Two-component in-situ crosslinking agent, degradable two-component hydrogel and application thereof

Abstract

This invention belongs to the field of biomaterials technology and discloses a two-component in-situ crosslinking agent, a biodegradable two-component hydrogel, and their applications. The two-component in-situ crosslinking agent disclosed in this invention has injectability and rapid gelation properties. The hydrogel formed after crosslinking has a low self-swelling rate, good elasticity, and is not easily crushed. It also has a suitable metal ion slow-release cycle and degradation cycle for preventing tissue adhesion or tissue repair. It is especially suitable for high-risk areas of postoperative adhesion such as the uterine cavity, abdominal cavity, and joints, and can also promote tissue regeneration.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials, specifically relating to a two-component in-situ crosslinking agent, a biodegradable two-component hydrogel, and their applications. Background Technology

[0002] Tissue adhesions refer to abnormal fibrous connections that form between different tissues or organs after injury or inflammation, and can occur after surgery, trauma, infection, or inflammation. Tissue adhesions can lead to organ dysfunction; for example, endometrial adhesions can cause female infertility, intestinal adhesions can cause intestinal obstruction, and tendon adhesions can cause joint stiffness. Current clinical treatment of tissue adhesions faces many challenges: traditional natural materials (such as hyaluronic acid) degrade too quickly to form an effective barrier; synthetic materials lack bioactivity and easily inhibit tissue regeneration, leading to poor tissue healing and fibrosis; furthermore, some materials (such as silicone membranes) are non-degradable and require secondary surgery for removal. There is an urgent need for a barrier material that is biocompatible, biodegradable, and can effectively prevent adhesions and promote tissue healing.

[0003] Hydrogels, as a class of biomaterials with a three-dimensional network structure, high water content, and similarity to the natural extracellular matrix, show broad application prospects in the field of soft tissue repair. Hydrogels can not only provide growth scaffolds for cells but also load and release bioactive factors, promoting tissue regeneration and functional reconstruction. Currently, hydrogel materials used for soft tissue repair mainly include natural polymer hydrogels and synthetic polymer hydrogels. Natural polymer hydrogels, such as hyaluronic acid, have good biocompatibility and biodegradability, but relatively weak mechanical strength; synthetic polymer hydrogels, such as polyvinyl alcohol and polyethylene glycol, have better mechanical properties, but relatively lower bioactivity. In the prior art, CN111437435A discloses a hydrogel cell scaffold, which is made by blending functional polymer materials with polyvinyl alcohol, adding a metal ion crosslinking agent for reaction, and then physically crosslinking to obtain a hydrogel, which is then inoculated with M2 macrophages to regulate the chronic inflammatory microenvironment. CN112107731A describes an injectable bilayer drug-loaded osteochondral repair hydrogel scaffold comprising methacrylamide polysaccharide and protein-based natural polymers, a UV photoinitiator, modified cyclodextrin, osteo- or cartilage differentiation-promoting drugs, a metal ion source, and seed cells, for osteochondral tissue repair.

[0004] Studies have shown that bioactive proteins, such as decellularized matrix, can reduce the incidence of adhesions by promoting the growth of healthy tissue and reducing scar formation after thyroid and cardiac surgeries. Metal ions such as Cu... 2+ Zn 2+It can mediate cell behavior, promote cell migration and tissue regeneration, or have antibacterial functions, and has been demonstrated in wound dressings and tissue regeneration applications. PEG (polyethylene glycol) and its derivatives are widely used in many medical scenarios, including tissue repair, tissue isolation, and drug delivery, due to their excellent biocompatibility, high hydrophilicity, and tunable physicochemical properties.

[0005] However, existing hydrogel materials still have some technical problems: First, many protein-containing hydrogel systems require complex preparation processes or special cross-linking conditions, resulting in long gelation times or the need for special equipment to induce gelation, making it difficult to achieve convenient in-situ gelation in clinical practice; second, unchelated metal ion hydrogels often face the problem of local toxicity caused by the acute release of metal ions, affecting cell growth and tissue regeneration; third, most hydrogel systems lack effective angiogenesis and cell recruitment capabilities, leading to poor tissue integration and regeneration after implantation; in addition, most anti-adhesion hydrogels are slow-degrading bioinert materials, which can easily trigger immune responses, induce aseptic inflammation, or form fibrotic tissue; finally, most anti-adhesion hydrogels often contain added drugs, making it difficult to balance hydrogel cytotoxicity and therapeutic efficacy. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide a two-component in-situ crosslinking agent. This two-component in-situ crosslinking agent is injectable, rapidly gelling, and the hydrogel formed after crosslinking has a low self-swelling rate, good elasticity, and is not easily crushed. It also possesses a suitable metal ion slow-release cycle and degradation cycle for preventing tissue adhesion or tissue repair.

