A rapamycin-g4h nanocapsule, a preparation method and application thereof

Abstract

The application relates to a rapamycin-G4H nanocapsule, a preparation method and application thereof. The preparation method of the rapamycin-G4H nanocapsule comprises the following steps: S1. preparing a rapamycin micelle by adopting a film hydration method; S2. modifying a dibenzocyclooctyne amplification primer on the surface of the rapamycin micelle through a click chemistry reaction to obtain a rapamycin micelle modified with the amplification primer; S3. taking a linear DNA molecule as a template, and synthesizing a circular amplification template through an intramolecular cyclization reaction; and S4. incubating the circular amplification template, the rapamycin micelle modified with the amplification primer, a thermophilic DNA polymerase, a pyrophosphatase, dNTP and Co-G4-haem DNAzyme under the condition of a thermophilic DNA polymerase reaction buffer. The rapamycin-G4H nanocapsule can play an efficient antioxidant stress and immune inflammatory regulation function, can improve the intraocular microenvironment of a glaucoma patient, and can inhibit the continuous apoptosis of retinal ganglion cells.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a rapamycin-G4H nanocapsule, its preparation method, and its application. Background Technology

[0002] Acute glaucoma is a major blinding eye disease that can lead to rapid and irreversible vision loss. Its core danger lies in the mechanical compression and ischemic damage to the optic nerve axons and retinal ganglion cells (RGCs) caused by the rapidly increasing intraocular pressure in a short period of time.

[0003] Currently, the main clinical treatment for acute glaucoma is emergency reduction of intraocular pressure (such as medication, laser, and surgery). While this can salvage some vision, it has significant limitations: it only addresses the initial factor of "pressure" and cannot effectively block the ongoing neuronal apoptosis process after intraocular pressure normalizes, making it difficult to fundamentally reverse visual impairment. Research has found that oxidative stress and the immune inflammatory microenvironment are key intrinsic mechanisms driving neurodegenerative changes in glaucoma. Acute high intraocular pressure leads to mitochondrial dysfunction, generating large amounts of reactive oxygen species (ROS), triggering severe oxidative stress, and directly damaging lipids, proteins, and DNA in rhabdomyosarcomas (RGCs). Simultaneously, the release of damage-associated molecular patterns (DAMPs) activates immune cells such as microglia and promotes the secretion of large amounts of cytokines / chemokines, forming a chronic pro-inflammatory immune microenvironment. Activated microglia not only lose their neuroprotective function but also further exacerbate oxidative stress by releasing pro-inflammatory factors such as TNF-α and IL-1β, creating a vicious cycle that accelerates RGC death. Therefore, single antihypertensive therapy has the limitation of "treating the symptoms but not the root cause," and combining it with improving the local oxidative stress and immune inflammatory microenvironment in glaucoma has become a highly promising neuroprotective strategy.

[0004] The development of novel bionanomaterials offers new hope for overcoming the bottlenecks of traditional glaucoma treatments and achieving more effective optic nerve protection. Currently, researchers have constructed a series of functionalized biodelivery systems to improve the oxidative stress and immune-inflammatory microenvironment in glaucoma. These biodelivery systems include polymer hydrogels, targeted liposomes, and nanomicelles formed by the self-assembly of block polymers. By encapsulating antioxidants (such as NAC, melatonin, and metallo-nanozymes) or anti-inflammatory drugs (such as verteporfin and rapamycin), they exert sustained drug effects through localized sustained release at the posterior end of the eye.

[0005] However, existing biotherapy strategies have the following limitations: 1) Small molecule antioxidants are easily inactivated, cannot be regenerated after consumption, and have a narrow range of functions, so their effects are often short-lived and passive; 2) Although metal nanozymes can effectively scavenge reactive oxygen species by mimicking the activity of superoxide dismutase or catalase, their long-term biosafety is questionable, and they may produce nanotoxicity that induces inflammatory responses, which is contrary to the treatment goal; 3) Most existing delivery systems only carry single-function drugs (such as immunosuppressants or antioxidants), which are difficult to simultaneously address the complex pathological microenvironment formed by the mutual promotion of oxidative stress and inflammation, resulting in poor overall treatment effects. Summary of the Invention

[0006] In view of this, the present invention provides a rapamycin-G4H nanocapsule, its preparation method and application, to solve the above problems.

[0007] To achieve the above solution, the technical solution of the present invention is as follows: This application provides a method for preparing rapamycin-G4H nanocapsules (rapamycin-G4H nanocapsules (RGNc)), the preparation method comprising the following steps: S1. Rapamycin micelles were prepared using a thin-film hydration method; S2. By using a click chemical reaction, a dibenzocyclooctyne amplification primer (DBCO-Amplification Primer) was modified on the surface of rapamycin micelles to obtain primer-modified rapamycin micelles (RAPA RMs modified with amplification primer). S3. Using linear DNA molecules as templates, circular amplification templates are synthesized through intramolecular cyclization reactions; S4. Under the conditions of thermophilic DNA polymerase reaction buffer, the circular amplification template, rapamycin micelles modified with amplification primers, thermophilic DNA polymerase, pyrophosphatase, dNTPs and Co-G4-hemin DNAzyme are incubated to prepare the rapamycin-G4H nanocapsules.

[0008] Optionally, the preparation of rapamycin micelles using the thin-film hydration method includes: adding rapamycin and a nonionic surfactant to an anhydrous ethanol water bath to obtain a mixture, rotary evaporating the mixture, adding a buffer solution, and reconstituted by ultrasonication in a water bath to obtain rapamycin micelles.

