Injectable hydrogel pre-gel solution based on ligand regulation and preparation method and application thereof

Abstract

The application relates to the field of biomaterials, and provides an injectable hydrogel pre-gel solution based on ligand regulation as well as a preparation method and application thereof. The pre-gel solution comprises the following components: an alginic acid compound; a multivalent metal ion; and a small molecule ligand; wherein the alginic acid compound comprises alginic acid or an alginic acid salt and a derivative thereof; the molar ratio of the small molecule ligand to the multivalent metal ion is denoted as L / M; the minimum L / M for maintaining the system in a sol state at a set temperature and ion strength is defined as a critical ligand ratio, denoted as CLR; and in the pre-gel solution, L / M >= CLR. The pre-gel solution can be smoothly injected through a fine needle, can form a gel shell at the interface to prevent leakage after being injected into an aqueous environment, and has the performance of continuously self-reinforcing, and thus has a wide application range.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, in particular, relates to an injectable hydrogel pre-gel solution based on ligand regulation and a preparation method and application thereof. BACKGROUND

[0002] In recent years, injectable hydrogels have shown great application potential in the fields of tissue engineering, regenerative medicine and drug delivery due to their minimally invasive drug delivery characteristics and excellent tissue shape adaptability, and are considered as a promising biomaterial platform. Alginate (Alg) as a widely available, biocompatible and biodegradable natural polysaccharide can form a three-dimensional network structure hydrogel through ion crosslinking with multivalent metal ions (such as Ca 2+ , Ba 2+ , Sr 2+ , etc.), under mild conditions, and thus has attracted much attention.

[0003] At present, the gelation strategies of alginate-based injectable hydrogels mainly include two types: exogenous gelation and endogenous gelation. Among them:

[0004] (1) The exogenous gelation method is usually to directly contact the alginate solution with the external multivalent metal ion solution (such as CaCl2 solution), which can quickly induce gel formation. Although this method is simple to operate, the crosslinking reaction rate is too fast, which easily leads to uneven gel structure; in addition, the application often needs to use a double-barreled syringe and other devices to deliver the two components separately, which increases the complexity of the operation and limits its application in situations requiring precise injection forming.

[0005] (2) The endogenous gelation technology usually disperses insoluble metal salts (such as CaCO3, calcium malate, calcium sodium ethylenediaminetetraacetate, calcium oxalate and calcium gluconate, etc.) and acidic precursors (such as gluconic acid-delta-lactone, guluronic acid, etc.) in the alginate solution, as disclosed in patent CN 118267527 A, etc., which utilizes the low solubility of insoluble metal salts in water, and the particles in the solution slowly release metal ions under acidolysis conditions to achieve in-situ crosslinking. Although this method can improve the gel uniformity, the metal ion release process is slow and irreversible, which leads to poor initial stability of the material after injection and easy leakage; at the same time, the presence of insoluble particles in the system increases the risk of fine needle blockage, which restricts its application in minimally invasive interventional therapy and other scenarios.

[0006] In addition, there are strategies in the prior art to prepare low modulus gels by reducing the degree of crosslinking, to prepare physically crosslinked "injectable" cryogels by freeze-thaw cycles, or to rely on external fields such as light and magnetism to trigger gelation. However, these methods often have difficulty in balancing mechanical properties, shape maintenance ability, universality of triggering conditions, and convenience of clinical application, especially in simultaneously achieving excellent fine needle injectability, immediate shape fixation after injection, and long-term mechanical property self-enhancement.

[0007] Therefore, there is an urgent need for a new alginate hydrogel system that can maintain smooth fine needle injection while achieving immediate interfacial gelation after injection and obtaining long-term stability and enhanced mechanical properties through subsequent crosslinking densification, thereby meeting the application requirements of tissue engineering, regenerative repair, and precise drug delivery. SUMMARY

[0008] In view of the above deficiencies in the prior art, the core purpose of the present application is to solve the technical problems of poor fine needle injectability, insufficient stability after injection, and poor mechanical properties commonly existing in existing alginate hydrogel systems.

[0009] The present application is achieved by the following technical solutions:

[0010] An injectable hydrogel pre-gel solution based on ligand regulation, comprising the following components:

[0011] alginate compounds; multivalent metal ions; small molecule ligands;

[0012] Among them, the alginate compounds include alginic acid or alginate and their derivatives;

[0013] The molar ratio of the small molecule ligand to the multivalent metal ion is denoted as L / M; the minimum L / M that maintains the system in sol state at a given temperature, a given alginate compound concentration, and a given multivalent metal ion concentration is defined as the critical ligand ratio, denoted as CLR;

[0014] In the pre-gel solution, L / M≥CLR.

