Glutamine-induced injectable hydrogel and preparation method thereof

Abstract

The invention belongs to the technical field of hydrogel preparation, and discloses glutamine-induced injectable hydrogel and a preparation method thereof, the glutamine-induced injectable hydrogel comprises sodium alginate hydrogel of a hydroxyethyl methylacrylate matrix, a cross-linking agent and an initiator; the hydroxyethyl methylacrylate matrix sodium alginate hydrogel is prepared from the following raw materials: hydroxyethyl methylacrylate, sodium alginate, L-glutamine and anhydrous magnesium chloride. According to the invention, the poly (hydroxyethyl methacrylate) which is anti-swelling, stable and good in biocompatibility is used as a hydrogel matrix, L-glutamine is utilized to induce rapid gelation, remove active oxygen and activate cells, and meanwhile, a physical and ionic cross-linked network formed by sodium alginate and magnesium ions is introduced; the stable vagina supporting material which is injectable, resistant to swelling and tough in mechanical property is prepared.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogel preparation, and more particularly to a glutamine-induced injectable hydrogel and a preparation method thereof. Background Art

[0002] Müllerian agenesis (Mazer-Rokitanskz-Küster-Hauser, MRKH syndrome) refers to the absence or occlusion of the vagina caused by the failure or incomplete development of both mesonephric ducts (Müllerian ducts) during embryogenesis. This syndrome is often accompanied by uterine and upper genitourinary tract malformations. Patients with MRKH syndrome often face severe challenges such as abdominal pain, amenorrhea, and sexual dysfunction, making it a complex gynecological condition.

[0003] Currently, the primary treatment strategy for MRKH syndrome is vaginoplasty, which aims to reconstruct the vaginal structure. However, postoperative complications such as poor wound healing, scar formation, and vaginal stenosis are common, severely impacting treatment efficacy. Therefore, vaginal dilation is often required to achieve a normal vaginal structure after vaginoplasty. Clinically, vaginal dilation is commonly performed using vaginal molds. Patients must wear rigid silicone vaginal molds for extended periods of time. Larger vaginal wounds can also slow wound healing, with some patients requiring the molds for six months to a year, causing pain and inconvenience. Furthermore, prolonged mold compression can easily lead to localized vaginal tissue ischemia, hypoxia, cell deformation, and death, ultimately causing tissue necrosis. Furthermore, silicone molds cannot be customized in clinical practice, making it difficult to perfectly fit the vaginal structure of individual patients. Hydrogels with high water content, excellent biocompatibility, and adjustable mechanical properties can effectively address the challenges faced by vaginal molds.

[0004] The paper "Dual-crosslinked bioactive hzdrogel scaffold for accelerated repair of genital tract defects" developed a 3D-printed hydrogel scaffold composed of chemical crosslinking of methacrylated gelatin and ionic crosslinking of carrageenan with magnesium ions. This scaffold promotes cell proliferation, migration, and angiogenesis through the sustained release of magnesium ions. Implantation in a rat model showed that it repaired penetrating vaginal wounds to near-normal levels within a week. However, this hydrogel still faces challenges such as complex preparation (photoinitiated reaction followed by immersion in magnesium chloride solution) and low mechanical strength (tensile strength < 60 kPa, compressive strength < 50 N), making it difficult to support the vaginal structure. Therefore, the development of an injectable, highly flexible, and mechanically stable hydrogel support mold is particularly important for treating vaginal wounds after MRKH surgery. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention proposes a glutamine-induced injectable hydrogel and its preparation method, which uses poly(hydroxyethyl methacrylate) which is anti-swelling, stable and biocompatible as the hydrogel matrix, utilizes the rapid gelation and toughening effect induced by L-glutamine, and introduces sodium alginate and magnesium ions (SA / Mg 2+ ) to form a physical cross-linked network, which can be used to prepare an injectable, anti-swelling and mechanically strong stable vaginal support material.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] In one aspect, the present invention provides a glutamine-induced injectable hydrogel, comprising a sodium alginate hydrogel with a hydroxyethyl methacrylate matrix, a crosslinking agent, and an initiator. The glutamine-induced injectable hydrogel is named PHSG. y -Mg 2+ Hydrogel, "H" stands for hydroxyethyl methacrylate (HEMA), "S" stands for sodium alginate, "G" stands for L-glutamine, and "y" stands for L-glutamine in PHSG m -Mg 2+ Mass percentage in hydrogel, 1.5% ≥ y ≥ 0.1%,

[0008] The raw materials for preparing the sodium alginate hydrogel with a hydroxyethyl methacrylate matrix include hydroxyethyl methacrylate, L-glutamine, sodium alginate and anhydrous magnesium chloride.

