Hydrogel capable of forming gel under driving of body fluid and response as well as preparation method and application of hydrogel

Abstract

The invention relates to the technical field of biological materials and medical products, in particular to hydrogel capable of forming gel through body fluid driving response, a preparation method and application of the hydrogel. The crosslinking based on the Michael addition reaction of the four-arm polyethylene glycol mercaptan and the four-arm polyethylene glycol maleimide developed by the invention has the characteristic of sensitive body fluid triggering gelling, so that the hydrogel has multiple purposes; the proven applications comprise various applications in contact with body fluid, such as liver hemostasis, subcutaneous injection, intramuscular injection, cornea surface filming, tooth extraction groove repair and the like. Compared with a traditional material, the material has sensitive body fluid response performance and injectability, and has remarkable advantages in application in the scenes of irregular wounds, invisible wounds, irregular tiny wounds and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomaterial technology and medical product technology, and in particular to a hydrogel material that forms a gel in response to body fluid drive, a preparation method, and its application in liver hemostasis, subcutaneous injection, intramuscular injection, corneal surface gelation, and filling of tissues such as tooth extraction sockets. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Stimuli-responsive hydrogels triggered by biological signals can provide targeted, controllable and adaptive responses on demand, making them invaluable for improving therapeutic effects, promoting tissue and wound repair, enabling real-time diagnosis and promoting personalized medicine. These biological triggers include pH, enzymes, redox agents or other chemicals, which usually require the presence of specific corresponding parts in the hydrogel. Therefore, they are highly selective for specific signals, which sometimes limits their wide application. So far, there have been limited studies on responses to tissue environments such as biological fluids. Body fluids (including blood, saliva, tears, etc.) account for more than 55% of body weight and play a vital role in almost all biological activities. Therefore, the development of hydrogel systems that respond to body fluids will have widely valuable applications in various biological scenarios. However, reports on body fluid-responsive biomedical materials are still very rare. Body fluids are buffered aqueous liquids rich in salt ions, such as Cl - 、HCO3、Na + Etc. Using chemical reactions that can respond to these media can achieve response to body fluids. Michael addition reaction is such a reaction, and its reaction rate is highly related to the polarity of the reaction medium. Due to its applicability in water, Michael addition reaction has been widely used in the manufacture of various biomaterials, especially hydrogel materials, in the past few decades. One of them is polyethylene glycol connected by thiol and maleimide, which can directly form hydrogel after mixing. However, there has been no report on their research and application of response to body fluids so far.

[0004] Although traditional injectable hydrogels can be injected into wounds, due to the characteristics of polymers and hydrogels, their fluidity is relatively poor, making them difficult to apply to relatively small wounds to form a patch, and they may cause the risk of thrombosis; secondly, their injection resistance is generally relatively large, and when used with a small syringe, a relatively large thrust is often required, further limiting their convenience of use. The use of hydrogels that respond to body fluids can ensure the fluidity of the liquid before contacting the body fluids, and can better fit the tiny tissues of the wound. Secondly, due to their responsiveness to blood, they can coagulate the blood at the wound while contacting the blood to better protect the wound; more advantageously, since the hydrogel is a small molecule before contacting the body fluids, its hardness and modulus are more suitable for injection, and the pressure required for injection is relatively small, which is very suitable for use in minimally invasive surgery and can solve the limitations and defects of traditional ordinary materials.

[0005] Against the backdrop of the rapid rise of minimally invasive surgery, the clinical demand for injectable, multi-purpose, and easy-to-use fluid-responsive hydrogels has become particularly urgent. Summary of the invention

[0006] The purpose of the present invention is to provide a hydrogel material that responds to body fluids to form gels, a preparation method and its application. The material can form gels in response to blood, tears, saliva, PBS, etc. The material has very good fluidity and injectability before forming a hydrogel, and can quickly form a hydrogel when in contact with body fluids. This property gives the material excellent hemostatic properties, and it can form gels on irregular wounds and small wounds, and fit the wound surface to facilitate wound recovery; at the same time, its excellent injectability ensures that it forms hydrogels in various locations in the body, including subcutaneous, intramuscular, and corneal surfaces; based on these properties, the material is particularly suitable for filling tooth extraction sockets. Since tooth extraction will produce relatively large wounds, and accompanied by symptoms such as bleeding and inflammation, the material can be beneficial to the healing of tooth extraction socket wounds and reduce the occurrence of side effects. Specifically, a body fluid-driven responsive gel hydrogel is characterized in that a hydrogel precursor material with polyethylene glycol as the main body and connecting thiol and maleimide forms a hydrogel through a Michael addition reaction of thiol and maleimide under body fluid-driven conditions to generate crosslinking points.