[0007] In a first aspect, the present invention provides a two-component in-situ crosslinking agent, wherein the in-situ crosslinking agent is composed of a first precursor solution and a second precursor solution, wherein the first precursor solution is prepared by dissolving thiolated polyethylene glycol PEGSH and protein in a buffer solution and adjusting the pH to 7.2 with NaOH; and the second precursor solution is prepared by dissolving CuSO4 in deionized water.

[0008] The number-average molecular weight of the thiolized polyethylene glycol is 200~800000, and the thiolized polyethylene glycol is one or a mixture of 2-arm-PEGSH, 3-arm-PEGSH, 4-arm-PEGSH, 6-arm-PEGSH and 8-arm-PEGSH;

[0009] The structural formula of 2-arm-PEGSH is as follows:

[0010] ;

[0011] The structural formula of 3-arm-PEGSH is as follows:

[0012]

[0013] The structural formula of 4-arm-PEGSH is as follows:

[0014] ;

[0015] The structural formula of 6-arm-PEGSH is as follows:

[0016]

[0017] The 8-arm-PEGSH structure can be any one of the following structures:

[0018]

[0019] .

[0020] In the first precursor solution, the concentration of thiolated polyethylene glycol is no higher than 8%; the concentration of protein is 0.2% to 20%.

[0021] The concentration of Cu ions in the second precursor solution is 0.01%~20% (w / v);

[0022] The proteins include, but are not limited to, gelatin, decellularized matrix, collagen, silk fibroin, recombinant human protein, polypeptides, and derivatives of the above proteins; wherein the decellularized matrix is ​​derived from tissues of any animal, such as humans, cattle, pigs, horses, sheep, or fish; and derivatives of the above proteins include, for example, protein thiolated derivatives.

[0023] As a preferred embodiment of the two-component in-situ crosslinking agent described in the first aspect of the present invention, in the first precursor solution, the thiolated polyethylene glycol is 4-arm-PEGSH with a molecular weight of 20kDa and a concentration of 8%; the protein is decellularized matrix (SIS) with a concentration of 1%; and in the second precursor solution, the Cu ion concentration is 0.0615%~0.246%.

[0024] As another preferred embodiment of the two-component in-situ crosslinking agent described in the first aspect of the present invention, in the first precursor solution, the thiolated polyethylene glycol is 4-arm-PEGSH with a molecular weight of 20kDa and a concentration of 8%; the protein is gelatin with a freeze strength of 300g bloom and a concentration of 1%; in the second precursor solution, the Cu ion concentration is 0.0615%~0.246%.

[0025] Secondly, based on the aforementioned two-component in-situ crosslinking agent, the present invention also provides a biodegradable two-component hydrogel, wherein the hydrogel is prepared by crosslinking a first precursor liquid and a second precursor liquid in the two-component in-situ crosslinking agent described in the first aspect, that is, it is formed by metal coordination crosslinking of thiolized polyethylene glycol, metal ions and proteins.

[0026] In protein-based hydrogel in-situ crosslinking agents, active proteins endow the hydrogel with good biocompatibility and induce cell migration to the wound site, promoting cell adhesion and tissue regeneration. Metal ions participate in the construction of the gel network and simultaneously achieve long-term low-concentration release, coordinating inflammatory responses and tissue repair processes. In addition, PEG, as the main material, can inhibit cell growth into the hydrogel, avoiding adhesion caused by excessive proliferation.