[0009] Preferably, the mass percentage concentration of the precursor solution of the sodium alginate hydrogel based on the hydroxyethyl methacrylate is M, M=(15.6+z) / (35.6+z)*100%, 0.1≤z≤0.4; the mass ratio of the hydroxyethyl methacrylate, L-glutamine, sodium alginate and anhydrous magnesium chloride is 15:0.1~0.4:0.1:0.5.

[0010] Preferably, the initiator is potassium persulfate, and the initiator is in PHSG m -Mg 2+ The mass percentage in the hydrogel is 0.10%~0.15%.

[0011] Preferably, the cross-linking agent is N,N,N',N'-tetramethylethylenediamine, and the volume-to-mass ratio of the cross-linking agent to the initiator is 2-3 μL:4 mg.

[0012] On the one hand, the present invention also provides a method for preparing a glutamine-induced injectable hydrogel, comprising dissolving hydroxyethyl methacrylate, L-glutamine, sodium alginate and anhydrous magnesium chloride in ultrapure water to obtain a composite solution, stirring it evenly and then deoxidizing it, and adding a crosslinking agent and an initiator to prepare PHSG.m -Mg 2+ hydrogel.

[0013] Preferably, the mass ratio of the hydroxyethyl methacrylate, L-glutamine, sodium alginate and anhydrous magnesium chloride is 15:0.1-0.4:0.1:0.5, and the mass concentration of L-glutamine in the composite solution is 0.28%-1.15%.

[0014] Preferably, the initiator is potassium persulfate, and the initiator is in PHSG m -Mg 2+ The mass percentage in the hydrogel is 0.10%~0.15%.

[0015] Preferably, the cross-linking agent is N,N,N',N'-tetramethylethylenediamine, and the volume-to-mass ratio of the cross-linking agent to the initiator is 2-3 μL:4 mg.

[0016] In one aspect, the present invention further provides use of the above-mentioned injectable hydrogel or the injectable hydrogel prepared by the above-mentioned preparation method as a support mold material for preparing a vaginal support mold.

[0017] Preparation of PHSG by the present invention y -Mg 2+ The mechanism of hydrogel is that single amino acid small molecules with -NH2 and -COOH (such as L-serine, L-glutamine, etc.) can exist in the form of zwitterions in the reaction system of hydrogel free radical polymerization, acting as a catalyst to promote the rapid generation of free radicals and induce rapid gelation of the hydrogel precursor solution. L-glutamine can accelerate the generation rate of free radicals, and at the same time build multiple hydrogen bond interactions with the hydrogel matrix, thereby achieving rapid cross-linking and curing of the hydrogel and enhancing the mechanical properties. At the same time, L-glutamine can act as an endogenous therapeutic factor to activate basal cells, fibroblasts and vascular endothelial cells in the wound, accelerate the formation of blood vessels, extracellular matrix and epithelium in the tissue, and thus promote vaginal wall repair without the need for additional drugs. In addition, sodium alginate and Mg 2+ The formed coordination bonds introduce a physical complexation cross-linking network, which can further improve the mechanical properties of the hydrogel.

[0018] A noteworthy caveat: Natural L-glutamine (Gln), the amide of glutamic acid (Glu), is a conditionally essential amino acid with numerous biological benefits. Through various metabolic pathways, Gln can directly influence energy metabolism, nucleotide and protein synthesis, as well as cellular redox status and signaling. Its intermediate metabolite, glutathione (GSH), acts as an antioxidant by binding to intracellular free radicals, combating oxidative stress. Studies have shown that Gln can activate signaling pathways such as mTOR, ERK, Wnt / β-Catenin, and PI3K-AKT, while simultaneously downregulating TGF-β and HCK signaling pathways. This can regulate reactive oxygen species (ROS) balance, promote cell proliferation and survival, and inhibit programmed cell death, including autophagy, apoptosis, and ferroptosis. These studies suggest that introducing Glu, Gln, or the downstream metabolite GSH into vaginal hydrogels may help regulate oxidative stress in damaged tissues, activate basal cells, fibroblasts, and endothelial cells within the wound, accelerate the formation of blood vessels, extracellular matrix (ECM), and epithelium within the tissue, and promote vaginal wall repair. However, in the redox reaction system of chemically synthesized hydrogels, GSH in its reduced state is highly unstable and easily degraded. In contrast, Gln is not easily degraded in aqueous solutions and can remain stable for extended periods. Furthermore, compared to using GSH directly, selecting Gln to construct a hydrogel system not only increases the number of downstream metabolites but also supports cell survival and growth by influencing cell signaling. Therefore, the present invention selects Gln for constructing a hydrogel system primarily composed of water.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. L-glutamine can promote the rapid gelation and toughening of hydrogels, while SA / Mg 2+ The introduction of PHSG forms a physical / chemical interpenetrating network. Compared with traditional molds, PHSG 0.2 -Mg 2+ Hydrogel can not only be injected to obtain a customized vaginal mold, but also has the mechanical characteristics of low modulus, high strength and high toughness, which can not only greatly reduce the patient's discomfort, but also provide sufficient support to obtain a normal vaginal structure.