[0007] On the other hand, the present invention provides a method for preparing a hydrogel that responds to body fluid-driven gelation, wherein a hydrogel precursor material with polyethylene glycol as the main component and thiol and maleimide connected to form a hydrogel under body fluid-driven conditions by Michael addition reaction of thiol and maleimide to generate crosslinking points. Specifically, the method comprises the following steps: S1. Weigh a certain amount of four-arm polyethylene glycol-thiol and add it to an aqueous solvent with a low salt ion concentration to fully dissolve it; S2, weighing a certain amount of four-arm polyethylene glycol-maleimide and adding it to an aqueous solvent with a low salt ion concentration to fully dissolve; S3. Keep the two solutions in a weakly acidic environment; S4, mixing the two solutions in a certain ratio to obtain a precursor solution of a hydrogel; S5. Add the mixed solution into an environment where body fluids are present for use.

[0008] Furthermore, the aqueous solvent with low salt ion concentration in S1 and S2 is generally deionized water, and high concentration of salt ions can accelerate gelation ( Figure 4 A) It is not conducive to the response function of body fluids.

[0009] Furthermore, the concentration of four-arm polyethylene glycol-thiol and four-arm polyethylene glycol-maleimide in S1 and S2 is generally 1% to 10%.

[0010] Furthermore, the weakly acidic environment in S3 is generally controlled to have an acidic pH value below 4.0 in order to prevent the mixed solution from reacting too quickly.

[0011] Furthermore, the mixing ratio of the two solutions in S4 is generally 1:1.

[0012] Furthermore, the mixed solution in S5 must be used within 10 minutes.

[0013] Another aspect of the present invention provides a blood-responsive gelling hydrogel for use in hemostasis in different body fluid scenarios.

[0014] Further, liver hemostasis, subcutaneous hemostasis, and muscle hemostasis are achieved.

[0015] Another aspect of the present invention provides a use of a blood-responsive gelling hydrogel in corneal eye drops.

[0016] Another aspect of the present invention provides a blood-responsive gelling hydrogel for use in restoring an extraction socket after tooth extraction.

[0017] In another aspect, the present invention provides a fluid-responsive gelling hydrogel for administration by injection, specifically injection into the liver surface, subcutaneous injection, intramuscular injection, corneal surface of the eye, and injection into any site of bleeding and presence of body fluids.

[0018] Another aspect of the present invention provides a use of a hydrogel that gels in response to body fluids in tissue filling.

[0019] Further, subcutaneous tissue filling, muscle tissue filling, and wound filling.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) The advantages of the present invention are that the preparation process is simple, the raw materials used are commercially available, and there is no need for excessive chemical modification and modification. It only needs to be prepared into a suitable solution according to the instructions, which is easy to prepare on a large scale and market promotion. The operation process of the present invention is simple. Before use, it is only necessary to mix the two solutions and inject them. There is no cumbersome preparation process, which is convenient for medical staff to operate and quickly get started.

[0021] (2) The hydrogel that can be triggered by body fluids provided by the present invention has a wide range of uses. The hydrogel has excellent fluidity and sensitive responsiveness before being triggered, and can quickly gel after being triggered by body fluids. Proven implementation cases include: 1) Rapid hemostasis, especially suitable for tiny irregular wounds and invisible conditions in the body; 2) subcutaneous injections and intramuscular injections, etc., which require precise and low-resistance injections; 3) Uniform gelation on the corneal surface triggered by tears; 4) Small wounds such as oral extraction sockets and scenarios that require close fitting of wounds. This invention is particularly suitable for minimally invasive surgery and has great value and significance for clinical surgery.

