Preparation of a complex nanoparticle loaded with a ripk1 inhibitor and its polymer carrier and use in ophthalmic drugs

Abstract

The application belongs to the field of medicine, and particularly relates to a polymer carrier and a RIPK1 inhibitor composite nanoparticle loaded by the polymer carrier. The application further provides preparation of the carrier and the nanoparticle and application of the carrier and the nanoparticle in ophthalmic drugs. The carrier and the nanoparticle have good ROS scavenging capacity, can inhibit a significant up-regulation of a necroptosis pathway, reduce death of RGCs, and effectively protect visual function of mice.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals, specifically relating to the field of drug carrier materials. Background Technology

[0002] Glaucoma is currently the leading cause of irreversible blindness worldwide, threatening the vision health of tens of millions of people. Its typical pathological feature is the progressive death of retinal ganglion cells (RGCs) and the loss of their axons. Currently, it is believed that abnormally elevated oxidative stress in RGCs leads to mitochondrial dysfunction, producing large amounts of oxygen free radicals (ROS), which then attack cells and tissues, activating a series of death signaling pathways, including necrosis and apoptosis, resulting in the death of a large number of RGCs and ultimately causing the pathological damage of glaucoma.

[0003] Necrotic apoptosis, a recently proposed cell death model characterized by programmed necrosis, is closely related to the occurrence and development of various clinical diseases. Its typical characteristic is the activation of key cellular pathways RIPK1 / RIPK3 / MLKL, leading to multi-organelle damage and necrosis, disruption of cell membrane integrity, and the release of large amounts of inflammatory factors, resulting in further cell death. Numerous studies have shown that RIPK1 is a key regulator of apoptosis, necrosis, and inflammatory pathways, and it has become one of the effective targets for the treatment of various diseases, including glaucoma, neurodegenerative diseases, autoimmune diseases, and inflammation.

[0004] To improve the treatment efficacy of glaucoma, two main approaches have been developed to protect rhabdomyosarcoma cells (RGCs) and reduce their damage, thereby treating glaucoma, targeting the aforementioned key pathogenic factors. One approach involves using small-molecule drugs or nanoparticle-based drug delivery systems to consume excess reactive oxygen species (ROS) within RGCs. However, these small-molecule drugs are generally easily metabolized in vivo, have poor water solubility, and are difficult for cells to take up, limiting their widespread application in vivo. Furthermore, while a range of novel nanomaterials, such as inorganic materials, organic polymers, and nanozymes, can consume ROS within RGCs and thus protect them from damage, their complex mass chemical synthesis, difficulty in quality control, singular mechanisms of action, and potential long-term toxicity limit their protective effect on RGCs, making them less of a first choice.

[0005] Secondly, gene therapy or small molecule drugs are used to selectively inhibit necroptosis in RGCs. On the one hand, viral vectors are used to deliver short hairpin RNA (shRNA) into RGCs, silencing key molecules RIPK1 or RIPK3, thereby inhibiting necroptosis and protecting damaged RGCs. However, viral vectors pose certain biosafety risks. On the other hand, through screening a large number of compounds, researchers have discovered that small molecule compounds such as Necrostatins can directly act on various cells, selectively inhibiting key proteins in the necroptosis pathway. Among these inhibitors, Necrostatin-1 (NEC) is a selective allosteric inhibitor of the death domain receptor-associated adapter kinase RIP1. It can inhibit RIPK1 activity, reduce the formation of RIPK1 / RIPK3 necrosomes, leading to reduced MLKL phosphorylation, and thus alleviating cell membrane rupture and other cascade reactions of necroptosis, making it a representative inhibitor of highly effective necroptosis inhibition. In addition, NEC has been widely reported to inhibit cell necroptosis in conditions such as acute kidney injury, neurodegenerative diseases, and myocardial diseases, exerting a protective effect. However, to date, there are few specific reports on the use of NEC to inhibit necrotizing apoptosis and protect RGCs for the treatment of glaucoma. NEC is expected to become a potential clinical treatment drug for glaucoma.