[0027] Thirdly, the present invention also provides the application of the above-mentioned biodegradable two-component hydrogel in the preparation of formulations for preventing tissue adhesion or tissue repair. The hydrogel forms a hydrogel network through coordination crosslinking of thiol-metal ions. Proteins endow the hydrogel with good biocompatibility and provide bioactive sites to promote cell adhesion and tissue regeneration at the tissue repair site. Furthermore, based on the specific component hydrogel provided by the present invention, the release period of the metal ions and the long degradation period of the hydrogel are conducive to the slow repair of tissues. The metal ions can coordinate the inflammatory response and recruit cells to accelerate the repair process.

[0028] Beneficial effects:

[0029] The two-component in-situ crosslinking agent provided by this invention has an extremely short gelation time, achieving complete gelation within 3 seconds. The resulting biodegradable two-component hydrogel is based on thiolized polyethylene glycol (PEGSH) as the main chain donor, which, together with metal ions and proteins, constructs a network in which proteins are uniformly distributed. This composite hydrogel combines high bioactivity with reversible gelation. The hydrogel provided by this invention has a low swelling rate, is less likely to cause additional pressure on tissues, and has good elasticity, making it less prone to breakage under tissue compression. The reasonable degradation cycle can also promote tissue repair, inhibit scar tissue formation, and prevent excessive tissue proliferation. Attached Figure Description

[0030] Figure 1 PEGSH / Cu in Example 1 2+ Stress-strain curves of composite hydrogels;

[0031] Figure 2 PEGSH / Cu in Example 1 2+ Young's modulus of composite hydrogels;

[0032] Figure 3The preparation steps of the decellularized small intestinal matrix described in Example 2;

[0033] Figure 4 The results of gelation detection of the decellularized matrix in Example 2;

[0034] Figure 5 The histological staining of the small intestine and decellularized small intestine matrix in Example 2;

[0035] Figure 6 PEGSH / Cu in Example 2 2+ Stress-strain curves of decellularized matrix (SIS) composite hydrogels;

[0036] Figure 7 PEGSH / Cu in Example 2 2+ Young's modulus of decellularized matrix (SIS) composite hydrogel;

[0037] Figure 8 PEGSH / Cu in Example 2 2+ / Ion release from decellularized matrix (SIS) composite hydrogel;

[0038] Figure 9 PEGSH / Cu in Example 2 2+ Swelling properties of decellularized matrix (SIS) composite hydrogels;

[0039] Figure 10 PEGSH / Cu in Example 3 2+ Stress-strain curve of gelatin composite hydrogel;

[0040] Figure 11 PEGSH / Cu in Example 3 2+ Young's modulus of gelatin composite hydrogel;

[0041] Figure 12 PEGSH / Cu in Example 3 2+ Ion release from gelatin composite hydrogels;

[0042] Figure 13 PEGSH / Cu in Example 3 2+ Swelling properties of gelatin composite hydrogels;

[0043] Figure 14 The endometrial injury repair status of each group in Example 1;

[0044] Figure 15 PEGSH / Cu in Example 2 2+ / Degradation cycle test results of decellularized matrix (SIS) composite hydrogel;

[0045] Figure 16 PEGSH / Cu in Example 3 2+ Degradation cycle test results of gelatin composite hydrogel. Detailed Implementation

[0046] The present invention will be further illustrated by the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0047] It should be noted that the w / v ratios used in this application refer to the mass ratio of solute to the volume ratio of solvent; the percentages mentioned below also refer to the mass ratio of solute to the volume ratio of solvent.

[0048] Main raw material sources: Small intestine tissue is from adult Large White pigs; PEGSH is 4-arm-PEGSH with a molecular weight of 20kDa, purchased from MCE (MedChemExpress), specification HY-W1048849C; Gelatin is Aladdin gelatin with a freeze strength of 300g bloom; Copper ions are sourced from anhydrous copper sulfate.

[0049] Example 1: PEGSH / Cu 2+ Composite hydrogel

[0050] Weigh out PEGSH with a molecular weight of 20 kDa and dissolve it in PBS. Stir magnetically for 30 min until clear to prepare solution 1 with a PEGSH concentration of 8%. Weigh out CuSO4 and dissolve it in deionized water. Adjust the pH to 7.2 with 1 M NaOH to prepare solution 2 with a CuSO4 concentration of 0.123%. Using a dual syringe, mix solution 1 and solution 2 in equal volume ratio, extrude at room temperature (25°C), and observe the gelation time.