[0015] The present application is in the face of exogenous gelation needs two components to be delivered to the body, endogenous gelation is difficult to realize the existing technology problem of stable delivery of fine needle, thinks of using small molecule ligand and multivalent metal ion to form reversible complex, so that before injection, the crosslinking of alginic acid compound and multivalent metal ion is inhibited, so that the system can maintain a uniform single-phase sol system, which is convenient for smooth injection of fine needle; and after injection into the body water environment, small molecule ligand will diffuse from high concentration area (pre-gel solution inside) to low concentration area (external body fluid) under the driving of concentration gradient, break the complex balance and release multivalent metal ions, the released multivalent metal ions are quickly captured by the carboxyl groups of alginate, and a gradual crosslinking process from outside to inside is formed at the injection point. Due to the preferential diffusion of small molecule ligand outside, the metal ions outside are released first, and the outer gel shell is formed first, then the small molecule ligand inside diffuses gradually, the metal ions inside are released gradually, so that the inside is gradually densified, thereby realizing instant stabilization and subsequent mechanical self-reinforcement after injection.

[0016] More importantly, the present application defines a parameter of critical ligand ratio CLR, which is defined as the minimum L / M that maintains the system in sol state under the condition of a set temperature, a set alginic acid compound concentration and a set multivalent metal ion concentration. Moreover, the present application limits the molar ratio L / M of small molecule ligand to multivalent metal ion in pre-gel solution to be greater than or equal to CLR. Further, by controlling the value of L / M, it can be ensured that the pre-gel solution is always in sol state, and it can be ensured that the small molecule ligand can preferentially complex with the multivalent metal ions in the solution system, thereby avoiding excessive crosslinking between the multivalent metal ions and alginate and causing gelation in the pre-gel solution.

[0017] Further, the present application provides a method for measuring the critical ligand ratio (CLR) of any small molecule ligand A and any multivalent metal ion B under the condition of a set temperature T, a set alginic acid compound concentration C and a set multivalent metal ion concentration M, which comprises the following steps:

[0018] (1) Under a set temperature T, prepare a plurality of groups of alginic acid compound aqueous solutions with a concentration of C;

[0019] (2) Add different amounts of the small molecule ligand A to the plurality of groups of alginic acid compound aqueous solutions of step (1) respectively, mix uniformly, and obtain a plurality of groups of uniform solutions;

[0020] (3) to each of the uniform solution of step (2), add a fixed concentration of polyvalent metal ion B, and add an appropriate amount of water to make the solution constant, so that the concentration of the alginic compound obtained is C, the molar concentration of the polyvalent metal ion 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 group of solution systems; when the system changes from gel state to sol state, L / M is the CLR of the small molecule ligand A and the polyvalent metal ion B under the condition of setting temperature T, setting alginic compound concentration C, and setting polyvalent metal ion concentration M.

[0021] In practice, since the pre-gel system of the present application is used in the field of biological medicine, based on the limitation of its use field, the setting temperature T is generally selected to be 20-37℃; it has been verified in practice that within this temperature range, the CLR value of the small molecule ligand A and the polyvalent metal ion B under the condition of setting alginic compound concentration C and setting polyvalent metal ion concentration M changes very little within an acceptable range, so in practice, the influence of temperature on CLR is often ignored.

[0022] Further preferably, in step (3), the difference between L / M when the system changes from gel state to sol state and L / M corresponding to the concentration of the previous small molecule ligand A (in gel state) is less than 15%, so as to ensure that the accuracy of the finally measured CLR is as high as possible, and if the difference is greater than 15%, the concentration gradient gap of the small molecule ligand A is further narrowed to determine a more accurate CLR.

[0023] As a preferred, the stability constant of the complex formed by any of the small molecule ligands A and any of the polyvalent metal ions B is K A-B For the CLR under the condition of setting temperature, setting alginic compound concentration, and setting polyvalent metal ion concentration, it is proportional to 1 / K A-B .

[0024] It has been found through research that CLR and the reciprocal of the stability constant of the complex formed by the small molecule ligand A and the polyvalent metal ion B (1 / K A-B ) have a good linear correlation; based on this correlation, a mapping / regression model of CLR-1 / K A-B can be established, according to which the corresponding CLR value under the condition of setting temperature, setting alginic compound concentration, and setting polyvalent metal ion concentration can be calculated according to the stability constant of the complex formed by the selected small molecule ligand and the selected polyvalent metal ion, and the L / M value of the corresponding pre-gel solution can be designed, so that rapid formula design across ligands and metal ions can be realized, providing a predictable design path for rapid screening and engineering amplification of combinations of different small molecule ligands and polyvalent metal ions.

[0025] As preferred, the alginate salt comprises sodium alginate, potassium alginate or ammonium alginate; the alginate salt derivative comprises but is not limited to the alginate salt that can be chemically / biologically modified; preferably the alginate salt is modified by esterification, amidation, grafting of polypeptide or grafting of polymer.

[0026] As preferred, the polyvalent metal ion is one or more of divalent metal ion or trivalent metal ion; preferably the divalent metal ion is selected from one or more of Ca2+, Mg2+, Ba2+, Sr2+, Cu2+, Zn2+, Mn2+, Co2+, Ni2+, Fe2+; the trivalent metal ion is selected from one or more of Fe3+, Al3+. In practice, the polyvalent metal ion can be one or more of divalent metal ion, can be one or more of trivalent metal ion, or can be any combination of one or more of divalent metal ion and one or more of trivalent metal ion. The selection criterion is that the polyvalent metal ion is capable of forming cross-linking reaction with the alginate compound.