[0009] Optionally, the mass ratio of rapamycin to nonionic surfactant is 2-4:20-30, preferably 2.5-4:25-30.

[0010] Nonionic surfactants include substances such as polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymers (poloxam F127) and azide-modified polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymers (N3-F127).

[0011] Optionally, the ratio of rapamycin to anhydrous ethanol is 2-4 mg: 2-4 mL, preferably 2.5-4 mg: 2.5-4 mL.

[0012] Optionally, the prepared rapamycin micelles can be stored at 3-5°C.

[0013] Optionally, during the rotary evaporation process, the temperature is 35-45℃, preferably 38-45℃; the rotation speed is 40-60 rpm, preferably 50-60 rpm; and the evaporation time is 8-15 min, preferably 9-15 min.

[0014] Optionally, the surface of rapamycin micelles is modified with dibenzocyclooctylene amplification primers by click chemistry, including: mixing rapamycin micelles with the modified dibenzocyclooctylene amplification primers in a molar ratio of 0.5-1.5:0.5-1.5 and reacting at 35℃-40℃ for 0.5-1.5 h.

[0015] Optionally, the molar ratio of rapamycin micelles to dibenzocyclooctyne amplification primers is 0.5-2:0.5-2, preferably 0.8-2:0.8-2.

[0016] Optionally, the synthesis of the circular amplification template includes: co-incubating the linear template, ligation primers and T4 DNA ligase in T4 DNA ligase reaction buffer, and then inactivating the T4 DNA ligase to obtain the T4 DNA ligase.

[0017] Optionally, the molar ratio of the linear template to the ligation primer is 0.5-2:0.5-2, preferably 0.8-2:0.8-2.

[0018] Optionally, the ratio of the linear template to T4 DNA ligase is 0.5-2 mol: 8-15 U, preferably 0.8-2 mol: 9-15 U.

[0019] Optionally, the nucleotide sequence of the linear template is as shown in SEQ ID NO.1.

[0020] Optionally, the nucleotide sequence of the ligation primer is shown in SEQ ID NO.2.

[0021] Optionally, the co-incubation temperature is 20-25℃, preferably 21-25℃; the co-incubation time is 1-3h, preferably 1.5-3h.

[0022] During co-incubation, T4 DNA ligase catalyzes the ligation of the two ends of a phosphorylated linear template mediated by primers to form a circular DNA molecule.

[0023] Optionally, the inactivation temperature is 55-65℃, preferably 58-65℃; the inactivation time is 10-20 min, preferably 18-20 min.

[0024] Optionally, the molar ratio of the circular amplification template to the rapamycin micelles modified with amplification primers is 90-110:450-550.

[0025] Optionally, the molar ratio of the circular amplification template to Co-G4-heme DNAase is 0.08-0.12:0.5-2, preferably 0.09-0.12:0.8-2.

[0026] Optionally, the ratio of the thermophilic DNA polymerase is 80-120 nM: 4-6 U, preferably 80-120 nM: 4-6 U.

[0027] Optionally, the ratio of the thermophilic DNA polymerase to the pyrophosphatase is 4-6U:8-12U, preferably 4-6U:9-12U.

[0028] Optionally, the thermophilic DNA polymerase is selected from phi29 DNA polymerase.

[0029] Optionally, the molar ratio of the circular amplification template to dNTPs is 80-120 nM: 0.5-2 mM, preferably 90-120 nM: 0.8-2 mM.

[0030] Optionally, the incubation temperature is 30-40℃, preferably 35-40℃; the incubation time is 1-3h, preferably 1.5-3h.

[0031] During incubation, Co-G4-heme DNAase hybridizes with the amplified single-stranded complementary sequence.

[0032] The present invention also provides a rapamycin-G4H nanocapsule prepared according to the method described above.

[0033] The present invention also provides the use of the rapamycin-G4H nanocapsules as described above in the preparation of a medicament for the treatment of acute glaucoma.

[0034] As described above, the present invention has the following beneficial effects: This application synthesizes a rapamycin-G4H nanocapsule, which can exert highly efficient antioxidant stress and immune inflammation regulation functions, improve the intraocular microenvironment of glaucoma patients, and inhibit the continuous apoptosis of retinal ganglion cells. Attached Figure Description

