Hydrogen-releasing nanogel for ovarian transplantation protection, preparation method and application thereof

Abstract

The application discloses a hydrogen-releasing nanogel for protecting ovary transplantation, a preparation method and application thereof, and belongs to the technical field of biomedical materials and reproductive medicine, and comprises the following steps: step 1, preparing KBH4 nanoparticles; step 2, preparing a temperature-sensitive hydrogel solution; and step 3, mixing the KBH4 nanoparticles in step 1 and the temperature-sensitive hydrogel solution in step 2 at low temperature to obtain KBH4@Gel. The KBH4 nanoparticles prepared by adopting an oil-in-water microemulsion method and coated by liposomes are combined with the PLGA-PEG-PLGA hydrogel having a temperature-sensitive sol-gel transition characteristic, so that the release of hydrogen is changed from burst release to slow release, the residence time in the transplantation site is significantly prolonged, and a local, continuous and controllable hydrogen release process is ensured. Due to the structural design of the composite system, active oxygen can be effectively removed in an ischemic and slightly acidic environment, the ultrastructure of mitochondria is protected, energy metabolism is recovered, and angiogenesis is promoted, so that the survival rate of ovary grafts and the functional recovery level are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical materials and reproductive medical technology, and particularly relates to a hydrogen-releasing nanogel for protecting ovary transplantation, a preparation method and application thereof. BACKGROUND

[0002] In the prior art, ischemia-reperfusion injury generally exists after ovary tissue cryopreservation and recovery transplantation, which leads to excessive accumulation of reactive oxygen species (ROS), mitochondrial dysfunction and insufficient angiogenesis, thereby causing a large number of primordial follicle loss and reduced function of the graft. In order to alleviate the above problems, researchers attempt to apply antioxidant drugs (such as N-acetylcysteine, vitamin E), but their half-life is short and their effect is limited; angiogenic factors or exosomes can improve local blood supply, but they have poor stability and high cost. Hydrogen gas has attracted attention because it can specifically scavenge hydroxyl radicals, but it is difficult to achieve stable and sustained release in the local graft, which limits its clinical application.

[0003] Therefore, how to achieve controllable and sustained release of hydrogen gas at the ovary transplantation site and effectively restore mitochondrial energy metabolism is a bottleneck problem that needs to be broken through in the prior art. SUMMARY

[0004] The present application aims to solve the problems of ischemia-reperfusion injury in the process of ovary tissue cryopreservation and recovery transplantation, excessive generation of reactive oxygen species (ROS), impaired mitochondrial energy metabolism, insufficient angiogenesis, and the difficulty of conventional hydrogen intervention to achieve stable and sustained release in the local graft, which are difficult to effectively protect the ovary tissue, and provides a hydrogen-releasing nanogel for protecting ovary transplantation, a preparation method and application thereof.

[0005] In order to achieve the above technical purpose, the technical scheme provided by the present application is as follows:

[0006] A preparation method of a hydrogen-releasing nanogel for protecting ovary transplantation, comprising the following steps:

[0007] Step 1: preparing KBH4 nanoparticles;

[0008] Step 2: preparing a temperature-sensitive hydrogel solution;

[0009] Step 3: mixing the KBH4 nanoparticles of step 1 and the temperature-sensitive hydrogel solution of step 2 at low temperature to obtain KBH4@Gel.

[0010] Further, the mass concentration of the KBH4 nanoparticles in the temperature-sensitive hydrogel system is 80-320 µg / mL.

[0011] The mass concentration of the KBH4 nanoparticles in the temperature-sensitive hydrogel system is preferably 160 µg / mL, and the property reversibility is not affected at this concentration.

[0012] Further, in step 1, the KBH4 nanoparticles are prepared by using an oil-in-water microemulsion method.

[0013] Further, the specific preparation method of the oil-in-water microemulsion method comprises the following steps:

[0014] Step 1.1: Dissolve potassium oleate and oleylamine in a mixed solvent of n-hexane and anhydrous ethanol, add tetramethylammonium borohydride after ultrasonic treatment, stir at room temperature for a certain time, and then centrifuge and wash to obtain hydrophobic KBH4 nanoparticles;

[0015] Step 1.2: Disperse the hydrophobic KBH4 nanoparticles in n-hexane, drop the chloroform solution containing phosphatidylcholine dioleoylphosphatidyl ethanolamine and cholesterol, and redisperse and ultrasonic using PBS buffer to obtain hydrophilic KBH4 nanoparticles.