[0021] 2. PHSG 0.2 -Mg 2+ The hydrogel exhibited excellent anti-swelling properties, mechanical stability, and biocompatibility, significantly promoting vaginal epithelial cell proliferation and enabling rapid and complete healing of vaginal wounds in rats without the need for additional therapeutic agents. Therefore, this injectable, anti-swelling, mechanically strong, and stable vaginal support mold offers a new approach for treating vaginal wounds following vaginoplasty in MRKH syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 PHSG 0.2 -Mg 2+ Molecular and network structure design of hydrogels;

[0023] Figure 2 Characterize the effect of each component on PHSG for rheological tests x -Mg 2+ Effect of hydrogel gelation time ((a) is pH, PHS-Mg 2+ , PHSG 0.2 and PHSG 0.2 -Mg 2+ The gelation time of hydrogel at 37 °C, (b) is PHSG 0.2 -Mg 2+ Time scanning curve of hydrogel at 37℃, (c) is PHSG x -Mg 2+ The gelation time of the hydrogel at 37 °C, (d) is PHSG 0.2 -Mg 2+ gelation process of hydrogel);

[0024] Figure 3 PH, PHS-Mg 2+ , PHSG 0.2 and PHSG 0.2 -Mg 2+ Infrared spectra of hydrogels;

[0025] Figure 4 Rheological frequency sweep test to characterize the network structure of different hydrogels ((a) PH, PHS-Mg 2+ , PHSG 0.2 and PHSG 0.2 -Mg 2+ Frequency scanning curve of hydrogel, (b) is PHSG x -Mg 2+ Frequency sweep curve of hydrogel);

[0026] Figure 5 PH, PHS-Mg 2+ , PHSG 0.2 and PHSG 0.2 -Mg 2+ Mechanical properties test results of hydrogels ((a), (c), and (e) are the tensile strain curves, tensile stress, and corresponding mechanical parameters, respectively; (b), (d), and (f) are the compressive stress-strain curves, compressive stress, and corresponding mechanical parameters, respectively);

[0027] Figure 6 PHSGx -Mg 2+ Mechanical properties test results of hydrogels ((a), (c), and (e) are the tensile strain curves, tensile stress, and corresponding mechanical parameters, respectively; (b), (d), and (f) are the compressive stress-strain curves, compressive stress, and corresponding mechanical parameters, respectively);

[0028] Figure 7 For silicone molds and PHSG 0.2 -Mg 2+ Mechanical properties test results of hydrogels ((a), (c), and (e) are the tensile strain curves, tensile stress, and corresponding mechanical parameters, respectively; (b), (d), and (f) are the compressive stress-strain curves, compressive stress, and corresponding mechanical parameters, respectively);

[0029] Figure 8 PHSG 0.2 -Mg 2+ Results of continuous loading-unloading compression tests on the hydrogel ((a) is the 1000-cycle compression curve at a maximum strain of 20%, and (b) is the 100-cycle compression curve at a maximum strain of 50%).

[0030] Figure 9 PHSG 0.2 -Mg 2+ Swelling ratio curve of hydrogel;

[0031] Figure 10 PHSG 0.2 -Mg 2+ Degradation behavior of the hydrogel ((a) is a physical image of the accelerated degradation process in vitro, (b) is the accelerated degradation behavior in vitro);

[0032] Figure 11 As control group, PHSG 0.2 Group and PHSG 0.2 -Mg 2+ The corresponding cell viability of the hydrogel group measured in the CCK-8 assay after co-culture with human vaginal epithelial cells for 1, 2, and 3 days, respectively (ns indicates no significant difference, **p < 0.01, ****p < 0.0001);

[0033] Figure 12 PHSG 0.2 -Mg 2+ The therapeutic effect of the hydrogel on the rat vaginal wound model ((a) is a representative image of rat vaginal wound healing, (b) is the rat vaginal wound area, and (c) is the rat vaginal wound closure rate);

[0034] Figure 13 PHSG 0.2 -Mg 2+Schematic diagram of the therapeutic mechanism of hydrogel for treating vaginal wounds. DETAILED DESCRIPTION

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0036] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0037] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0038] Example

[0039] 1 Experimental content

[0040] 1.1 Experimental Materials and Reagents

[0041] The experimental materials and reagents are shown in Table 1.

