Injectable hydrogel pre-gelatinizing solution based on ligand regulation and control as well as preparation method and application of injectable hydrogel pre-gelatinizing solution

Abstract

The invention relates to the field of biological materials, and provides an injectable hydrogel pre-gelatinizing solution based on ligand regulation and control and a preparation method and application of the injectable hydrogel pre-gelatinizing solution. A multivalent metal ion; a small molecule ligand; wherein the alginic acid compound comprises alginic acid or alginate and a derivative thereof; the molar ratio of the micromolecule ligand to the multivalent metal ions is marked as L / M; defining the minimum L / M for keeping the system in a sol state at the set temperature and ion strength as a critical ligand ratio, and recording the critical ligand ratio as CLR; in the pre-gelatinizing solution, L / M is greater than or equal to CLR. The pre-gelatinizing solution can be smoothly injected through a fine needle, can quickly form a gel shell at an interface to prevent leakage after being injected to a water-containing environment, has continuous self-enhancement performance, and is wide in application range.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and more specifically, to an injectable hydrogel pregel solution based on ligand regulation, its preparation method, and its application. Background Technology

[0002] In recent years, injectable hydrogels have shown great application potential in tissue engineering, regenerative medicine, and drug delivery due to their minimally invasive drug delivery characteristics and excellent tissue shape adaptability, and are considered a promising biomaterial platform. Alginate (Alg), as a widely available, biocompatible, and biodegradable natural polysaccharide, can interact with multivalent metal ions (such as Ca²⁺) to form hydrogels. 2+ Ba 2+ 、Sr 2+ Hydrogels that undergo ionic cross-linking (such as those containing ions) and form a three-dimensional network structure under mild conditions have attracted much attention.

[0003] Currently, the gelation strategies of injectable hydrogels based on alginate are mainly divided into two categories: exogenous gelation and endogenous gelation. Among them: (1) The exogenous gelation method usually involves directly contacting the alginate solution with an external polyvalent metal ion solution (such as CaCl2 solution) to rapidly induce gel formation. Although this method is simple to operate, the cross-linking reaction rate is too fast, which can easily lead to uneven gel structure. In addition, it is often necessary to use devices such as double-barreled syringes to deliver the two components separately, which increases the complexity of the operation and limits its application in situations requiring precise injection molding.

[0004] (2) Endogenous gelation technology typically involves dispersing sparingly soluble metal salts (such as CaCO3, calcium malate, calcium sodium EDTA, calcium oxalate, and calcium gluconate) and acidic precursors (such as gluconate-δ-lactone and guluronic acid) in an alginate solution, as disclosed in patent CN 118267527 A. This method utilizes the low solubility of sparingly soluble metal salts in water, allowing particles in the solution to slowly release metal ions under acidic conditions, thus achieving in-situ cross-linking. While this method can improve gel uniformity, the slow and irreversible release of metal ions results in poor initial stability and easy leakage of the injected material. Furthermore, the presence of suspended sparingly soluble particles in the system increases the risk of needle blockage, hindering its widespread application in minimally invasive interventional therapies and other scenarios.

[0005] In addition, existing technologies include strategies such as preparing low-modulus gels by reducing the degree of crosslinking, preparing physically crosslinked "injectable" cryogels using freeze-thaw cycles, or relying on external fields such as light and magnetism to trigger gelation. However, these methods often fail to simultaneously meet multiple requirements such as mechanical properties, shape retention ability, universality of triggering conditions, and convenience of clinical application. In particular, they still have significant shortcomings in achieving excellent needle injectability, immediate shape fixation after injection, and long-term self-reinforcing mechanical properties.

[0006] Therefore, there is an urgent need for a new alginate hydrogel system that can achieve immediate gelation at the interface after injection while maintaining smooth injection through fine needles, and obtain long-term stability and enhanced mechanical properties through subsequent cross-linking and densification, thereby meeting the application needs of tissue engineering, regeneration and repair, and precision drug delivery. Summary of the Invention

[0007] In view of the above-mentioned shortcomings in the prior art, the core objective of this invention is to solve the technical problems commonly found in existing alginate hydrogel systems, such as poor needle injectability, insufficient post-injection stability, and poor mechanical properties.

[0008] This invention is achieved through the following technical solution: A ligand-regulated injectable hydrogel pregel solution comprising the following components: Alginic acid compounds; polyvalent metal ions; small molecule ligands; The alginic acid compounds include alginic acid or alginate and their derivatives; The molar ratio of the small molecule ligand to the multivalent metal ion is denoted as L / M; the minimum L / M required to maintain the system in a sol state under set temperature, set alginate compound concentration, and set multivalent metal ion concentration is defined as the critical ligand ratio, denoted as CLR. In the pregel solution, L / M ≥ CLR.

[0009] In response to the existing technical challenges of exogenous gelation requiring separate delivery of two components into the body and endogenous gelation struggling to achieve stable delivery via fine needles, this invention proposes a method that utilizes small-molecule ligands and multivalent metal ions to form reversible complexes. This inhibits cross-linking between alginate compounds and multivalent metal ions before injection, maintaining a homogeneous single-phase sol system that facilitates smooth fine needle injection. Upon injection into the body's aqueous environment, the small-molecule ligands, due to their small molecular weight and strong hydrophilicity, diffuse from high-concentration regions (inside the pregel solution) to low-concentration regions (external body fluids) driven by a concentration gradient. This disrupts the complexation equilibrium, releasing multivalent metal ions. These released ions are rapidly captured by the carboxyl groups of alginate, forming a gradual cross-linking process from the outside in at the injection site. Because the external small-molecule ligands diffuse preferentially, the external metal ions are released first, forming an outer gel shell. Subsequently, the internal small-molecule ligands gradually diffuse, releasing internal metal ions, leading to gradual densification of the interior. This achieves immediate post-injection stability and subsequent mechanical self-reinforcement.