[0051] Mechanical Experiment: The newly prepared hydrogel was mechanically tested using a universal mechanical testing instrument. The hydrogel compression rate was 1 cm / min. Figure 1 The results showed that the maximum stress in the hydrogel compression test was 198.38 kPa, and the strain was 43.6%. Figure 2 As shown, the Young's modulus of the hydrogel is 65.875 kPa.

[0052] Example 2: PEGSH / Cu 2+ / Decellularized Matrix (SIS) Composite Hydrogel

[0053] Preparation of decellularized matrix SIS: such as Figure 3As shown, fresh small intestine tissue from healthy pigs was selected, and the mesentery, adipose tissue, and attached blood vessels were quickly removed. The intestine was then repeatedly rinsed inside and outside the lumen with water to remove food residue and surface impurities. The treated small intestine was cut into uniform 2 cm segments for later use. Cells were lysed by soaking in 0.5% sodium dodecyl sulfate (SDS) solution for 12 h. The SIS was then soaked in a 1% Triton X-100 / 1% NH3•H2O composite solution at room temperature for 72 h to remove lipids and some cell debris. The SIS was then transferred to a methanol / chloroform mixture (volume ratio 1:1) for defatting treatment for 24 h. After each treatment step, the small intestine matrix was washed with deionized water for 10 min. The decellularized small intestine matrix was frozen at -20°C overnight, then freeze-dried in a freeze dryer for 48 h, and then stored at 4°C for later use.

[0054] Gel formation detection of decellularized matrix: such as Figure 4 As shown, SIS was weighed and dissolved in 0.01 M HCl containing 1 mg / ml pepsin at a concentration of 1% w / v. The solution was stirred overnight at room temperature at 300 rpm to obtain a homogeneous solution. The pH was then adjusted to 7.0 using 0.1 M NaOH, and the solution was incubated at 37°C for 30 min. Figure 4 As shown in the middle left image, its gelation is visible; for comparison, as... Figure 4 As shown in the middle right figure, the SIS solution without added NaOH does not form a gel, indicating that the prepared SIS has good activity.

[0055] like Figure 5 As shown, the stained images of fresh small intestine clearly show a large number of cell nuclei, with the tissue arranged tightly, revealing a multi-layered structure of the small intestine; the stained images of SIS show no visible cell nuclei, with the tissue arranged loosely, and the material has a single-layered structure; scale bar 200 μm.

[0056] PEGSH / Cu 2+ Preparation of decellularized matrix (SIS) composite hydrogel: SIS powder was dissolved in 0.01 M HCl containing 1 mg / ml pepsin to prepare a 1% w / v solution. The solution was stirred overnight at 300 rpm at room temperature to obtain a homogeneous solution. The pH was adjusted to 7.2 with 1 M NaOH. PEGSH with a molecular weight of 20 kDa was dissolved in the above solution at an 8% w / v ratio. The solution was magnetically stirred for 30 min until clear to obtain solution 1. CuSO4 was dissolved in deionized water to prepare solution 2. Solutions 1 and 2 were mixed according to the different proportions in Table 1 and injected into a double syringe. The mixture was extruded at room temperature (25°C) to obtain PEGSH / CuSO4 composite hydrogel. 2+ / A decellularized matrix (SIS) composite hydrogel was used, and the gelation time of each group was observed. The results are shown in Table 1. Figure 4The figure shows the gelation of solutions with different concentrations. It can be seen that hydrogels based on three different copper ion concentrations can all form stable gels.

[0057] Table 1: PEGSH / Cu 2+ / Formulation ratio of decellularized matrix (SIS) composite hydrogel

[0058]

[0059] Mechanical Experiment: The newly prepared hydrogel was mechanically tested using a universal mechanical analyzer. The hydrogel compression rate was 1 cm / min. Figure 6 The results showed that the maximum stress in the hydrogel compression test was 61.82 kPa, and the strain was 41.3%. Figure 7 As shown, its Young's modulus is 54.535 kPa.

