in an oscillatory mode with a frequency of 1 Hz and a strain amplitude of 0.01%.

[0216] The degradation performance of the biocompatible polymer hydrogel of the present invention is measured by incubating it in hyaluronidase at 37°C for a certain period of time. The biocompatible polymer hydrogel is prepared in a mold 9 mm wide and 1.6 mm high to obtain a cylindrical sample. The cylindrical sample (gel disc) of the biocompatible polymer hydrogel is then placed in phosphate-buffered saline and incubated overnight to reach a balanced swelling state. After balanced swelling, the biocompatible polymer hydrogel is weighed. The phosphate-buffered saline is then removed, and the biocompatible polymer hydrogel is incubated in a 5 U / mL hyaluronidase solution at 37°C. The degradation of the biocompatible polymer hydrogel is evaluated by weighing the biocompatible polymer hydrogel at different time points and calculating the degradation as the residual weight percentage of the biocompatible polymer hydrogel. The degradation calculation formula is: wt / wi×100%, where wt is the weight of the biocompatible polymer hydrogel at the target time point and wi is the initial weight of the biocompatible polymer hydrogel after balanced swelling.

[0217] The inventors unexpectedly discovered that the biocompatible polymer hydrogel of the present invention obtained by the above reaction shows little susceptibility to mechanical erosion between two movable joints.

[0218] The biocompatible polymer hydrogel of the present invention hardly swells or swells very little and has a good density, so that chondrocytes can migrate and grow into the biocompatible polymer hydrogel of the present invention.

[0219] As measured by an oscillatory rheology apparatus, the shear storage modulus of the characterized biocompatible polymer hydrogel of the present invention is between 10-100 kPa, preferably between 15-90 kPa, more preferably between 20-70 kPa, and most preferably between 30-60 kPa; and its shrinkage rate α is determined by the above formula (I) between -20% and 20%, preferably between -15% and 15%, and more preferably between -10% and 10%.

[0220] In addition, the polymer hydrogel of the present invention degrades slowly and can provide sufficient support at the defect site, thereby providing a longer time for the migration and growth of chondrocytes to promote cartilage regeneration.

[0221] After incubation in hyaluronidase at a concentration of 5 units / ml for 30 days, the polymer hydrogel of the present invention exhibits an in vitro degradation rate between 10% and 50%, preferably between 10% and 40%, more preferably between 10% and 30%, and even more preferably between 10% and 20%.

[0222] The term "units / ml" in relation to hyaluronidase refers to the concentration of hyaluronidase expressed in "activity units" related to enzyme activity, which causes a turbidity-reducing effect equivalent to that caused by 1.0 unit of the international standard preparation.

[0223] The present invention also relates to the use of the biocompatible polymer hydrogel of the present invention as a medicament for preventing or inhibiting painful joint conditions in humans or animals, wherein the painful conditions are selected from the group consisting of inflammatory pain, arthritis pain, osteoarthritis pain, osteochondritis dissecans pain, traumatic defects caused by trauma, accidents, slips or falls.

[0224] As used herein, "a medicament for treating or preventing a painful joint condition" refers to a therapeutic method intended to prevent or minimize joint pain caused by tissue damage or loss, wherein the polymer hydrogel of the present invention is provided to improve regeneration of damaged or lost tissue and alleviate pain. Preferably, the damaged or lost tissue is cartilage tissue.

[0225] This painful condition can be caused by diseases such as inflammation, arthritis, osteoarthritis, or osteochondritis, or it can result from a traumatic defect caused by trauma, accidents, slips, or falls.

[0226] The polymer hydrogel may further comprise a drug or medicament that can be released after a sustained or extended release mechanism. Thus, the biocompatible polymer hydrogel can be used as a monotherapy or as an adjuvant therapy, or in combination with other known therapies.

[0227] The present invention also relates to a method for treating or preventing joint pain in humans or animals, which comprises administering a therapeutically effective amount of the biocompatible polymer hydrogel of the present invention.

[0228] A therapeutically effective amount of the biocompatible polymer hydrogel of the present invention can be applied to the surgical site or incision site, typically by injecting the therapeutically effective amount of the hybrid polymer composition of the present invention within a time period of between 2-10 seconds.