[0010] More importantly, this invention defines the critical ligand ratio (CLR) parameter, which is defined as the minimum L / M required to maintain the system in a sol state under set temperature, set alginate compound concentration, and set polyvalent metal ion concentration. Furthermore, this invention limits the molar ratio of small molecule ligands to polyvalent metal ions in the pregel solution to be L / M ≥ CLR. Thus, by controlling the value of L / M, it is ensured that the pregel solution is always in a sol state, ensuring that small molecule ligands can preferentially complex polyvalent metal ions in the solution system, and avoiding excessive cross-linking between polyvalent metal ions and alginate, which would lead to gelation in the pregel solution.

[0011] Furthermore, the present invention provides a method for determining the critical ligand ratio (CLR). Under set temperature T, set alginate compound concentration C, and set polyvalent metal ion concentration M, the method for determining the CLR of any of the small molecule ligands A and any of the polyvalent metal ions B includes the following steps: (1) Prepare multiple groups of aqueous solutions of alginate compounds with a concentration of C at a set temperature T; (2) Add different amounts of the small molecule ligand A to the multiple groups of alginate compounds aqueous solutions in step (1), mix them evenly, and obtain multiple groups of homogeneous solutions; (3) Add a fixed concentration of polyvalent metal ions B to each of the homogeneous solutions in step (2), and add an appropriate amount of water to make the solution volume up so that the concentration of the resulting alginate compound is C, the molar concentration of polyvalent metal ions B is M, and the molar concentration L of the small molecule ligand A changes from low to high according to the gradient; observe the morphology of each solution system; the L / M when the system changes from gel state to sol state is the CLR of the small molecule ligand A and the polyvalent metal ions B under the set temperature T, the set concentration of alginate compound C, and the set concentration of polyvalent metal ions M.

[0012] In practice, since the pregel system of this invention is used in the biomedical field, the temperature T is generally set to 20℃-37℃ due to the limitations of its application field. Through actual verification, within this temperature range, the CLR values ​​of small molecule ligand A and multivalent metal ion B vary very little under set alginate compound concentration C and multivalent metal ion concentration M, which is within an acceptable range. Therefore, in practice, the effect of temperature on CLR is often negligible.

[0013] More preferably, in step (3), the difference between the L / M when the system changes from gel state to sol state and the L / M corresponding to the previous small molecule ligand A concentration (gel state) is less than 15%, so as to ensure that the accuracy of the final measured CLR is as high as possible. If the difference between the two is greater than 15%, the concentration gradient gap of small molecule ligand A is further reduced to obtain a more accurate CLR.

[0014] Preferably, the stability constant of the complex formed by any of the small molecule ligands A and any of the polyvalent metal ions B is set to K. A-B For a given temperature, a set concentration of alginate compounds, and a set concentration of polyvalent metal ions, the CLR ∝ 1 / K A-B .

[0015] Studies have found that the stability constant of the complex formed by CLR and the small molecule ligand A-polyvalent metal ion B is the reciprocal of (1 / K) A-B The two signals exhibit a strong linear correlation; based on this correlation, a CLR–1 / K relationship can be established. A-B The mapping / regression model can be used to calculate the corresponding CLR value at a set temperature, a set concentration of alginate compound, and a set concentration of multivalent metal ions based on the stability constant of the complex formed by the selected small molecule ligand and the selected multivalent metal ion. This allows for the design of the corresponding L / M value of the pregel solution, thereby enabling rapid formulation design across ligands and metal ions. This provides a predictable design path for the rapid screening and engineering scale-up of different combinations of small molecule ligands and multivalent metal ions.

[0016] Preferably, the alginate includes sodium alginate, potassium alginate, or ammonium alginate; the alginate derivatives include, but are not limited to, chemically / biologically modified alginates; preferably, alginates modified by esterification, amidation, grafting peptides, or grafting polymers.

[0017] Preferably, the multivalent metal ion is one or more of divalent or trivalent metal ions; preferably, the divalent metal ion is selected from one or more of Ca²⁺, Mg²⁺, Ba²⁺, Sr²⁺, Cu²⁺, Zn²⁺, Mn²⁺, Co²⁺, Ni²⁺, and Fe²⁺; the trivalent metal ion is selected from one or more of Fe³⁺ and Al³⁺. In practice, the multivalent metal ion can be one or more of various divalent metal ions, one or more trivalent metal ions, or any combination of one or more divalent metal ions and one or more trivalent metal ions. The selection criterion is the ability to achieve a cross-linking reaction with the alginate compound.

[0018] Preferably, the small molecule ligand contains at least one functional group that can form a coordination bond with the multivalent metal ion, wherein the functional group is selected from one or more of carboxyl, carboxylate, hydroxyl, amino, amine, phosphonic acid, pyridyl, and imidazole groups.