[0060] Ion release experiment: After blotting the surface moisture of the hydrogel with sterile filter paper, the initial mass (W0) was weighed using an electronic balance and recorded. The sample was placed in a centrifuge tube, and 5 times the mass of the hydrogel was added to PBS buffer (pH 7.4). The mixture was incubated at 37°C and the solution was collected at specific time points. The solution was then replaced with a new batch of the same mass of PBS buffer (pH 7.4). The amount of copper ions released was detected using a UV spectrophotometer. Figure 8 The results showed that the cumulative release rate of copper ions in each proportion of hydrogels gradually increased with the increase of sustained release time. At 28 days, the release rate of copper ions was between 3% and 22%, with the S3 group, which had the highest copper ion content, having the lowest release rate and the S1 group having the highest release rate.

[0061] Swelling experiment: After blotting the surface moisture of the hydrogel with sterile filter paper, the initial mass (W0) was weighed using an electronic balance and recorded. The sample was placed in a centrifuge tube, and 5 times the mass of the hydrogel was added to PBS buffer (pH 7.4). The tube was incubated at 37°C for a specific time and then removed. The hydrogel was gently handled with tweezers, and the surface liquid was aspirated before weighing the wet weight (Wt). The swelling rate was calculated using the formula: Swelling rate = [(Wt - W0) / W0] × 100%. The experimental results are as follows: Figure 9 As shown, the results indicate that the swelling rate of all hydrogel samples in Example 1 remained within 200% over 28 days, with minimal swelling variation.

[0062] In vitro degradation experiment: Record the initial gel mass m0. Add 10 times the initial gel mass of PBS (pH=7.4) to a vial and place it in a water bath shaker at 37℃ and 40 rpm for in vitro degradation. Replace the PBS periodically and record the gel mass m after degradation periodically. n The formula for calculating the degree of gel degradation on day n is as follows: 100% * (m n -m0) / m0. Experimental results are as follows: Figure 15As shown, the results indicate that none of the hydrogel samples in Example 1 degraded within 30 days, demonstrating a relatively long degradation period.

[0063] Example 3: PEGSH / Cu 2+ / Gelatin composite hydrogel

[0064] Preparation of composite solutions: Gelatin powder was weighed and dissolved in PBS, and heated at 50°C to obtain a solution with a concentration of 1%. PEGSH (molecular weight 20 kDa) was weighed and dissolved in the cooled solution to obtain a concentration of 8%, and magnetically stirred for 30 min to obtain solution 1. CuSO4 was weighed and dissolved in deionized water, and the pH was adjusted to 7.2 with 1 M NaOH to obtain solution 2.

[0065] Gel formation experiment: Using a dual syringe, mix PEGSH, gelatin, and Cu according to the following group ratios in Table 2. 2+ The solution was extruded at room temperature (25°C), and the gelation time was observed. The results are shown in Table 2. It can be seen that hydrogels based on three different copper ion concentrations can all form stable gels.

[0066] Table 2: PEGSH / Cu 2+ / Formulation ratio of gelatin composite hydrogel

[0067]

[0068] Mechanical experiment: The operating procedures are the same as in Example 1. Figure 10 The results showed that the maximum stress of the hydrogel was 38.5 kPa, and the maximum strain was 48.6%. Figure 11 As shown, the Young's modulus is 31.7 kPa.

[0069] Ion release experiment: The procedure is the same as in Example 2. Figure 12 The results showed that the cumulative release rate of copper ions in hydrogels of different proportions gradually increased with the increase of sustained release time. At 28 days, the release rate of copper ions was 2% to 7%, with the G3 group, which had the highest copper ion content, having the lowest release rate and the G1 group having the highest release rate.

[0070] Swelling experiment: The procedure is the same as in Example 2. Figure 13 The results showed that the swelling rate of all hydrogels remained within 200% over 28 days, with minimal swelling changes.

[0071] In vitro degradation experiment: The procedure was the same as in Example 2. The experimental results are as follows: Figure 16 As shown, the results indicate that none of the hydrogel samples in Example 3 degraded within 30 days, demonstrating a relatively long degradation period.

[0072] Application Example 1: Tissue Repair Experiment

[0073] Mature female SD rats (8 weeks old, weighing approximately 250g) were selected for the experiment. Rats were anesthetized by intraperitoneal injection of chloral hydrate. After anesthesia, a vertical incision was made along the midline of the abdominal cavity to confirm the location of the uterus. A horizontal incision (0.2cm) was made 0.5cm above the uterine bifurcation. Under aseptic conditions, the endometrium was damaged using a physical mechanical injury method, with the endometrium scraped away with a curette. After injury, the cavity was flushed with physiological saline to drain any residual fluid from the uterine horn. Treatment solutions were then injected: formulation G2, formulation S2, and 0.123w / v% copper sulfate solution. The injection volume was 0.2mL. The cavity was then sutured and closed. The rats were euthanized 7 days post-surgery, and the uterus was removed to assess the degree of repair of the injury model. The results are as follows: Figure 14 As shown.