[0022] (3) The polyethylene glycol main material used in the present invention is a pharmaceutical excipient approved for use by the U.S. Food and Drug Administration (FDA). It has low biological toxicity, good biocompatibility, and is safe to use, and can meet the needs of application in different scenarios. At the same time, due to the antioxidant properties of thiol, it can also play an anti-inflammatory role and promote wound repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0024] Figure 1 : Schematic diagram of fluid-triggered hydrogel: (A) Chemical molecular structure of each component of the hydrogel; (B) Configuration and usage of the hydrogel.

[0025] Figure 2 :The huge difference in reaction rate makes the hydrogel able to be triggered by body fluids. (A) The hydrogel gels slowly in pure water (>10 min), and can gel quickly (<20 s) when 20 μL of blood is added; (B) The reaction unit structure of the hydrogel; (C) The change of UV absorption with chemical reaction; (D) The change of UV absorption at 218 nm with time; (E) The second-order reaction rate equation; (FG) The fitting of the rate equation in water (F) and PBS (G): The reaction rate in body fluid is 33.90 times that in deionized water.

[0026] Figure 3: Rheological test of hydrogel. The gelation time in pure water is about 30 minutes (A), and it has already gelled at the moment of detection in PBS (B).

[0027] Figure 4 :Comparison of gelation speed under different environments. (A) Gelation time in pure water, saline, PBS, saliva, and blood; (B) Effect of pH on gelation time; (C) Effect of blood volume on gelation time; (D) Effect of concentration on gelation time.

[0028] Figure 5 : Comparison of the morphology of hydrogels injected into water (left) and PBS (right) over time.

[0029] Figure 6 :The liquid hydrogel precursor was injected into the subcutaneous tissue of mice to form a gel (A), and the weight of the hydrogel remained roughly unchanged over time (B).

[0030] Figure 7 : The hydrogel precursor solution can be successfully injected into the muscle of rats to form gel.

[0031] Figure 8 :The hydrogel precursor solution is dripped onto the corneal surface and comes into contact with the tear fluid to form a uniform gel on the corneal surface. (A) Schematic diagram of hydrogel gelation on the corneal surface; (B) AS-OCT detection of hydrogel before and after dripping.

[0032] Fig. 9 :The hydrogel's gelling property when in contact with blood is used for hemostasis in mouse liver. (A) Schematic diagram of hydrogel liver hemostasis; (B) Picture of hydrogel bleeding volume changing over time; (C) Statistics of bleeding volume under different treatment methods; (D) Statistics of the amount of hydrogel and surgiflo® materials used in hemostasis applications.

[0033] Fig.10 :Hydrogel has excellent antioxidant and anti-inflammatory effects. (A) Results of hydrogel antioxidant test in vitro; (B) Antioxidant effect of hydrogel in cells; (C) Anti-inflammatory effect of hydrogel in cells.

[0034] Fig.11 :Application of humoral responsive hydrogel in tooth extraction socket filling. (A) Schematic diagram of tooth extraction socket animal experiment; (B) Macroscopic photo of the tooth extraction socket after two weeks of treatment; (C) Micro-CD imaging detection of tooth extraction socket restoration; (D) HE staining.

[0035] Fig.12 : Immunohistochemical staining of CD31 (A) and CD68 (B) markers showed that the hydrogel can achieve a similar effect to gelatin sponge. DETAILED DESCRIPTION

[0036] The present invention proposes a method for preparing and using a hydrogel medical material based on controlling the rate of Michael addition reaction to achieve rapid gelation triggered by body fluids. In order to make the advantages and technical solutions of the present invention clearer and more specific, the present invention is described in detail below in conjunction with specific embodiments.

[0037] First, the main raw materials and experimental equipment required for the present invention are described in detail.

[0038] Experimental instruments: electronic balance, ultrasonic mixer, vacuum oven, magnetic stirrer, pH meter, high-speed centrifuge, -80 ℃ medical freezer, freeze dryer, vacuum drying oven, water purification system, syringe, UV spectrophotometer.