[0042] Table 1 Experimental reagents 1.2

[0044] 1.3 Preparation of hydrogel

[0045] Hydroxyethyl methacrylate (HEMA), L-glutamine, sodium alginate (SA) and anhydrous magnesium chloride were dissolved in ultrapure water at room temperature, stirred evenly and then deoxidized in a deoxidizer. Potassium persulfate (KPS) and N,N,N',N'-tetramethylethylenediamine (TEMED) were added to prepare PHSG. x -Mg 2+ Hydrogel. Among them, "H" stands for HEMA, "S" stands for SA, "G" stands for L-glutamine, x ” represents the content of L-glutamine.

[0046] For comparison, a hydrogel containing only hydroxyethyl methacrylate was prepared under the same conditions and named PH hydrogel. 0.2 Hydrogel and PHS-Mg 2+ The hydrogels were prepared by adding sodium alginate, L-glutamine, sodium alginate, and anhydrous magnesium chloride to the pH hydrogel. The formulas of the hydrogels are shown in Table 2.

[0047] Table 2 pH, PHS-Mg 2+ , PHSG 0.2 and PHSG x -Mg 2+Composition of hydrogel

[0048]

[0049] Experimental Example 1 Design and structural characterization of hydrogel

[0050] In the embodiment, L-glutamine and SA / Mg 2+ A PHEMA-based hydrogel (PHSG) with good biocompatibility, anti-swelling and long-term stability prepared by physical cross-linking network 0.2 -Mg 2+ Molecular and network structure design of hydrogels Figure 1 The hydrogel prepared in the example was subjected to rheological test and Fourier transform infrared spectroscopy test.

[0051] (1) Time sweep test: The gelation time of the hydrogel precursor solution (liquid before the hydrogel) prepared in the examples was measured using an Anton Paar MCR 302 rheometer (Anton Paar GmbH). The test frequency was constant at 1 Hz, the strain was fixed at 1%, and the test temperature was 37°C. The time corresponding to the intersection of the storage modulus (G′) and the loss modulus (G′′) was defined as the gelation time.

[0052] (2) Frequency sweep test: An Anton Paar MCR 302 rheometer was used to perform a frequency sweep on a hydrogel sample with a diameter of 25 mm and a thickness of 2 mm. The test frequency was 0.1–100 Hz, and the fixed strain was 0.01%.

[0053] (3) Fourier transform infrared spectroscopy: Thermo Scientific Nicolet iS50 Fourier transform infrared spectrometer (Thermo Fisher Scientific) was used to measure the -1 The chemical structure of the hydrogel was characterized in total reflection mode within a wide range.

[0054] The rheological test was used to characterize the effect of each component on PHSG x -Mg 2+ The effect of hydrogel gelation time, the results are as follows Figure 2 Compared with PH hydrogel, after the introduction of L-glutamine and sodium alginate, PHSG 0.2 The gelation time of the hydrogel at 37 °C was significantly shortened from 32.8 min to 16.3 min (see Figure 2 (a) in the figure shows the rapid gelation of L-glutamine. 2+ Under the synergistic effect of the physical cross-linking network, PHSG 0.2 -Mg 2+The gelation time of the hydrogel at 37 °C was 20.2 min (see Figure 2 (b) in the figure) not only ensures that the hydrogel has good injectability when treating vaginal wounds, but also has good operability. In addition, PHSG x -Mg 2+ The gelation time of the hydrogel was also affected by the L-glutamine content. As the amount of L-glutamine added increased from 0.1 g to 0.4 g, the gelation time of the hydrogel (37 °C) gradually decreased from 21 min to 18.3 min (see Figure 2 (c) in the figure). Figure 2 (d) shows the PHSG 0.2 -Mg 2+ Gelation process of hydrogel.

[0055] Chemical structure analysis of hydrogels Figure 3 As shown, with the addition of L-glutamine and SA / Mg 2+ With the introduction of physical cross-linking network, the peak of -OH is increased from 3336 cm -1 Gradually shifted to 3326 cm -1 , and the peak of -C=O from 1644 cm -1 Gradually shifted to 1638 cm -1 , which indicates that -NH2 and -COOH in L-glutamine and -OH in sodium alginate are able to form hydrogen bond interactions with the PHEMA hydrogel network.

