A glutamine-induced injectable hydrogel and a method of preparing the same

Abstract

The application belongs to the technical field of hydrogel preparation, and discloses a glutamine-induced injectable hydrogel and a preparation method thereof, which comprises a sodium alginate hydrogel of a hydroxyethyl methacrylate matrix, a crosslinking agent and an initiator; and the preparation raw materials of the sodium alginate hydrogel of the hydroxyethyl methacrylate matrix comprise hydroxyethyl methacrylate, sodium alginate, L-glutamine and anhydrous magnesium chloride. The polyhydroxyethyl methacrylate which is anti-swelling, stable and has good biocompatibility is used as the hydrogel matrix, L-glutamine is used for inducing rapid gelation and removing active oxygen and activating cells, and a physical and ionic crosslinking network formed by sodium alginate and magnesium ions is introduced, so that a stable vaginal support material which is injectable, anti-swelling and mechanically strong and tough is prepared.

Description

TECHNICAL FIELD

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

[0002] Mullerian duct aplasia syndrome (Mazer-Rokitanskz-Küster-Hauser, MRKH syndrome) refers to the absence or aplasia of the vagina caused by the failure of the bilateral paramesonephric duct (Mullerian duct) to develop during the embryonic period, usually accompanied by malformations of the uterus and upper urinary tract. MRKH syndrome patients often face severe challenges such as abdominal pain, amenorrhea and sexual life disorders, and it is a complex disease in the field of gynecology.

[0003] At present, the treatment strategy for MRKH syndrome is mainly vaginoplasty, aiming to reconstruct the structure of the vagina, but postoperative wound healing is poor, scar formation and vaginal stenosis often occur, which seriously affects the treatment effect. Therefore, after vaginoplasty, the vagina usually needs to be expanded to obtain a normal vaginal structure. Clinically, a vaginal mold is usually used to achieve the effect of vaginal expansion, and the patient needs to wear a hard silicone vaginal mold for a long time, and when the vaginal wound is large, the vaginal wound healing also becomes slow, and some patients even need to wear the mold for 6 months to 1 year, causing pain and inconvenience. In addition, long-term compression of the mold can easily lead to local vaginal tissue ischemia, hypoxia, cell deformation and death, and further cause tissue necrosis. In addition to this, the silicone mold cannot be customized in clinical application, and it is difficult to perfectly fit the vaginal structure of different patients. A hydrogel with high water content, good biocompatibility and adjustable mechanics can well solve the difficulties faced by the vaginal mold.

[0004] In the literature "Dual-crosslinked bioactive hydrogel scaffold for accelerated repair of genital tract defect", a 3D printed hydrogel scaffold based on chemical crosslinking of methacrylated gelatin and ionic crosslinking of carrageenan and magnesium ions was developed, which promoted cell proliferation, migration and angiogenesis through the sustained release of magnesium ions. After implantation into a rat model, it can repair a penetrating vaginal wound to near normal level within a week. However, this hydrogel still faces problems such as complex preparation (soaking in magnesium chloride solution after photo-initiated reaction), low mechanical strength (tensile strength < 60 kPa, compression strength < 50 N) and difficulty in supporting the vaginal structure. Therefore, it is particularly important to develop an injectable, highly flexible and mechanically stable hydrogel support mold for the treatment of vaginal wounds after vaginoplasty for MRKH syndrome. SUMMARY

[0005] To solve the above technical problems, the present application provides a glutamine-induced injectable hydrogel and a preparation method thereof, taking anti-swelling, stable and biocompatible polyhydroxyethyl methacrylate as the hydrogel matrix, using L-glutamine-induced rapid gelation and toughening effect, and introducing a physical crosslinking network formed by sodium alginate and magnesium ions (SA / Mg 2+ ) to prepare a stable vaginal support material which is injectable, anti-swelling and mechanically tough.

