Nano-drug for enhancing transplantation curative effect of stem cell-derived retinal ganglion cells as well as preparation method and application of nano-drug
By combining lithium ions and epicatechin gallate self-assembled nanomedicines with ganglion cells derived from embryonic stem cells, the problem of retinal ganglion cell damage in glaucoma under oxidative stress microenvironment was solved, improving survival rate and neuroprotective effect, and realizing the restoration of visual function.
Patent Information
- Application Number
- CN202511346048.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-19
- Publication Date
- 2026-01-02
AI Technical Summary
In glaucoma, retinal ganglion cells are easily damaged in the oxidative stress microenvironment, resulting in low transplant survival rates. Current treatments cannot reverse ganglion cell damage, thus limiting the recovery of visual function.
A nanomedicine was formed by the self-assembly of lithium ions and epicatechin gallate, which combined with ganglion cells derived from embryonic stem cells to form a stable metal-polyphenol network structure, thereby enhancing cell survival rate and neuroprotective effect.
It significantly improves the survival rate of retinal ganglion cells and the protection of neurites under oxidative stress, achieving more effective neuroprotection and functional reconstruction, and is suitable for the treatment of retinal degenerative diseases such as glaucoma.
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Figure CN121243221A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological medicine, in particular to a nano-drug for enhancing the curative effect of stem cell-derived retinal ganglion cell transplantation, a preparation method and application thereof. BACKGROUND
[0002] Glaucoma is a complex progressive neurodegenerative disease and the leading cause of irreversible blindness, affecting more than 80 million people. The main pathological manifestations of the disease are the progressive loss of retinal ganglion cells and their axons, ultimately leading to visual field defects and irreversible decline in vision. Current clinical treatment mainly focuses on reducing intraocular pressure, including drug intervention and surgical treatment, which can effectively delay disease progression, but cannot reverse the damage to ganglion cells, and the recovery of visual function is limited, which seriously limits the treatment effect. The pathogenesis of glaucoma is complex, and oxidative stress is one of the core pathogenic mechanisms. Excessive accumulation of a large number of active oxygen in the retinal microenvironment damages mitochondrial function, leading to ganglion cell apoptosis and axon degeneration. At the same time, impaired mitochondrial energy metabolism affects nerve conduction and cell survival, accelerating neurodegeneration. The oxidative stress microenvironment not only exacerbates disease progression, but also poses a great challenge to neuroprotection and cell therapy.
[0003] In recent years, ganglion cell replacement therapy has become a research hotspot in the treatment of glaucoma, aiming to replace damaged ganglion cells with exogenous ganglion cells with good function to restore visual function. Among them, embryonic stem cell-derived ganglion cells are considered to be a cell source with application and transformation value due to their high differentiation potential and stable genetic background. However, the preparation of embryonic stem cell-derived ganglion cells has a long cycle, the resources are scarce, and the survival rate in the pathological microenvironment of glaucoma after transplantation is low, making it difficult to restore synaptic reconstruction and nerve function, which is a key bottleneck restricting the development of this technology. SUMMARY
[0004] The purpose of the present application is to solve the technical problem of retinal ganglion cell damage in glaucoma under oxidative stress microenvironment and low survival rate after transplantation, and to provide a new therapeutic drug for retinal degenerative diseases such as glaucoma.
[0005] In order to achieve the above purpose, the present application provides a nano-drug for enhancing the curative effect of stem cell-derived retinal ganglion cell transplantation, which is formed by self-assembly of metal ions and epicatechin gallate, and the phenolic hydroxyl or o-diphenol group of the metal ions and epicatechin gallate is combined by coordination bond.
[0006] Optionally, the metal ions at least include any one of lithium ions, zinc ions and magnesium ions.
[0007] Optionally, the average particle size of the nano-drug is 460 nm.
[0008] The application further provides a preparation method of the nanodrug, comprising at least the following steps:
[0009] Step S1, dissolving lithium chloride monohydrate and epicatechin gallate in ethanol to obtain a mixed solution;
[0010] Step S2, adjusting the pH of the mixed solution to 7 by sodium carbonate and stirring at room temperature;
[0011] Step S3, after the stirring is completed, collecting the product by centrifugation, and obtaining the self-assembled nanomaterial after vacuum drying and purification of the product.
[0012] Optionally, in step S1, the molar ratio of lithium chloride monohydrate to epicatechin gallate is (2-5):1.
[0013] Optionally, in step S2, the stirring is performed at 37℃ for 24 hours.