[0056] Rheological frequency sweep tests characterized the network structure of the hydrogels. Figure 4 As shown, SA / Mg 2+ The physical cross-linking network brings a denser network structure, PHS-Mg 2+ The storage modulus (G') of the hydrogel was the highest. However, with the introduction of L-glutamine and sodium alginate, PHSG 0.2 The G' of the hydrogel is significantly lower than that of the pH hydrogel, which is because the sodium alginate introduces inert long chains, diluting the cross-linking density of the hydrogel network. In addition, as the concentration of L-glutamine increases, the pHSG x -Mg 2+ The G' of the hydrogel gradually increased, which indicates that in the range of 0.1~0.4 g, the increase of L-glutamine can introduce more hydrogen bonding interactions and form a stronger and denser hydrogel network (see Figure 4 (b) in the figure).

[0057] In summary, L-glutamine can build multiple hydrogen bond interactions with the hydrogel matrix to achieve rapid cross-linking and curing of the hydrogel and enhance its mechanical properties. 2+The formed coordination bonds introduce a physical complexation cross-linking network, which can further improve the mechanical properties of the hydrogel.

[0058] Test Example 2 Mechanical properties test of hydrogel

[0059] Ideal hydrogel mold materials must be stable and tough to form an effective scaffold, promoting vaginal wound repair while also supporting a healthy vaginal structure. Therefore, in this study, the mechanical properties of the hydrogels were evaluated through tensile and compression testing to determine suitable hydrogel mold materials. Specifically, an Instron 3367 universal testing machine (Instron Instruments) was used to systematically test the hydrogel mechanical properties. In the tensile test, dumbbell-shaped hydrogel samples (20 mm long, 4 mm wide, 1 mm thick) were stretched at a rate of 100 mm / min until fracture. In the compression test, cylindrical hydrogel samples (20 mm diameter, 20 mm height) were compressed at a rate of 5 mm / min to a maximum strain of 80%. Cyclic compression tests were then performed at a rate of 200 mm / min to evaluate the hydrogel's fatigue resistance, with maximum strains of 20% and 50%, respectively.

[0060] like Figure 5 As shown, with the SA / Mg 2+ The introduction of physical cross-linking network, PHS-Mg 2+ The tensile strength of the hydrogel increased from 0.38 MPa to 0.46 MPa, the compressive strength increased from 1.6 MPa to 1.9 MPa, and the toughness increased from 0.94 MJ / m 3 Increased to 1.05MJ / m 3 , which indicates that SA / Mg 2+ The physical / chemical interpenetrating cross-linking network brought by the physical cross-linking network significantly improves the strength and toughness of the hydrogel. However, with the introduction of sodium alginate and L-glutamine, PHSG 0.2 The mechanical properties of the hydrogel are slightly lower than those of the pH hydrogel, which is due to the introduction of inert long chains by sodium alginate, which dilutes the network cross-linking density. It is worth noting that the multiple hydrogen bonds introduced by L-glutamine and the SA / Mg 2+ Under the synergistic effect of the physical cross-linking network, PHSG 0.2 -Mg 2+ Hydrogels have a tight and strong three-dimensional cross-linked network, thus exhibiting optimal mechanical properties.

[0061] Effect of L-glutamine content on the mechanical properties of hydrogels Figure 6As shown. When the content of L-glutamine increases from 0.1g to 0.2g, the mechanical properties of the hydrogel are significantly improved due to the multiple hydrogen bonds introduced by L-glutamine. However, if the concentration of L-glutamine continues to increase (> 0.2g), the cross-linking density will be too high, resulting in a decrease in mechanical strength and toughness (see Figure 6 (a)-(c) in the figure). In the compression behavior, as the L-glutamine content increases, the compression modulus, compression strength and toughness of the hydrogel increase monotonically (see Figure 6 (d)-(f) in the above). In summary, it is necessary to select a PHSG with a suitable gelation time and both strength and toughness. 0.2 -Mg 2+ hydrogel for subsequent studies.

[0062] Multiple hydrogen bonds introduced in L-glutamine and SA / Mg 2+ Under the synergistic effect of the physical cross-linking network, PHSG 0.2 -Mg 2+ The hydrogel exhibited suitable mechanical properties, combining strength and toughness, an advantage that was verified by comparison with existing silicone molds used for the treatment of MRKH syndrome. 0.2 -Mg 2+ Although the strength and toughness of the hydrogel are weaker than those of the commercially available silicone molds, its tensile and compression moduli are also much lower than those of the silicone molds (see Figure 7 ), which indicates that PHSG 0.2 -Mg 2+ Hydrogel is much softer and more elastic than silicone molds, which greatly reduces the discomfort of patients wearing support molds, which also makes PHSG 0.2 -Mg 2+ When treating vaginal wounds, hydrogel not only provides support, but also adapts well to the vaginal environment that is constantly under stress.