[0027] As preferred, the small molecule ligand contains at least one functional group that is capable of forming coordination bond with the polyvalent metal ion, the functional group is selected from one or more of carboxyl, carboxylate, hydroxyl, amino, amine, phosphonic acid, pyridyl, imidazolyl.

[0028] As preferred, the small molecule ligand is biocompatible organic acid and / or salt of organic acid.

[0029] As preferred, 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, phytic acid. The salt of organic acid is selected from one or more of lactate, sodium citrate, malate, tartrate, acetate, acetic alcoholate, succinate, gluconate, glycinate, aspartate, glutamate, pantothenate, sorbate, threonate, phytate.

[0030] In practice, any one or more small molecule compound that is capable of forming coordination bond with the polyvalent metal ion, is biocompatible, and is highly hydrophilic can be selected.

[0031] As preferred, the mass fraction of the alginate compound is 0.1-30 w / v%; further preferred, the mass fraction of the alginate compound is 0.5-6 w / v%; more further preferred, the mass fraction of the alginate compound is 1-5 w / v%.

[0032] As preferred, the molar concentration of the polyvalent metal ions is 0.1-500 mM. Further preferably, the molar concentration of the polyvalent metal ions is 0.2-200 mM; more preferably, the molar concentration of the polyvalent metal ions is 1-100 mM; more preferably, the molar concentration of the polyvalent metal ions is 5-50 mM.

[0033] The present application also provides a preparation method of the injectable hydrogel pre-gel solution based on ligand regulation, comprising the following steps: uniformly mixing the alginic compound, the donor providing the polyvalent metal ions, and the small molecule ligand in a solvent; obtaining the pre-gel solution; the solvent is an aqueous solvent; the donor providing the polyvalent metal ions is a metal ion compound dissolved in the aqueous solvent.

[0034] As preferred, the aqueous solvent mentioned above refers to a solvent whose continuous phase is water-based, which is distinguished from an organic phase solvent whose continuous phase is an organic solvent (such as petroleum ether, acetone), and is preferably water, physiological saline, or PBS buffer.

[0035] As preferred, the donor providing the polyvalent metal ions mentioned above is a metal ion compound dissolved in water, preferably a metal ion compound with high solubility in water, including CaCl2, Ca(NO3)2, CuCl2, CuSO4, MgCl2, Mg(NO3)2, ZnCl2, Zn(NO3)2, etc.

[0036] As preferred, the preparation method of the injectable hydrogel pre-gel solution based on ligand regulation of the present application comprises the following steps:

[0037] (1) dissolving the alginic compound in the aqueous solvent to obtain a uniform solution;

[0038] (2) adding the small molecule ligand to the uniform solution obtained in step (1) and mixing uniformly; then adding the donor providing the polyvalent metal ions and mixing again; or dissolving the small molecule ligand and the donor providing the polyvalent metal ions in the aqueous solvent to obtain a mixed solution, and adding the mixed solution to the uniform solution obtained in step (1); obtaining the pre-gel solution.

[0039] In practice, for the pre-gel solution with a higher requirement for the concentration of the alginic compound, a uniform solution with a higher concentration of the alginic compound can be prepared in step (1), and after step (2) is completed, the aqueous solvent is continuously added to adjust the volume and concentration of the solution to the target value, and the pre-gel solution is obtained.

[0040] The application adopts two priority steps: one is to mix small molecule ligands with polyvalent metal ions to form a complex, and the other is to introduce small molecule ligands into alginic acid compounds in advance. The above-mentioned methods can avoid the direct cross-linking of polyvalent metal ions and alginic acid compounds into glue, thereby ensuring the uniform distribution of small molecule ligands and realizing the rapid and efficient construction of the pre-gel system.

[0041] The application also provides an application of the injectable hydrogel pre-gel solution based on ligand regulation.

[0042] The technical scheme of the embodiment of the application has at least the following advantages and beneficial effects:

[0043] (1) Excellent fine needle injectability: the system shows significant shear thinning characteristics, and can be smoothly extruded through a 34G ultrafine needle without blockage and flow interruption.

[0044] (2) Immediate stability after injection: ligand diffusion releases polyvalent metal ions, and a "gel shell" is formed at the interface instantaneously, effectively preventing material leakage.

[0045] (3) In-situ self-enhanced mechanical properties: internal cross-linking gradually densifies over time, G' is significantly improved, and the material maintains stable morphology for a long time.

[0046] (4) Formulation predictability: by establishing the correlation between CLR and the stability constant K of small molecule ligand-polyvalent metal ion, the gelation behavior can be predictively designed, which is suitable for the extension of different ligands and metal ions.

[0047] (5) Wide application range: can be used as a tissue engineering scaffold filling material, a regeneration and repair carrier, a drug / cell delivery system and a three-dimensional printing ink, and has good biocompatibility and clinical transformation potential. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 It is a schematic diagram of the linear correlation between the critical ligand ratio (CLR) and the reciprocal of the ligand-metal ion stability constant (1 / K) in experimental example 1.