[0035] Figure 1 The characterization results are shown in the following figures: 1A is the electron microscopy scan image, 1B is the diameter analysis result image, 1C is the elemental energy dispersive spectroscopy analysis result image, 1D is the Zeta potential analysis result image, 1E is the infrared spectroscopy analysis result image, and 1F is the circular dichroism spectroscopy characterization result image. Zeta Potential represents Zeta potential, Absorbance represents absorbance, Wave number represents wavenumber, Wavelength represents wavelength, and Diameter represents diameter. Figure 2 For RG Nc to effectively regulate R28 Glu The experimental results of cell apoptosis and inflammation are shown in the figure. Figure 2 A shows the results of the CCK8 assay; 2B shows the results of the expression level detection of the apoptosis-related gene Bax; 2C shows the results of the expression level detection of the apoptosis-related gene Bcl2; 2D shows the results of the expression level detection of the apoptosis-related gene Caspase3; 2E shows the results of the expression level detection of the inflammation-related gene TLR4; 2F shows the results of the expression level detection of the inflammation-related gene TNFα; 2G shows the results of the expression level detection of the inflammation-related gene IL6; 2H shows the results of the flow cytometry analysis; 2I shows the results of the immunofluorescence analysis of the TRL4 expression level. Relative cell activity indicates relative cell activity; Fold changes indicate fold changes; 3D surface plot indicates a three-dimensional surface plot, and the same applies below. Figure 3 RG Nc can effectively alleviate R28 Glu The results of the oxidative stress assay are shown in the following figures: 3A shows the ROS detection results; 3B shows the detection results of the ability of RG Nc to alleviate the mitochondrial membrane potential imbalance in R28Glu cells; 3C shows the detection results of the expression level of the oxidative stress-related gene NRF2; 3D shows the detection results of the expression level of the oxidative stress-related gene SOD2; and 3E shows the detection results of the expression level of the oxidative stress-related gene APOC3. Merge indicates merging, and aggregates indicate aggregates. Figure 4The figures show the experimental results of how RG Nc can effectively alleviate the rapid apoptosis of RGCs in NMDA-induced glaucoma model mice. 4A represents the results of the opto-motor assay, 4B the results of the minefield assay, 4C the statistical data of Brn3a fluorescence staining (scale bar 5µm), 4D the results of Brn3a fluorescence staining, 4E the results of H&E staining, and 4F the results of biological transmission electron microscopy characterization. OMR assays represent the opto-motor assay, Optornotor response represents the opto-motor response, Forward followtime represents the forward following time, Rekative follow time represents the relative following time, Open-field test represents the minefield assay, Ambulatory distance represents the distance traveled, Ambulatory time represents the time traveled, Stereotypic time represents the stereotypic time, Numbers represents the number of organisms, Mitochondria represents mitochondria, Center represents the center, Middle represents the middle, and Retina represents the retina. Detailed Implementation

[0036] The present invention will be further illustrated by specific examples below. However, it should be noted that the specific material ratios, process conditions and results described in the embodiments of the present invention are only for illustrating the present invention and cannot be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

[0037] This application describes the synthesis of azide-modified poloxamer F127 micelles for encapsulating poorly soluble rapamycin as an immunomodulator.

[0038] Synthetic route of azido-modified poloxamer F127 micelles: 100 mg of F-127-NHS was weighed and dissolved in an appropriate amount of chloroform. Azide propylamine (2.5 eq., where eq. represents equivalent amount) and triethylamine (2.5 eq.) were added and dissolved completely. The reaction was carried out at room temperature for 0.5 h. The solvent was removed by vacuum distillation. A large amount of ice-cold diethyl ether was added to precipitate the product. The product was collected by filtration to obtain F-127-N3. Based on this, nucleic acid primers are modified around the micelles using mild click chemistry, and a stable nucleic acid hydrogel shell is formed based on the constructed micromineralized rolling circle amplification system, thereby forming a novel core-shell nanocapsule structure to achieve continuous release of encapsulated molecules.

[0039] A novel covalently coupled G4-Hemin DNAzyme (Co-G4H) can be loaded into a nucleic acid hydrogel shell via base complementary pairing. Co-G4H, by covalently crosslinking the Hemin catalytic core to the G4 sequence end, highly mimics horseradish peroxidase activity and can act as a highly efficient ROS scavenger, effectively improving local oxidative stress. Through this novel nanocapsule co-delivery system, Co-G4H and rapamycin can be continuously released intraocularly, synergistically remodeling the optic nerve protective microenvironment in glaucoma and significantly alleviating the rapid apoptosis of RGCs.

[0040] The present invention will be described in detail below through specific examples and embodiments. It should also be understood that the following embodiments are only for specific illustration of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0041] (I) Synthesis of Rapamycin-G4H Nanocapsules (RG Nc) The specific steps for synthesizing rapamycin-G4H nanocapsules (RG Nc) are as follows: S1. Weigh 100 mg of F-127-NHS and dissolve it in an appropriate amount of chloroform. Add 2.5 eq. of azidopropylamine and 2.5 eq. of triethylamine and dissolve completely. React at room temperature for 0.5 h. Remove the solvent by vacuum distillation, add a large amount of ice-cold diethyl ether to precipitate, filter and collect the product to obtain F-127-N3. Rapamycin micelles were prepared by thin-film hydration method: 3 mg rapamycin and 25 mg F-127-N3 were added to an EP tube, and 3 mL of anhydrous ethanol was added to the EP tube. The mixture was sonicated in a water bath until completely dissolved to obtain a rapamycin / poloxam 127 ethanol solution. Subsequently, the rapamycin / poloxam 127 ethanol solution in the EP tube was transferred to a 50 mL round-bottom flask, and the round-bottom flask was placed in a rotary evaporator and rotary evaporated for 10 min at a temperature of 40 °C and a rotation speed of 50 rpm to completely remove ethanol from the system. Then, add 3 mL of 1×PBS buffer to a round-bottom flask, sonicate in a water bath to reconstitute, and obtain rapamycin micelles, which are then stored at 4°C for later use.