[0014] The application further provides an application of the nanodrug, and the nanodrug is applied to the preparation of a product for treating retinal neurodegenerative diseases.
[0015] Optionally, the product is a drug.
[0016] Optionally, the product is a product obtained by transplanting the nanodrug and embryonic stem cell-derived ganglion cells jointly.
[0017] Optionally, the retinal neurodegenerative diseases at least include any one of glaucoma, retinitis pigmentosa and age-related macular degeneration.
[0018] Compared with the prior art, the application has at least the following beneficial effects:
[0019] The present application forms a lithium-epigallocatechin gallate nanodrug through self-assembly of metal ions lithium ions and epigallocatechin gallate, which is not a simple physical mixture, but a stable network structure formed through metal-polyphenol complexation. Lithium ions not only have neuroprotective effects themselves, can effectively regulate various cell signaling pathways, promote neuron survival and axon regeneration, but also can change the molecular configuration of epigallocatechin gallate, significantly enhancing its antioxidant activity. At the same time, the coordination of epigallocatechin gallate to lithium ions can reduce the adverse reactions caused by free lithium ions and improve its stability in the biomaterial system. The nanodrug formed by the combination of the two is more stable in structure, can delay the degradation of epigallocatechin gallate, and at the same time realize multiple effects such as mitochondrial protection and oxidative stress inhibition, thus showing a comprehensive therapeutic effect far exceeding that of a single component, and showing a unique and irreplaceable advantage in neuroprotection; further, the lithium-epigallocatechin gallate self-assembled nanodrug combined with fetal stem cell-derived ganglion cells can significantly improve the survival rate and neurite protection under oxidative stress, thereby achieving more effective neuroprotection and functional reconstruction in the treatment of retinal degenerative diseases such as glaucoma. BRIEF DESCRIPTION OF DRAWINGS
[0020] Figure 1 A characterization image of the lithium-epigallocatechin gallate nanodrug of the present application.
[0021] Figure 2 An XPS spectrum of the lithium-epigallocatechin gallate nanodrug of the present application.
[0022] Figure 3 An ABTS· scavenging capacity determination result graph of the lithium-epigallocatechin gallate nanodrug of the present application. + A determination result graph of the ABTS· scavenging capacity of the lithium-epigallocatechin gallate nanodrug of the present application.
[0023] Figure 4 A DPPH· scavenging capacity determination result graph of the lithium-epigallocatechin gallate nanodrug of the present application.
[0024] Figure 5 An ·OH scavenging capacity determination result graph of the lithium-epigallocatechin gallate nanodrug of the present application.
[0025] Figure 6 An H2O2 scavenging capacity determination result graph of the lithium-epigallocatechin gallate nanodrug of the present application.
[0026] Figure 7 A 24-hour biocompatibility evaluation of the lithium-epigallocatechin gallate nanodrug of the present application.
[0027] Figure 848 hours biocompatibility evaluation of the lithium-epigallocatechin gallate nanodrug of the application.
[0028] Figure 9 Schematic diagram of protecting embryonic stem cell-derived ganglion cell neurite injury by the lithium-epigallocatechin gallate nanodrug of the application.
[0029] Figure 10 Schematic diagram of protecting mitochondria morphology by the lithium-epigallocatechin gallate nanodrug of the application.
[0030] Figure 11 Protective effect diagram of the lithium-epigallocatechin gallate nanodrug of the application combined with embryonic stem cell-derived ganglion cells on the function and structure of the retina of an acute glaucoma model.
[0031] Figure 12 Result diagram of immunostaining of a retina paraffin section after the lithium-epigallocatechin gallate nanodrug of the application combined with embryonic stem cell-derived ganglion cells. DETAILED DESCRIPTION
[0032] The technical solutions of the application are further described below in combination with the drawings and examples.
[0033] The experimental methods not mentioned in the application are conventional experimental methods in the art, and the experimental materials not mentioned in the application are conventional commercially available experimental materials.
[0034] In order to solve the technical problems that the retinal ganglion cells in glaucoma are easily damaged under an oxidative stress microenvironment and have a low survival rate after transplantation, the application, starting from the pathological mechanism, combines a natural antioxidant polyphenol-epigallocatechin gallate (Shanghai Maikelin Biochemical Technology Co., Ltd., E885861) with lithium ions having a neuroprotective effect to form a metal-polyphenol network, constructs a functionalized nanoplatform-lithium-epigallocatechin gallate self-assembled nanodrug, and further combines the nanodrug with embryonic stem cell-derived ganglion cells for transplantation to synergistically improve the survival rate of the transplanted cells, functional integration and the protective effect on endogenous ganglion cells.