[0016] Further, in step 1.1, the reaction time is 40-80 min.

[0017] Further, the temperature-sensitive hydrogel uses a PLGA-PEG-PLGA triblock copolymer.

[0018] The application also provides a hydrogen-releasing nanogel for protecting ovarian transplantation.

[0019] The application also provides an application of the hydrogen-releasing nanogel for protecting ovarian transplantation in preparing a material for protecting ovarian transplantation.

[0020] The application has the following beneficial effects:

[0021] Traditional antioxidant drugs have a short half-life in the body and do not have a long-lasting effect. Although hydrogen intervention has the advantage of specifically removing free radicals, it is difficult to achieve stable and controllable release of the graft in a conventional manner, resulting in the accumulation of reactive oxygen species and mitochondrial damage after ovarian transplantation. The KBH4 nanoparticles prepared by the oil-in-water microemulsion method and coated with liposomes, combined with the PLGA-PEG-PLGA hydrogel having a temperature-sensitive sol-gel transition property, change the release of hydrogen from a burst to a slow release, significantly prolonging the residence time in the transplantation site and ensuring a local, continuous and controllable hydrogen release process. It is precisely due to the structural design of this composite system that it can effectively remove reactive oxygen species, protect the ultrastructure of mitochondria, restore energy metabolism and promote angiogenesis in an ischemic and slightly acidic environment, thereby improving the survival rate and functional recovery level of the ovarian graft. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1For the structural characterization of nanoparticles, Figure (A) is the SEM characterization of KBH4 NPs; (B) is the SEM characterization of pure hydrogel; (C) is the SEM characterization of KBH4@Gel; (D) is the DLS test chart; (E) is the FTIR spectrum detection chart; (F) is the XRD analysis chart;

[0023] Figure 2 For the hydrogen release rate chart of KBH4@Gel and KBH4 NPs;

[0024] Figure 3 For the degradation chart of KBH4@Gel and KBH4 NPs, Figure (A) is the fluorescence imaging analysis chart; (B) is the fluorescence imaging chart;

[0025] Figure 4 For the chart of KBH4@Gel improving early follicle survival and promoting angiogenesis of transplanted ovarian tissue, (A) is the experimental procedure and grouping, (B) is the representative HE and TUNEL staining of ovarian grafts on day 7 after transplantation, (C) is the VEGF immunohistochemistry, (D) is the CD34 immunohistochemistry, (E) is the quantitative analysis of the number of primordial follicles, follicle apoptosis rate, VEGF positive area and microvessel density, ns, no significant; P<0.05; *P<0.01; **P<0.001; ***P<0.0001, the scale bar is 50 μm;

[0026] Figure 5 For the chart of the effect of KBH4@Gel on the structure of mitochondria, oxidative stress and endocrine function of transplanted ovary, (A) is the scanning electron microscope chart of oocytes and granulosa cells, (B-E) is the biochemical analysis of ATP, T-AOC, H2O2 and MDA levels in ovarian grafts, (F-H) is the serum AMH, E2 and FSH levels before and after transplantation in each group, ns, no significant; P<0.05; *P<0.01; **P<0.001; ***P<0.0001;

[0027] Figure 6To verify the key molecules in the AMPK-TFAM-OXPHOS pathway by qPCR and Western blot, (A) is a schematic diagram of mitochondrial energy metabolism remodeling through the AMPK-TFAM-OXPHOS signaling axis, (B) is a Western blot to detect PRKAA2, TFAM, NDUFAB1, COX4I1, and ATP5E, with β-actin as a normalization control for Western blot, (C) measures the protein expression levels of PRKAA2, TFAM, NDUFAB1, COX4I1, and ATP5E by qRT-PCR, with data expressed as mean ± standard deviation, n = 5-7. *p<0.05, **p<0.01, ***p<0.001; (D) measures the mRNA expression levels of PRKAA2, TFAM, NDUFAB1, COX4I1, and ATP5E by qRT-PCR, with data expressed as mean ± standard deviation, n = 5-7. *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0028] The technical solutions of the present application will be described clearly and completely below with reference to the drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0029] In addition, the technical features involved in different embodiments of the present application described below can be combined with each other as long as they do not conflict with each other. EMBODIMENT