[0229] It will be understood that the therapeutically effective amount of the biocompatible polymer hydrogel or mixed polymer composition according to the present invention will be determined, for example, by the volume of the area of ​​tissue to be repaired or replaced, but will also depend on the swelling behavior and degradation characteristics of the polymer hydrogel, as well as whether the polymer hydrogel is used as a monotherapy or in combination therapy. If the treatment is also used to provide for the release of a drug, the amount will also depend on the dose of the drug incorporated into the polymer hydrogel, the pharmacokinetics of the drug, and the progression of the disease condition.

[0230] For example, known methods routinely used in the pharmaceutical industry (eg, in vivo experiments, clinical trials, etc.) can be used to determine the therapeutically effective amount and treatment regimen (eg, dosage and administration frequency) of the biocompatible polymer hydrogel or mixed polymer composition of the present invention.

[0231] The invention will now be illustrated in a non-limiting manner by the following examples.

[0232] Example

[0233] abbreviation

[0234] Dex (dextran); PNC (p-nitrophenyl chloroformate); LiCl (lithium chloride); TA (tyramine); DMF (N,N-dimethylformamide); TA·HCl (tyramine hydrochloride); DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine hydrochloride); NaCl (sodium chloride); HA (hyaluronic acid); PBS (phosphate-buffered saline, pH approximately 7.4); H2O2 (hydrogen peroxide); NMR (nuclear magnetic resonance); wt% (weight percentage); Eq. (molar equivalent).

[0235] Example 1

[0236] Synthesis of Dex-TA

[0237] Synthesis of Dextran-p-Nitrophenyl Carbonate (Dextran-PNC)

[0238] Lithium chloride (4.0 g, dried at 115°C) and dextran (5.00 g, 30.8 mmol repeating units (ru)) were weighed into a 500 mL three-necked round-bottom flask equipped with a magnetic stirrer. The flask was evacuated and filled with nitrogen three times, then placed under vacuum at 95°C for 1.5 hours. After sufficient drying, nitrogen was filled into the flask and 200 mL of anhydrous DMF was added through a cannula while stirring. The flask was then equipped with a thermometer and the mixture was heated to 95°C while stirring the solution. Once the dextran was completely dissolved, the solution was cooled to 0°C and anhydrous pyridine (2.0 mL, 25.8 mmol) was added. Next, while maintaining the temperature below 2°C, freshly sublimed p-nitrophenyl chloroformate (2.5 g, 12.4 mmol) was added in batches. After 1 hour, the reaction mixture was poured into 1 L of ice ethanol, and the precipitate (Por 4) was filtered off and washed with cold ethanol (3×100 mL) and ether (3×100 mL) in sequence. After vacuum drying, the product was obtained as a white powder (6.00 g, 30.7 mmol ru, yield 99%, DS 20%). 1H-NMR (400 MHz): δ (ppm) = 3.0-4.0 (sugar ring proton, m, 6H); 4.2-5.8 (anomeric proton and hydroxyl proton, m, 4H); 7.58 (Ar o-CH, d, 2H); 8.34 (Ar m-CH, d, 2H).

[0239] Synthesis of Dextran-Tyramine

[0240] Dextran-PNC (6.00 g, 30.7 mmol repeating units, 6.15 mmol p-nitrophenyl carbonate) was weighed into a 250 mL three-necked round-bottom flask equipped with a magnetic stirrer. The flask was evacuated and filled with nitrogen three times, then the flask was filled with nitrogen and 100 mL of anhydrous DMF was added through a cannula while stirring. Once the dextran was completely dissolved, tyramine (1.69 g, 12.3 mmol) was added. After 1 hour, the reaction mixture was poured into 1 L of ice ethanol. The precipitate (Por 4) was filtered off and washed with cold ethanol (3 × 100 mL) and ether (3 × 100 mL). The crude product was obtained as a white powder after vacuum drying. The crude product was dissolved in 75 mL of Milli-Q water, dialyzed against Milli-Q water for 3 days (MWCO 3500 Da), sterilized by filtration, and freeze-dried to give a white foamy product (5.04 g, 28.0 mmol, 92% yield, DS 10%). 1H-NMR (400 MHz): δ (ppm) = 3.0-4.0 (sugar ring protons, m, 6H); 4.8-5.2 (anomeric protons and hydroxyl protons, m, 4H); 6.7-7.1 (Ar-CH, d, 2H); 6.9-7.3 (Ar o-CH, d, 2H).