[0006] To achieve the above-mentioned purposes, the present application provides the following technical solutions:

[0007] In one aspect, the present application provides a glutamine-induced injectable hydrogel, which comprises a sodium alginate hydrogel of a hydroxyethyl methacrylate matrix, a crosslinking agent and an initiator, and is named as PHSG y -Mg 2+ hydrogel, "H" represents hydroxyethyl methacrylate (HEMA), "S" represents sodium alginate, "G" represents L-glutamine, and y represents the mass percentage of L-glutamine in the PHSG m -Mg 2+ hydrogel, 1.5%≥y≥0.1%,

[0008] The preparation raw materials of the sodium alginate hydrogel of the 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 of the hydroxyethyl methacrylate matrix is M, M=(15.6+z) / (35.6+z)*100%, 0.1≤z≤0.4; and 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 mass percentage of the initiator in the PHSG m -Mg 2+ hydrogel is 0.10%~0.15%.

[0011] Preferably, the crosslinking agent is N,N,N',N'-tetramethyl ethylenediamine, and the volume-mass ratio of the crosslinking agent and the initiator is 2~3μL:4mg.

[0012] In one aspect, the present application also provides a preparation method of a glutamine-induced injectable hydrogel, which comprises dissolving hydroxyethyl methacrylate, L-glutamine, sodium alginate and anhydrous magnesium chloride in ultrapure water to obtain a composite solution, deoxygenating after uniform stirring, and adding a crosslinking agent and an initiator to prepare PHSGm -Mg 2+ The 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 complex solution is 0.28%~1.15%.

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

[0015] Preferably, the crosslinking agent is N,N,N',N'-tetramethyl ethylenediamine, and the volume-mass ratio of the crosslinking agent and initiator is 2~3 μL:4 mg.

[0016] In one aspect, the application also provides the use of the injectable hydrogel or the injectable hydrogel prepared by the preparation method in the preparation of a vaginal support mold.

[0017] The PHSG y -Mg 2+ The mechanism of the hydrogel is that the 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 radical polymerization of the hydrogel, promote the rapid generation of radicals as catalysts, and induce the rapid gelation of the hydrogel precursor solution. L-glutamine can accelerate the generation rate of radicals, and at the same time, interact with the hydrogel matrix to construct multiple hydrogen bonds, thereby achieving the rapid crosslinking and solidification of the hydrogel and the enhancement of the mechanical properties. At the same time, L-glutamine can activate the basal cells, fibroblasts and vascular endothelial cells in the wound as an endogenous therapeutic factor, accelerate the formation of blood vessels, extracellular matrix and epithelium in the tissue, and thus promote the repair of the vaginal wall without the need of additional drugs. In addition, the coordination bond formed between sodium alginate and Mg 2+ The coordination bond formed between sodium alginate and Mg

[0018] It should also be noted herein that: natural L-glutamine (L-glutamine, Gln) is the amide of glutamate (Glutamate, Glu), a conditionally essential amino acid, and has multiple biological effects. Through different metabolic pathways, Gln can directly affect energy metabolism, nucleotide and protein synthesis, as well as the redox state and signal transduction of cells, among which the intermediate metabolite glutathione (GSH) can play an antioxidant role by combining with intracellular free radicals to resist oxidative stress. Studies have shown that Gln can activate mTOR, ERK, Wnt / β-Catenin, PI3K-AKT signaling pathways, while down-regulating TGF-β, HCK signaling pathways, regulating reactive oxygen species (ROS) balance, promoting cell proliferation and survival, and inhibiting programmed cell death such as autophagy, apoptosis and ferroptosis. The above studies suggest that the introduction of Glu, Gln or downstream metabolic molecules GSH into the vaginal hydrogel may help the damaged tissue to regulate oxidative stress, activate the basal cells, fibroblasts and vascular endothelial cells in the wound, accelerate the formation of blood vessels, extracellular matrix (ECM) and epithelium in the tissue, and promote the repair of the vaginal wall. However, in the redox reaction system of chemically synthesized hydrogel, GSH in the reduced state is highly unstable and easily degradable; while Gln is not easily degradable in aqueous solution and can exist stably for a long time. In addition, compared with the direct use of GSH, the selection of Gln to construct the hydrogel system not only increases the downstream metabolic molecules, but also supports cell survival and growth by affecting cell signal transduction. Therefore, the present application selects Gln for constructing the hydrogel system with water as the main component.