[0049] Figure 2 It is a shear rate-viscosity curve diagram of the pre-gel solution of example 1 and the sodium alginate aqueous solution of comparative example 1 in experimental example 2.

[0050] Figure 3 It is a bolus injection force-time curve diagram of the pre-gel solution of example 1 under different needle gauges (25G-32G) in experimental example 3.

[0051] Figure 4The injection force-internal diameter relationship curve of the pre-gel solution of Example 1 in Experimental Example 3 under different needle gauges (25G-32G).

[0052] Figure 5 The display diagram of the pre-gel solution of Example 1 in Experimental Example 3 smoothly injected through a 34G needle.

[0053] Figure 6 The rheological result schematic diagram of the pre-gel solution of Example 1 in Experimental Example 4 before and after soaking in PBS.

[0054] Figure 7 The state diagram of the pre-gel solution of Example 1, the pre-gel solution of Example 6 and the hydrogel of Comparative Example 2 in Experimental Example 5 after preparation and 24h of static state.

[0055] Figure 8 The state diagram of the pre-gel solution of Example 1 in Experimental Example 6 at the time of injection, after injection and 1 week after injection. DETAILED DESCRIPTION

[0056] Example 1

[0057] The present embodiment provides an injectable hydrogel pre-gel solution based on ligand regulation, comprising the following components: sodium alginate; calcium ions; sodium lactate; wherein the mass volume concentration of sodium alginate is 2w / v%; the molar concentration of calcium ions is 25.2mM.

[0058] The present embodiment first carries out the critical molar ratio CLR determination of sodium lactate-calcium ions under the condition that the temperature is 25℃, the mass volume concentration of sodium alginate is 2w / v%, and the molar concentration of calcium ions is 25.2mM. The specific determination method of the critical molar ratio CLR of sodium lactate-calcium ions in the present embodiment under the above condition includes:

[0059] (1) At 25℃, 5 groups of 3 w / v% sodium alginate solution, each 2 mL, were prepared;

[0060] (2) 0.212 g, 0.254 g, 0.296 g, 0.339 g, 0.382 g of sodium lactate was respectively added to the 5 groups of 3 w / v% sodium alginate aqueous solution of step (1), and the mixture was fully mixed to form a uniform solution;

[0061] (3) Under stirring, 168 μL of 5 w / v% CaCl2 solution was added into each of the 5 groups of homogeneous sodium lactate-containing solutions obtained in step 2, and deionized water was added to make the total volume 3 mL, so that the final w / v% 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 concentration of sodium lactate was 630.6 mM, 755.5 mM, 880.5 mM, 1008.4 mM and 1133.3 mM, respectively. The system morphology of each group of solutions was observed; among them, the system of the experimental group with a sodium lactate concentration of 1133.3 mM changed from a gel state to a sol state, at this time L / M was 45, and the L / M corresponding to the sodium lactate solubility was 45, the sodium lactate concentration (1008.4 mM) of the previous gel state corresponded to L / M = 40, and the difference between the two was less than 15% ((45-40) / 40=12.5%); therefore, it was determined that the critical molar ratio of sodium lactate-calcium ions under the above conditions was CLR=45, and if the difference was greater than 15%, the concentration gradient range of sodium lactate would be further narrowed to determine a more accurate CLR.

[0062] Based on the above results of CLR=45, the molar concentration of sodium lactate in this embodiment was selected to be 1133.3 mM, i.e. L / M=45.

[0063] This embodiment also provides a preparation method of the above injectable hydrogel pre-gel solution based on ligand regulation, comprising the following steps:

[0064] (1) Dissolve sodium alginate (SA) in deionized water in proportion, and magnetically stir at room temperature overnight to obtain a homogeneous sodium alginate solution with a w / v% of 3 w / v%;

[0065] (2) Add 0.382 g of sodium lactate to 2 mL of the sodium alginate solution obtained in step (1), and stir thoroughly until completely dissolved to obtain a sodium alginate + sodium lactate mixed solution;

[0066] (3) Under vigorous stirring, 168 μL of 5 w / v% CaCl2 solution was added into each of the 5 groups of homogeneous sodium lactate-containing solutions obtained in step 2, and deionized water was added to make the total volume 3 mL, so that the final w / v% 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 concentration of sodium lactate was 630.6 mM, 755.5 mM, 880.5 mM, 1008.4 mM and 1133.3 mM, respectively. The system morphology of each group of solutions was observed; among them, the system of the experimental group with a sodium lactate concentration of 1133.3 mM changed from a gel state to a sol state, at this time L / M was 45, and the L / M corresponding to the sodium lactate solubility was 45, the sodium lactate concentration (1008.4 mM) of the previous gel state corresponded to L / M = 40, and the difference between the two was less than 15% ((45-40) / 40=12.5%); therefore, it was determined that the critical molar ratio of sodium lactate-calcium ions under the above conditions was CLR=45, and if the difference was greater than 15%, the concentration gradient range of sodium lactate would be further narrowed to determine a more accurate CLR. 2+ reversible complexation occurs between sodium lactate and Ca

[0067] (4) An appropriate amount of deionized water was added to adjust the total volume to 3 mL to obtain a sodium alginate + sodium lactate pre-gel solution of this embodiment with a w / v% of 2 w / v% (calculated based on SA).