[0019] Preferably, the small molecule ligand is a biocompatible organic acid and / or a salt of an organic acid; Preferably, the organic acid is selected from one or more of lactic acid, citric acid, malic acid, tartaric acid, acetic acid, acetic alcohol, succinic acid, gluconic acid, glycine, aspartic acid, glutamic acid, pantothenic acid, sorbic acid, threonic acid, and phytic acid. The salt of the organic acid is selected from one or more of lactate, sodium citrate, malate, tartrate, acetate, acetic alcohol, succinate, gluconate, glycine, aspartate, glutamate, pantothenate, sorbate, threonic acid, and phytate.

[0020] In practice, any one or more small molecule compounds that can form coordination bonds with multivalent metal ions, are biocompatible, and are highly hydrophilic can be selected.

[0021] Preferably, the alginate compound has a mass fraction of 0.1~30 w / v; more preferably, the alginate compound has a mass fraction of 0.5~6 w / v; and even more preferably, the alginate compound has a mass fraction of 1~5 w / v.

[0022] Preferably, the molar concentration of the polyvalent metal ion is 0.1~500 mM. More preferably, the molar concentration of the polyvalent metal ion is 0.2~200 mM; even more preferably, the molar concentration of the polyvalent metal ion is 1~100 mM; even more preferably, the molar concentration of the polyvalent metal ion is 5~50 mM.

[0023] The present invention also provides a method for preparing an injectable hydrogel pregel solution based on ligand regulation, comprising the following steps: uniformly mixing the alginate compound, a donor providing the multivalent metal ions, and the small molecule ligand in a solvent in a certain proportion; obtaining the pregel solution; wherein the solvent is an aqueous solvent; and the donor providing the multivalent metal ions is a metal ion compound soluble in the aqueous solvent.

[0024] Preferably, the aqueous solvent refers to a solvent in which the continuous phase is water-based, as opposed to an organic solvent (such as petroleum ether or acetone) in which the continuous phase is organic. Water, physiological saline, or PBS buffer are preferred.

[0025] Preferably, the donor for providing polyvalent metal ions is a water-soluble metal ion compound, and more preferably a metal ion compound with high water solubility, including CaCl2, Ca(NO3)2, CuCl2, CuSO4, MgCl2, Mg(NO3)2, ZnCl2, Zn(NO3)2, etc.

[0026] Preferably, the method for preparing the ligand-regulated injectable hydrogel pregel solution of the present invention includes the following steps: (1) Dissolve the alginate compound in an aqueous solvent to obtain a homogeneous solution; (2) Add a small molecule ligand to the homogeneous solution obtained in step (1) and mix evenly; then add a donor that provides the multivalent metal ions and mix evenly again; or dissolve the small molecule ligand and the donor that provides the multivalent metal ions in an aqueous solvent to prepare a mixed solution, and add the mixed solution to the homogeneous solution obtained in step (1); to obtain the pregel solution.

[0027] In practice, for pregel solutions requiring a high concentration of alginate compounds, a homogeneous solution with a high concentration of alginate compounds can be prepared in step (1), and an aqueous solvent can be added after step (2) to adjust the solution volume and concentration to the target value, thereby obtaining the pregel solution.

[0028] This invention employs two preferred steps: first, mixing small molecule ligands with multivalent metal ions to form a complex; second, introducing small molecule ligands into alginate compounds. Both methods avoid direct cross-linking of multivalent metal ions and alginate compounds, thus ensuring uniform distribution of small molecule ligands and enabling rapid and efficient construction of a pregel system.

[0029] This invention also provides applications of the above-mentioned ligand-regulated injectable hydrogel pregel solutions for the preparation of materials for tissue engineering, regenerative medicine, drug delivery, cell delivery, 3D printing, or imaging.

[0030] The technical solutions of the embodiments of the present invention have at least the following advantages and beneficial effects: (1) Excellent fine needle injectability: The system exhibits significant shear thinning characteristics and can be smoothly extruded even through a 34G ultrafine needle without clogging or flow interruption.

[0031] (2) Immediate stabilization after injection: The ligand diffuses and releases multivalent metal ions, which instantly form a "gel shell" at the interface, effectively preventing material leakage.

[0032] (3) In-situ self-reinforcing mechanical properties: The internal cross-linking gradually densifies over time, G′ is significantly improved, and the material maintains a stable morphology over a long period of time.

[0033] (4) Formulation predictability: By establishing the correlation between CLR and the stability constant K of small molecule ligands-multivalent metal ions, the predictable design of gelation behavior can be achieved, which is applicable to the expansion of different ligands and metal ions.

[0034] (5) Wide range of applications: It can be used as a tissue engineering scaffold filling material, regeneration and repair carrier, drug / cell delivery system and 3D printing ink, with good biocompatibility and clinical translation potential. Attached Figure Description

[0035] Figure 1 This is a schematic diagram showing the linear correlation between the critical ligand ratio (CLR) and the reciprocal of the ligand-metal ion stability constant (1 / K) in Experiment Example 1.

[0036] Figure 2 The shear rate-viscosity curves are for the pregel solution of Example 1 in Experimental Example 2 and the sodium alginate aqueous solution of Comparative Example 1.

[0037] Figure 3 The figure shows the injection force-time curves of the pregel solution of Example 1 in Experiment 3 under different needle gauges (25G-32G).