[0035] Epigallocatechin gallate, as the main active ingredient in tea, has been confirmed by a large number of studies to have excellent free radical scavenging ability, and is natural in source and high in safety; lithium ions, as a widely used neuroprotective factor in clinical practice, can effectively regulate various cell signaling pathways at a low dose, promote neuron survival and axon regeneration, and are safe and controllable. The application combines the two to form a stable metal-polyphenol structure, which not only has the advantages of double neuroprotection, but also can improve the stability and treatment effect of the drug.
[0036] The application breaks through the traditional "single cell transplantation" method, realizes the functional synergy of nanodrugs and cells, combines metal-polyphenol structure with fetal stem cell-derived ganglion cell transplantation, and thus realizes more effective neuroprotection and functional reconstruction in the treatment of retinal degenerative diseases such as glaucoma.
[0037] I. Preparation of lithium-epicatechin gallate nanodrug
[0038] Lithium monohydrate chloride and epicatechin gallate were dissolved in ethanol at a molar ratio of 4:1 to obtain a mixed solution, and the final concentrations were 4 mmol / L and 1 mmol / L, respectively. After complete dissolution, the pH of the mixed solution was adjusted to 7 with sodium carbonate. The adjusted mixed solution was continuously stirred at 37℃ for 24 hours to promote the formation of lithium-epicatechin gallate self-assembled nanodrug. After the reaction was completed, the product was collected by centrifugation and washed with ethanol several times. After vacuum drying and purification, the lithium-epicatechin gallate nanodrug was obtained, which was a light brown powder in appearance.
[0039] II. Characterization of lithium-epicatechin gallate nanodrug
[0040] The lithium-epicatechin gallate nanodrug was observed under a transmission electron microscope, a scanning electron microscope and an atomic force microscope, as shown in Figure 1 , Figure 1 A is a transmission electron micrograph, B is a scanning electron micrograph, and C is an atomic force micrograph. As can be seen from the figure, the lithium-epicatechin gallate nanodrug has a spherical structure, and the average particle size is 460 nm.
[0041] As shown in Figure 2 , the XPS spectrum of the prepared lithium-epicatechin gallate nanodrug shows the presence of O, C and Li elements, indicating that lithium ions have successfully entered the lithium-epicatechin gallate nanodrug.
[0042] III. Antioxidant properties of lithium-epicatechin gallate nanodrug
[0043] ABTS· + scavenging capacity determination
[0044] 0.2 mL of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) solution (7.4 mmol / L) was mixed with 0.2 mL of potassium persulfate (K2S2O8) solution (2.6 mmol / L), and the reaction was carried out in the dark for 24 hours to generate ABTS· +Radicals. The resulting solution was then diluted with PBS. Next, 1.8 mL of aqueous solutions of epigallocatechin gallate lithium nanomedicine at different concentrations (0, 25, 50, 75, 100 μg / mL) were mixed with 0.20 mL of ABTS· + solution. After the reaction, the absorbance was measured at 734 nm using a UV-Vis spectrophotometer. The results showed that the absorbance gradually decreased as the concentration of epigallocatechin gallate lithium nanomedicine increased, indicating that the ABTS· Figure 3 radical scavenging ability of epigallocatechin gallate lithium nanomedicine was enhanced, and its antioxidant activity was improved. +
[0045] DPPH·radical scavenging ability determination:
[0046] A 0.05 mg / mL DPPH ethanol solution was prepared, and 2 mL of this solution was mixed with 2 mL of epigallocatechin gallate lithium nanomedicine solutions at different concentrations (0, 50, 100, 200, 400 μg / mL). After 30 minutes of reaction, the absorbance was measured at 519 nm using a UV-Vis spectrophotometer. The results showed that the absorbance gradually decreased as the concentration of epigallocatechin gallate lithium nanomedicine increased ( Figure 4 ), indicating that the DPPH·radical scavenging ability of epigallocatechin gallate lithium nanomedicine was enhanced, and its antioxidant activity was improved.
