Injectable hydrogel combined reagent, self-curing conductive hydrogel and application of self-curing conductive hydrogel

Abstract

The invention belongs to the technical field of biological medicine, and discloses an injectable hydrogel combined reagent, self-curing conductive hydrogel and application thereof. The injectable hydrogel combined reagent provided by the invention comprises a solution A and a solution B, the solution A is a first precursor solution formed by dissolving modified poloxamer PFUDAm and ammonium persulfate in deionized water; the solution B is a second precursor solution formed by dissolving N-isopropylacrylamide NIPAM, ferrous gluconate and PEDOT: PSS in deionized water, after the injectable hydrogel combined reagent is mixed in a duplex injection mode, PFUDAm, poly (N-isopropylacrylamide) and PEDOT: PSS are subjected to an oxidation-reduction reaction of ammonium sulfate and ferrous gluconate to initiate polymerization, and the self-curing conductive hydrogel is constructed. The self-curing conductive hydrogel has controllable curing time, and the self-curing conductive hydrogel has excellent swelling resistance, conductivity and good elasticity and toughness, and is expected to be combined with a cardiac pacemaker to be applied to treatment of arrhythmia.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to an injectable hydrogel combination reagent, a self-curing conductive hydrogel and application thereof. BACKGROUND

[0002] Scar tissue in the heart can be formed for a variety of reasons, including myocardial infarction, surgery, inflammation, etc. These scar tissues can interrupt the normal conduction of electrical signals in the heart, causing delays or blockages in the propagation of electrical signals. When the electrical signals near the scar tissue undergo a reentry phenomenon, i.e., the electrical signals repeatedly turn back and stimulate the myocardial cells during the propagation process, it can cause arrhythmia. Reentry is an important mechanism of arrhythmia, especially in some types of tachycardia. When the reentry phenomenon occurs, the propagation path of the electrical signals in the heart forms a loop, causing the myocardial cells to be repeatedly stimulated, thereby producing a persistent rapid rhythm. This reentry loop can be formed at any part of the heart, including the ventricle, atrium or atrioventricular node, etc. For this arrhythmia caused by scar tissue and reentry, the injectable conductive hydrogel is injected into the venous blood vessels of the scar tissue, which, in combination with the traditional pacemaker, can achieve pacing similar to the natural conduction of the heart, thereby effectively mimicking the natural electrical rhythm of the heart and providing painless defibrillation to eliminate arrhythmia.

[0003] Elizabeth Cosgriff-Hernandez et al. (Nat Commun., 2024, 15, 64) of the University of Texas at Austin developed a self-curing injectable conductive hydrogel, which can be delivered intravenously, fill the epicardial coronary veins and tributaries, and convert them into flexible electrodes that can extend to the middle of the myocardium. However, its swelling rate at body temperature is relatively high (swelling rate of about 200%), and in the case of long-term use, it is likely that the mechanical strength will be insufficient and the expansion will cause excessive mechanical pressure on the fragile cardiovascular system, thereby causing inflammation and tissue damage. In addition, the high swelling easily leads to a decrease in the concentration of salt ions in the conductive component and a slow loss into the tissue, affecting the long-term stability of the hydrogel's conductivity, or causing a loss of function due to its rapid degradation or dissolution in the body environment. Therefore, it is a current hot research direction to develop a conductive hydrogel with low swelling rate (or no swelling), stable material conductivity, and good mechanical properties. SUMMARY

[0004] The present application aims to provide an injectable hydrogel combination reagent, which has controllable curing time, and the self-curing conductive hydrogel formed after injection has excellent anti-swelling performance, conductivity, and good elasticity and toughness, and is expected to be applied to the treatment of arrhythmia in combination with a cardiac pacemaker.

[0005] In a first aspect, the present application provides an injectable hydrogel combination reagent, which comprises solution A and solution B; the solution A is a first precursor solution formed by dissolving modified poloxamer PFUDAm and ammonium persulfate in deionized water; the solution B is a second precursor solution formed by dissolving N-isopropyl acrylamide NIPAM, ferrous gluconate and PEDOT: PSS in deionized water; wherein the chemical structural formula of the modified poloxamer PFUDAm is as follows:

[0006]

[0007] In the solution A, the mass of PFUDAm is 20-25wt% of the total mass of solution A, and the concentration of ammonium persulfate is 2.5-7.5mM; in the solution B, the mass of NIPAM is 5-10wt% of the total mass of solution B, the concentration of ferrous gluconate is 2.5-7.5mM, and the mass of PEDOT: PSS is 1-1.3wt% of the total mass of solution B.