[0038] Figure 4 The graph shows the injection force-inner diameter relationship curves of the pregel solution of Example 1 in Experiment 3 under different needle gauges (25G-32G).

[0039] Figure 5 This is a diagram showing the successful injection of the pregel solution from Example 1 in Experiment 3 using a 34G needle.

[0040] Figure 6 This is a schematic diagram showing the rheological results of the pregel solution of Example 1 in Experiment 4 before and after immersion in PBS.

[0041] Figure 7 The images show the state of the pregel solution of Example 1 in Experimental Example 5, the pregel solution of Example 6, and the hydrogel of Comparative Example 2 after preparation and after standing for 24 hours.

[0042] Figure 8 The images show the state of the pregel solution of Example 1 in Experimental Example 6 during injection, after injection, and one week after injection. Detailed Implementation

[0043] Example 1 This embodiment provides an injectable hydrogel pregel solution based on ligand regulation, comprising the following components: sodium alginate; calcium ions; sodium lactate; wherein the mass-volume concentration of sodium alginate is 2 w / v; and the molar concentration of calcium ions is 25.2 mM.

[0044] This embodiment first determined the critical molar ratio (CLR) of sodium lactate to calcium ions at a temperature of 25°C, a sodium alginate concentration of 2 w / v%, and a calcium ion molar concentration of 25.2 mM. The specific method for determining the CLR of sodium lactate to calcium ions under the above conditions in this embodiment includes: (1) Prepare 5 groups of 3 w / v% sodium alginate solutions at 25℃, 2 mL of each; (2) Add 0.212 g, 0.254 g, 0.296 g, 0.339 g and 0.382 g of sodium lactate to the 5 groups of 3 w / v% sodium alginate aqueous solutions in step (1) respectively, and mix thoroughly to form a homogeneous solution; (3) Under stirring conditions, 168 µL of calcium chloride (CaCl2) solution with a mass volume concentration of 5 w / v% was added to each of the five homogeneous solutions containing sodium lactate obtained in step 2, and water was added to make up to 3 mL, so that the final mass volume concentration of sodium alginate in the system was 2 w / v%. At this time, the molar concentration of calcium ions in each group of solutions was 25.2 mM, and the molar concentrations of sodium lactate were 630.6 mM, 755.5 mM, 880.5 mM, 1008.4 mM and 1133.3 mM, respectively. Observe the system morphology of each group of solutions; among them, the experimental group with sodium lactate concentration of 1133.3mM changed from gel state to sol state, at which time L / M was 45, and the L / M corresponding to this sodium lactate solubility was 45. The L / M corresponding to the previous gel state sodium lactate concentration (1008.4mM) was 40. The difference between the two is less than 15% ((45-40) / 40=12.5%). Therefore, under the above conditions, the critical molar ratio CLR of sodium lactate-calcium ions is determined to be 45. If the difference between the two is greater than 15%, the concentration gradient range of sodium lactate will be further narrowed to determine a more accurate CLR.

[0045] Based on the above result of CLR=45, the molar concentration of sodium lactate was selected as 1133.3 mM in this embodiment, that is, L / M=45.

[0046] This embodiment also provides a method for preparing the above-mentioned ligand-regulated injectable hydrogel pregel solution, including the following steps: (1) Sodium alginate (SA) was dissolved in deionized water in proportion and stirred magnetically overnight at room temperature to obtain a homogeneous sodium alginate solution with a mass-volume concentration of 3 w / v%. (2) Add 0.382 g of sodium lactate to 2 mL of sodium alginate solution obtained in step (1) and stir thoroughly until completely dissolved to obtain a sodium alginate + sodium lactate mixed solution; (3) Under vigorous stirring, add 168 µL of calcium chloride (CaCl2) solution with a mass-volume concentration of 5 w / v% to the mixed solution obtained in step (2) dropwise, so that sodium lactate reacts with CaCl2. 2+ Reversible complexation occurs; (4) Add an appropriate amount of deionized water to adjust the total volume to 3 mL to obtain a sodium alginate + sodium lactate pregel solution with a mass volume concentration of 2 w / v% (calculated as SA) in this embodiment.

[0047] Example 2 This embodiment provides an injectable hydrogel pregel solution based on ligand regulation. The difference between this solution and the one in Example 1 is that sodium lactate is replaced with sodium citrate.

[0048] In this embodiment, the critical molar ratio (CLR) of sodium citrate to calcium ions was first determined at a temperature of 25°C, a sodium alginate concentration of 2 w / v%, and a calcium ion molar concentration of 25.2 mM. The determination method was the same as in Example 1, except that in step (2), anhydrous sodium citrate of 9.76 mg, 11.71 mg, 13.66 mg, 15.61 mg, and 17.56 mg was added to the five groups of 3 w / v% sodium alginate aqueous solutions from step (1), respectively. After volume adjustment, the sodium citrate concentrations were 12.6 mM, 15.12 mM, 17.64 mM, 20.16 mM, and 22.68 mM, respectively. Finally, the critical molar ratio (CLR) of sodium citrate to calcium ions under the conditions of this embodiment was determined to be 0.8.

[0049] Based on the above result of CLR=0.8, the molar concentration of sodium citrate in this embodiment is selected as 22.3mM, that is, L / M=0.85.

[0050] The same method for preparing the injectable hydrogel pregel solution based on ligand regulation is used in this embodiment. The difference from Example 1 is that the amount of sodium citrate added in step (2) is 0.017g, and the pregel solution prepared in this embodiment is sodium alginate + sodium citrate pregel solution.