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

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

[0021] 2. The PHSG 0.2 -Mg 2+ hydrogel shows excellent anti-swelling, mechanical stability and biocompatibility, and can significantly promote the proliferation of vaginal epithelial cells, so that the rat vaginal wound can be quickly and completely healed without the need for additional therapeutic drugs. Therefore, this injectable, anti-swelling, mechanically strong and tough stable vaginal support mold provides a new idea for the treatment of vaginal wounds after MRKH syndrome vaginoplasty. BRIEF DESCRIPTION OF DRAWINGS

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

[0023] Figure 2 Rheological tests to characterize the effect of each component on PHSG x -Mg 2+ Effect of hydrogel gelation time (a) for PH, PHS-Mg 2+ , PHSG 0.2 and PHSG 0.2 -Mg 2+ Gelation time of hydrogels at 37 °C, (b) for PHSG 0.2 -Mg 2+ Time sweep curves of hydrogels at 37 °C, (c) for PHSG x -Mg 2+ Gelation time of hydrogels at 37 °C, (d) for PHSG 0.2 -Mg 2+ Gelation process of hydrogels);

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

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

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

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

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

[0029] Figure 8 PHSG 0.2 -Mg 2+ Continuous loading-unloading compression test results of hydrogels ((a) is 1000 cycles of compression curve at a maximum strain of 20%, (b) is 100 cycles of compression curve at a maximum strain of 50%);

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

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

[0032] Figure 11 Control group, PHSG 0.2 group and PHSG 0.2 -Mg 2+ Cell viability measured in CCK-8 test after hydrogel group and human vaginal epithelial cells were co-cultured 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+ Therapeutic effect of hydrogels on rat vaginal wound model ((a) is a representative image of rat vaginal wound healing, (b) is rat vaginal wound area, (c) is rat vaginal wound closure rate);

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

[0035] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0036] The materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified.

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

[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 method of hydrogel

[0045] Hydroxyethyl methacrylate (HEMA), L-glutamine, sodium alginate (SA) and anhydrous magnesium chloride were dissolved in ultrapure water at room temperature, stirred uniformly and then deoxygenated in a deoxygenator, and then potassium persulfate (KPS) and N, N, N', N'-tetramethyl ethylenediamine (TEMED) were added to prepare PHSG x -Mg 2+ hydrogel. Among them, "H" represents HEMA, "S" represents SA, "G" represents L-glutamine, and "Mg" represents anhydrous magnesium chloride. x

[0046] For comparison, a hydrogel containing only hydroxyethyl methacrylate was prepared under the same conditions and named PH hydrogel. While the PHSG 0.2 hydrogel and the PHS-Mg 2+ hydrogel were prepared by adding sodium alginate, L-glutamine and sodium alginate, anhydrous magnesium chloride into the PH hydrogel, respectively. The formulations 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 the hydrogel

[0048]

[0049] Design and structure characterization of the hydrogel

[0050] The effect of L-glutamine and SA / Mg 2+ physical crosslinking network on the gelation time of PHSG 0.2 -Mg 2+ The molecules and network structure design of the hydrogel are shown in Figure 1 The hydrogels prepared in the examples were respectively subjected to rheological test and Fourier transform infrared spectroscopy test.

[0051] (1) Time sweep test: Anton Paar MCR 302 rheometer (Anton Paar GmbH) was used to determine the gelation time of the hydrogel precursor solution (liquid before the hydrogel gels) prepared in the examples. The test frequency was constant at 1 Hz, the fixed strain was 1%, and the test temperature was 37 ℃. The time corresponding to the cross point of the storage modulus (G') and the loss modulus (G'') was defined as the gelation time.

[0052] (2) Frequency sweep test: Anton Paar MCR 302 rheometer was used to carry out frequency sweep on the 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 test: Thermo Scientific Nicolet iS50 Fourier transform infrared spectrometer (Thermo Fisher Scientific) was used to characterize the chemical structure of the hydrogel in the full reflection mode in the range of 4000-500 cm -1 .