[0063] The vaginal mold that is subjected to external forces for a long time should have good fatigue resistance, so further research on PHSG 0.2 -Mg 2+ The hydrogel was subjected to continuous loading-unloading compression tests. Under the condition of maximum strain of 20%, even after 1000 cycles of compression, PHSG 0.2 -Mg 2+ The compressive strength of the hydrogel remained basically stable, decreasing by only 0.009 MPa (see Figure 8 (a) in Figure 3) is 88.4% of the initial compressive strength. Correspondingly, under the condition that the maximum strain reaches 50%, after 100 cycles of compression, the PHSG 0.2 -Mg 2+The decrease in the compressive strength of the hydrogel was only 0.03 MPa (see Figure 8 (b) in Figure 3) still maintains 91% of the initial compressive strength. This shows that under cyclic compression at low strain (20%) and high strain (50%), PHSG 0.2 -Mg 2+ The hydrogels showed good fatigue resistance and mechanical stability, which indicated that PHSG 0.2 -Mg 2+ When used as a vaginal support mold, the hydrogel can show good adaptability, allowing it to maintain structural integrity under the action of external forces, avoiding fragmentation caused by stress concentration, and showing the potential for long-term use as a vaginal support mold.

[0064] Test Example 3 PHSG 0.2 -Mg 2+ Hydrogel swelling performance test

[0065] For non-degradable hydrogel materials used in aqueous environments in vivo, it is crucial to have excellent anti-swelling properties. Therefore, this study investigated the PHSG 0.2 -Mg 2+ Swelling behavior of hydrogels.

[0066] The PHSG with an initial weight of W0 0.2 -Mg 2+ The hydrogel samples (25 mm in diameter and 2 mm in thickness) were immersed in PBS buffer at 37 °C. Samples were taken and weighed on days 1, 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, and 30, and the weight was recorded as W. t , the calculation formula of swelling ratio SR is as follows:

[0067] .

[0068] The results are as follows Figure 9 As shown, PHSG 0.2 -Mg 2+ The hydrogel rapidly absorbed water to 117.73% of its initial mass on the first day of swelling, and then the swelling ratio gradually decreased and basically reached swelling equilibrium on the 7th day of swelling (swelling ratio of 102.04%). After 30 days of swelling, the swelling ratio of the hydrogel was still maintained at 101.96%, which indicates that PHSG 0.2 -Mg 2+ The hydrogel will not absorb excessive water and swell in an environment containing excess aqueous solution. 0.2 -Mg 2+ The hydrogel's rapid water absorption on the first day and then stable swelling behavior may be caused by the change in ion concentration inside the hydrogel and in the PBS buffer solution. Figure 9 As can be seen in the illustration, after immersion in PBS buffer solution at 37 °C for 30 days, PHSG 0.2 -Mg 2+ The macroscopic scale of the hydrogel also has almost no change. The excellent anti-swelling property makes PHSG 0.2 -Mg 2+ Even under the osmotic pressure of body fluids, the hydrogel can avoid morphological changes caused by excessive water absorption. This property enables it to be placed in the vagina for a long time and used as a vaginal support mold.

[0069] Test Example 4 PHSG 0.2 -Mg 2+ Degradation performance test of hydrogel

[0070] A suitable vaginal support mold should not only resist swelling but also have good stability. 0.2 -Mg 2+ The stability of hydrogel as vaginal support mold was investigated by in vitro accelerated degradation experiment. 0.2 -Mg 2+ Degradation properties of hydrogels.

[0071] A dumbbell-shaped (length: 20 mm, width: 4 mm, thickness: 1 mm) PHSG 0.2 -Mg 2+ The hydrogel sample was placed in a 100°C oven and vacuum dried to a constant weight, which was recorded as the initial weight W0. Subsequently, the dried hydrogel sample was immersed in a PBS buffer solution (37°C) and stirred to accelerate the degradation process. Samples were taken at 0, 1, 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, and 30 days, and placed in a 100°C oven and vacuum dried to a constant weight, and the weight after degradation was obtained as W0. t , the calculation formula of the quality retention rate Q is as follows:

[0072] .