[0068] Example 2

[0069] The present example provides a ligand-regulated injectable hydrogel pre-gel solution, which is different from the formula of Example 1 in that sodium lactate is replaced by sodium citrate.

[0070] The present example also first carries out the determination of the critical molar ratio CLR of sodium citrate-calcium ions at a temperature of 25°C, a mass-volume concentration of sodium alginate of 2 w / v%, and a molar concentration of calcium ions of 25.2 mM. The determination method is the same as in Example 1, except that in step (2), 9.76 mg, 11.71 mg, 13.66 mg, 15.61 mg, and 17.56 mg of anhydrous sodium citrate are respectively added to the 5 groups of 3 w / v% sodium alginate aqueous solutions of step (1); after constant volume, the concentrations of sodium citrate are 12.6 mM, 15.12 mM, 17.64 mM, 20.16 mM, and 22.68 mM, respectively. Finally, it is determined that the critical molar ratio CLR of sodium citrate-calcium ions under the conditions of the present example is 0.8.

[0071] Based on the above result of CLR=0.8, the present example selects a molar concentration of sodium citrate of 22.3 mM, i.e., L / M=0.85.

[0072] The present example also provides a preparation method of the above-mentioned ligand-regulated injectable hydrogel pre-gel solution, which is different from Example 1 in that the amount of sodium citrate added in step (2) is 0.017 g. The pre-gel solution prepared in the present example is a sodium alginate + sodium citrate pre-gel solution.

[0073] Example 3

[0074] The present example provides a ligand-regulated injectable hydrogel pre-gel solution, which is different from the formula of Example 1 in that sodium lactate is replaced by disodium malate.

[0075] The present example also first carries out the determination of the critical molar ratio CLR of disodium malate-calcium ions at a temperature of 25°C, a mass-volume concentration of sodium alginate of 2 w / v%, and a molar concentration of calcium ions of 25.2 mM. The determination method is 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 are respectively added to the 5 groups of 3 w / v% sodium alginate aqueous solutions of step (1); after constant volume, the concentrations of disodium malate are 176.4 mM, 201.6 mM, 226.8 mM, 252.0 mM, and 277.2 mM, respectively. Finally, it is determined that the critical molar ratio CLR of disodium malate-calcium ions under the conditions of the present example is 11.

[0076] Based on the above result of CLR=11, the molar concentration of disodium malate in this embodiment is selected as 300 mM, i.e. L / M=11.5.

[0077] This embodiment also provides the preparation method of the injectable hydrogel pre-gel solution based on ligand regulation, which is different from that of Example 1 in that the amount of disodium malate added in step (2) is 0.161 g, and the pre-gel solution prepared in this embodiment is a sodium alginate + disodium malate pre-gel solution.

[0078] Example 4

[0079] This embodiment provides an injectable hydrogel pre-gel solution based on ligand regulation, which is different from that of Example 1 in that sodium lactate is replaced by sodium tartrate.

[0080] This embodiment also first carries out the determination of the critical molar ratio CLR of sodium tartrate-calcium ions when the temperature is 25 ℃, the mass-volume concentration of sodium alginate is 2 w / v%, and the molar concentration of calcium ions is 25.2 mM. The determination method is the same as that of Example 1, and the difference lies in that in step (2), 322.76 mg, 351.98 mg, 381.21 mg, 410.43 mg and 439.75 mg of anhydrous sodium tartrate are respectively added to the 5 groups of 3 w / v% sodium alginate aqueous solution of step (1); after constant volume, the concentrations of sodium tartrate are 554.4 mM, 604.8 mM, 655.2 mM, 705.6 mM and 756.0 mM, respectively. Finally, it is determined that the critical molar ratio CLR of sodium tartrate-calcium ions under the conditions of this embodiment is 28.

[0081] Based on the above result of CLR=28, the molar concentration of sodium tartrate in this embodiment is selected as 706 mM, i.e. L / M=28.

[0082] This embodiment also provides the preparation method of the injectable hydrogel pre-gel solution based on ligand regulation, which includes the following steps:

[0083] (1) Dissolve sodium alginate (SA) in deionized water in proportion, and magnetically stir at room temperature overnight to obtain a uniform sodium alginate solution with a mass-volume concentration of 3 w / v%;

[0084] (2) Mix 0.411 g of sodium tartrate with 168 μL of calcium chloride (CaCl2) solution with a mass-volume concentration of 5 w / v%, and add water to 1 mL and stir thoroughly until uniform;

[0085] (3) Under the condition of vigorous stirring, the mixed solution obtained in step (2) is added into 2 mL of the sodium alginate solution obtained in step (1) to obtain a sodium alginate + sodium tartrate pre-gel solution with a mass-volume concentration of 2 w / v% (calculated based on SA) in this example.