[0241] The degree of substitution (DS) of dextran-TA and dextran-PNC was calculated based on the integral from 4.8 to 5.2 ppm (corresponding to the four anomeric protons of dextran) compared to the integral of the aromatic protons of tyramine (6.7-7.1 and 6.9-7.3) or p-nitrobenzene (7.3-7.7 and 8.10-8.50). The degree of substitution (OS) of dextran is expressed as the percentage of modified sugar units in the dextran.

[0242] Example 2

[0243] Synthesis of HA-TA

[0244] Sodium hyaluronate (5.00 g, 12.5 mmol) was dissolved in 500 mL of Milli-Q water (relative volume 100) placed in a 1 L round-bottom flask equipped with a stirring magnet. DMTMM (3.46 g, 12.5 mmol, molar equivalent of 1) and TAHCl (2.17 g, 12.5 mmol, molar equivalent of 1) were added sequentially while stirring at room temperature. DMTMM and TAHCl were added repeatedly after 24 and 48 hours. After 72 hours of reaction, 40 mL of sodium chloride solution (relative volume 8, saturated) was added to the reaction mixture and the reaction mixture was poured into 2.5 L of cold ethanol (relative volume 500). The crude product was separated by centrifugation at 5000 rpm and then vacuum dried. The crude product was dissolved in 75 mL of Milli-Q water and dialyzed for 3 days (MWCO 1000 Da) with Milli-Q water. After filtration sterilization and freeze-drying, the HA-TA product was obtained as a white foam (5.10 g, 12.4 mmol, yield 99%, DS 10%).

[0245] pass 1 H-NMR confirmed that HA-TA was successfully synthesized. The specific method is as follows:

[0246] Baseline correction: Spline method

[0247] The peaks at 1.7-2.1 ppm, 6.7-7.1 ppm, and 6.9-7.3 ppm were integrated;

[0248] The peak area of ​​the peak at 1.7–2.1 ppm was set to 3000;

[0249] DS calculation: add the peak areas of 6.7-7.1 ppm and 6.9-7.3 ppm and divide by 40.

[0250] 1 H-NMR (400 MHz, D2O): δ (ppm) = 1.98 (acetyl-CH3, s, 3H); 2.75 (2-CH2, s, 2H); 2.90 (1-CH2, s, 2H); 3.2-4.2 (sugar ring proton, m, 10H); 4.34 (s, 1H); 4.43 (d, 1H); 6.84 (Ar m-CH, d, 2H); 7.16 (Ar o-CH, d, 2H).

[0251] Example 3

[0252] Hydrogel kit suitable for preparing biocompatible polymer hydrogel formulations using a 4:1 ratio premix

[0253] Equal amounts of Dex-TA and HA-TA were added to PBS (phosphate-buffered saline or a buffer solution with a pH of approximately 7.4) to obtain a polymer solution with a concentration ranging from 11.0 to 13.0 wt%. HRP (horseradish peroxidase) was dissolved in PBS to obtain a 30 U / mL HRP (stock solution). 30 wt% H2O2 was diluted with PBS to obtain a 0.25-0.5 wt% H2O2 solution.

[0254] Premix the Dex-TA / HA-TA polymer solution with the HRP solution at a 4:1 volume ratio. Premix the Dex-TA / HA-TA polymer solution with the H2O2 solution at a 4:1 volume ratio. Then, combine the two premixes at a 1:1 volume ratio.

[0255] Example 4

[0256] Hydrogel kit suitable for preparing biocompatible polymer hydrogel formulations using a 7:1 ratio premix

[0257] Equal amounts of Dex-TA and HA-TA were added to PBS (phosphate-buffered saline or a buffer solution with a pH of approximately 7.4) to obtain a polymer solution with a concentration ranging from 11.0 to 13.0 wt%. HRP (horseradish peroxidase) was dissolved in PBS to obtain a 30 U / mL HRP (stock solution). 30 wt% H2O2 was diluted with PBS to obtain a 0.25-0.5 wt% H2O2 solution.

[0258] Premix the Dex-TA / HA-TA polymer solution with the HRP solution at a volume ratio of 7:1. Mix this premix with H2O2 at a ratio of 4:1.

[0259] method

[0260] Several approaches have been employed to explore the properties, structure, processability, and performance of the mixed polymer compositions and biocompatible polymer hydrogels of the present invention.