[0054] The effect of each component on the gelation time of PHSG x -Mg 2+ was characterized by rheological test, and the results are shown in Figure 2 Compared with PH hydrogel, after the introduction of L-glutamine and sodium alginate, the gelation time of PHSG 0.2 hydrogel at 37 ℃ was significantly shortened from 32.8 min to 16.3 min (see (a) in Figure 2 ), which embodied the rapid gelation effect of L-glutamine. Under the synergistic effect of L-glutamine and SA / Mg 2+ physical crosslinking network, the gelation time of PHSG 0.2 -Mg 2+The gelation time of the hydrogel at 37 °C was 20.2 min (see...). Figure 2 (b) in the text not only ensures the hydrogel's good injectability in treating vaginal wounds but also its 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 (37 ℃) of the hydrogel gradually decreased from 21 min to 18.3 min (see...). Figure 2 (c) in the middle. Figure 2 (d) in the middle shows the PHSG 0.2 -Mg 2+ The gelation process of hydrogels.

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

[0056] Rheological frequency scanning tests characterized the network structure of the hydrogel, such as Figure 4 As shown, SA / Mg 2+ Physical cross-linking networks result in a denser network structure, PHS-Mg 2+ The hydrogel exhibits the highest storage modulus (G'). However, with the introduction of L-glutamine and sodium alginate, PHSG... 0.2 The G' of the hydrogel was significantly lower than that of the pH hydrogel. This is because sodium alginate introduces inert long chains, diluting the crosslinking density of the hydrogel network. Furthermore, with increasing L-glutamine concentration, the pHSG... x -Mg 2+ The G' of the hydrogel gradually increases, indicating that within the range of 0.1–0.4 g, an increase in L-glutamine can introduce more hydrogen bond interactions, forming a stronger and denser hydrogel network (see...). Figure 4 (b) in the middle.

[0057] In summary, L-glutamine can form multiple hydrogen bond interactions with the hydrogel matrix, achieving rapid cross-linking and curing of the hydrogel and enhanced mechanical properties. Furthermore, sodium alginate and Mg... 2+The resulting coordination bonds introduce a physical complex cross-linking network, which can further enhance the mechanical properties of the hydrogel.

[0058] Experimental Example 2: Mechanical Property Testing of Hydrogels

[0059] Ideal hydrogel mold materials need to be stable and tough to form an effective scaffold, promoting vaginal wound healing while constructing a normal vaginal structure. Therefore, this experiment evaluated the mechanical properties of the hydrogel through tensile and compression tests to determine a suitable hydrogel mold material. Specifically, an Instron 3367 universal testing machine (Instron Instruments Ltd.) was used to systematically test the mechanical properties of the hydrogel. In the tensile test, a dumbbell-shaped hydrogel sample (dimensions: length 20 mm, width 4 mm, thickness 1 mm) was stretched at a rate of 100 mm / min until fracture. In the compression test, a cylindrical hydrogel sample (dimensions: diameter 20 mm, height 20 mm) was compressed at a rate of 5 mm / min to 80% of its maximum strain. Cyclic compression tests were performed on the hydrogel sample at a rate of 200 mm / min to evaluate its fatigue resistance, with maximum strains of 20% and 50%, respectively.

[0060] like Figure 5 As shown, with SA / Mg 2+ The introduction of physical cross-linking networks, 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 also increased from 0.94 MJ / m. 3 Increased to 1.05 MJ / m 3 This indicates that SA / Mg 2+ Physical cross-linking networks, resulting in physical / chemical interpenetrating cross-linking networks, significantly improve the strength and toughness of hydrogels. 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. This is because sodium alginate introduces inert long chains, diluting the network crosslinking density. It is worth noting the multiple hydrogen bonds introduced by L-glutamine and the SA / Mg... 2+ Under the synergistic effect of the resulting physical cross-linking network, PHSG 0.2 -Mg 2+ Hydrogels possess a tight, robust three-dimensional cross-linked network, thus exhibiting optimal mechanical properties.