[0086] Example 5

[0087] This example provides an injectable hydrogel pre-gel solution based on ligand regulation, which is different from Example 1 in that sodium lactate is replaced by disodium malate, and the mass-volume concentration of sodium alginate is 5 w / v%, and the molar concentration of calcium ions is 50 mM.

[0088] This example also first carries out the determination of the critical molar ratio CLR of disodium malate-calcium ions under the condition of a temperature of 25°C, a mass-volume concentration of sodium alginate of 5 w / v%, and a molar concentration of calcium ions of 50 mM. The determination method is the same as in Example 1, except that:

[0089] In step (1), 5 groups of 2.5 mL of a 6 w / v% sodium alginate aqueous solution are prepared;

[0090] In step (2), 0.168 g, 0.185 g, 0.202 g, 0.219 g, and 0.235 g of disodium malate are added to the 5 groups of 6 w / v% sodium alginate aqueous solutions in step (1) respectively, and the solutions are mixed thoroughly to form uniform solutions;

[0091] In step (3), 332.7 µL of a 5 w / v% calcium chloride (CaCl2) solution is added to each of the 5 groups of uniform solutions in step (2), and water is added to make the final volume 3 mL, so that the final mass-volume concentration of sodium alginate in the system is 5 w / v%. At this time, the molar concentration of calcium ions in each group of solutions is 50 mM, and the molar concentration of disodium malate is 500 mM, 550 mM, 600 mM, 650 mM, and 700 mM, respectively.

[0092] Finally, it is determined that the critical molar ratio CLR of disodium malate-calcium ions under the conditions of this example is 13.

[0093] Based on the above result of CLR=13, this example selects a molar concentration of disodium malate of 660 mM, i.e., L / M=13.2.

[0094] This example also provides a preparation method of the above injectable hydrogel pre-gel solution based on ligand regulation, which is different from Example 1 in that:

[0095] In step (1), a uniform sodium alginate solution with a mass-volume concentration of 6 w / v% is prepared;

[0096] In step (2), 0.222 g of disodium malate was added to 2.5 mL of the sodium alginate solution obtained in step (1);

[0097] In step (3), 332.7 μL of a 5 w / v% calcium chloride (CaCl2) solution was added dropwise to the mixed solution obtained in step (2);

[0098] In step (4), an appropriate amount of deionized water was added to adjust the total volume to 3 mL, thereby obtaining a sodium alginate + disodium malate pregel solution with a mass / volume concentration of 5 w / v% (calculated based on SA) in this example.

[0099] Example 6

[0100] This example provides an injectable hydrogel pregel solution based on ligand regulation, which is different from the formula of Example 1 in that sodium lactate is replaced by sodium tartrate, the mass / volume concentration of sodium alginate is 1 w / v%, and the molar concentration of calcium ions is 5 mM.

[0101] This example also first carried out the determination of the critical molar ratio CLR of sodium tartrate-calcium ions under the conditions of a temperature of 20°C, a mass / volume concentration of sodium alginate of 1 w / v%, and a molar concentration of calcium ions of 5 mM. The determination method is the same as in Example 1, except that:

[0102] In step (1), five groups of 2 mL of a 1.5 w / v% sodium alginate aqueous solution were prepared;

[0103] In step (2), 0.0931 g, 0.0989 g, 0.1048 g, 0.1106 g, and 0.1164 g of sodium tartrate were added to the five groups of 1.5 w / v% sodium alginate aqueous solutions of step (1), respectively, and the solutions were mixed thoroughly to form uniform solutions;

[0104] In step (3), 20.8 μL of an 8 w / v% calcium chloride (CaCl2) solution was added to each of the five uniform solutions of 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 1 w / v%. At this time, the molar concentration of calcium ions in each group of solutions was 5 mM, and the molar concentration of sodium tartrate was 160 mM, 170 mM, 180 mM, 190 mM, and 200 mM, respectively. Finally, it was determined that the critical molar ratio CLR of sodium tartrate-calcium ions under the conditions of this example was 36.

[0105] Based on the above result of CLR=36, this example selected a molar concentration of sodium tartrate of 190 mM, i.e., L / M=38.

[0106] The preparation method of the injectable hydrogel pre-gel solution based on ligand regulation in this embodiment is the same as the above, and the difference from Example 1 is that:

[0107] In step (1), a uniform sodium alginate solution with a mass-volume concentration of 1.5 w / v% was prepared;

[0108] In step (2), 0.1106 g of sodium tartrate was added to 2 mL of the sodium alginate solution obtained in step (1);

[0109] In step (3), 20.8 µL of a calcium chloride (CaCl2) solution with a mass-volume concentration of 8 w / v% was added dropwise to the mixed solution obtained in step (2);

[0110] In step (4), an appropriate amount of deionized water was added to adjust the total volume to 3 mL, and a sodium alginate + sodium tartrate pre-gel solution with a mass-volume concentration of 1 w / v% (based on SA) was obtained.

[0111] Comparative Example 1

[0112] This comparative example provides a sodium alginate aqueous solution; specifically, a sodium alginate aqueous solution with a mass-volume concentration of 2 w / v%.