[0008] It should be noted that in the actual configuration process, NIPAM and ferrous gluconate are dissolved in the directly purchased raw material PEDOT: PSS aqueous solution to serve as solution B, wherein the mass ratio of PEDOT: PSS in the raw material PEDOT: PSS aqueous solution is 1-1.3wt%.

[0009] As a preferred embodiment of the injectable hydrogel combination reagent of the present application, in the solution A, the mass of PFUDAm is 20wt% of the total mass of solution A, and the concentration of ammonium persulfate is 5mM; in the solution B, the mass of NIPAM is 10wt% of the total mass of solution B, the concentration of ferrous gluconate is 5mM, and the mass of PEDOT: PSS is 1wt% of the total mass of solution B.

[0010] As a preferred preparation method of the modified poloxamer PFUDAm, the preparation method comprises the following steps: firstly, dissolving N,N'-carbonyl diimidazole and poloxamer in anhydrous dichloromethane respectively, adding the poloxamer solution into the N,N'-carbonyl diimidazole solution dropwise under the protection of nitrogen, stirring at room temperature, precipitating the product by excessive diethyl ether, filtering and vacuum drying to obtain the activated poloxamer intermediate; dissolving the obtained product poloxamer intermediate in anhydrous dichloromethane, and performing condensation reaction with ethylenediamine under the protection of nitrogen, then performing water washing, drying and ice-ether precipitation in sequence, filtering and vacuum drying to obtain the amino-functionalized modified poloxamer derivative; dissolving the above product amino-functionalized modified poloxamer derivative in anhydrous dichloromethane, adding triethylamine, and performing acylation reaction by dropwise adding acryloyl chloride solution under ice bath condition, and then performing ice-ether precipitation, filtering and vacuum drying to obtain the final product PFUDAm.

[0011] Preferably, the molar ratio of the N,N'-carbonyl diimidazole to the poloxamer is (10-20):1; the molar amount of the ethylenediamine is (10-20) times of the molar amount of the poloxamer intermediate; and the molar amount of the acryloyl chloride is (3-5) times of the molar amount of the amino-functionalized modified poloxamer derivative.

[0012] In a second aspect, the present application further provides a self-curing conductive hydrogel, which is prepared by mixing solution A and solution B in a volume ratio of 1:1 and then curing in situ.

[0013] In a third aspect, the present application provides application of the self-curing conductive hydrogel in preparation of a conductive hydrogel fiber for treating arrhythmia.

[0014] Beneficial effects:

[0015] The injectable hydrogel combination reagent provided in the first aspect of the present application has the advantages of Figure 7 As shown in the figure, the main component in solution A is modified poloxamer PFUDAm, and poloxamer itself is a temperature-sensitive nanomicelle which forms a heat-responsive fragment by forming a hydrophobic collapse to offset the swelling trend of the hydrogel, and has excellent anti-swelling performance at a normal human body temperature of 37℃. The modified poloxamer PFUDAm provided in the present application is modified by hydrolysis-resistant urethane and amide groups on the original poloxamer structure, and cooperates with N-isopropyl acrylamide in solution B, so that the hydrogel material formed by self-curing after mixing has extremely low swelling rate and excellent mechanical properties, and is more stable in a body fluid environment.

[0016] The injectable hydrogel composition provided in the first aspect of the application uses PEDOT:PSS as a conductive polymer, which can stably exist in the self-curing formed hydrogel, and there is no loss of conductive substances caused by swelling and the like of conventional hydrogels; in addition, the degradation period of PEDOT:PSS in the body can be adjusted between several months and several years, and PEDOT:PSS has good stability in a biological environment, such as under electrical stimulation, and can resist oxidative degradation, and the in-vivo service life of partially modified PEDOT:PSS, such as the addition of tannic acid or hydrophobic materials, can be further prolonged, possibly to several months or even longer, which can ensure that the self-curing conductive hydrogel has relatively stable conductive performance for a long time, and in combination with the good elasticity and toughness of the self-curing conductive hydrogel, the structure can remain intact under the external force of heart beating in application, and has good fatigue resistance, and thus is a very potential conductive hydrogel fiber material that can be used for the treatment of arrhythmia.