[0039] Experimental drugs: Four-arm polyethylene glycol thiol (4-Arm-PEG-SH, molecular weight 5000 Da) was purchased from Xiamen Sinopharm Biotechnology Co., Ltd., and four-arm polyethylene glycol maleimide (4-Arm-PEG-Mal, molecular weight 5000 Da) was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. Physiological saline, sodium hydroxide (NaOH), and phosphate-balanced saline (PBS buffer) were purchased from Sinopharm Chemical Reagent Co., Ltd. N -(2-Hydroxyethyl)maleimide and sodium 2-mercaptoethane sulfonate were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0040] Example 1: Preparation of hydrogel precursor solution triggered by body fluid Use an analytical balance to weigh a certain amount of 4-Arm-PEG-SH and 4-Arm-PEG-Mal (see the schematic diagram for the specific chemical structure) Figure 1 A) Dissolve in an aqueous solvent with low salt ion concentration. In this example, double distilled deionized water is used. Ultrasonic wave is used to assist dissolution. Each solution (10%) is prepared. Add a drop of 0.02 mol / L hydrochloric acid to 4-Arm-PEG-SH to ensure that the pH of the solution is maintained below 4 ( Figure 1 B).

[0041] Example 2: Principle of body fluid-triggered hydrogel use N -(2-Hydroxyethyl)maleimide and sodium 2-mercaptoethane sulfonate were used as template compounds to explore the reaction rates of thiol and maleimide crosslinking sites (see Figure 2 ). Prepared in water and PBS respectively. N-(2-Hydroxyethyl)maleimide and sodium 2-mercaptoethane sulfonate (0.1 mM). The two solutions were mixed in a 1:1 ratio, and the ultraviolet absorption of the solution at different times was detected by a UV spectrophotometer, and a curve of the absorption intensity at a wavelength of 218 nm versus time was plotted. The curve was simulated using the second-order reaction rate equation, and the reaction rate constant at this concentration was calculated based on the curve. K . It was found through calculation that K (PBS) / K (H2O) = 33.90, which is similar to the measured gelation rate, proving that the hydrogel is indeed a body fluid-triggered mechanism caused by the difference in reaction rate in body fluids.

[0042] Example 3: Sensitivity test of body fluid-triggered hydrogels in different body fluid environments Take 150 uL of each of the 4-Arm-PEG-SH and 4-Arm-PEG-Mal solutions (10%) prepared in Example 1, mix them in a 2 mL glass bottle, and add 20 uL of blood, saline, artificial saliva, PBS buffer, etc. as body fluid signals to trigger the solution to gel. Use rheological test ( Figure 3 ) and the inverted bottle experiment ( Figure 4 ) and record the gelation time. The measurement results are as follows Figure 4 As shown in the figure: it takes about 10 minutes to gel in pure water, about 6 minutes to gel in physiological saline, about 20 seconds to gel in PBS buffer, and only about 15 seconds to gel in blood and artificial saliva. This shows that salt ion concentration affects the gelation time, but pH has a greater impact on the gelation time. The gelation speed is faster at high pH ( Figure 4 B), which also proves that the system provided by the invention has a very sensitive triggering effect to body fluids.

[0043] Example 4: Application of body fluid-triggered hydrogel in hemostasis of mouse liver Healthy 6-week-old female mice were selected, with 4 mice in each group, and divided into three groups: the experimental group selected 10% 4-Arm-PEG-SH and 4-Arm-PEG-Mal solutions, the positive control group used Surgiflo®, and the negative control group did not treat the wound. After the mice were anesthetized by intraperitoneal injection of 1% w / v sodium pentobarbital solution, the chest hair was removed, and the liver was carefully and fully exposed and placed on a pre-weighed filter paper. Then, a surgical blade was used to make an incision 3 mm in length and 2 mm in depth on the surface of the liver to create a liver bleeding model. Subsequently, the gel was immediately covered on the wound with a syringe, and the hemostasis time was recorded and the amount of bleeding was calculated (bleeding amount = postoperative weight of the filter paper - preoperative weight). The hemostatic effect is as follows: Fig. 9It can be seen that the body fluid-triggered hydrogel prepared in the present invention has good hemostatic effect, convenient operation, rapid hemostasis and the like, and the usage amount is significantly less than that of the positive control group Surgiflo®.