[0051] Example 3 This embodiment provides an injectable hydrogel pregel solution based on ligand regulation. The difference between this solution and the one in Example 1 is that sodium lactate is replaced with disodium malate.

[0052] In this embodiment, the critical molar ratio (CLR) of disodium malate to calcium ions was first determined at a temperature of 25 °C, a sodium alginate concentration of 2 w / v%, and a calcium ion molar concentration of 25.2 mM. The determination method was the same as in Example 1, except that in step (2), 94.22 mg, 107.69 mg, 121.15 mg, 134.61 mg, and 148.07 mg of disodium malate were added to the five groups of 3 w / v% sodium alginate aqueous solutions from step (1), respectively. After volume adjustment, the concentrations of disodium malate were 176.4 mM, 201.6 mM, 226.8 mM, 252.0 mM, and 277.2 mM, respectively. Finally, the critical molar ratio (CLR) of disodium malate to calcium ions under the conditions of this embodiment was determined to be 11.

[0053] Based on the above result of CLR=11, the molar concentration of disodium malate in this embodiment is selected as 300mM, that is, L / M=11.5.

[0054] The same method for preparing injectable hydrogel pregel solution based on ligand regulation is used in this embodiment. The difference from Example 1 is that the amount of disodium malate added in step (2) is 0.161g. The pregel solution prepared in this embodiment is sodium alginate + disodium malate pregel solution.

[0055] Example 4 This embodiment provides an injectable hydrogel pregel solution based on ligand regulation. The difference between this solution and the one in Example 1 is that sodium lactate is replaced with sodium tartrate.

[0056] In this embodiment, the critical molar ratio (CLR) of sodium tartrate-calcium ions was first determined at a temperature of 25 °C, a sodium alginate concentration of 2 w / v%, and a calcium ion molar concentration of 25.2 mM. The determination method was the same as in Example 1, except that in step (2), 322.76 mg, 351.98 mg, 381.21 mg, 410.43 mg, and 439.75 mg of anhydrous sodium tartrate were added to the five groups of 3 w / v% sodium alginate aqueous solutions from step (1), respectively. After volume adjustment, the sodium tartrate concentrations were 554.4 mM, 604.8 mM, 655.2 mM, 705.6 mM, and 756.0 mM, respectively. Finally, the critical molar ratio (CLR) of sodium tartrate-calcium ions under the conditions of this embodiment was determined to be 28.

[0057] Based on the above result of CLR=28, the molar concentration of sodium tartrate selected in this embodiment is 706mM, that is, L / M=28.

[0058] This embodiment also uses the above-described method for preparing the injectable hydrogel pregel solution based on ligand regulation, including the following: (1) Sodium alginate (SA) was dissolved in deionized water in proportion and stirred magnetically overnight at room temperature to obtain a homogeneous sodium alginate solution with a mass-volume concentration of 3 w / v%. (2) Take 0.411 g of sodium tartrate and 168 µL of calcium chloride (CaCl2) solution with a mass volume concentration of 5 w / v%, mix them, add water to 1 mL and stir thoroughly until the mixture is homogeneous; (3) Under vigorous stirring conditions, the mixed solution obtained in step (2) is added to 2 mL of sodium alginate solution obtained in step (1) to obtain a sodium alginate + sodium tartrate pregel solution with a mass volume concentration of 2 w / v% (calculated as SA) in this embodiment.

[0059] Example 5 This embodiment provides an injectable hydrogel pregel solution based on ligand regulation. The difference between this solution and the one in Example 1 is that sodium lactate is replaced with disodium malate, the mass-volume concentration of sodium alginate is 5 w / v, and the molar concentration of calcium ions is 50 mM.

[0060] In this embodiment, the critical molar ratio (CLR) of disodium malate to calcium ions was also determined at a temperature of 25°C, a sodium alginate concentration of 5 w / v%, and a calcium ion molar concentration of 50 mM. The determination method was the same as in Example 1, with the difference being: In step (1), prepare 5 groups of sodium alginate aqueous solutions with a concentration of 6 w / v, each 2.5 mL; In step (2), 0.168 g, 0.185 g, 0.202 g, 0.219 g and 0.235 g of disodium malate were added to the five groups of 6 w / v% sodium alginate aqueous solutions in step (1) respectively, and the solutions were thoroughly mixed to form a homogeneous solution. In step (3), 332.7 µL of calcium chloride (CaCl2) solution with a mass-volume concentration of 5 w / v% was added to each of the five homogeneous solutions from step (2), and water was added to bring the volume to 3 mL, so that the final mass-volume concentration of sodium alginate in the system was 5 w / v%. At this point, the molar concentration of calcium ions in each group of solutions was 50 mM, and the molar concentrations of disodium malate were 500 mM, 550 mM, 600 mM, 650 mM, and 700 mM, respectively.

[0061] The critical molar ratio (CLR) of disodium malate-calcium ions was ultimately determined to be 13 under the conditions of this embodiment.

[0062] Based on the above result of CLR=13, the molar concentration of disodium malate in this embodiment is selected as 660 mM, that is, L / M=13.2.