[0006] However, NECs need to enter RGCs (Retinal Gestational Cells) to exert their efficacy effectively. Therefore, delivering NECs into the ocular RGCs via a delivery system is essential, but this strategy has always faced a series of problems. On the one hand, ocular drug delivery suffers from poor corneal-conjunctival permeability and the blood-eye barrier, resulting in a very limited amount of drug reaching the retina. On the other hand, intraocularly injected drugs have short retention times, unsatisfactory cell absorption rates, and short durations of action, leading to the need for more frequent injections, which in turn causes greater trauma to the eye and increases the risk of infection. Furthermore, excessive drug dosage can cause significant toxic side effects. Therefore, developing novel NEC drug delivery systems that can efficiently remove ROS within RGCs, inhibit necrosis and apoptosis, and increase drug retention within the eye, enabling efficient entry into RGCs and sustained release, can better protect RGCs and alleviate glaucoma symptoms—a pressing need in current glaucoma treatment research. Summary of the Invention

[0007] In view of the problems of unsatisfactory efficacy and short duration of action of active ingredients in ophthalmic drugs such as RIP1 inhibitors, the primary objective of this invention is to provide a novel drug-loaded polymer designed to improve the efficacy of active ingredients in ophthalmic drugs.

[0008] A second objective of this invention is to provide a method for preparing the drug-loaded polymer.

[0009] A third objective of this invention is to provide the application of the drug-loaded polymer in pharmaceutical manufacturing, particularly in ophthalmic drugs.

[0010] The fourth objective of this invention is to provide the drug-loaded polymer-RIPK1 inhibitor composite nanoparticles.

[0011] The fifth objective of this invention is to provide the preparation of the drug-loaded polymer-RIPK1 inhibitor composite nanoparticles and their application in pharmaceuticals.

[0012] The sixth objective of this invention is to provide a medicament for treating glaucoma comprising the aforementioned drug-loaded polymer-RIPK1 inhibitor composite nanoparticles.

[0013] A drug-loaded polymer having the structural formula of Formula 1:

[0014]

[0015] Formula 1

[0016] The n mentioned is an integer from 1 to 250;

[0017] x is an integer from 1 to 100;

[0018] The y mentioned is an integer from 1 to 100;

[0019] The z mentioned is an integer from 1 to 100.

[0020] This invention provides a novel drug-loaded polymer. Studies have found that this novel drug-loaded polymer possesses excellent RGC-targeting and ROS-scavenging capabilities, mitigating oxidative stress damage. Furthermore, it significantly upregulates the necroptosis pathway, reducing RGC death and effectively protecting visual function. Additionally, its use as a carrier for drugs, particularly ophthalmic drugs, facilitates synergistic effects.

[0021] Preferably, n is 80-150, and more preferably 100-120.

[0022] The value of x is 1-14, preferably 1-6.

[0023] The value of y is 1-14, preferably 1-6.

[0024] The value of z is 1-35, preferably 2-30.

[0025] This invention also provides a method for preparing the drug-loaded polymer, wherein formulas 2, 3, 4, and 5 are subjected to a polycondensation reaction, followed by an end-capping reaction with formula 6 to obtain the ophthalmic drug-loaded polymer:

[0026]

[0027] In the preparation method of the present invention, the molar ratio of Formulas 2, 3, 4, and 5 is 2-3:1-1.2:1-1.2:0.1-0.5; preferably 2.4-2.6:1-1.1:1-1.1:0.3-0.4.

[0028] Preferably, the weight ratio of Formula 2 to Formula 6 is 1:1 to 2; more preferably, it is 1:1.6 to 1.9.

[0029] Preferably, the solvent for the reaction is at least one of DMF and DMSO;

[0030] Preferably, the reaction temperature is 30–60°C;

[0031] Preferably, the drug-loaded polymer is obtained by dialysis after the reaction.

[0032] The present invention also provides the application of the drug-loaded polymer as a carrier of active pharmaceutical ingredients for the preparation of drugs.

[0033] In a preferred application of this invention, it is used as a carrier for the active ingredient of ophthalmic drugs in the preparation of ophthalmic drugs;

[0034] Preferably, it is used as a carrier for RIPK1 inhibitors in the preparation of ophthalmic drugs for treating progressive RGC death and axonal loss. In this invention, the polymer carrier and the RIPK1 inhibitor are combined to achieve a synergistic effect, which can synergistically improve the ROS of RGCs and reduce progressive RGC death and axonal loss.

[0035] Preferably, the RIPK1 inhibitor is at least one of Necrostatins, RIPA-56, and RIPK1-IN-7; other related RIPK1 inhibitors have problems such as limited efficacy and potential biotoxicity risks.

[0036] Preferably, the ophthalmic drug is a drug for treating glaucoma.

[0037] The present invention also provides a drug-loaded polymer-RIPK1 inhibitor composite nanoparticle, comprising a carrier and the RIPK1 inhibitor loaded thereon; wherein the carrier is the drug-loaded polymer.