[0113] Comparative Example 2

[0114] This comparative example provides a hydrogel, which includes 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.

[0115] The preparation method of the hydrogel of this comparative example includes:

[0116] (1) Dissolve sodium alginate (SA) in deionized water, and magnetically stir at room temperature overnight to obtain a uniform sodium alginate solution with a mass-volume concentration of 3 w / v%;

[0117] (2) Add 0.3235 g of sodium lactate to 2 mL of the sodium alginate solution obtained in step (1), and stir thoroughly until completely dissolved to obtain a sodium alginate + sodium lactate mixed solution;

[0118] (3) Under vigorous stirring, add 168 µL of a calcium chloride (CaCl2) solution with a mass-volume concentration of 5 w / v% to the mixed solution obtained in step (2) to allow reversible complexation of sodium lactate and Ca 2+

[0119] ​(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.

[0120] Experimental Example 1

[0121] 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 1 It 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.

[0122] Experimental Example 2

[0123] 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.

[0124] 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.

[0125] Experimental Example 3

[0126] The alginate + sodium lactate pre-gel solution prepared in Example 1 was used as the experimental sample. The sample was loaded into a 1 mL syringe and equipped with different needle gauges. A universal material testing machine (Model: UniVert, CellScale Biomaterials Testing, Canada) was used to test the sample in compression mode at room temperature. The injection flow rate was set to 1 mL / min, and the injection force changes under different needle gauges were recorded; the test results are shown in Figure 3 , Figure 4 and Figure 5 .

[0127] wherein Figure 3 is the injection force-time curve of the sodium alginate + sodium lactate pre-gel solution system of Example 1 under different needle gauges (25G-32G), and the results show that the system still maintains acceptable injection force under a thin needle (32G). Figure 4 is the injection force-internal diameter relationship curve of the sodium alginate + sodium lactate pre-gel solution system of Example 1 under different needle gauges (25G-32G), indicating that the injection force increases with the decrease of the needle internal diameter, but still within the clinical operable range. Figure 5 is a display diagram of the sodium alginate + sodium lactate pre-gel solution system of Example 1 smoothly injected through a 34G needle. Rhodamine was added to the solution to facilitate visual observation.

[0128] The results of this experimental example show that the pre-gel solution has good thin needle passability and injectability, meeting the requirements of convenient operation in minimally invasive surgery applications.

[0129] Experimental Example 4

[0130] The sodium alginate + sodium lactate pre-gel solution obtained in Example 1 was poured into a high-density polyethylene (HDPE) mold (diameter 30 mm, cylindrical), and the sample thickness was about 2 mm. The sample was degassed by room temperature standing for 1 h or centrifugation at 1000 rpm for 5 min to remove bubbles. Subsequently, phosphate buffered saline (PBS) was slowly added to the mold to avoid disturbing the sample surface, and the sample was immersed at 37°C for a total of 1 h, and the sample was carefully turned over during the immersion process to ensure full contact with PBS. The sample before and after PBS immersion for 1 h was tested using a physical rheometer (Model: MCR300, Anton Paar, Austria) equipped with a parallel plate with a diameter of 25 mm at 37°C. A strain of 0.1% was scanned in the frequency range of 0.1-10 rad / s to further characterize the rheological properties of the sample; the test results are shown in Figure 6 .

[0131] Figure 6In the middle, G' is the storage modulus, G" is the loss modulus; soaking PBS solution simulates the pre-gel solution into the body environment; from the comparison of the data before and after soaking can be known: before soaking, the storage modulus (G') and the loss modulus (G") of the pre-gel solution prepared by example 1 are relatively low in overall value, and with the change of frequency, the growth of G' and G" is relatively gentle. This shows that before soaking, the elasticity and viscosity response of the sample formed by sodium alginate + sodium lactate pre-gel solution are weak, and no crosslinking reaction occurs. After soaking for 1 hour, the G' value increases significantly, and is basically stable at a high level (about 100 Pa) in the whole frequency range, G' is much larger than G", which shows that the elasticity of the sample after soaking dominates, and a stable elastic network structure is formed, reflecting strong self-reinforcing ability.

[0132] Experimental example 5

[0133] Rhodamine was added in the preparation process of sodium alginate-sodium lactate pre-gel solution of example 1, sodium alginate + sodium tartrate pre-gel solution of example 6 and sodium alginate + sodium lactate hydrogel of comparative example 2 respectively to realize visualization. The system state after just being successfully prepared and after being static for 24h was observed.

[0134] The results are shown in Figure 7 From Figure 7 it can be known that the pre-gel solution prepared by example 1 and the pre-gel solution prepared by example 6 both maintain stable sol system state after just being successfully prepared and after being static for 24h, and the solution is uniform without impurities under naked eye observation; the hydrogel prepared by comparative example 2 has obvious gel state after just being successfully prepared, and still maintains gel state after being static for 24h.

[0135] Experimental example 6

[0136] Rhodamine was added in the sodium alginate-sodium lactate pre-gel solution of example 1 to realize visualization. After the solution was injected into PBS solution, the state during injection, after injection and one week after injection was observed, and the results are shown in Figure 8 .