[0063] This embodiment uses the same method as the above-described preparation method of the injectable hydrogel pregel solution based on ligand regulation, but differs from Example 1 in that: In step (1), a homogeneous sodium alginate solution with a mass-volume concentration of 6 w / v% is prepared; In step (2), 0.222 g of disodium malate is added to 2.5 mL of sodium alginate solution obtained in step (1); In step (3), 332.7 µL of calcium chloride (CaCl2) solution with a mass-volume concentration of 5 w / v% is added dropwise to the mixed solution obtained in step (2); In step (4), add an appropriate amount of deionized water to adjust the total volume to 3 mL, and obtain a sodium alginate + disodium malate pregel solution with a mass volume concentration of 5 w / v% (calculated as SA) in this embodiment.

[0064] Example 6 This embodiment provides an injectable hydrogel pregel solution based on ligand regulation. The difference between this solution and the one in Example 1 is that sodium lactate is replaced with sodium tartrate, the mass-volume concentration of sodium alginate is 1 w / v, and the molar concentration of calcium ions is 5 mM.

[0065] In this embodiment, the critical molar ratio (CLR) of sodium tartrate-calcium ions was also determined at a temperature of 20 °C, a sodium alginate concentration of 1 w / v%, and a calcium ion molar concentration of 5 mM. The determination method was the same as in Example 1, with the difference being: In step (1), prepare 5 groups of sodium alginate aqueous solutions with a concentration of 1.5 w / v%, 2 mL for each group; In step (2), sodium tartrate 0.0931 g, 0.0989 g, 0.1048 g, 0.1106 g and 0.1164 g were added to the five groups of 1.5 w / v% sodium alginate aqueous solutions in step (1) respectively, and they were mixed thoroughly to form a homogeneous solution; In step (3), 20.8 µL of calcium chloride (CaCl2) solution with a mass-to-volume concentration of 8 w / v% was added to each of the five homogenized solutions from step (2), and water was added to bring the volume to 3 mL, so that the final mass-to-volume concentration of sodium alginate in the system was 1 w / v%. At this point, the molar concentration of calcium ions in each group of solutions was 5 mM, and the molar concentrations of sodium tartrate were 160 mM, 170 mM, 180 mM, 190 mM, and 200 mM, respectively. Finally, the critical molar ratio (CLR) of sodium tartrate-calcium ions was determined to be 36 under the conditions of this embodiment.

[0066] Based on the above result of CLR=36, the molar concentration of sodium tartrate selected in this embodiment is 190 mM, that is, L / M=38.

[0067] This embodiment uses the same method as the above-described preparation method of the injectable hydrogel pregel solution based on ligand regulation, but differs from Example 1 in that: In step (1), a homogeneous sodium alginate solution with a mass-volume concentration of 1.5 w / v% is prepared; In step (2), 0.1106 g of sodium tartrate is added to 2 mL of sodium alginate solution obtained in step (1); In step (3), 20.8 µL of calcium chloride (CaCl2) solution with a mass-volume concentration of 8 w / v% is added dropwise to the mixed solution obtained in step (2); In step (4), add an appropriate amount of deionized water to adjust the total volume to 3 mL, and obtain a sodium alginate + sodium tartrate pregel solution with a mass volume concentration of 1 w / v% (calculated as SA) in this embodiment.

[0068] Comparative Example 1 This comparative example provides an aqueous solution of sodium alginate; specifically, it is an aqueous solution of sodium alginate with a mass-volume concentration of 2 w / v%.

[0069] Comparative Example 2 This comparative example provides a hydrogel comprising the following components by weight percentage: sodium alginate; calcium ions; sodium lactate; wherein the mass-volume concentration of sodium alginate is 2 w / v%; the molar concentration of calcium ions is 25.2 mM; and the molar concentration of sodium lactate is 960 mM.

[0070] The preparation method of this comparative hydrogel includes: (1) Dissolve sodium alginate (SA) in deionized water and stir magnetically overnight at room temperature to obtain a homogeneous sodium alginate solution with a mass-volume concentration of 3 w / v. (2) Add 0.3235 g of sodium lactate to 2 mL of sodium alginate solution obtained in step (1) and stir thoroughly until completely dissolved to obtain a sodium alginate + sodium lactate mixed solution; (3) Under vigorous stirring, add 168 µL of calcium chloride (CaCl2) solution with a mass-volume concentration of 5 w / v% to the mixed solution obtained in step (2) dropwise, so that sodium lactate reacts with CaCl2. 2+ Reversible complexation occurs; (4) Add an appropriate amount of deionized water to adjust the total volume to 3 mL to obtain sodium alginate + sodium lactate hydrogel with a mass volume concentration of 2 w / v% (calculated as SA) in this embodiment.

[0071] Experimental Example 1 Examples 1-4 determined the critical molar ratio (CLR) of different small molecule ligands (sodium lactate, sodium citrate, disodium malate, and tartaric acid) to calcium ions at 25°C, a sodium alginate concentration of 2 w / v, and a calcium ion molar concentration of 25.2 mM. Further analysis revealed that under specific conditions (25°C, sodium alginate concentration of 2 w / v, and calcium ion molar concentration of 25.2 mM), the reciprocal of the stability constant of the complexes formed by the small molecule ligands and metal ions was positively correlated with the CLR. Plotting the reciprocal of the stability constants of the small molecule ligand-metal complexes in Examples 1-4 on the x-axis and their corresponding CLRs on the y-axis yielded the following results: Figure 1 The schematic diagram shown is composed of Figure 1It can be seen that there is a linear correlation between the critical ligand ratio (CLR) and the reciprocal of the ligand-metal ion stability constant (1 / K), indicating that the CLR can be predicted by the K value, and the formulation designability across ligands and across metal ions can be realized.