[0073] The results showed that PHSG 0.2 -Mg 2+ The hydrogel maintains its morphology well (see Figure 10 (a) in the figure shows good stability. 0.2 -Mg 2+ The mass retention rate of the hydrogel decreased slowly, and on the 30th day of degradation, its mass retention rate still remained at 93.5% (see Figure 10 (b) in the figure shows that the degradation is not obvious, which shows good stability. The excellent anti-swelling performance and good stability of PHSG 0.2-Mg 2+ The hydrogel provides a reliable basis for long-term placement in the vagina as a supporting mold material.

[0074] Test Example 5 Hydrogel Biocompatibility Test

[0075] To study PHSG 0.2 -Mg 2+ The feasibility of the hydrogel as a support mold material for treating vaginal wounds was investigated, and its biocompatibility was evaluated by culturing human vaginal epithelial cells in vitro and performing CCK-8 assay.

[0076] PHSG in a volume of 1 mL 0.2 and PHSG 0.2 -Mg 2+ The hydrogel was immersed in 30 mL of RPMI 1640 culture medium and cultured for 12 h to obtain the hydrogel extract. Subsequently, human vaginal epithelial cells were seeded into a tissue culture-treated 96-well plate at a cell density of 100 μL per well and cultured for 12 h. Then, 16 μL of PHSG was added. 0.2 and PHSG 0.2 -Mg 2+ The hydrogel extracts were added to wells containing human vaginal epithelial cells, while an equal volume of RPMI 1640 basal medium was added to the control group. After 1, 2, and 3 days of co-culture with the different extracts, the cell viability of the human vaginal epithelial cells in different groups was assessed using the Cell Counting Kit-8 (CCK-8).

[0077] like Figure 11 As shown in Figure 2, within 3 days of culture, the cell viability of each group of cells increased significantly with the extension of culture time. 0.2 The cell viability of the hydrogel group was significantly higher than that of the control group and was significantly higher than that of the PHSG group. 0.2 -Mg 2+ The cell viability of the hydrogel group cells remained the same, but on the second and third days of culture, the PHSG 0.2 and PHSG 0.2 -Mg 2+ The cell viability of the hydrogel group was significantly higher than that of the control group, and there was no significant difference between the two groups. 0.2 and PHSG 0.2 -Mg 2+ The hydrogels can promote the proliferation of human vaginal epithelial cells and maintain their vitality, showing excellent biocompatibility, making them show great potential in treating vaginal wounds as vaginal support materials.

[0078] Experimental Example 6 Establishment of Rat Vaginal Wound Model and PHSG 0.2 -Mg 2+Evaluation of hydrogel treatment effects

[0079] To evaluate PHSG 0.2 -Mg 2+ The actual therapeutic effect of hydrogel on vaginal wounds. A vaginal wound model was established in rats and treated with hydrogel.

[0080] All animal experimental procedures in this study were reviewed and approved by the Ethics Committee of West China Second Hospital, Sichuan University (Approval No. 20240192). Female Sprague-Dawlez (SD) rats (180-220 g, 6-8 weeks old) were obtained from Chengdu Dashuo. The animals were housed in a pathogen-free environment with free access to food and water. The experimental rats were randomly divided into three groups: control group (rats that underwent modeling but did not receive any treatment), PHSG group (rats that underwent modeling but did not receive any treatment), and PHSG group (rats that underwent modeling but did not receive any treatment). 0.2 Group (modeling and accepting PHSG 0.2 hydrogel-treated rats) and PHSG 0.2 -Mg 2+ Group (modeling and accepting PHSG 0.2 -Mg 2+ In the vaginal wound model control group, a skin punch with a diameter of 3 mm was used to perforate the distal end of the rat vagina to establish a standard vaginal wound model. 0.2 Group and PHSG 0.2 -Mg 2+ Group, after establishing vaginal wound model in rats, PHSG 0.2 and PHSG 0.2 -Mg 2+ The hydrogel precursor solution was injected into the vagina to treat the vaginal wound after modeling. Within 4 days after the modeling surgery, the condition of the vaginal wound of the rats was observed and photographed every day.