[0042] S2. Modification of rapamycin micelles with dibenzocyclooctylene amplification primers (DBCO-Amplification Primer) via click chemistry reaction. The specific steps are as follows: rapamycin micelles and dibenzocyclooctylene-modified amplification primers are mixed in a 1:1 molar ratio and reacted at 37°C for 1 h to obtain rapamycin micelles modified with amplification primers (RAPA RMs modified with amplification primers). S3. Using linear DNA molecules as templates, a circular amplification template is synthesized through an intramolecular circularization reaction: 1µM linear template, 1µM ligation primer, and 10µM T4 DNA ligase are incubated together at 22℃ for 2 hours in 1×T4 DNA ligase reaction buffer (containing 66mM Tris-HCl, 6.6mM MgCl2, and 10mM DTT, where DTT is dithiothreitol, pH=7.6). During this process, T4 DNA ligase catalyzes the ligation of the two ends of the phosphorylated linear template mediated by the primers to form a circular DNA molecule. Then, the system was treated at 60°C for 15 min to inactivate the T4 DNA ligase, and then stored at 4°C for later use. The nucleotide sequences of the linear template and the ligation primers are shown in Table 1: Table 1 Sequence List S4. Under 1×phi29 DNA polymerase reaction buffer conditions (33mM Tris-Cl (pH=7.9, 37℃), 10mM MgCl2, 66mM KCl, 0.1% (v / v) Tween 20, 1mM DTT, Tween 20 is Tween-20, DTT is 1,4-dithiothreitol), 100nM circular amplification template and 400nM Amplification primer-modified RAPARMs, 5U phi29 DNA polymerase, 10U pyrophosphatase, and 1 mM dNTPs were thoroughly mixed. Additionally, 1µM Co-G4-Hemin DNAzyme was added to hybridize with the amplified single-stranded complementary sequence. The above components constituted a micromineralization-amplification system, which was incubated at 37℃ for 2 h to obtain rapamycin-G4H nanocapsules (RG Nc). All concentrations refer to final concentrations. The prepared RG Nc was stored at 4℃ for later use.

[0043] Electron microscopy was performed on untreated rapamycin (Ms), rapamycin micelles modified with amplification primers (RAPA RMs), and rapamycin-G4H nanocapsules (RG Nc), and the results are as follows: Figure 1 As shown in A; The diameter of rapamycin-G4H nanocapsules (RG Nc) was analyzed using transmission electron microscopy, and the results are as follows: Figure 1 As shown in B; Elemental energy dispersive spectroscopy (EDS) analysis of rapamycin-G4H nanocapsules (RG Nc) was performed using transmission electron microscopy. The results are as follows: Figure 1 As shown in C; Zeta potential analysis of rapamycin-G4H nanocapsules (RG Nc) was performed using a particle size analyzer, and the results are as follows: Figure 1 As shown in D; Infrared spectroscopy analysis of rapamycin-G4H nanocapsules (RG Nc) was performed using an infrared spectrometer, and the results are as follows: Figure 1 As shown in E; The rapamycin-G4H nanocapsules (RG Nc) were characterized by circular dichroism spectroscopy, and the results are as follows: Figure 1 As shown in F.

[0044] Depend on Figure 1 The constructed nanocapsules exhibit a complete core-shell structure and are loaded with Co-G4H and rapamycin. This result demonstrates the successful construction of the nanocapsules.

[0045] (ii) RG Nc effectively regulates R28 Glu Cell apoptosis and inflammatory state Rat retinal progenitor cell line (R28 cells) was used to construct a glaucoma neuroexcitotoxicity model (i.e., R28 cells) by glutamate stimulation of NMDA receptors. Glu (Cell model), the specific steps are: initial density 1.5×10 4 Cells / well were seeded into 96-well plates and cultured in glutamine-free, low-glucose DMEM medium containing 10 mM glutamate at 37°C and 5% CO2 for 6 h.