[0072] Experiment Example 2 The alginate + sodium lactate pregel solution prepared in Example 1 was used as the experimental sample, and the sample prepared using only 2 w / v% sodium alginate aqueous solution in Comparative Example 1 was used as the control sample. A physical rheometer (model: MCR300, Anton Paar, Austria) equipped with parallel plates of 25 mm diameter was used for testing at 37°C. The sample was placed between the upper and lower plates, the gap was adjusted to 1.0 mm, and excess sample was removed with an edge trimmer. The shear rate was measured from 0.1 to 1000 s⁻¹. -1 Continuous scanning was performed within the range to evaluate the shear-thinning properties of the sample; the test results are as follows: Figure 2 As shown.

[0073] Depend on Figure 2 Analysis shows that the alginate-sodium lactate pregel solution prepared in Example 1 exhibits significant "shear-thinning" behavior, that is, the apparent viscosity decreases with increasing shear rate. As the shear rate further increases, the viscosity of the alginate-sodium lactate pregel solution in Example 1 gradually approaches the viscosity of the sodium alginate aqueous solution in Comparative Example 2. This indicates that the pregel solution has not undergone a cross-linking reaction of sodium alginate, and the entire system has good injectability, especially suitable for fine needle injection.

[0074] Experimental Example 3 The alginate + sodium lactate pregel solution prepared in Example 1 was used as the experimental sample. The sample was loaded into a 1 mL syringe, and injection needles of different gauges were used. The test was conducted at room temperature in compression mode using a universal testing machine (Model: UniVert, CellScale Biomaterials Testing, Canada). The injection flow rate was set to 1 mL / min, and the changes in injection force under different needle gauge conditions were recorded; the test results are shown below. Figure 3 , Figure 4 and Figure 5 As shown.

[0075] in Figure 3 The injection force-time curves of the sodium alginate + sodium lactate pregel solution system in Example 1 under different needle gauges (25G-32G) show that the system still maintains an acceptable injection force under fine needles (32G). Figure 4 The injection force-inner diameter relationship curves for the sodium alginate + sodium lactate pregel solution system in Example 1 under different needle gauges (25G-32G) show that the injection force increases as the needle inner diameter decreases, but is still within the clinically operable range. Figure 5This image shows the successful injection of the sodium alginate + sodium lactate pregel solution system of Example 1 through a 34G needle. Rhodamine was added to the solution for easier visualization.

[0076] The results of this experiment show that the pregel solution has good needle penetration and injectability, meeting the requirements for ease of operation in minimally invasive surgical applications.

[0077] Experiment Example 4 The sodium alginate + sodium lactate pregel solution obtained in Example 1 was poured into a high-density polyethylene (HDPE) mold (30 mm in diameter, cylindrical) to make the sample thickness approximately 2 mm. The sample was degassed by standing at room temperature for 1 hour or centrifuging at 1000 rpm for 5 minutes to remove air bubbles. Then, phosphate-buffered saline (PBS) was slowly added to the mold, avoiding disturbance of the sample surface, and the sample was immersed at 37°C for a total time of 1 hour. During immersion, the sample was carefully turned over to ensure full contact with the PBS. Samples before and after 1 hour of PBS immersion were tested using a physical rheometer (Model: MCR300, Anton Paar, Austria) equipped with a 25 mm diameter parallel plate at 37°C. The rheological properties of the samples were further characterized by scanning at 0.1% strain within a frequency range of 0.1–10 rad / s; the test results are shown below. Figure 6 As shown.

[0078] Figure 6 In this context, G′ represents the storage modulus, and G′′ represents the loss modulus. Immersion in PBS solution simulates the pregel solution entering the in vivo environment. Comparative analysis of data before and after immersion reveals that before immersion, the overall values ​​of the storage modulus (G′) and loss modulus (G′′) of the pregel solution prepared in Example 1 are relatively low, and the increases in G′ and G′′ with frequency are relatively gradual. This indicates that before immersion, the sample formed by the sodium alginate + sodium lactate pregel solution has weak elastic and viscous responses, and no cross-linking reaction has occurred. After 1 hour of immersion, the value of G′ increases significantly and remains relatively stable at a high level (approximately 100 Pa) across the entire frequency range. G′ is much larger than G′′, indicating that after immersion, the elasticity of the sample is dominant, and cross-linking forms a stable elastic network structure, reflecting a strong self-reinforcing ability.

[0079] Experimental Example 5 Rhodamine was added during the preparation of the sodium alginate-sodium lactate pregel solution in Example 1, the sodium alginate + sodium tartrate pregel solution in Example 6, and the sodium alginate + sodium lactate hydrogel in Comparative Example 2 to achieve visualization. The state of the system was observed immediately after preparation and after standing for 24 hours.

[0080] The results are as follows Figure 7 As shown, from Figure 7 It can be seen that the pregel solutions prepared in Example 1 and Example 6 both maintained a stable sol system state immediately after preparation and after standing for 24 hours. The solutions were uniform and free of impurities when observed with the naked eye. The hydrogel prepared in Comparative Example 2 had an obvious gel state immediately after preparation and maintained the gel state after standing for 24 hours.