[0047] ·OH radical scavenging ability determination:
[0048] The ·OH radical scavenging ability of epigallocatechin gallate lithium nanomedicine was detected using the salicylic acid method. The basic principle is that hydroxyl radicals (·OH) have strong oxidizing properties and can specifically react with salicylic acid to generate 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA). These products have characteristic absorption peaks at specific wavelengths (usually around 510 nm), and the amount of ·OH generated in the system can be indirectly calculated by measuring the absorbance value. First, ·OH was generated by mixing (0.1 mL) FeSO4 (2 mM) with (0.1 mL) H2O2 (5 mM) and reacting for 5 minutes using the Fenton reaction. Then, different concentrations (0, 50, 100, 200, 400 μg / mL) of epigallocatechin gallate lithium nanomedicine solution (1.7 mL) were added to the reaction system, followed by the addition of (0.1 mL) salicylic acid ethanol solution (10 mM), and the entire solution system was thoroughly shaken and continued to react for 15 minutes. Finally, the absorbance of 2,3-dihydroxybenzoic acid, which is the product of the oxidation of salicylic acid by ·OH, was detected at 510 nm using a UV-Vis spectrophotometer. The results showed that the absorbance decreased in a concentration-dependent manner as the concentration of epigallocatechin gallate lithium nanomedicine increased ( Figure 5 ), indicating that its ·OH scavenging ability is enhanced and the antioxidant performance is improved.
[0049] H2O2 scavenging ability determination:
[0050] Hydrogen peroxide (H2O2) was detected by titanium salt colorimetric method (Titanium Sulfate Spectrophotometric method, TSS). Titanium salt reacts with H2O2 to generate yellow peroxotitanium complex, which can be dissolved in strong acid solution and has a characteristic absorption peak at 410 nm. The absorbance intensity is proportional to the concentration of H2O2 within a certain range. When preparing the H2O2 detection solution, 1.25 g of Ti(SO4)2 was dissolved in 8 mL of H2SO4 and diluted to 100 mL with deionized water. Then, 200 μL of H2O2 (10 mM) was mixed with 1.2 mL of detection solution, and 2 mL of different concentrations (0, 25, 50, 100, 200 μg / mL) of lithium-epigallocatechin gallate nanomedicine solution was added. After 1 hour of reaction, the absorbance was measured at 410 nm. As shown in Figure 6 , with the increase of the concentration of lithium-epigallocatechin gallate nanomedicine, the absorbance gradually decreased, indicating that the total antioxidant capacity was enhanced and the removal effect of H2O2 was significant.
[0051] Four. Evaluation of the biocompatibility of lithium-epigallocatechin gallate nanomedicine
[0052] The biocompatibility of lithium-epigallocatechin gallate nanomedicine at different concentrations was evaluated by CCK-8 method. Embryonic stem cell-derived ganglion cells were digested with collagenase type II and plated in 96-well plates at equal amounts. After treatment with different concentrations (0, 5, 10, 25, 50, 100, 200, 400 μg / mL) of lithium-epigallocatechin gallate nanomedicine for 24 hours Figure 7 ) and 48 hours Figure 8 ), the absorbance was measured at 450 nm wavelength using a microplate reader. The results showed that lithium-epigallocatechin gallate nanomedicine at a concentration of 25 μg / mL or below had no inhibitory effect on the survival of embryonic stem cell-derived ganglion cells, so a concentration of 25 μg / mL was selected for subsequent drug efficacy testing.
[0053] Five. Lithium-epigallocatechin gallate nanomedicine protects embryonic stem cell-derived ganglion cells from neurite injury and mitochondrial morphology
[0054] The neuroprotective effect of lithium-epigallocatechin gallate nanomedicine was explored at the cellular level by evaluating the neurite integrity and mitochondrial morphology of embryonic stem cell-derived ganglion cells. After the embryonic stem cell-derived ganglion cells formed a neural network after 5 days of culture, they were first treated with hydrogen peroxide to induce oxidative stress, and then 25 μg / mL of epigallocatechin gallate or lithium-epigallocatechin gallate nanomedicine was added for further culture. As shown in Figure 9 , compared with epigallocatechin gallate, lithium-epigallocatechin gallate nanomedicine had a more significant protective effect on neurite morphology, with more cell survival, increased number of neurites, and thicker neurite diameter.
[0055] As shown in Figure 10 , ultrastructure analysis using a transmission electron microscope showed that the embryonic stem cell-derived ganglion cells in the hydrogen peroxide group had obvious mitochondrial swelling and destruction of the cristae structure. In contrast, lithium-epigallocatechin gallate nanomedicine treatment significantly protected mitochondrial morphology and structural integrity, with significantly reduced mitochondrial swelling and increased cristae density, demonstrating good mitochondrial protection.
[0056] VI. Protective effect of lithium-epigallocatechin gallate nanomedicine combined with embryonic stem cell-derived ganglion cells on retinal function and structure in an acute glaucoma model
[0057] In an ischemia / reperfusion (I / R)-induced acute pathological glaucoma injury mouse model, lithium-epigallocatechin gallate nanomedicine was used in combination with embryonic stem cell-derived ganglion cells for treatment.