[0046] In R28 Glu Based on the cell model, the following groups were set up: Normal R28 cell group (WT): No treatment was performed; R28 Glu Model cell group: R28 cells were induced with glutamate according to the above system; Free Co-G4H treatment group (Co-G4H): 1 μM of free Co-G4H was added to the model cells for treatment; this refers to the final concentration. Rapamycin micelles (RMs) treatment group: Rapamycin micelles were added to the model cells and the final concentration of rapamycin micelles was 1 μM. RG Nc treatment group: RG Nc was added to the model cells for treatment, and the final concentration of RG Nc was 1 μM. Then, the RG Nc alleviation of R28 in different groups of cells was analyzed using the CCK8 assay. Glu The apoptosis capacity of the cell model was determined by the following steps: R28 cells were initially placed at a density of 1.5 × 10⁶ cells / year. 4 R28 cells were seeded per well in 96-well plates and cultured in glutamine-free, low-glucose DMEM medium containing 10 mM glutamate at 37°C and 5% CO2 for 6 h. Subsequently, R28 cells were treated with the sample from each group at a 1:9 volume ratio. Finally, a quantitative CCK solution was added, and the plates were incubated at 37°C for 1 h. Absorbance was measured at 450 nm using a multi-functional microplate luminescence detector. The anti-apoptotic activity of each group was assessed by observing cell viability. Results are shown below. Figure 2 As shown in A; Table 2. Nucleotide sequences of primers The expression level of the apoptosis-related gene Bax in different groups of cells was detected using qPCR. The specific steps were as follows: R28 cells were collected, and cell lysis and total RNA extraction were performed using a commercially available SteadyPure kit. RNA concentration was determined using a nano-titer spectrometer. Reverse transcription was then performed according to the Evo M-MLV kit. Finally, qPCR detection was performed according to the kit instructions, using GADPH as an internal control gene. The nucleotide sequences of the primers used are shown in Table 2. The results are as follows. Figure 2 As shown in B; The expression level of the apoptosis-related gene Bcl2 in different groups of cells was detected by qPCR. The specific steps were as follows: R28 cells were collected, and cell lysis and total RNA extraction were performed using a commercially available SteadyPure kit. RNA concentration was determined using a nano-titer spectrometer. Reverse transcription was then performed according to the Evo M-MLV kit. Finally, qPCR detection was performed according to the kit instructions, using GADPH as an internal control gene. The nucleotide sequences of the primers used are shown in Table 2. The results are as follows. Figure 2 As shown in C; The expression level of Caspase3, a cell apoptosis-related gene, in different groups of cells was detected by qPCR. The specific steps were as follows: R28 cells were collected, and cell lysis and total RNA extraction were performed using a commercially available SteadyPure kit. RNA concentration was determined using a nano-titer spectrometer. Reverse transcription was then performed according to the Evo M-MLV kit. Finally, qPCR detection was performed according to the kit instructions, using GADPH as an internal control gene. The nucleotide sequences of the primers used are shown in Table 2. The results are as follows. Figure 2 As shown in D; The expression level of the inflammation-related gene TLR4 in different groups of cells was detected by qPCR. The specific steps were as follows: R28 cells were collected, and cell lysis and total RNA extraction were performed using a commercially available SteadyPure kit. RNA concentration was determined using a nano-titer spectrometer. Reverse transcription was then performed according to the Evo M-MLV kit. Finally, qPCR detection was performed according to the kit instructions, using GADPH as an internal control gene. The nucleotide sequences of the primers used are shown in Table 2. The results are as follows. Figure 2 As shown in E; The expression levels of the inflammation-related gene TNFα in different groups of cells were detected by qPCR. The specific steps were as follows: R28 cells were collected, and cell lysis and total RNA extraction were performed using a commercially available SteadyPure kit. RNA concentration was determined using a nano-titer spectrometer. Reverse transcription was then performed according to the Evo M-MLV kit. Finally, qPCR detection was performed according to the kit instructions, using GADPH as an internal control gene. The nucleotide sequences of the primers used are shown in Table 2. The results are as follows. Figure 2 As shown in F; The expression level of the inflammation-related gene IL6 in different groups of cells was detected by qPCR. The specific steps were as follows: R28 cells were collected, and cell lysis and total RNA extraction were performed using a commercially available SteadyPure kit. RNA concentration was determined using a nano-titer spectrometer. Reverse transcription was then performed according to the Evo M-MLV kit. Finally, qPCR detection was performed according to the kit instructions, using GADPH as an internal control gene. The nucleotide sequences of the primers used are shown in Table 2. The results are as follows. Figure 2 As shown in G; The ability of RG Nc to alleviate apoptosis in an R28Glu cell model was analyzed by flow cytometry. The specific steps were as follows: R28 cell apoptosis was detected using the commercially available Alexa Fluor 488 Annexin V / PI dead cell apoptosis detection kit. Specifically, R28 cells from each group were collected after treatment and stained with Alexa Fluor 488 Annexin and PI at room temperature for 15 minutes. The stained cells were analyzed by flow cytometry, and fluorescence intensity was measured using FlowJo 10.10 software (Tree Star, Ashland, OR, USA). Results are as follows: Figure 2 As shown in H; Immunofluorescence analysis was performed on the expression levels of TRL4 in cells from different groups. The specific steps were as follows: a coverslip was placed at the bottom of each well of a 24-well plate, and R28 cells were introduced at an initial density of 1.0 × 10⁶ cells / well. 5R28 cells / well were seeded in 24-well plates and cultured in glutamine-free, low-glucose DMEM medium containing 10 mM glutamate at 37°C and 5% CO2 for 6 h. Then, the samples from each group were added to the medium at a 1:9 volume ratio to treat R28 cells. After treatment, the cells were fixed with 4% paraformaldehyde for 15 min. Following washing with PBS buffer, the cells were permeabilized with 0.1% Trixton-X-100 for 10 min. Finally, the cells were blocked with 4% donkey serum for 30 min, and then incubated overnight at 4°C with a diluted primary antibody solution containing 5% BSA (the primary antibody was a TLR4 monoclonal antibody, 10 μL, prepared by a 1:300 dilution). After washing three times with PBS, the cells were incubated with a secondary antibody (Alexa Fluor 594-labeled rabbit anti-mouse IgG, 10 μL) at 37°C for 60 min. The sample was then washed three times with PBS and stained with DAPI; images were then acquired using a confocal laser scanning microscope (Zeiss LSM880, Germany), and the results are shown below. Figure 2 As shown in Figure I.

[0047] Depend on Figure 2 As shown in A and 2H, glutamate stimulation reduced R28 cell viability to approximately 30% compared to the WT group. However, the addition of RGNc significantly restored cell viability, approaching the WT group level (2A). Flow cytometry results revealed that Model cells exhibited significant apoptosis, with 64% of cells undergoing late apoptosis. After treatment, the proportion of late apoptotic cells in the Co-G4 and RMs groups decreased to 35.2% and 40.8%, respectively, while the RG Nc group showed the lowest apoptosis rate at only 29.4%, approaching the WT group level. Early and late apoptosis showed the same trend (2H).