[0017] The self-curing conductive hydrogel provided in the application has controllable curing time, which is adjusted by adjusting the concentration of ferrous gluconate and ammonium persulfate, because the two only play a role in providing an initiation reaction, and the mass concentration in the hydrogel is low, and the influence on other performance can be ignored; the self-curing conductive hydrogel provided in the application has controllable conductive strength, which is adjusted by adjusting the concentration of PEDOT:PSS, because its viscosity is low, and the mass concentration in the hydrogel is low, and the influence on other performance can be ignored; the self-curing conductive hydrogel precursor solution provided in the application has controllable viscosity, within the concentration range of the precursor solution defined in the application, by changing the concentration of PFDAm and NIPAM, the mechanical and degradation properties of the hydrogel are adjusted in a small range to adapt to different technical requirements, which has functional expansion, and has great application potential in the biomedical field. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 PFUDAm nuclear magnetic resonance hydrogen spectrum in Example 1;

[0019] Figure 2A Swelling curve of sample 2 in PBS buffer within 60h in Example 2;

[0020] Figure 2B Swelling curve of sample 1 in PBS buffer within 60h in Example 2;

[0021] Figure 3 Mechanical properties of hydrogels with different PFUDAm and NIPAM contents in Example 3;

[0022] Figure 4 Rheological properties of hydrogels using different concentrations of ammonium persulfate and ferrous gluconate in Example 4;

[0023] Figure 5 Conductivity of the hydrogels with different PEDOT:PSS content in Example 5;

[0024] Figure 6 Schematic representation of the in vitro characterization of the in situ solidification of the blood vessels in Example 6;

[0025] Figure 7 Schematic representation of the formation of the hydrogel after injection of the injectable hydrogel composition according to the present application. DETAILED DESCRIPTION

[0026] The application will be further described with reference to the following examples. It should be understood that these examples are intended to illustrate the application and are not intended to limit the scope of the application. The examples described are only a few of the embodiments of the application, not all of the embodiments. Based on the examples in the present application, all other embodiments obtained by those skilled in the art without creative work are within the scope of the present application.

[0027] The main raw materials involved in the following examples are as follows:

[0028] PEDOT:PSS aqueous solution was purchased from Clevios TM PH1000 Heraeus;

[0029] Poloxamer was purchased from Macklin (item number 768502);

[0030] NIPAM was purchased from Macklin (item number N811777).

[0031] Example 1: Preparation and structural characterization of PFUDAm

[0032] PFUDAm was synthesized using poloxamer as raw material, and the structure of the polymer was characterized by Bruker AVANCE III HD 600 MHz. 1 H NMR (CDCI3). The specific preparation process is as follows:

[0033] Dissolve 24.3225 g of N,N'-carbonyldiimidazole in 100 ml of anhydrous dichloromethane, and dissolve 126 g of poloxamer in 840 ml of anhydrous dichloromethane; slowly drop the poloxamer solution into the N,N'-carbonyldiimidazole solution under the protection of nitrogen atmosphere, and continuously stir the reaction at room temperature for 12 hours; after the reaction is completed, add excess ethyl ether into the mixture to fully precipitate the product, separate the precipitate, and dry the precipitate under vacuum at 40°C for 6 hours to obtain the activated poloxamer intermediate; dissolve 129.243 g of the intermediate in 1000 ml of anhydrous dichloromethane; dissolve 9.015 g of ethylenediamine in 60 ml of anhydrous dichloromethane, and slowly drop the intermediate solution into the ethylenediamine solution under the protection of nitrogen, and continuously stir the reaction at room temperature for 12 hours; after the reaction is completed, wash the reaction liquid with 3 L of deionized water for 3 times, then add 500 g of anhydrous sodium sulfate to dry the organic phase, and remove the drying agent by filtration; concentrate the filtrate, slowly drop it into 10 times volume of ice ethyl ether, collect the precipitate, and dry the precipitate under vacuum at 40°C for 6 hours to obtain the amino-functionalized poloxamer derivative. Dissolve the above product in 1000 ml of anhydrous dichloromethane, and add 2.204 g of anhydrous triethylamine; slowly drop 3.62 g of acryloyl chloride diluted with 30 ml of dichloromethane under the condition of ice water bath, and continuously react at room temperature for 12 hours. After the reaction is completed, slowly pour the mixture into 10 times volume of ice ethyl ether to precipitate white flocculent precipitate, filter and vacuum dry to collect the product PFUDAm, and store the product in dark at room temperature.