[0081] Experimental Example 6 Rhodamine was added to the sodium alginate-sodium lactate pregel solution in Example 1 to achieve visualization. After injecting this solution into PBS solution, the condition was observed at the time of injection, after injection, and one week after injection. The results are as follows: Figure 8 As shown.

[0082] from Figure 8 It can be seen that the pregel system can be successfully injected, and the system can maintain its shape well after injection, indicating that a gel shell can be formed rapidly on its surface, thereby effectively preventing material leakage. After soaking in PBS for 1 week, the system still maintains its intact shape, proving that it can gradually complete gelation after injection and maintain long-term structural stability.

[0083] In summary, the pregel solution proposed in this invention not only possesses excellent injectability and operational stability, but also achieves a transformation from sol to gel after injection, accompanied by a continuous enhancement of mechanical properties. This effectively solves the problem that traditional alginate hydrogels struggle to simultaneously achieve injectability, immediate stability, and long-term mechanical properties.

Claims

1. A ligand-regulated injectable hydrogel pregel solution, characterized in that, It includes the following components: Alginic acid compounds; polyvalent metal ions; Small molecule ligands; The alginic acid compounds include alginic acid or alginate and their derivatives; the small molecule ligand contains at least one functional group that can form a coordination bond with the multivalent metal ion, and the functional group is selected from one or more of carboxyl, carboxyl, hydroxyl, amino, amine, phosphonic, pyridyl, and imidazole groups. The molar ratio of the small molecule ligand to the multivalent metal ion is denoted as L / M; the minimum L / M required to maintain the system in a sol state under set temperature, set alginate compound concentration, and set multivalent metal ion concentration is defined as the critical ligand ratio, denoted as CLR. In the pregel solution, L / M ≥ CLR.

2. The ligand-regulated injectable hydrogel pregel solution according to claim 1, characterized in that, Let K be the stability constant of the complex formed by any of the small molecule ligands A and any of the polyvalent metal ions B. A-B For a given temperature, a set concentration of alginate compounds, and a set concentration of polyvalent metal ions, the CLR ∝ 1 / K A-B .

3. The injectable hydrogel pregel solution based on ligand regulation according to claim 1, characterized in that, The method for determining the CLR of any of the small molecule ligands A and any of the multivalent metal ions B, under a set temperature T, a set concentration of alginate C, and a set concentration of multivalent metal ions M, includes the following steps: (1) Prepare multiple groups of aqueous solutions of alginate compounds with a concentration of D at a set temperature T; (2) Add different amounts of the small molecule ligand A to the multiple groups of alginate compounds aqueous solutions in step (1), mix them evenly, and obtain multiple groups of homogeneous solutions; (3) Add a fixed concentration of polyvalent metal ions B to each of the homogeneous solutions in step (2), and add an appropriate amount of water to make the solution volume up so that the concentration of the resulting alginate compound is C, the molar concentration of polyvalent metal ions B is M, and the molar concentration L of the small molecule ligand A changes from low to high according to the gradient; observe the morphology of each solution system; the L / M when the system changes from gel state to sol state is the CLR of the small molecule ligand A and the polyvalent metal ions B under the set temperature T, the set concentration of alginate compound C, and the set concentration of polyvalent metal ions M.

4. The ligand-regulated injectable hydrogel pregel solution according to claim 1, characterized in that, The alginate includes sodium alginate, potassium alginate, or ammonium alginate; the alginate derivatives include alginate modified by esterification, amidation, grafting of polypeptides, or grafting of polymers.

5. The ligand-regulated injectable hydrogel pregel solution according to claim 1, characterized in that, The multivalent metal ion is one or more of a divalent or trivalent metal ion.

6. The ligand-regulated injectable hydrogel pregel solution according to claim 1, characterized in that, The small molecule ligand is a biocompatible organic acid and / or a salt of an organic acid.

7. The ligand-regulated injectable hydrogel pregel solution according to claim 1, characterized in that, The mass-volume concentration of the alginate compound is 0.1~30 w / v; the molar concentration of the polyvalent metal ion is 0.1~500 mM.

8. The method for preparing the ligand-regulated injectable hydrogel pregel solution according to any one of claims 1 to 7, characterized in that, The process includes the following steps: uniformly mixing the alginate compound, the donor of the multivalent metal ions, and the small molecule ligand in a solvent in a certain proportion to obtain the pregel solution; the solvent is an aqueous solvent; and the donor of the multivalent metal ions is a metal ion compound soluble in the aqueous solvent.

9. The method for preparing an injectable hydrogel pregel solution based on ligand regulation according to claim 8, characterized in that, Includes the following steps: (1) Dissolve the alginate compound in an aqueous solvent to obtain a homogeneous solution; (2) Add a small molecule ligand to the homogeneous solution obtained in step (1) and mix evenly; then add a donor that provides the multivalent metal ions and mix evenly again; or dissolve the small molecule ligand and the donor that provides the multivalent metal ions in an aqueous solvent to prepare a mixed solution, and add the mixed solution to the homogeneous solution obtained in step (1) to obtain the pregel solution.

10. The application of the ligand-regulated injectable hydrogel pregel solution according to any one of claims 1 to 7, for the preparation of tissue engineering materials, regenerative medicine materials, drug delivery materials, cell delivery materials, three-dimensional printing materials, or imaging tracer materials.

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