[0061] The effect of L-glutamine content on the mechanical properties of hydrogels, such as Figure 6As shown, when the L-glutamine content increased from 0.1 g to 0.2 g, the mechanical properties of the hydrogel were significantly improved due to the multiple hydrogen bonding introduced by L-glutamine. However, further increases in L-glutamine concentration (> 0.2 g) resulted in excessively high crosslinking density, leading to a decrease in mechanical strength and toughness (see...). Figure 6 (a)-(c)). In terms of compression behavior, with increasing L-glutamine content, the compressive modulus, compressive strength, and toughness of the hydrogel all increase monotonically (see [reference]). Figure 6 (d)-(f)). In summary, PHSG with suitable gelation time and both strength and toughness should be selected. 0.2 -Mg 2+ Further research will be conducted on the hydrogel.

[0062] Multiple hydrogen bonds introduced by L-glutamine and SA / Mg 2+ Under the synergistic effect of the resulting physical cross-linking network, PHSG 0.2 -Mg 2+ The hydrogel exhibits suitable mechanical properties, combining strength and toughness, an advantage that can be validated by comparison with existing silicone molds used for the treatment of MRKH syndrome. The results show that PHSG... 0.2 -Mg 2+ Although hydrogels are weaker and less resilient than commercially available silicone molds, their tensile and compressive moduli are also significantly lower (see...). Figure 7 This indicates that PHSG 0.2 -Mg 2+ Hydrogel is far softer and more elastic than silicone molds, greatly reducing the discomfort patients experience when wearing support molds. This also makes PHSG more effective. 0.2 -Mg 2+ Hydrogels not only provide support when treating vaginal wounds, but also adapt well to the constantly stressed vaginal environment.

[0063] Vaginal molds subjected to prolonged external forces should possess good fatigue resistance; therefore, further research on PHSG is necessary. 0.2 -Mg 2+ The hydrogel underwent continuous load-unload compression tests. Even after 1000 cycles of compression at a maximum strain of 20%, the PHSG... 0.2 -Mg 2+ The compressive strength of the hydrogel remained essentially stable, decreasing by only 0.009 MPa (see...). Figure 8 In (a) of the figure, the initial compressive strength is 88.4%. Correspondingly, under the condition that the maximum strain reaches 50%, after 100 cycles of compression, the PHSG... 0.2 -Mg 2+The decrease of compressive strength of the hydrogel is only 0.03 MPa (see Figure 8 (b) in the present application, which is still 91% of the initial compressive strength. This indicates that the PHSG 0.2 -Mg 2+ hydrogels all exhibit good fatigue resistance and mechanical stability, which indicates that the PHSG 0.2 -Mg 2+ hydrogels can exhibit good adaptability when used as a vaginal support mold, so that they can still maintain the integrity of the structure under external force, avoid the phenomenon of fragmentation caused by stress concentration, and exhibit the potential for long-term use as a vaginal support mold.

[0064] Test Example 3 PHSG 0.2 -Mg 2+ Swelling performance test of hydrogel

[0065] For non-degradable hydrogel materials used in the in vivo aqueous environment, it is essential to have excellent anti-swelling performance. Therefore, the present test example investigates the anti-swelling performance of the PHSG 0.2 -Mg 2+ Swelling behavior of hydrogel.

[0066] PHSG 0.2 -Mg 2+ The hydrogel sample (diameter 25 mm, thickness 2 mm) was immersed in a 37 °C PBS buffer. The sample was weighed at 1, 2, 3, 4, 5, 6, 7, 10, 15, 20, 25 and 30 days, and the weight was recorded as W t The formula for calculating the swelling ratio SR is as follows:

[0067] .