[0261] Viscosity measurement

[0262] Viscosity measurements were performed on a TA Instruments Discovery HR20 instrument (equipped with double-gap geometry). During the measurements, the temperature was kept constant at 25°C and the shear rate was 10 s -1, lasting 205 seconds, and the test was repeated twice. The viscosity value was calculated using software (Trios software, TA Instruments). The viscosity values ​​within the last 105 seconds of each measurement were averaged. The average of the two repeated tests was taken as the final viscosity. This method was used to determine the average dynamic (shear) viscosity of the biocompatible polymer hydrogel.

[0263] A. Viscosity Measurement Geometry

[0264] Double gap device

[0265] Inner cup diameter: 40.0 mm

[0266] Inner column diameter: 40.79 mm

[0267] Outer column diameter: 43.88 mm

[0268] Outer cup diameter: 44.76 mm

[0269] Inner column height: 55.0 mm

[0270] Immersion height: 53 mm

[0271] Operating clearance: 2 mm

[0272] Bob material: Aluminum

[0273] Peltier heating

[0274] B. Measurement Setup

[0275] 4-step measurement has been performed.

[0276] 1. Shear scan (not used for data processing, only for additional inspection)

[0277] T=25.0±0.1℃

[0278] Shear scan 0.01-100Hz

[0279] 10 points per decade

[0280] 5 seconds per point measurement

[0281] 2. Time scan (for data processing)

[0282] T=25.0±0.1℃

[0283] ·Measurement time: 205 seconds

[0284] Shear rate = 10 Hz

[0285] Sampling interval = 5 seconds

[0286] 3. Shear scan (not used for data processing, only for additional inspection)

[0287] T=25.0±0.1℃

[0288] Shear scan 0.01-100Hz

[0289] 10 points / order of magnitude

[0290] 5 seconds per point measurement

[0291] 4. Time scan (for data processing)

[0292] T=25.0±0.1℃

[0293] ·Measurement time: 205 seconds

[0294] Shear rate = 10 Hz

[0295] Sampling interval = 5 seconds

[0296] C. Rheometer Data Processing

[0297] Only the data obtained in steps 2 and 4 are used for data processing to obtain the viscosity of the solution. The viscosity data obtained in the second half of step 2 (i.e., the last 105 seconds) are averaged. The viscosity data obtained in the second half of step 4 (i.e., the last 105 seconds) are averaged. The average values ​​of steps 2 and the last 105 seconds of step 4 are averaged again to obtain the viscosity of the solution.

[0298] Rheological measurements

[0299] 1. Preparation of hydrogel discs using rheological molds

[0300] use Figure 5 Assemble the mold shown to prepare hydrogel discs.

[0301] Place the mold piece in the mold block, place a glass slide on top, and secure with a clamp. Inject the mixture of the present invention through the 1.5 mm hole on the left side of the mold and allow it to gel in the mold. Then, remove the mold from the mold block and press the gel disc out of the mold belt. Incubate the gel disc in PBS at 2-8°C for 12-168 hours before measurement.

[0302] 2. Rheological measurement details

[0303] A. Rheometer Measurement Geometry

[0304] Parallel plate device

[0305] mm axis

[0306] Stainless steel

[0307] Peltier heating

[0308] B. Rheometer Measurement Setup

[0309] Lower the spindle onto the gel disc until a normal force (axial force) of 0.05 ± 0.005 N is reached. After the above normal force is reached, record the gap size.

[0310] The measurements were performed in oscillation mode using the following settings:

[0311] Strain amplitude = 0.01%

[0312] Frequency = 1 Hz

[0313] Measurement time = 120 seconds

[0314] Sampling interval = 5 seconds

[0315] Temperature = 20 ± 0.1 °C

[0316] C. Rheometer Data Processing

[0317] The storage modulus data of the second half of the measurement (60-120 seconds) were averaged.

[0318] Swelling / shrinkage

[0319] The swelling / shrinkage behavior of biocompatible polymer hydrogels was determined using an oscillatory rheometer (TA Instruments-Discovery HR20).

[0320] Gel discs were prepared in 1.6 mm thick molds, placed in PBS at 2-7°C for 12-168 hours, dried and placed in an oscillatory rheology apparatus equipped with an 8 mm diameter spindle.

[0321] The spindle was lowered onto the aligned gel disc until a normal force of 0.05 ± 0.005 N was recorded.

[0322] The shrinkage rate α is determined according to formula (I):

[0323]

[0324] Where, α is the shrinkage of the gel disc, in %,

[0325] Among them, h mold is the mold height, 1.6 mm, and

[0326] Among them, h disc is the height of the gel disc measured in an oscillatory rheometer at a normal force of 0.05 N, in millimeters.