[0034] As shown in the middle nuclear magnetic resonance hydrogen spectrum, the new characteristic proton peaks corresponding to the acryloyl chloride double bond are respectively at about 5.6 ppm, 6.1 ppm and 6.3 ppm, indicating that the double bond is successfully grafted onto the poloxamer, and the PFUDAm is synthesized. Figure 1

[0035] Example 2: Test on the swelling behavior of the hydrogel

[0036] The sample 1 is prepared as follows: dissolve 2.0 g of PFUDAm and 0.01 g of ammonium persulfate in 10 mL of deionized water, and stir uniformly; dissolve 1.0 g of NIPAM and 0.024 g of ferrous gluconate in 10 mL of deionized water, and stir uniformly. Inject the two precursor solutions obtained above into a double-barreled syringe, and extrude to form a self-curing hydrogel.

[0037] The sample 2 is prepared as follows: dissolve 2.0 g of PFUDAm and 0.01 g of ammonium persulfate in 10 mL of deionized water, and stir uniformly; dissolve 0.024 g of ferrous gluconate in 10 mL of deionized water, and stir uniformly. Inject the two precursor solutions obtained above into a double-barreled syringe, and extrude to form a self-curing hydrogel.

[0038] ​The weight of the above hydrogel sample is weighed and recorded as W0, then the sample is immersed in a centrifuge tube containing 10 mL of PBS buffer, the environmental temperature is controlled at 4℃ or 37℃, three parallel groups are set, and the sample weight is weighed and recorded as W at a certain time t The swelling rate curve is plotted by the weight change at different time points, and the swelling rate (SR) at time t is calculated according to the following formula: SR (%) = (W t -W0) / W0x100%.

[0039] As shown in Figure 2A , the swelling performance of sample 2 in PBS is shown in Figure 2B , the swelling performance of sample 1 in PBS is shown, it can be seen that the swelling rate of the hydrogel in sample 1 and sample 2 at different temperatures gradually increases with the increase of immersion time; the swelling rate of the hydrogel containing only PFUDAm in sample 2 is about 6% after immersion at 37℃ for 60h; the swelling rate is about 160% after immersion at 4℃ for 60h; the swelling rate of the hydrogel containing PFUDAm and NIPAM in sample 1 is about 24% after immersion in PBS at 37℃ for 60h; the swelling rate is about 200% after immersion at 4℃ for 60h. The results show that the low swelling performance of the hydrogel mainly depends on the PFUDAm component, but the addition of NIPAM has little effect on the swelling of the hydrogel, therefore, the above hydrogel materials all have excellent anti-swelling performance, and the anti-swelling performance is more excellent at 37℃. The hydrogel provided by the present application has excellent anti-swelling performance, which is different from 4℃, the swelling rate of the hydrogel at human body temperature 37℃ is low, which is beneficial to the implantation application of the hydrogel.

[0040] It should be noted that the hydrogel prepared in the present application is prepared by the network framework of the copolymerization of PFUDAM and NIPAM, in the precursor solution, NIPAM and ferrous gluconate are dissolved by using PEDOT:PSS aqueous solution, in the present embodiment, the mass ratio of PEDOT:PSS itself in the raw material PEDOT:PSS aqueous solution is only 1%, and PEDOT:PSS is a high molecular polymer which has completed copolymerization, under the condition of mixing the two precursor solutions according to the present application, it does not have the ability to react with PFUDAM and NIPAM, therefore, the influence of PEDOT:PSS on the other gel properties and curing properties of the self-curing conductive hydrogel prepared after mixing the two precursor solutions except for the conductivity can be ignored. Therefore, the swelling properties of the self-curing hydrogel prepared in the present embodiment can represent the swelling properties of the self-curing hydrogel according to the present application.