[0058] Visual evoked potential (VEP) results showed that I / R injury significantly reduced the P1-N1 amplitude, reflecting a decrease in the response of the visual cortex to visual stimuli Figure 9 . Retinal electrogram (ERG) detection results showed that the amplitudes of the a and b waves were significantly reduced after I / R injury, indicating impaired retinal function. After treatment with embryonic stem cell-derived ganglion cells or in combination with epigallocatechin gallate, these electrophysiological responses were partially restored, while the lithium-epigallocatechin gallate nanomedicine combined with embryonic stem cell-derived ganglion cells had the most significant effect on the recovery of VEP and ERG signals Figure 11 . These results indicate that the combination of lithium-epigallocatechin gallate nanomedicine and embryonic stem cell-derived ganglion cells can provide stronger neuroprotection and effectively repair visual pathway function.
[0059] As shown in Figure 12As shown, the results of retinal paraffin section immunostaining showed that the Tuj1 (ganglion cell marker) positive expression (white) of lithium-epicatechin gallate nanodrug combined with embryonic stem cell-derived ganglion cells was significantly increased, which supported that the combination therapy could enhance the survival of ganglion cells at the level of retinal structure. The above results showed that lithium-epicatechin gallate nanodrug combined with embryonic stem cell-derived ganglion cell therapy could protect the function and structure of the retina of an acute glaucoma mouse model.
[0060] In summary, the lithium ion has a neuroprotective effect, and can effectively regulate multiple cell signaling pathways, promote neuron survival and axon regeneration at a low dose. The epicatechin gallate has excellent free radical scavenging ability. The lithium-epicatechin gallate nanodrug formed by self-assembly of the two has stable structure, strong oxidation and mitochondrial protection. The two are organically coordinated in structure and function, not only have the advantages of double neuroprotection, but also can improve the stability and therapeutic effect of the drug. Further, the lithium-epicatechin gallate nanodrug combined with embryonic stem cell-derived ganglion cell transplantation can improve the survival rate, functional integration and protection of endogenous ganglion cells of the transplanted cells, and is suitable for cell transplantation therapy for various retinal neurodegenerative diseases such as age-related macular degeneration and retinitis pigmentosa.
[0061] Although the content of the present application has been described in detail by the above preferred embodiments, it should be recognized that the above description should not be considered as a limitation of the present application. After reading the above content, various modifications and alternatives of the present application will be apparent to those skilled in the art. Therefore, the protection scope of the present application should be defined by the appended claims.
Claims
1. A nanomedicine for enhancing the therapeutic effect of stem cell-derived retinal ganglion cell transplantation, characterized in that, The nanomedicine is formed by the self-assembly of metal ions and epicatechin gallate, wherein the metal ions and the phenolic hydroxyl groups or catechol groups of epicatechin gallate are linked by coordination bonds.
2. The nanomedicine as described in claim 1, characterized in that, The metal ions include at least one of lithium ions, zinc ions, and magnesium ions.
3. The nanomedicine as described in claim 1, characterized in that, The average particle size of the self-assembled nanomaterial is 460 nm.
4. A method for preparing a nanomedicine as described in any one of claims 1 to 3, characterized in that, Include at least the following steps: Step S1: Dissolve lithium chloride monohydrate and epicatechin gallate in ethanol to obtain a mixed solution; Step S2: Adjust the pH of the mixed solution to 7 with sodium carbonate and stir at room temperature; Step S3: After stirring, the product is collected by centrifugation, and the product is vacuum dried and purified to obtain the nanomedicine.
5. The preparation method according to claim 4, characterized in that, In step S1, the ratio of lithium chloride monohydrate to epicatechin gallate is (2-5):1, measured by molar ratio.
6. The preparation method according to claim 4, characterized in that, In step S2, stir at 37°C for 24 hours.
7. An application of a nanomedicine as described in any one of claims 1 to 3, characterized in that, The application of the nanomedicine in the preparation of products for treating retinal neurodegenerative diseases.
8. The application as described in claim 7, characterized in that, The product in question is a medicine.
9. The application as described in claim 7, characterized in that, The product is obtained by transplanting nanomedicines in combination with ganglion cells derived from embryonic stem cells.
10. The application as described in claim 7, characterized in that, The aforementioned retinal neurodegenerative diseases include at least one of glaucoma, retinitis pigmentosa, and age-related macular degeneration.