[0327] For example, if the height recorded at a normal force of 0.05 N is, for example, 1.543 mm, this indicates a contraction of the biocompatible polymer hydrogel of 5.7% [(1.600-1.543) / 1.600 x 100%).

Claims

1. A kit for preparing a biocompatible polymer hydrogel, the kit comprising at least two containers, each container containing an aqueous composition comprising at least one of tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide, wherein the components are capable of forming the biocompatible polymer hydrogel when mixed. in, Peroxidase and hydrogen peroxide are each present in different containers. wherein the total weight ratio of Dex-TA to HA-TA is between 75:25 and 25:75, preferably between 60:40 and 40:60, and more preferably between 55:45 and 45:55; wherein the concentration of Dex-TA is between 2 wt % and 7 wt % relative to the total weight of the aqueous composition in the kit; wherein the concentration of HA-TA is between 2 wt % and 7 wt % relative to the total weight of the aqueous composition in the kit; wherein the DEX-TA comprises unsubstituted glucan repeating units and TA-substituted glucan repeating units, wherein the amount of TA-substituted repeating units in DEX-TA (DS) relative to the total amount of unsubstituted repeating units and TA-substituted repeating units according to proton nuclear magnetic resonance is DEX ) between 5 mol % and 20 mol %; Wherein, the HA-TA comprises unsubstituted hyaluronic acid repeating units and TA-substituted hyaluronic acid repeating units, wherein the amount of TA-substituted repeating units in HA-TA (DS) relative to the total amount of unsubstituted repeating units and TA-substituted repeating units according to proton nuclear magnetic resonance is HA ) between 5 mol % and 20 mol %; wherein the DEX-TA has a weight average molecular weight (Mw) between 15 and 650 kDa; wherein the HA-TA has a weight average molecular weight (Mw) between 15 and 550 kDa; Wherein, relative to the total weight of the aqueous composition in the kit, the peroxidase is present in a concentration between 0.25 units / ml and 10 units / ml, and the hydrogen peroxide is present in a concentration between 0.01% by weight and 0.10% by weight.

2. The kit according to claim 1, wherein The first container contains an aqueous composition A comprising a. Tyramine functionalized dextran (Dex-TA), b. Tyramine functionalized hyaluronic acid (HA-TA), c. Peroxidase, And, wherein the second container contains an aqueous composition B, which comprises d. hydrogen peroxide, and e. Optionally, at least one of tyramine-functionalized dextran (Dex-TA) and tyramine-functionalized hyaluronic acid (HA-TA), preferably, both Dex-TA and HA-TA, wherein the total weight ratio of Dex-TA to HA-TA is between 75:25 and 25:75, preferably between 60:40 and 40:60, and more preferably between 55:45 and 45:55; wherein the concentration of Dex-TA is between 2 wt % and 7 wt % relative to the total weight of the aqueous composition in the kit; wherein the concentration of HA-TA is between 2 wt % and 7 wt % relative to the total weight of the aqueous composition in the kit; Wherein, the DEX-TA comprises unsubstituted glucan repeating units and TA-substituted glucan repeating units, wherein according to proton nuclear magnetic resonance, the amount of TA-substituted repeating units in DEX-TA (DS DEX ) is between 5 mol% and 20 mol%; Wherein, the HA-TA comprises unsubstituted hyaluronic acid repeating units and TA-substituted hyaluronic acid repeating units, wherein according to proton nuclear magnetic resonance, the amount of TA-substituted repeating units in HA-TA (DS HA ) is between 5 mol% and 20 mol%; wherein the Dex-TA has a weight average molecular weight (Mw) between 15 kDa and 650 kDa; wherein the HA-TA has a weight average molecular weight (Mw) between 15 kDa and 550 kDa; Wherein, relative to the total weight of the aqueous composition in the kit, the peroxidase is present in a concentration between 0.25 units / ml and 10 units / ml, and the hydrogen peroxide is present in a concentration between 0.01% by weight and 0.10% by weight.

3. The kit according to claim 1 or 2, wherein The peroxidase is selected from horseradish peroxidase (HRP), soybean peroxidase, myeloperoxidase, lactoperoxidase, Nitrospira recombinant peroxidase and Coleoptera recombinant lysyl oxidase.