[0041] Example 3: Mechanical property test of hydrogel

[0042] The sample for testing in this embodiment 3 was prepared in the same way as sample 1 in embodiment 2, except that, in order to facilitate the tensile test, the double-barreled syringe was directly extruded into a standard 3 dumbbell-shaped polytetrafluoroethylene mold to form a self-curing hydrogel, and then a universal material testing machine was used to measure the breaking strength and breaking elongation of the hydrogel.

[0043] As shown in Figure 3 , the breaking stress of the hydrogel provided by the present application is 1.66 MPa when the experimental concentration of PFUDAm is 20 wt% and the content of poly N-isopropyl acrylamide is 10 wt%, and the elongation at break is 274.5%, which has good elasticity and toughness.

[0044] Embodiment 4: Rheological property test of hydrogel

[0045] The sample for testing in this embodiment 4 was prepared in the same way as sample 1 in embodiment 2, except that the concentration of ammonium persulfate and ferrous gluconate in the precursor solution was different, and three samples with different concentrations were prepared. The Anton Paar MCR 301 rheometer was used to test the viscosity of the precursor solution and the self-crosslinking curing time, and then two precursor solutions were loaded into a double-barreled syringe and injected into a 37℃ parallel plate configuration through a mixing head. During the test, mineral oil was used to seal the edges of the hydrogel to prevent water evaporation during the test. In the constant frequency and constant strain oscillation test mode, the strain was set to 0.5%, the constant frequency was 10 rad / s, the complex modulus was measured every 3s, and the gap was 1mm. The complex viscosity change is less than 1% and the crosslinking network is considered to be completely formed.

[0046] As shown in Figure 4 , the viscosity of the hydrogel precursor solution of the three samples in this embodiment 4 is about 200 Pa·s, and the self-curing time is relatively short, for example, the sample with a concentration of 5mM of ammonium persulfate and ferrous gluconate takes about 4min to completely form a gel network, which can meet the clinical application requirements.

[0047] Clevios TM The viscosity of 1wt% PEDOT:PSS solution provided by Clevios PH1000 Heraeus is 50cP, i.e. 50mPa·s, which is much lower than the viscosity of 200Pa·s of the hydrogel precursor solution, so its influence on the viscosity of the solution can be ignored. The sample prepared in this embodiment 4 can represent the rheological properties of the injectable hydrogel combination reagent of the present application.

[0048] Embodiment 5: Conductivity test of hydrogel

[0049] The self-curing conductive hydrogel sample prepared in Example 5 differs from sample 1 prepared in Example 2 only in that different proportions of PEDOT:PSS solution were added, namely 0.6wt%, 0.8wt%, and 1wt%, respectively, thus preparing three kinds of self-curing conductive hydrogel samples. The conductivity of the three hydrogel samples prepared in Example 5 was measured using a four-probe tester. The conductivity σ was calculated as follows: the reciprocal of the resistance multiplied by the gel thickness L, and then divided by the area A, i.e., σ = L / (R*A).

[0050] To activate the myocardium beyond single-point contact in vivo, the conductivity of the hydrogel must be higher than that of the myocardium (0.1–6.0 mS / cm). For example... Figure 5 As shown, when the PEDOT:PSS solution is added at a ratio of 1 wt%, the hydrogel conductivity is 12.0 ± 0.8 mS / cm, which meets the application requirements.

[0051] Example 6: In vitro in situ vascular solidification characterization test

[0052] The self-curing conductive hydrogel sample prepared in Example 5 differs from sample 1 prepared in Example 2 only in that 1 wt% PEDOT:PSS solution is added, and in vitro in situ vascular curing characterization is performed based on porcine heart. A 16G vascular catheter is inserted into the central venous vein (MCV), the proximal end is sutured, a double-barreled syringe is connected to the vascular catheter, and the hydrogel precursor solution is injected into the central venous vein. After curing, the heart epidermis is peeled off, and the self-curing hydrogel is observed to form in the central venous vein. After removal, it is photographed and recorded.