[0030] A preparation method of hydrogen-releasing nanogel for ovarian transplantation protection, KBH4 nanoparticles (KBH4 NPs) are prepared by an oil-in-water microemulsion method, and the specific method is as follows: potassium oleate (20 mg) and oleylamine (0.5 mL) are dissolved in a mixed solvent of n-hexane (10 mL) and anhydrous ethanol (0.75 mL), a uniform phase is formed after ultrasonic treatment, and then tetramethylammonium borohydride (TMAB, 22.2 mg) is added, the product is collected by centrifugation and washed with n-hexane three times to obtain hydrophobic KBH4 NPs; then the KBH4 NPs are dispersed in n-hexane (80 μg / mL), a chloroform solution (50 μL) containing phosphatidylcholine dioleoylphosphatidylethanolamine (DOPE, 10 mg / mL) and cholesterol (5 mg / mL) is slowly added dropwise, ultrasonic treatment and rotary evaporation are performed to remove the organic solvent, and then the product is redispersed in PBS (10 mL) and ultrasonicated to obtain hydrophilic KBH4 NPs.

[0031] The temperature-sensitive hydrogel is a commercially available PLGA-PEG-PLGA triblock copolymer (Shanghai Yuanye Biotechnology Co., Ltd., batch number JS274386), which is a light yellow to brown viscous solid, dissolved in 4 ℃ pre-cooled sterile deionized water according to the recommended proportion of the manufacturer, and stirred daily under light-proof conditions until completely dissolved, to obtain a hydrogel solution.

[0032] When in use, 1 mL of the hydrogel solution is mixed with the synthesized KBH4 NPs under low temperature conditions, and the obtained KBH4@Gel can be quickly converted from a liquid state to a gel state at 37 ℃ for about 5 min, realizing in-situ solidification and fixation at the implantation site.

[0033] The obtained nanoparticle structure characterization is shown in Figure 1 Figure (A) SEM characterization: KBH4 NPs are spherical, smooth in surface, uniform in size and without aggregation; Figure (B.C) SEM characterization: the pure hydrogel has a porous structure, while after loading KBH4 NPs, the particles are distributed in the pores and make the surface morphology more dense, proving successful encapsulation; Figure (D) DLS analysis: the hydrodynamic average diameter of KBH4 NPs is measured to be 154.2 ± 38.7 nm, and the particle size is mainly distributed in the range of 100-200 nm; Figure (E) FTIR analysis: the characteristic peaks (B-H bond) of KBH4 and the characteristic absorption bands of DOPE and cholesterol are detected on the coated particles at the same time, confirming that the lipids are successfully coated on the surface of the nanoparticles; Figure (F) XRD analysis: the diffraction peak of KBH4 NPs matches the standard KBH4 crystal phase card, proving that the synthesis and encapsulation process does not change its crystal structure.

[0034] Test Example 1

[0035] To investigate whether the hydrogen-releasing nanogel KBH4@Gel prepared in the examples of the present application can slow down the release of hydrogen.

[0036] The results are shown in Figure 2 Gas chromatography shows that the hydrogen release rate of KBH4@Gel is slower than that of KBH4 NPs without adding hydrogel, and after 2 hours, the total amount of hydrogen released by KBH4@Gel is not less than that of pure KBH4 NPs.

[0037] As Figure 3To verify the sustained-release effect, DiD-labeled KBH4 NPs were incubated under weakly acidic conditions, and fluorescence imaging was performed at 0.5, 1, and 2 hours, respectively (Fig. A-B). The fluorescence signal of the non-wrapped group was significantly weakened after 0.5 hours, while the hydrogel-wrapped group still maintained a strong signal within 2 hours, and the fluorescence intensity was significantly higher than that of the control group (P<0.01). This result is consistent with the gas chromatography detection data, confirming that the temperature-sensitive hydrogel can effectively prolong the retention time of nanoparticles and achieve stable hydrogen release under acidic conditions.

[0038] Test Example 2

[0039] This test example explores the effect of KBH4@Gel on neovascularization and apoptosis of primordial follicles.

[0040] The present application measures active oxygen such as hydrogen peroxide, MDA and T-AOC, ATP in the graft by an active oxygen detection kit, observes the ultrastructure of mitochondria by transmission electron microscopy, detects the expression of angiogenesis-related proteins VEGF and CD34 by immunohistochemical staining, detects the number of primordial follicles and apoptosis by HE and TUNEL staining, and detects serum hormone levels by ELISA.

[0041] The results are shown in Figure 4 .

[0042] Figure 4 (A) is a flow chart of the experiment.