[0044] Example 5: Comparison of in vitro injectability of hydrogels triggered by body fluids Prepare 4 mL PBS buffer and deionized water in a 4 mL glass bottle. Take 150 uL of 4-Arm-PEG-SH and 4-Arm-PEG-Mal solutions, mix them evenly, and inject them into PBS buffer and deionized water at a certain speed. It can be observed that the mixed solution injected into deionized water enters the liquid surface in the form of water flow, and then the solution stays at the bottom; the mixed solution injected into PBS buffer will immediately form a cluster on the top of the liquid to form a hydrogel ( Figure 5 ). This indicates that liquid hydrogels can be injected into body fluids and quickly gel.

[0045] Example 6: Test of injectable gel formation of hydrogel triggered by body fluid in mice Healthy 6-week-old female mice were selected and intraperitoneally injected with 1% w / v pentobarbital solution. After the mice were anesthetized, 200 uL of a mixed solution of 4-Arm-PEG-SH and 4-Arm-PEG-Mal (10%, 1:1) was injected subcutaneously into the abdomen of the mice, and the mice were immediately dissected to observe the state of the hydrogel injected subcutaneously. Three other mice were dissected every other week and weighed to observe the state of the hydrogel. The experiment found that the liquid could immediately gel under the mouse skin ( Figure 6 ), and the shape of the gel is sheet-like, indicating that the liquid has a certain flow under the skin and forms a gel after fully contacting the subcutaneous liquid. After observing the subcutaneous hydrogel for 4 weeks, it was found that the volume of the gel did not change much, indicating that the gel can also play a filling role.

[0046] Example 7: Test of hydrogel formation triggered by body fluids after intramuscular injection in rats Healthy 10-week-old female rats were selected and anesthetized by intraperitoneal injection of 1% w / v pentobarbital solution. 200 uL of hydrogel precursor solution (4-Arm-PEG-SH and 4-Arm-PEG-Mal 10%, 1:1) was injected into the tibia muscle of the rats using a 1 mL syringe. The muscles were immediately dissected 2 min later to observe the state of the hydrogel in the muscle ( Figure 7 ). The experiment found that even in muscles with relatively large resistance, the material of the present invention can be injected relatively easily and smoothly, and the liquid material turns into hydrogel in the muscles of rats. This shows that the hydrogel can not only play the role of filling muscle tissue but also be used as a potential drug-loaded material to achieve controlled release.

[0047] Example 8: Body fluid-triggered hydrogel uniformly gelled on the surface of mouse cornea Healthy 6-week-old female mice were selected and anesthetized with 1% w / v pentobarbital solution injected intraperitoneally. After the mice were anesthetized, the upper and lower eyelids of the mice were separated to fully expose the eyeballs. A hydrogel precursor solution (4-Arm-PEG-SH and 4-Arm-PEG-Mal 10%, 1:1) was prepared. Use a pipette to draw 2.5 μL of gel precursor solution and drop it onto the ocular surface. Gently close the upper and lower eyelids of the mouse to ensure that the gel is in full contact with the tears and adheres to the ocular surface. After the liquid gel is converted into a solid gel after contact with the tears and successfully adheres to the ocular surface, an anterior segment optical coherence tomography (AS-OCT) scanner is used to install the anterior lens and perform an anterior corneal scan to observe the adhesion of the gel to the ocular surface. Experiments have found that the precursor solution can be uniformly gelled on the surface of the mouse cornea ( Figure 8 Right), and no adverse effects of adhesion to the eyelids were found after gelation.