[0074] Group 1 (Endometrial injury model group): The uterus was typically symmetrically Y-shaped, with symmetrical corners and a smooth surface. The interior was opaque, indicating uterine adhesions due to tissue hyperplasia. This result suggests that some damage still existed in the uterus 7 days after the physical trauma.

[0075] Group 2 (material group, S2): The uterine morphology was similar to that of Group 1, with an intact Y-shaped structure, symmetrical corners, smooth surface, and uniform transparent color, without bleeding or abnormal swelling. This result indicates that Group S2 can repair uterine wounds after physical trauma.

[0076] Group 3 (material group, G2): The uterus also maintained a good Y-shaped structure, with symmetrical corners, a relatively smooth surface, and a transparent and uniform color, without obvious bleeding points or tissue residue. This result indicates that the G2 group can repair uterine wounds after physical trauma.

[0077] Group 4 (material group, CuSO4 solution): The uterus showed obvious abnormalities in morphology: overall tortuous and deformed, irregular Y-shaped structure, obvious swelling of one corner accompanied by a dark red bleeding area, rough surface, blood clots or tissue residue, uneven color, showing alternating local pallor and congestion. This indicates poor repair in the copper sulfate-only group, and there may be persistent inflammation, adhesions or healing disorders.

Claims

1. A two-component in-situ crosslinking agent, wherein the in-situ crosslinking agent is composed of a first precursor solution and a second precursor solution, wherein the first precursor solution is prepared by dissolving thiolated polyethylene glycol (PEGSH) and protein in a buffer solution and adjusting the pH to 7.2 with NaOH; the second precursor solution is prepared by dissolving CuSO4 in deionized water; wherein the number average molecular weight of the thiolated polyethylene glycol is 200-800,000, and the thiolated polyethylene glycol is one or a mixture of 2-arm-PEGSH, 3-arm-PEGSH, 4-arm-PEGSH, 6-arm-PEGSH, and 8-arm-PEGSH.

2. The two-component in-situ crosslinking agent as described in claim 1, characterized in that, In the first precursor solution, the concentration of thiolated polyethylene glycol is no higher than 8%; the concentration of protein is 0.2% to 20%.

3. The two-component in-situ crosslinking agent as described in claim 1, characterized in that, The concentration of Cu ions in the second precursor solution is 0.01%~20%.

4. The two-component in-situ crosslinking agent as described in claim 1, characterized in that, The protein is any one of gelatin, decellularized matrix, collagen, silk fibroin, recombinant human protein, polypeptide, and derivatives of the above proteins; wherein the decellularized matrix is ​​derived from tissues of any animal, such as human, cattle, pig, horse, sheep, or fish.

5. The two-component in-situ crosslinking agent as described in claim 4, characterized in that, The material derivative of the protein is a protein thiolated derivative.

6. The two-component in-situ crosslinking agent as described in claim 1, characterized in that, In the first precursor solution, the thiolated polyethylene glycol is 4-arm-PEGSH with a molecular weight of 20 kDa and a concentration of 8%; the protein concentration is 1%; in the second precursor solution, the Cu ion concentration is 0.0615%~0.246%.

7. The two-component in-situ crosslinking agent as described in claim 6, characterized in that, The protein is either decellularized matrix SIS or gelatin with a freeze strength of 300g bloom.

8. A biodegradable two-component hydrogel, characterized in that, The hydrogel is prepared by cross-linking the first and second precursor solutions of the two-component in-situ cross-linking agent described in claim 1, that is, it is formed by metal coordination cross-linking of thiolized polyethylene glycol, metal ions and proteins.

9. The use of the biodegradable two-component hydrogel of claim 8 in the preparation of formulations for preventing tissue adhesions.

10. The use of the biodegradable two-component hydrogel of claim 8 in the preparation of formulations for tissue repair.

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