[0053] like Figure 6 As shown, from left to right, Figure 1 on the left represents the insertion of the injection cannula into the cardiovascular system; Figure 2 on the left represents the in-situ injection of the injectable hydrogel combination reagent to form a prepolymer solution; Figure 3 on the left represents the in-situ formation of hydrogel from the prepolymer solution; Figure 4 on the left represents the removal of the hydrogel; Example 6 completed the self-curing characterization of conductive hydrogel in the myocardial vein of a pig heart.

[0054] Based on the above test results, the self-curing conductive hydrogel provided by this invention has excellent anti-swelling properties and is stable in body fluid environments; it has good elasticity and toughness, and can withstand the external force of heartbeats to maintain structural integrity; the curing time, conductivity, and precursor solution viscosity are controllable and can be adjusted to meet different technical requirements, and it is expected to be combined with cardiac pacemakers for application in the biomedical field.

[0055] The preferred embodiments of the present invention have been described above, but the scope of protection of the present invention is not limited thereto. Various modifications and substitutions to the present invention made by those skilled in the art within the scope of the technology disclosed herein should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. An injectable hydrogel combination reagent, characterized in that, The injectable hydrogel combination reagent includes solution A and solution B; solution A is a first precursor solution formed by dissolving modified poloxamer PFUDAm and ammonium persulfate in deionized water; solution B is a second precursor solution formed by dissolving N-isopropylacrylamide NIPAM, ferrous gluconate, and PEDOT:PSS in deionized water. The chemical structural formula of the modified poloxamer PFUDAm is shown below: In solution A, the mass of PFUDAm is 20-25 wt% of the total mass of solution A, and the concentration of ammonium persulfate is 2.5-7.5 mM; in solution B, the mass of NIPAM is 5-10 wt% of the total mass of solution B, the concentration of ferrous gluconate is 2.5-7.5 mM, and the mass of PEDOT:PSS is 1-1.3 wt% of the total mass of solution B.

2. The injectable hydrogel combination reagent according to claim 1, characterized in that, In solution A, the mass of PFUDAm is 20 wt% of the total mass of solution A, and the concentration of ammonium persulfate is 5 mM; in solution B, the mass of NIPAM is 10 wt% of the total mass of solution B, the concentration of ferrous gluconate is 5 mM, and the mass of PEDOT:PSS is 1 wt% of the total mass of solution B.

3. The injectable hydrogel combination reagent according to claim 1, characterized in that, The preparation method of the modified poloxamer PFUDAm includes the following steps: First, N,N'-carbonyldiimidazole and poloxamer are dissolved separately in anhydrous dichloromethane. Under nitrogen protection, the poloxamer solution is added dropwise to the N,N'-carbonyldiimidazole solution. After stirring at room temperature, the product is precipitated with excess diethyl ether, filtered, and vacuum dried to obtain the activated poloxamer intermediate. The obtained poloxamer intermediate is dissolved in anhydrous dichloromethane and condensed with ethylenediamine under nitrogen protection. After the reaction is completed, the product is washed with water, dried, and precipitated with ice-cold diethyl ether. After filtration and vacuum drying, an amino-functionalized poloxamer derivative is obtained. The amino-functionalized poloxamer derivative is dissolved in anhydrous dichloromethane, triethylamine is added, and an acylation reaction is carried out by adding acryloyl chloride solution dropwise under ice bath conditions. After the reaction is completed, the product is precipitated with ice-cold diethyl ether, filtered, and vacuum dried to obtain the final product PFUDAm.

4. The injectable hydrogel combination reagent according to claim 3, characterized in that, The molar ratio of N,N'-carbonyldiimidazole to poloxamer is (10-20):

1.

5. The injectable hydrogel combination reagent according to claim 3, characterized in that, The molar amount of the ethylenediamine is (10 to 20) times the molar amount of the poloxamer intermediate.

6. The injectable hydrogel combination reagent according to claim 3, characterized in that, The molar amount of acryloyl chloride is (3 to 5) times the molar amount of the amino-functionalized poloxamer derivative.

7. A self-curing conductive hydrogel, characterized in that, The self-curing conductive hydrogel is a self-curing conductive hydrogel prepared by mixing solution A and solution B in a 1:1 volume ratio as described in claim 1 and then gelling them in situ.

8. The use of the self-curing conductive hydrogel of claim 7 in the preparation of conductive hydrogel fibers for the treatment of arrhythmia.

Images