[0048] Example 9: Application of body fluid-triggered hydrogel in tooth extraction sockets of rats Eight-week-old male SD rats were selected to establish a tooth extraction model. The rats were divided into three groups: the experimental group selected 10% 4-Arm-PEG-SH and 4-Arm-PEG-Mal solutions, the positive control group used colloidal silver sponge, and the negative control group did not treat the wound. After anesthesia with pentobarbital solution, the rats were fixed on the operating table, and the corners of the mouth were pulled open to expose the maxillary teeth. The gums of the maxillary first molar were carefully separated with a dental probe, and then the tooth was extracted with forceps. The hydrogel was immediately injected into the inner surface of the extraction socket to observe the hemostatic effect. Two weeks later, photos were taken to observe the recovery of the extraction socket, and the small animal in vivo three-dimensional tomography system (micro-CT) was used to image the extraction socket. H&E staining and pathological evaluation were performed after maxillary bone processing ( Fig.11 D), and CD31 and CD68 immunohistochemical staining was used to analyze the inflammatory response and recovery of the extraction socket ( Fig.12 ). After two weeks, the tissues of the extraction sockets of rats coated with the fluid-triggered hydrogel had recovered to a state similar to that of normal tissues, while the untreated extraction sockets still had a yellowish appearance. Micro-CT imaging revealed that the osteogenesis of the hydrogel group was better than that of the untreated group ( Fig.11C), which is similar to the positive control group. Further immunohistochemistry showed that neither the hydrogel nor the gelatin sponge affected the angiogenesis process during the healing of the alveolar bone. The expression of CD68 in the experimental group was reduced compared with the other two control groups. The presence of fewer CD68 macrophages in the late healing of the extraction socket led to a faster healing process and more complete bone healing, which can effectively protect the empty socket from infection and promote bone healing to a certain extent. The wound healing effect of this hydrogel may come from the excellent antioxidant and anti-inflammatory functions of the hydrogel ( Fig.10 ). These experiments show that the hydrogel can better fit the wound tissue, achieve wound filling, and better play the role of antioxidant and anti-inflammatory, and has great potential as an excellent extraction socket filling material after oral tooth extraction surgery.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A hydrogel that responds to body fluid-driven gelation, characterized in that: The hydrogel precursor material with polyethylene glycol as the main body and thiol and maleimide as the connecting materials forms a hydrogel through Michael addition reaction between thiol and maleimide under body fluid driving conditions to generate cross-linking points.

2. A method for preparing a hydrogel that responds to body fluid-driven gelation as claimed in claim 1, characterized in that: The specific steps include: S1. Weigh a certain amount of four-arm polyethylene glycol-thiol and add it to an aqueous solvent with a low salt ion concentration to fully dissolve it; S2, weighing a certain amount of four-arm polyethylene glycol-maleimide and adding it to an aqueous solvent with a low salt ion concentration to fully dissolve; S3. Keep the two solutions in a weakly acidic environment; S4, mixing the two solutions in a certain ratio to obtain a precursor solution of a hydrogel; S5. Add the mixed solution into an environment where body fluids are present for use.

3. A method for preparing a hydrogel that responds to body fluid-driven gelation as claimed in claim 2, characterized in that: The concentration of four-arm polyethylene glycol-thiol and four-arm polyethylene glycol-maleimide in S1 and S2 is 1% to 10%.

4. A method for preparing a hydrogel that responds to body fluid-driven gelation as claimed in claim 2, characterized in that: The S3 is a weakly acidic environment.

5. A method for preparing a hydrogel that responds to body fluid-driven gelation as claimed in claim 2, characterized in that: The mixing ratio of the two solutions in S4 is generally 1:

1.

6. A method for preparing a hydrogel that responds to body fluid-driven gelation as claimed in claim 2, characterized in that: The mixed solution in S5 must be used within 10 minutes.

7. Use of the blood-responsive gelling hydrogel as claimed in claim 1 for hemostasis in different body fluid scenarios.

8. The use according to claim 7, characterized in that: The hemostasis includes liver hemostasis, subcutaneous hemostasis and muscle hemostasis.

9. Use of the blood-responsive gelling hydrogel as claimed in claim 1 in corneal eye drops.

10. Use of the blood-responsive gelling hydrogel according to claim 1 for restoring an extraction socket after tooth extraction.

11. Use of the fluid-responsive gelling hydrogel as claimed in claim 1 in tissue filling.

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