[0048] Depend on Figure 2 From B to 2D, the expression levels of apoptosis-related genes were detected, showing that Bax, Bcl-2, and Caspase3, among other apoptosis-related genes, were present at R28. Glu The expression of RG Nc was significantly high in all cell models, but the expression level was significantly reduced after treatment with RG Nc (2B to 2D). This result indicates that RG Nc can effectively reverse the R28 expression pattern. Glu The apoptotic state of cells has the potential to protect optic nerve cells in glaucoma.

[0049] From 2E to 2G, the expression level detection results of the inflammation-related gene IL6 showed that R28 GluIn the cell model, the levels of TLR4, TNFα, and IL6 were significantly increased compared to the WT group, indicating a significant inflammatory state. After treatment with Co-G4H and RMs, the expression levels of inflammatory genes decreased, while the combined use of RG Nc achieved the best recovery effect (2E to 2G).

[0050] The above results indicate that the combined use of Co-G4H in RG Nc and rapamycin can effectively regulate R28. Glu The immune inflammatory state of cells is beneficial for rescuing cells from rapid apoptosis in the cell model.

[0051] (iii) RG Nc can effectively alleviate R28 Glu cellular oxidative stress Validation of intracellular ROS scavenging capacity: ROS levels in cells of each group were monitored using a 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. The specific steps were as follows: ROS levels in cells of different treatment groups were measured using a commercially available reactive oxygen species (ROS) detection kit. The specific conditions were as follows: a coverslip was placed at the bottom of each well of a 24-well plate, and R28 cells were introduced at an initial density of 1.0 × 10⁶ cells / well. 5 R28 cells were seeded per well in 24-well plates and cultured in glutamine-free, low-glucose DMEM containing 10 mM glutamate at 37°C and 5% CO2 for 6 h. Subsequently, R28 cells were treated with samples from each group at a 1:9 volume ratio. 1×PBS containing 10 μM DCFH-DA was added, and the cells were incubated at 37°C in the dark for 30 min. After washing, intracellular ROS accumulation was measured using a multi-functional microplate luminescence detector (λex = 492–495 nm; λem = 517–527 nm). Cell imaging was performed under a fluorescence microscope, and the results are shown below. Figure 3 As shown in A; The ability of RG Nc to alleviate mitochondrial membrane potential imbalance in R28Glu cells was verified using JC-1 fluorescent probe staining. The specific steps were as follows: ΔΨm was measured using the JC-1 mitochondrial membrane potential assay kit. The specific experimental conditions were as follows: a coverslip was placed at the bottom of each well of a 24-well plate, and R28 cells were introduced at an initial density of 1.0 × 10⁶ cells / well. 5 R28 cells were seeded per well in 24-well plates and cultured in glutamine-free, low-glucose DMEM medium containing 10 mM glutamate at 37°C and 5% CO2 for 6 h. Subsequently, R28 cells were treated with samples from each group at a 1:9 volume ratio; then 0.5 mL of JC-1 staining working solution was added, and the cells were incubated at 37°C for 20 min. After centrifugation and washing, the stained cells were imaged and photographed under a fluorescence microscope. The results are shown below. Figure 3 As shown in B; The expression level of NRF2, a gene related to cellular oxidative stress, was detected in different groups of cells. The specific steps were as follows: R28 cells were collected, and cell lysis and total RNA extraction were performed using a commercially available SteadyPure kit. RNA concentration was determined using a nano-titer spectrometer. Reverse transcription was then performed according to the Evo M-MLV kit. Finally, qPCR was performed according to the kit instructions, using GADPH as an internal control gene. The nucleotide sequences of the primers used are shown in Table 2. The results are as follows. Figure 3 As shown in C; The expression levels of SOD2, a gene related to cellular oxidative stress, were detected in different groups of cells. The specific steps were as follows: R28 cells were collected, and cell lysis and total RNA extraction were performed using a commercially available SteadyPure kit. RNA concentration was determined using a nano-titer spectrometer. Reverse transcription was then performed according to the Evo M-MLV kit. Finally, qPCR was performed according to the kit instructions, using GADPH as an internal control gene. The nucleotide sequences of the primers used are shown in Table 2. The results are as follows. Figure 3 As shown in D; The expression level of APOC3, a gene related to cellular oxidative stress, was detected in different groups of cells. The specific steps were as follows: R28 cells were collected, and cell lysis and total RNA extraction were performed using a commercially available SteadyPure kit. RNA concentration was determined using a nano-titer spectrometer. Reverse transcription was then performed according to the Evo M-MLV kit. Finally, qPCR was performed according to the kit instructions, using GADPH as an internal control gene. The nucleotide sequences of the primers used are shown in Table 2. The results are as follows. Figure 3 As shown in E.

[0052] Depend on Figure 3 As can be seen from A, compared to the WT group, R28 Glu The presence of a bright green fluorescent signal in the cells indicates a large accumulation of ROS molecules. However, the addition of Co-G4H and RG Nc significantly reduced the green fluorescent signal in R28 cells. This result demonstrates that the Co-G4H DNAzyme can exert highly efficient peroxidase-like activity within cells, effectively clearing accumulated ROS molecules. Furthermore, abnormal ROS accumulation induces the opening of mitochondrial bimembrane permeability pores, altering membrane permeability and leading to membrane potential imbalance, ultimately promoting apoptosis.