4. The kit according to claim 2 or 3, wherein The volume to volume ratio of Composition A:Composition B is in the range of 0.1:1 to 10:

1.

5. The kit according to claim 2, wherein Components A and B are fluid in the temperature range of 0°C to 37°C, and as required, components A and B have a viscosity between 1 mPa·s and 50 Pa·s, preferably between 1 mPa·s and 100 mPa·s, more preferably less than 50 mPa·s, and even more preferably less than 35 mPa·s, as measured in a rheometer using a double gap or cone-plate apparatus at a shear rate of 10 Hz at 25±0.1°C.

6. The kit according to claim 1, wherein The aqueous composition in each container comprises between 80% and 99.9% by weight of water or aqueous saline solution or phosphate buffered solution or phosphate buffered saline solution.

7. The kit according to claims 2 to 5, wherein Composition A and composition B each comprise between 80% and 99.9% by weight of water or aqueous saline solution or phosphate buffer solution or phosphate buffer saline solution.

8. A method for preparing a biocompatible polymer hydrogel, comprising the following steps: a. mixing an aqueous composition comprising tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide to obtain a mixed polymer composition, b. forming the biocompatible polymer hydrogel from the mixed polymer composition at a temperature between 15 and 38° C. for a period between 10 and 200 seconds, wherein the total weight ratio of Dex-TA to HA-TA is between 75:25 and 25:75, preferably between 60:40 and 40:60, and more preferably between 55:45 and 45:55; wherein the concentration of Dex-TA is between 2 wt % and 7 wt % relative to the mixed polymer composition, wherein the concentration of HA-TA is between 2% and 7% by weight relative to the mixed polymer composition, Wherein, the DEX-TA comprises unsubstituted glucan repeating units and TA-substituted glucan repeating units, wherein according to proton nuclear magnetic resonance, the amount of TA-substituted repeating units in DEX-TA (DS DEX ) is between 5 mol% and 20 mol%, Wherein, the HA-TA comprises unsubstituted hyaluronic acid repeating units and TA-substituted hyaluronic acid repeating units, wherein according to proton nuclear magnetic resonance, the amount of TA-substituted repeating units in HA-TA (D SHA ) is between 5 mol % and 20 mol %, wherein the DEX-TA has a weight average molecular weight (Mw) between 15 kDa and 650 kDa, wherein the HA-TA has a weight average molecular weight (Mw) between 15 kDa and 550 kDa, wherein the peroxidase is present at a concentration between 0.25 and 10 units / ml and the hydrogen peroxide is present at a concentration between 0.01% and 0.10% by weight relative to the mixed polymer composition.

9. A method for preparing a biocompatible polymer hydrogel, comprising the following steps: a. mixing the aqueous composition A and the aqueous composition B to obtain a mixed polymer composition, b. forming a biocompatible polymer hydrogel at a temperature between 15 and 38 ° C. and allowing the mixed polymer composition to form a biocompatible polymer hydrogel within a time period between 10 and 200 seconds; The aqueous composition A comprises tyramine-functionalized dextran (Dex-TA), tyramine-functionalized hyaluronic acid (HA-TA), peroxidase, and The aqueous composition B comprises hydrogen peroxide and optionally comprises at least one of tyramine-functionalized dextran (Dex-TA) and tyramine-functionalized hyaluronic acid (HA-TA), preferably comprises both Dex-TA and HA-TA. wherein the total weight ratio of Dex-TA to HA-TA is between 75:25 and 25:75, preferably between 60:40 and 40:60, more preferably between 55:45 and 45:55, wherein the concentration of Dex-TA is between 2 wt % and 7 wt % relative to the sum of composition A and composition B, wherein the concentration of HA-TA is between 2 wt % and 7 wt % relative to the sum of composition A and composition B, Wherein, the DEX-TA comprises unsubstituted glucan repeating units and TA-substituted glucan repeating units, wherein according to proton nuclear magnetic resonance, the amount of TA-substituted repeating units in DEX-TA (DS DEX ) is between 5 mol % and 20 mol %, Wherein, the HA-TA comprises unsubstituted hyaluronic acid repeating units and TA-substituted hyaluronic acid repeating units, wherein according to proton nuclear magnetic resonance, the amount of TA-substituted repeating units in HA-TA (DS HA ) is between 5 mol % and 20 mol %, Among them, Dex-TA has a weight average molecular weight (Mw) varying between 15 kDa and 650 kDa, Among them, HA-TA has a weight average molecular weight (Mw) varying between 15 kDa and 550 kDa, Wherein, relative to the total weight of aqueous composition A and aqueous composition B, the peroxidase is present in a concentration between 0.25 units / ml and 10 units / ml, and the hydrogen peroxide is present in a concentration between 0.01% by weight and 0.10% by weight.