[0068] The results are shown in Figure 9 , the PHSG 0.2 -Mg 2+ hydrogel rapidly absorbed water to 117.73% of the initial mass on the first day of swelling, and then the swelling ratio gradually decreased, and basically reached swelling equilibrium (swelling ratio of 102.04%) on the 7th day of swelling. After 30 days of swelling, the swelling ratio of the hydrogel could still be maintained at 101.96%, which indicates that the PHSG 0.2 -Mg 2+ hydrogel will not excessively absorb water and swell in an environment containing excess aqueous solution. The swelling behavior of the PHSG 0.2 -Mg 2+ hydrogel, which rapidly absorbs water on the first day and then tends to be stable, may be caused by changes in ion concentration in the hydrogel and the PBS buffer solution. FromFigure 9 The illustrations also show that after soaking in PBS buffer solution at 37 °C for 30 days, PHSG 0.2 -Mg 2+ The macroscopic scale of the hydrogel also remained almost unchanged. Excellent anti-swelling properties make PHSG... 0.2 -Mg 2+ Even under the influence of osmotic pressure from bodily fluids, hydrogels can avoid morphological changes due to excessive water absorption. This characteristic allows them to be placed in the vagina for extended periods as a vaginal support mold.

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

[0070] A suitable vaginal support mold not only needs to be resistant to swelling but also possess good stability. To evaluate PHSG... 0.2 -Mg 2+ To investigate the stability of hydrogel as a vaginal support mold, this experiment designed an in vitro accelerated degradation experiment to examine the PHSG. 0.2 -Mg 2+ Degradation properties of hydrogels.

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

[0072] .

[0073] The results showed that PHSG degraded over a period of up to 30 days. 0.2 -Mg 2+ Hydrogels can maintain their shape very well (see) Figure 10 (a) shows good stability. Meanwhile, PHSG 0.2 -Mg 2+ The mass retention of the hydrogel decreased slowly, and on day 30 of degradation, its mass retention remained at 93.5% (see [link to hydrogel description]). Figure 10 In (b) of the sample, degradation was not significant, demonstrating good stability. Excellent anti-swelling properties and good stability are characteristic of PHSG. 0.2-Mg 2+ The use of hydrogel as a support mold material for long-term placement inside the vagina provides reliable evidence.

[0074] Experimental Example 5: Biocompatibility Test of Hydrogels

[0075] In order to study PHSG 0.2 -Mg 2+ The feasibility of hydrogel as a support mold material for treating vaginal wounds was assessed by culturing human vaginal epithelial cells in vitro and performing CCK-8 assays to evaluate its biocompatibility.

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

[0077] like Figure 11 As shown, within 3 days of culture, the cell viability of each group of cells increased significantly with the extension of culture time. On day 1 of culture, PHSG... 0.2 The cell viability of the hydrogel group was significantly higher than that of the control group and compared with PHSG. 0.2 -Mg 2+ The cell viability of the hydrogel group remained the same, but on days 2 and 3 of culture, the PHSG group showed higher viability. 0.2 and PHSG 0.2 -Mg 2+ Cell viability in the hydrogel group was significantly higher than that in the control group, with no significant difference between the two. This indicates that PHSG... 0.2 and PHSG 0.2 -Mg 2+ Hydrogels can promote the proliferation of human vaginal epithelial cells and maintain their vitality, exhibiting excellent biocompatibility, which makes them great potential as vaginal support materials for the treatment of vaginal wounds.

[0078] Experimental Example 6: Establishment of a rat vaginal wound model and PHSG 0.2 -Mg 2+Evaluation of hydrogel therapeutic effect

[0079] To evaluate the PHSG 0.2 -Mg 2+ The actual therapeutic effect of hydrogel on vaginal wound was evaluated by establishing a vaginal wound model in rats and treating with hydrogel.

[0080] All animal experimental procedures in this test example have been approved by the Ethics Committee of the Second West China Hospital of Sichuan University (Approval No. 20240192). Female Sprague-Dawlez (SD) rats (180-220 g, 6-8 weeks old) were obtained from Chengdu Dasuo. The animals were raised in a pathogen-free environment and had free access to food and water. The experimental rats were randomly divided into three groups: control group (modeling but not receiving any treatment), PHSG 0.2 group (modeling and receiving PHSG 0.2 hydrogel treatment) and PHSG 0.2 -Mg 2+ group (modeling and receiving PHSG 0.2 -Mg 2+ hydrogel treatment), with 5 rats in each group. In the control group of the vaginal wound model, a 3-mm-diameter skin puncher was used to punch the distal end of the rat's vagina to establish a standard vaginal wound model. In the PHSG 0.2 group and the PHSG 0.2 -Mg 2+ group, after establishing the vaginal wound model in rats, PHSG 0.2 and PHSG 0.2 -Mg 2+ hydrogel precursor solution was injected into the vagina to form a hydrogel for treatment of the vaginal wound. Within 4 days after modeling surgery, the vaginal wound of the rats was observed and photographed every day.