[0053] Depend on Figure 3 As shown in Figure B, under normal cellular conditions (WT group), JC-1 accumulates in the mitochondrial matrix and produces strong red fluorescence. However, under oxidative stress (Model group), JC-1 exists only as monomers in the mitochondrial matrix, producing bright green fluorescence, indicating a decrease in mitochondrial membrane potential and loss of membrane function. The addition of RG Nc makes R28... GluThe cells exhibited bright red fluorescence, and the green effect produced by JC-1 monomers was significantly reduced, indicating that RG Nc protects mitochondria from damage through efficient ROS clearance and alleviates the resulting apoptosis.

[0054] Depend on Figure 3 From C to 3E, it can be seen that genes such as NRF2, SOD2, and SOD1 are present in R28. Glu The ROS molecules were significantly overexpressed in the cell model, but gradually returned to normal levels after treatment with RG Nc. This result indicates that the clearance of ROS molecules prevents compensatory gene expression in cells.

[0055] (iv) RG Nc can effectively alleviate the rapid apoptosis of RGCs in NMDA-induced glaucoma model mice. All mice were anesthetized by intraperitoneal injection of a saline mixture of chlorpromazine (80 mg / kg) and ketamine (16 mg / kg), and the surgical procedure was performed under a stereomicroscope. Mydriasis was achieved using 0.5% tropicamide and epinephrine eye drops, and local anesthesia was administered with 0.5% oflocaine hydrochloride eye drops. A 30-gauge needle was inserted along the limbus into the vitreous cavity, and 2 μL of N-methyl-D-aspartic acid (NMDA) was injected into each eye. Tobramycin-dexamethasone eye ointment was applied post-injection to prevent infection. Five days after injection, the mice were sacrificed, and the eyeballs were removed with forceps for subsequent studies.

[0056] Based on the NMDA mouse model, this application sets up the following groups: Normal mouse group: No treatment was given; NMDA model mouse group: NMDA injection-induced ocular neurotoxicity mice, the method is the same as above; Free Co-G4 / Hemin DNAzyme treatment group: Model mice were treated with free Co-G4 / Hemin DNAzyme. The specific steps were: NMDA injection was performed simultaneously with the injection of 1 μM Co-G4 / Hemin DNAzyme (this refers to the final concentration). Free rapamycin micelle treatment group: The model mice were treated with free rapamycin micelles. The specific steps were: NMDA injection was performed while simultaneously injecting 1 μM rapamycin micelles. RG Nc treatment group: The model mice were treated with RG Nc. The specific steps were: NMDA injection was performed while simultaneously injecting 1 μM RG Nc (final concentration). Observe the changes in behavior, visual function, and retinal RGCs in each group of mice. Specifically: Visuomotor tests were performed on mice in different groups, including parameters such as the visuomotor index, forward follow-up time, and reverse follow-up time. The specific steps were as follows: The visual ability of each group of mice was analyzed using the OptoTrack animal visuomotor response analysis system. The mice were placed on a central elevated platform and allowed to move freely, while vertical sinusoidal stripe patterns were projected onto surrounding LCD screens. These stripes rotated horizontally at a speed of 10° / s in randomly assigned clockwise or counterclockwise directions. Visual stimuli were presented at the optimal spatial frequency (0.2 cycles / °). Each stimulus was displayed for 45 seconds in a random order and direction. Each mouse underwent at least 10 trials, and the average performance value was used for analysis. Head movements in response to stimuli were recorded by a top-mounted camera and automatically tracked using the OptoTrack XR-OT101 system (OptoTrack Version 4, XINRUN). The results are as follows: Figure 4 As shown in A; Mice in different groups underwent open-field testing to measure parameters including active steps, activity level, and time spent in a stagnant state. The specific steps were as follows: Open-field behavior was assessed within an opaque cubic enclosure (43.2cm × 43.2cm × 30.5cm). Mice were acclimatized to the testing room for 30 minutes before the experiment to reduce stress from the new environment. Each animal was then gently placed in the center of the enclosure and allowed to explore freely for 5 minutes. Movement distance was automatically recorded using an infrared beam interruption system, and the motor activity was analyzed and evaluated using a behavior tracking system (SOF-842; MedAssociates Inc.). The enclosure was cleaned between each test to eliminate odor cues. Results are as follows: Figure 4 As shown in B; Brn3a fluorescent staining was performed on mouse retinal slices from different groups. The specific steps were as follows: The removed mouse eyeballs were fixed with 4% paraformaldehyde solution for 1 h at room temperature. After fixation, the eyeballs were equilibrated in fresh PBS buffer for 30 minutes. After separation of the retina, the eyeballs were blocked and permeabilized with blocking buffer (PBS containing 4% donkey serum and 0.5% Triton X-100) at room temperature for 1 h. Then, they were incubated overnight at 4 °C with primary antibody diluted with blocking buffer (the primary antibody was prepared by diluting the blocking buffer with Brn3a monoclonal antibody at a ratio of 1:300; the primary antibody was Brn3a monoclonal antibody, and the volume of the primary antibody was 10 μL). After washing three times with PBS, the slices were incubated with secondary antibody (specifically, rabbit anti-mouse IgG labeled with Alexa Fluor 488, and the volume of the secondary antibody was 10 μL) at room temperature for 1 h, and finally counterstained with DAPI. After the coverslip was mounted, images were acquired using a confocal laser scanning microscope (Zeiss LSM 880, Germany), including the fields of view of both the central and peripheral regions, as well as RGC cell statistics. The results are as follows: Figure 4 As shown in C, the staining results are as follows: Figure 4 As shown in D; Hematoxylin and eosin (H&E) staining was performed on retinal sections from different groups of mice. The specific steps were as follows: First, the eyeballs were completely removed within 15-30 minutes after mouse sacrifice and quickly fixed in 4% neutral paraformaldehyde for 48 hours. The fixed tissue was dehydrated with a gradient of ethanol (70%→100%), cleared with xylene, and then embedded in molten paraffin to form blocks. After freezing the paraffin blocks for 30 minutes, 7μm thin sections were cut using a paraffin microtome, spread in 40℃ warm water, retrieved from poly-L-lysine-treated slides, and baked at 60℃ for 2 hours. The sections were then dewaxed with xylene, rehydrated with a gradient of ethanol to distilled water, stained with hematoxylin (8 minutes), differentiated with 1% hydrochloric acid ethanol for a few seconds, blued with tap water for 10 minutes, stained with eosin for 5 minutes, dehydrated with ethanol, cleared with xylene, mounted with neutral resin, and observed under a microscope. The results are as follows: Figure 4 As shown in E; The retinas of different groups of mice were characterized using transmission electron microscopy (TEM). The results are as follows: Figure 4 As shown in F.