10. A hybrid polymer composition obtained by mixing an aqueous composition comprising tyramine functionalized dextran (Dex-TA), tyramine functionalized hyaluronic acid (HA-TA), peroxidase, and hydrogen peroxide, in, The total weight ratio of Dex-TA to HA-TA is between 75:25 and 25:75, preferably between 60:40 and 40:60, more preferably between 55:45 and 45:55; wherein the concentration of Dex-TA is between 2 wt % and 7 wt % relative to the mixed polymer composition, wherein the concentration of HA-TA is between 2% and 7% by weight relative to the mixed polymer composition, Wherein, the DEX-TA comprises unsubstituted glucan repeating units and TA-substituted glucan repeating units, wherein according to proton nuclear magnetic resonance, the amount of TA-substituted repeating units in DEX-TA (DS DEX ) is between 5 mol % and 20 mol %, Wherein, the HA-TA comprises unsubstituted hyaluronic acid repeating units and TA-substituted hyaluronic acid repeating units, wherein according to proton nuclear magnetic resonance, the amount of TA-substituted repeating units in HA-TA (DS HA ) is between 5 mol % and 20 mol %, wherein the DEX-TA has a weight average molecular weight (Mw) between 15 kDa and 650 kDa, wherein the HA-TA has a weight average molecular weight (Mw) between 15 kDa and 550 kDa, The peroxidase is present in a concentration of 0.25 units / ml to 10 units / ml, and the hydrogen peroxide is present in a concentration of 0.01 wt % to 0.10 wt % relative to the mixed polymer composition.

11. The mixed polymer composition according to claim 10, wherein The mixed polymer composition has a gel time of between 10 and 200 seconds at a temperature between 17 and 38°C.

12. The mixed polymer composition according to claim 10 or 11, wherein The viscosity of the mixed polymer composition at T0 at a shear rate of 10 Hz, measured at 25±0.1° C. in a double-gap or cone-plate rheometer, is between 15 and 25 mPa·s.

13. A biocompatible polymer hydrogel obtained by reacting tyramine-functionalized dextran (Dex-TA) with tyramine-functionalized hyaluronic acid (HA-TA) in the presence of peroxidase and hydrogen peroxide. in, The total weight ratio of Dex-TA to HA-TA is between 75:25 and 25:75, preferably between 60:40 and 40:60, more preferably between 55:45 and 45:55; Wherein, the DEX-TA comprises unsubstituted glucan repeating units and TA-substituted glucan repeating units, wherein according to proton nuclear magnetic resonance, the amount of TA-substituted repeating units in DEX-TA (DS DEX ) is between 5 mol % and 20 mol %, Wherein, the HA-TA comprises unsubstituted hyaluronic acid repeating units and TA-substituted hyaluronic acid repeating units, wherein according to proton nuclear magnetic resonance, the amount of TA-substituted repeating units in HA-TA (DS HA ) is between 5 mol % and 20 mol %, The biocompatible polymer hydrogel has a shear storage modulus between 10 kPa and 100 kPa as measured in an oscillatory rheology apparatus and exhibits a shrinkage α between -20% and 20%, wherein α is determined according to formula (I): Formula (I) Where, α is the shrinkage of the gel disc, in %, Among them, h mold is the height of the mold, which is 1.6 mm, and Among them, h disc is the height of the gel disc in millimeters, measured in an oscillatory rheometer at a normal force of 0.05 N.

14. The biocompatible polymer hydrogel according to claim 13, wherein The biocompatible polymer hydrogel had an in vitro degradation rate between 10% and 50% after 30 days in the presence of hyaluronidase at a concentration of 5 units / ml.

15. The biocompatible polymer hydrogel according to any one of claims 13 to 14, as a medicament for preventing or inhibiting joint pain conditions in humans or animals, wherein The pain condition is selected from the group consisting of inflammatory pain, arthritic pain, osteoarthritic pain, osteochondritis dissecans pain, traumatic defects caused by trauma, accidents, slips or falls.

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