[0043] Figure 4 (B) HE and TUNEL staining analysis, the results show: show the representative HE staining and TUNEL staining results of the ovary tissue on the 7th day after transplantation; analysis conclusion: HE staining shows that compared with the CT group, the primordial follicle in the ovary cortex of the Tx group has complete morphological structure and uniform cytoplasm, and the pathological changes are significantly reduced, and TUNEL staining further shows that the follicle apoptosis rate of the Tx group is significantly lower than that of the CT group, proving that KBH4@Gel can effectively inhibit follicle apoptosis in the early stage after transplantation and improve follicle survival rate.

[0044] Figure 4 (C) VEGF immunohistochemical analysis, the results show: show the immunohistochemical staining results of VEGF in the transplanted ovary tissue; analysis conclusion: the positive signal intensity and positive rate of VEGF in the Tx group are significantly higher than those in the CT group, and are in a diffuse distribution mode; this result suggests that KBH4@Gel can significantly up-regulate the expression of VEGF in the transplanted ovary, indicating that the angiogenesis process in the tissue is in an active state.

[0045] Figure 4(D) CD34 immunohistochemical analysis, the results are presented: shows the immunohistochemical staining results of CD34 (microvessel marker) in transplanted ovarian tissue; Analysis conclusion: The microvessel density of Tx group is significantly higher than that of CT group, and the newly formed microvessels are more evenly distributed in the central cortex and the junction of cortex and medulla, which indicates that KBH4@Gel effectively promotes the angiogenesis and reconstruction of transplanted ovaries and improves the local blood perfusion.

[0046] Figure 4 (E) Quantitative analysis of multiple indicators, the results are presented: The number of primary follicles, follicle apoptosis rate, VEGF positive area percentage and microvessel density were quantitatively analyzed; Analysis conclusion: Quantitative data consistently confirmed that compared with the CT group, the number of primary follicles, VEGF expression level and microvessel density in the Tx group were significantly increased, while the follicle apoptosis rate was significantly decreased, which comprehensively indicated that KBH4@Gel effectively improved the early health status of transplanted ovaries by synergistically promoting follicle survival and vascular reconstruction.

[0047] From the above test results, it can be concluded that KBH4@Gel can significantly promote the early vascular reconstruction of transplanted ovaries, thereby providing sufficient blood perfusion support for the subsequent follicle development and physiological function recovery of the graft.

[0048] Test Example 3

[0049] This test example explores the effects of KBH4@Gel on the mitochondrial structure, oxidative stress and endocrine function of transplanted ovaries.

[0050] The results are shown in Figure 5 .

[0051] Figure 5 (A) is the observation of the mitochondrial ultrastructure of the primary follicle in the ovary tissue 7 days after transplantation by transmission electron microscopy, the results show that the mitochondria in the CT group appear severe swelling, ridge rupture and vacuolization; while the mitochondrial structure in the Tx group is significantly repaired, the ridge structure is basically complete, which proves that KBH4@Gel can effectively maintain the structural integrity of mitochondria in transplanted ovaries.

[0052] Figure 5 (B-E) are the ATP content, total antioxidant capacity and oxidative stress marker levels of the ovary tissue by biochemical detection analysis (using the Biyun Tian kit to determine the oxidative stress related indicators: MDA content is determined by thiobarbituric acid method, total antioxidant capacity is detected by ABTS method, H2O2 level is quantified by colorimetric method. The specific operation steps are carried out according to the kit instructions.)

[0053] Results show that the ATP level and total antioxidant capacity of the Tx group are significantly higher than those of the CT group, while the hydrogen peroxide and malondialdehyde contents are significantly reduced, indicating that KBH4@Gel effectively improves the energy supply of the tissue and reduces oxidative stress damage by sustained hydrogen release.

[0054] Figure 5 (F-H) are the concentrations of AMH, E2 and FSH in serum detected by ELISA to evaluate the endocrine function of the transplanted ovary.

[0055] Results show that the Tx group exhibits significantly higher E2 levels and lower FSH levels, and AMH also shows a rebound trend, proving that KBH4@Gel intervention can effectively promote the recovery of the hypothalamus-pituitary-ovary axis feedback function and improve the endocrine function of the transplanted ovary.

[0056] Test Example 4

[0057] This test example verifies the key molecules in the AMPK-TFAM-OXPHOS pathway by qPCR and Western blot.

[0058] Results are shown in Figure 6 .

[0059] Figure 6 (A) is a schematic diagram of the AMPK-TFAM-OXPHOS signaling axis mediating mitochondrial energy metabolism remodeling.