[0038] In this invention, thanks to the combination of the drug-loaded polymer structure and the RIPK1 inhibitor, a synergistic effect is achieved. On the one hand, it can clear ROS in RGCs in acute glaucoma pathologically damaged cells and animal models, reducing oxidative stress damage. On the other hand, it can also significantly upregulate the necroptosis pathway, reduce RGC death, and effectively protect the visual function of mice.

[0039] In this invention, the content ratio of drug-loaded polymer and RIPK1 inhibitor can be adjusted as needed. For example, the weight ratio of drug-loaded polymer to RIPK1 inhibitor is 10-50:1-10.

[0040] In this invention, the drug-loaded polymer-RIPK1 inhibitor composite nanoparticles can be composited using conventional liquid-phase or solid-phase methods. For example, the RIPK1 inhibitor and the drug-loaded polymer can be dispersed in an organic solvent, followed by the addition of water, mixing, and then dialysis to obtain the final product.

[0041] The organic solvent mentioned is, for example, DMSO.

[0042] The present invention also provides the application of the aforementioned drug-loaded polymer-RIPK1 inhibitor composite nanoparticles in the preparation of ophthalmic drugs for treating progressive death of RGCs and axonal loss. Preferably, it is used to prepare ophthalmic drugs for treating glaucoma. More preferably, it is used to prepare injectable drug formulations for treating glaucoma.

[0043] The present invention also provides a medicament for treating glaucoma, comprising a pharmaceutically effective amount of the drug-loaded polymer-RIPK1 inhibitor composite nanoparticles;

[0044] Preferably, the medicament for treating glaucoma further comprises pharmaceutically acceptable excipients;

[0045] Preferably, the drug for treating glaucoma is a locally injectable preparation.

[0046] Beneficial effects

[0047] 1. This invention provides a novel drug-loaded polymer with a novel 1-structure, which, based on the combination of the polymer chain and structure, exhibits good ROS response and consumption performance.

[0048] 2. Combining the drug-loaded polymer with RIPK1 inhibitors such as Necrostatins can achieve synergistic effects, further inhibiting necrosis and apoptosis of cells and reducing cell death.

[0049] 3. The materials used in this invention are widely available, the preparation method is simple, and the resulting materials have better performance. Attached Figure Description

[0050] Figure 1 The molecular structure and H-NMR spectrum of the drug-loaded polymer (P1) prepared in Example 1 are shown; x is 4, y is 2, and z is 3.

[0051] Figure 2 The molecular structure and H-NMR spectrum of polymer P2 are shown.

[0052] Figure 3 The molecular structure and H-NMR spectrum of polymer P3 are shown.

[0053] Figure 4 The image shows a transmission electron microscope (TEM) image of the prepared nanoparticle NP1, and the particle size and zeta potential of NP1 and NP2 measured by DLS.

[0054] Figure 5 The study investigated the in vitro scavenging of reactive oxygen species, including H2O2 and ABTS, by the prepared NP1 nanoparticles.

[0055] Figure 6 Immunofluorescence imaging showed that the prepared nanoparticles NP1 possessed cell-targeting (adhesion) capabilities.

[0056] Figure 7 This study investigated the cell protection effects of small molecule inhibitors NP1, NP2, and NP3 on OGD modeling, as well as the effects of NP1 and the macromolecules P1, P2, and P3 alone on normal cells. Specifically, A shows the CCK8 results of R28 cells after OGD modeling with different concentrations of the small molecule inhibitor Necrostatin-1 (NEC); B shows the CCK8 results of R28 cells after OGD modeling with different concentrations of NP1, 100 μM NEC, NP2, and NP3; and C shows the CCK8 results of normal R28 cells after intervention with P1, P2, P3, and NP1, respectively.

[0057] Figure 8 Flow cytometry results for the ability of the prepared nanoparticles NP1 and NP2 to scavenge ROS levels in an oxygen-glucose deprivation cell model.

[0058] Figure 9 The distribution of the prepared NP1 nanoparticles in the retina of mice 1, 2, and 14 days after injection into the mouse eyeball.

[0059] Figure 10 The structure of the retina of normal mice and the retina of mice one week after the prepared nanoparticles NP1 were injected into the mouse eye were evaluated to assess the biosafety of the nanoparticles.