[0137] From Figure 8 it can be known that the pre-gel system can be smoothly injected, and the system can maintain good shape after injection, which shows that a gel shell layer can be quickly formed on the surface, thereby effectively avoiding material leakage. After PBS soaking for 1 week, the system still maintains complete morphology, proving that it can gradually complete gelation after injection and maintain long-term structural stability.

[0138] In conclusion, the pre-gel solution provided by the application not only has good fine-needle injectability and operation stability, but also can realize the transformation from sol to gel after injection and continuously enhance the mechanical property, effectively solving the problem that the traditional alginate hydrogel is difficult to balance the injectability, instant stability and long-term mechanical property.

Claims

1. An injectable hydrogel pre-gel solution based on ligand regulation, characterized in that, The pre-gel solution comprises the following components: an alginic acid compound; a multivalent metal ion; a small molecule ligand; The alginic acid compound comprises alginic acid or alginic acid salt and derivatives thereof; the small molecule ligand contains at least one functional group capable of forming a coordination bond with the multivalent metal ion, and the functional group is selected from one or more of carboxyl, hydroxyl, amino, phosphonic acid, pyridyl and imidazolyl; The molar ratio of the small molecule ligand to the multivalent metal ion is denoted as L / M; the minimum L / M that maintains the system in a sol state under a given temperature, a given concentration of the alginic acid compound and a given concentration of the multivalent metal ion is defined as the critical ligand ratio, denoted as CLR; In the pre-gel solution, L / M≥CLR.

2. The injectable ligand-regulated hydrogel precrosslinking solution of claim 1, wherein, The stability constant of the complex formed by any of the small molecule ligands A and any of the polyvalent metal ions B is K A-B For a given temperature, a given concentration of alginic compound and a given concentration of polyvalent metal ions, the CLR is proportional to 1 / K A-B .

3. The injectable ligand-regulated hydrogel precrosslinking solution of claim 1, wherein, The method for determining the CLR of any small molecule ligand A and any multivalent metal ion B under a given temperature T, a given concentration C of the alginic acid compound and a given concentration M of the multivalent metal ion comprises the following steps: (1) preparing a plurality of aqueous solutions of the alginic acid compound with a concentration D under the given temperature T; (2) adding different amounts of the small molecule ligand A to the plurality of aqueous solutions of the alginic acid compound in step (1) respectively, and mixing uniformly to obtain a plurality of uniform solutions; (3) adding a fixed concentration of the multivalent metal ion B to the plurality of uniform solutions in step (2) respectively, and adding an appropriate amount of water to make the solution constant volume, so that the concentration of the alginic acid compound is C, the molar concentration of the multivalent metal ion B is M, and the molar concentration L of the small molecule ligand A changes from low to high in gradient; observing the morphology of each solution system; when the system changes from a gel state to a sol state, the L / M is the CLR of the small molecule ligand A and the multivalent metal ion B under the given temperature T, the given concentration C of the alginic acid compound and the given concentration M of the multivalent metal ion.

4. The injectable ligand-regulated hydrogel precrosslinking solution of claim 1, wherein, The alginic acid salt comprises sodium alginate, potassium alginate or ammonium alginate; the derivative of the alginic acid salt comprises the alginic acid salt modified by esterification, amidation or grafting of polypeptides.

5. The injectable ligand-regulated hydrogel precrosslinking solution of claim 1, wherein, The multivalent metal ion is one or more of a divalent metal ion or a trivalent metal ion.

6. The injectable ligand-regulated hydrogel precrosslinking solution of claim 1, wherein, The small molecule ligand is a biocompatible organic acid and / or a salt of an organic acid.

7. The injectable ligand-regulated hydrogel precrosslinking solution of claim 1, wherein, The mass-volume concentration of the alginic acid compound is 0.1-30 w / v%; the molar concentration of the multivalent metal ion is 0.1-500 mM.

8. The method for preparing the injectable hydrogel pre-gel solution based on ligand regulation according to any one of claims 1-7, characterized in that, The method comprises the following steps: uniformly mixing the alginic acid compound, a donor providing the multivalent metal ion and the small molecule ligand in a solvent in proportion; obtaining the pre-gel solution; the solvent is an aqueous solvent; the donor providing the multivalent metal ion is a metal ion compound dissolved 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, The method comprises the following steps: (1) dissolving the alginic acid compound in the aqueous solvent to obtain a uniform solution; (2) adding a small molecule ligand to the homogeneous solution obtained in step (1) and mixing uniformly; then adding a donor providing the polyvalent metal ions and mixing again; or dissolving the small molecule ligand and the donor providing the polyvalent metal ions in an aqueous phase solvent to prepare a mixed solution, and adding the mixed solution to the homogeneous solution obtained in step (1) to obtain the pre-gel solution.

10. Use of the ligand-regulated injectable hydrogel pre-gel solution according to any one of claims 1 to 7 for preparing a tissue engineering material, a regenerative medicine material, a drug delivery material, a cell delivery material, a three-dimensional printing forming material or an image tracing material.

Images