[0057] Depend on Figure 4 As shown in A and 4B, the visual-motor index and positive follow-up time of NMDA-treated NMDA model mice were significantly lower than those in the WT group, while the negative follow-up time was longer. After RG Nc treatment, the visual-motor index and positive follow-up time of the mice were significantly restored, showing a clear difference from the Model group. The results of the mining experiment showed the same trend: the total number of steps and active time of RG Nc-treated mice were significantly higher than those in the Model group, while the time spent on stereotyped behaviors was significantly reduced. Behavioral data indicate that the behavior of mice treated with RG Nc was significantly improved compared to the NMDA model group and comparable to that of the WT group.

[0058] Depend on Figure 4 As shown in C to 4E, statistical analysis of cell counts clearly revealed a significant reduction in RGCs in the retina of the model mice compared to the WT group. RG Nc treatment significantly restored the number of RGCs, superior to the Co-G4H and RMs groups. Compared to the continuous and dense RGC cell layer in the WT group, NMDA treatment resulted in only scattered RGCs, while RG Nc treatment significantly restored RGC numbers. HE staining results also showed the same phenomenon. These results indicate that RG Nc treatment can significantly improve the rapid apoptosis of RGCs, effectively protecting optic nerve cells in a glaucoma model.

[0059] Depend on Figure 4As shown in Figure F, transmission electron microscopy revealed that NMDA-treated retinal RGCs mitochondria exhibited a vacuolated state, while RGCs-treated mitochondria maintained their cristae structure, indicating effective restoration of mitochondrial function. These results demonstrate that RG Nc exerts a highly effective antioxidant and immunomodulatory effect in vivo, effectively resisting the damage to mitochondrial function caused by ROS, thereby alleviating rapid RGC apoptosis and protecting visual function in glaucoma model mice.

[0060] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for preparing rapamycin-G4H nanocapsules, characterized in that, The preparation method includes the following steps: S1. Rapamycin micelles were prepared using a thin-film hydration method; S2. Rapamycin micelles were modified with dibenzocyclooctyne amplification primers by click chemistry to obtain primer-modified rapamycin micelles. S3. Using linear DNA molecules as templates, circular amplification templates are synthesized through intramolecular circularization reactions; S4. Under the conditions of thermophilic DNA polymerase reaction buffer, the circular amplification template, rapamycin micelles modified with amplification primers, thermophilic DNA polymerase, pyrophosphatase, dNTPs and Co-G4-heme DNAase are incubated to prepare the rapamycin-G4H nanocapsules.

2. The preparation method according to claim 1, characterized in that, The preparation of rapamycin micelles by thin-film hydration method includes: adding rapamycin and a nonionic surfactant to anhydrous ethanol to obtain a mixture, rotary evaporating the mixture, adding a buffer solution, and reconstituted by ultrasonication in a water bath to obtain rapamycin micelles.

3. The preparation method according to claim 2, characterized in that, The mass ratio of rapamycin to nonionic surfactant is 2-4:20-30.

4. The preparation method according to claim 2, characterized in that, During the rotary evaporation process, the temperature is 35-45℃, the rotation speed is 40-60rpm, and the evaporation time is 8-15min.

5. The preparation method according to claim 1, characterized in that, Modifying dibenzocyclooctylene amplification primers on the surface of rapamycin micelles via click chemistry includes: mixing rapamycin micelles with the modified dibenzocyclooctylene amplification primers at a molar ratio of 0.5-1.5:0.5-1.5 and reacting at 35℃-40℃ for 0.5-1.5 h.

6. The preparation method according to claim 1, characterized in that, The synthesis of the circular amplification template includes: co-incubating a linear template, ligation primers, and T4 DNA ligase in a T4 DNA ligase reaction buffer, followed by inactivation of the T4 DNA ligase to obtain the T4 DNA ligase.

7. The preparation method according to claim 6, characterized in that, The molar ratio of the linear template to the ligation primer is 0.5-2:0.5-2.

8. The preparation method according to claim 1, characterized in that, The molar ratio of the circular amplification template to the rapamycin micelles modified with amplification primers is 90-110:450-550.

9. A rapamycin-G4H nanocapsule prepared according to any one of claims 1-8.

10. The use of the rapamycin-G4H nanocapsules as described in claim 9 in the preparation of a medicament for the treatment of acute glaucoma.

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