[0081] The vaginal wounds of rats were recorded during the treatment (see Figure 12 The statistical results are as follows. Figure 12 As shown in (b) and (c), compared with the control group, the PHSG 0.2 After 4 days of hydrogel treatment, the vaginal wound area of rats was reduced from 2.5 mm 2 Down to 1.7 mm 2 . And through PHSG 0.2 -Mg 2+ After 4 days of hydrogel treatment, the vaginal wounds of rats were completely healed, with a wound closure rate of 100%, significantly higher than that of PHSG. 0.2 The experimental results show that PHSG 0.2Hydrogel can serve as a good vaginal support mold, isolate wounds, and promote wound healing to a certain extent. This is because sodium alginate, as a natural biomacromolecule, has good biocompatibility and can achieve the purpose of antibacterial and hemostasis by absorbing wound secretions and preventing secondary damage; at the same time, L-glutamine, as a precursor of protein synthesis, can support cell growth and repair by providing necessary amino acids and enhancing mucosal repair ability, thereby promoting cell regeneration and accelerating vaginal wound healing. At the same time, Mg 2+ It can scavenge free radicals, inhibit inflammatory responses and thus reduce tissue damage and pain. 2+ It can also promote collagen synthesis and promote vaginal wound healing by synthesizing new collagen fibers and blood vessels. L-glutamine and Mg 2+ Making PHSG 0.2 -Mg 2+ Hydrogel can achieve good therapeutic effects without the need for additional drugs. 0.2 -Mg 2+ The hydrogel showed the best therapeutic effect using PHSG 0.2 -Mg 2+ The hydrogel can completely heal the vaginal wound of rats after 4 days of treatment, and is a vaginal support mold material with great application potential. 0.2 -Mg 2+ The therapeutic mechanism of hydrogel for treating vaginal wounds Figure 13 shown.

[0082] In summary, the present invention is based on the strategy of L-glutamine-induced rapid gelation and toughening of hydrogels to construct an injectable, anti-swelling and mechanically strong PHEMA-based hydrogel, which can achieve efficient and comfortable vaginal wound treatment after MRKH syndrome vaginoplasty.

[0083] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. After reading this specification, those skilled in the art may make non-creative modifications to this embodiment as needed. However, as long as such modifications are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A glutamine-induced injectable hydrogel, characterized in that Named PHSG y -Mg 2+ hydrogel, y represents L-glutamine in PHSG y -Mg 2+ The mass percentage in the hydrogel, 1.5% ≥ y ≥ 0.10%, includes sodium alginate hydrogel with hydroxyethyl methacrylate matrix, crosslinking agent and initiator; The raw materials for preparing the sodium alginate hydrogel with a hydroxyethyl methacrylate matrix include hydroxyethyl methacrylate, L-glutamine, sodium alginate and anhydrous magnesium chloride.

2. The glutamine-induced injectable hydrogel according to claim 1, characterized in that The mass percentage concentration of the precursor solution of the sodium alginate hydrogel with a hydroxyethyl methacrylate matrix is M, M=(15.6+z) / (35.6+z)*100%, 0.1≤z≤0.4; the mass ratio of the hydroxyethyl methacrylate, L-glutamine, sodium alginate and anhydrous magnesium chloride is 15:0.1~0.4:0.1:0.

5.

3. The glutamine-induced injectable hydrogel according to claim 2, characterized in that: The initiator is potassium persulfate, and the initiator is in PHSG x -Mg 2+ The mass percentage in the hydrogel is 0.10%~0.15%.

4. The glutamine-induced injectable hydrogel according to claim 3, characterized in that The cross-linking agent is N,N,N',N'-tetramethylethylenediamine, and the volume mass ratio of the cross-linking agent to the initiator is 2-3 μL:4 mg.

5. The method for preparing a glutamine-induced injectable hydrogel according to any one of claims 1 to 4, characterized in that: Hydroxyethyl methacrylate, L-glutamine, sodium alginate and anhydrous magnesium chloride are dissolved in ultrapure water to obtain a composite solution, which is then stirred evenly and deoxidized, and a crosslinking agent and an initiator are added to prepare PHSG. y -Mg 2+ hydrogel.

6. The preparation method according to claim 5, characterized in that The mass ratio of the hydroxyethyl methacrylate, L-glutamine, sodium alginate and anhydrous magnesium chloride is 15:0.1-0.4:0.1:0.5, and the mass concentration of L-glutamine in the composite solution is 0.28%-1.15%.

7. The preparation method according to claim 6, characterized in that The initiator is potassium persulfate, and the initiator is in PHSG y -Mg 2+ The mass percentage in the hydrogel is 0.10%~0.15%.

8. The preparation method according to claim 7, characterized in that The cross-linking agent is N,N,N',N'-tetramethylethylenediamine, and the volume mass ratio of the cross-linking agent to the initiator is 2-3 μL:4 mg.

9. Use of the glutamine-induced injectable hydrogel according to any one of claims 1 to 4 or the injectable hydrogel prepared by the preparation method according to any one of claims 5 to 8 as a support mold material for preparing a vaginal support mold.

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