[0081] The vaginal wound of the rats during treatment was recorded (see (a) in Figure 12 ). The statistical results are shown in (b) and (c) in Figure 12 . Compared with the control group, the vaginal wound area of the rats treated with PHSG 0.2 hydrogel for 4 days was reduced from 2.5 mm 2 to 1.7 mm 2 . The vaginal wound of the rats treated with PHSG 0.2 -Mg 2+ hydrogel for 4 days had completely healed, with a wound closure rate of 100%, which was significantly higher than that of the PHSG 0.2 group. The experimental results show that PHSG 0.2The hydrogel can be used as a good vaginal support mold, isolate the wound, and promote wound healing to some extent, because sodium alginate as a natural biological macromolecule has good biocompatibility, can achieve the purposes of antibacterial and hemostasis by absorbing wound exudate 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 capacity, thereby promoting cell regeneration to accelerate the healing of vaginal wounds. At the same time, Mg 2+ can scavenge free radicals, can inhibit inflammatory response to reduce tissue damage and pain, Mg 2+ can also promote collagen synthesis, and promote vaginal wound healing by synthesizing new collagen fibers and blood vessels. L-glutamine and Mg 2+ make PHSG 0.2 -Mg 2+ The hydrogel can achieve good therapeutic effect without additional drugs. Therefore, PHSG 0.2 -Mg 2+ The hydrogel shows the best therapeutic effect, and PHSG 0.2 -Mg 2+ The hydrogel can completely heal the vaginal wound after 4 days of treatment, and is a vaginal support mold material with great application potential. PHSG 0.2 -Mg 2+ The hydrogel for treating vaginal wounds has a treatment mechanism as shown in Figure 13 .

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

[0083] The specific embodiments are only an explanation of the present application, and are not a limitation of the present application, and those skilled in the art can make modifications to the embodiments without creative contribution after reading the specification, as long as the modifications are within the scope of the claims of the present application.

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 of 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 of the hydroxyethyl methacrylate matrix include hydroxyethyl methacrylate, L-glutamine, sodium alginate and anhydrous magnesium chloride; the mass percentage concentration of the precursor solution of the sodium alginate hydrogel of the 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; The initiator is potassium persulfate, the initiator is in PHSG x -Mg 2+ The mass percentage in the hydrogel is 0.10%-0.15%; the crosslinking agent is N,N,N',N'-tetramethyl ethylenediamine, and the volume-mass ratio of the crosslinking agent and the initiator is 2-3 μL:4 mg; The injectable hydrogel is used as a vaginal support mold material for treating a vaginal wound.

2. A method of preparing a glutamine-induced injectable hydrogel according to claim 1, wherein, Hydroxyethyl methacrylate, L-glutamine, sodium alginate and anhydrous magnesium chloride are dissolved in ultrapure water to obtain a composite solution, which is stirred uniformly and deoxygenated, and then a crosslinking agent and an initiator are added to prepare PHSG y -Mg 2+ Hydrogel.

3. The production method according to claim 2, characterized by, The mass ratio of the hydroxyethyl methacrylate, L-glutamine, sodium alginate and anhydrous magnesium chloride is 15:0.2:0.1:0.5, and the mass percentage of L-glutamine in the composite solution is 0.56%.

4. The production method according to claim 2, characterized by, The initiator is potassium persulfate, and the mass ratio of the initiator to the composite solution is 0.04:35.

8.

5. The preparation method according to claim 2, characterized in that, The crosslinking agent is N,N,N',N'-tetramethyl ethylenediamine, and the volume-mass ratio of the crosslinking agent to the initiator is 2-3 μL:4 mg.

6. Use of the glutamine-induced injectable hydrogel of claim 1 or the injectable hydrogel prepared by the preparation method of any one of claims 2-5 as a support mold material for preparing a vaginal support mold.

Images