[0060] Figure 6 (B) is the Western blot detection result of PRKAA2, TFAM, NDUFAB1, COX4I1 and ATP5E (β-actin as internal reference). The results show that the expression of the above proteins in the KBH4@Gel treatment group is significantly up-regulated compared with the model group, indicating that the system can activate the expression of key proteins in the AMPK-TFAM-OXPHOS pathway.

[0061] Figure 6 (C) is the qRT-PCR detection of the protein expression levels of PRKAA2, TFAM, NDUFAB1, COX4I1 and ATP5E (data are expressed as mean ± SD, n=5-7; p<0.05, p<0.01, p<0.001). The results show that the expression of each gene in the KBH4@Gel group is significantly increased, further verifying that the pathway is activated at the transcription level.

[0062] Based on the above experimental results, it is proposed that KBH4@Gel promotes mitochondrial energy metabolism remodeling by activating the AMPK-TFAM-OXPHOS axis. This activation process enhances electron transport chain activity and ATP generation, ultimately alleviating oxidative stress and promoting the structural and functional recovery of the transplanted ovary tissue.

[0063] From the above results, it is shown that KBH4@Gel can reprogram mitochondrial metabolism by activating the AMPK-TFAM-OXPHOS axis, thereby reconstructing mitochondrial function and energy networks. This activation enhances the activity of the electron transport chain and ATP production, ultimately alleviating oxidative stress and promoting structural repair and functional recovery of transplanted ovarian tissue.

[0064] Therefore, the present application significantly improves the stability and effectiveness of hydrogen intervention; economically, the raw material source is simple, the preparation process is controllable, and the cost is lower than that of biological factor drugs; socially, this method is expected to be popularized to the prevention and treatment of other ischemia-reperfusion injury diseases, reduce the reproductive and health burden of female ovarian function decline, and has good clinical transformation prospects and social application value.

[0065] Although embodiments of the present application have been shown and described, it will be understood by those having ordinary skill in the art that various changes, modifications, substitutions and alterations can be made thereto without departing from the principles and spirit of the present application, the scope of which is defined by the appended claims and their equivalents.

[0066] The above specific embodiments are detailed descriptions of the present application, which cannot be considered as limiting the specific embodiments of the present application to these descriptions. For those skilled in the art to which the present application belongs, a number of simple deductions and substitutions can be made without departing from the concept of the present application, which should be considered as falling within the scope of protection of the present application.

Claims

1. A method for preparing hydrogen-releasing nanogels for ovarian transplant protection, characterized in that, Includes the following steps: Step 1: Preparation of KBH4 nanoparticles; In step 1, KBH4 nanoparticles are prepared using an oil-in-water microemulsion method. The specific preparation method of the oil-in-water microemulsion method includes the following steps: Step 1.1: Dissolve potassium oleate and oleylamine in a mixed solvent of n-hexane and anhydrous ethanol, sonicate, add tetramethylammonium borohydride, stir at room temperature for a certain time, and then centrifuge and wash to obtain hydrophobic KBH4 nanoparticles. Step 1.2: Disperse hydrophobic KBH4 nanoparticles in n-hexane, add dropwise a chloroform solution containing dioleoylphosphatidylethanolamine and cholesterol, remove the organic solvent by sonication and rotary evaporation, redisperse with PBS buffer and sonicate to obtain hydrophilic KBH4 nanoparticles. Step 2: Prepare a temperature-sensitive hydrogel solution; Thermosensitive hydrogel is made of PLGA–PEG–PLGA triblock copolymer; Step 3: Mix the KBH4 nanoparticles from Step 1 and the thermosensitive hydrogel solution from Step 2 at low temperature to obtain hydrogen-releasing nanogel KBH4@Gel.

2. The method for preparing a hydrogen-releasing nanogel for ovarian transplantation protection according to claim 1, characterized in that, The mass concentration of the KBH4 nanoparticles in the thermosensitive hydrogel system is 80-320 µg / mL.

3. The method for preparing a hydrogen-releasing nanogel for ovarian transplantation protection according to claim 2, characterized in that, The mass concentration of the KBH4 nanoparticles in the thermosensitive hydrogel system is 160 µg / mL.

4. The method for preparing a hydrogen-releasing nanogel for ovarian transplantation protection according to claim 1, characterized in that, In step 1.1, the reaction time is 40-80 min.

5. A hydrogen-releasing nanogel for ovarian transplantation protection prepared by any one of the preparation methods described in claims 1-4.

6. The application of a hydrogen-releasing nanogel for ovarian transplant protection prepared by any one of the preparation methods described in claims 1-4 in the preparation of ovarian transplant protection materials.

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