[0060] Figure 11The effects of the small molecule inhibitor NEC and the prepared nanoparticles NP1 and NP2 on the retinal protection of mice after IR modeling are shown in Figure A. Figure A shows the HE staining results of the retina of mice after IR modeling treated with NEC, NP1, and NP2, respectively; Figure B shows the survival rate of RGCs in the retina of mice after IR modeling treated with NEC, NP1, and NP2, respectively. Detailed Implementation Plan

[0061] This study used seven-week-old (20-25g) male C57BL / 6 mice free of specific pathogens. The mouse retinal ischemia-reperfusion (IR) model was established according to previously reported international standard procedures. In short, after puncturing the anterior chamber of the mouse with a 30G needle, physiological saline was infused at a constant pressure difference of 120 mmHg for 40 min.

[0062] I. Case Studies of Polymer Preparation

[0063] M1 Synthesis

[0064] 2,2,5-Trimethyl-1,3-dioxane-5-carboxylic acid (TDCA, 0.5 mmol, 87.1 mg), cholesterol (0.5 mmol, 193.3 mg), DMAP (0.25 mmol, 30 mg), and EDCI (0.75 mmol, 144 mg) were dissolved in 2.5 mL of LDCM and stirred for 15 minutes. Cholesterol was then added in portions to the reaction mixture and the reaction was allowed to proceed overnight. After the reaction was complete, the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography using hexane / ethyl acetate (v:v = 20 / 1) as the eluent to give compound M1.

[0065] M2 Synthesis

[0066] M1 (0.48 mmol, 260 mg) and Amberlyst resin (100 mg) were added to 2.5 mL of DCM and stirred for 12 hours. The solvent was then removed by rotary evaporator. The crude product was purified by silica gel column chromatography using hexane / ethyl acetate (v:v = 2 / 1) as the eluent to give compound M2. ¹H NMR (400 MHz, CDCl₃), δ 5.39 (¹H, m), 4.69 (¹H, m), 3.88 (2H, d, J: 9.6 Hz), 3.70 (2H, d, J: 10.4 Hz), 2.90 (¹H, m), 2.34 (2H, d, J: 7.6 Hz), 1.05 (3H, s), 1.02 (3H, s), 0.91 (3H, d, J: 8.0 Hz), 0.87 (3H, d, J: 2.0 Hz), 0.85 (3H, d, J: 2.0 Hz), 0.67 (3H, s). 28.01, 27.68, 24.29, 23.87, 22.83, 22.58, 21.05, 19.35, 18.74, 17.17, 11.86.

[0067] Example 1

[0068] The synthesis circuit is as follows:

[0069]

[0070] Synthesis of P1

[0071] 2,2'-(propane-2,2-diylbis(thionidyl))bis(ethan-1-ol) (DSB, 0.21 mmol, 41.2 mg), M2 (0.075 mmol, 37.7 mg), DTD (0.21 mmol, 32.0 mg), and CHTA (0.52 mmol, 117.6 mg) were dissolved in 5 mL of anhydrous DMF. After magnetic stirring at 50 °C for 24 hours, mPEG5k-OH (220 mg) was added to the reaction system, and the reaction was allowed to proceed for another 24 hours. Subsequently, the mixture was placed in a dialysis bag (MWCO: 8000 Da) and dialyzed against deionized water for 48 hours. After 48 hours, the solution was freeze-dried under reduced pressure to obtain P1, which was analyzed by 1H NMR.

[0072] P1 Cy5.5 Synthesis

[0073] DSB (0.21 mmol, 41.2 mg), M2 (0.075 mmol, 37.7 mg), DTD (0.21 mmol, 32.0 mg), N-Boc-serine (0.005 mmol, 1 mg), and CHTA (0.52 mmol, 117.6 mg) were dissolved in 5 mL of anhydrous DMF. After magnetic stirring at 50 °C for 24 hours, mPEG5k-OH (220 mg) was added to the reaction system, and the reaction was allowed to proceed for another 24 hours. Subsequently, the mixture was placed in a dialysis bag (MWCO: 8000 Da) and dialyzed against deionized water for 48 hours. After 48 hours, the solution was freeze-dried under reduced pressure to obtain an intermediate. The intermediate (200 mg) was dissolved in 2 mL of anhydrous DMF containing 0.5 mL of TFA. After stirring at room temperature for 4 hours, the mixture was dialyzed against deionized water (MWCO: 8000 Da) and freeze-dried. The obtained polymer was dissolved in 1 ml of DMSO, and then Cy5.5-NHS was added. The solution was stirred at room temperature in the dark for 6 hours, and then dialyzed against deionized water (MWCO: 8000 Da) for 12 hours. After 12 hours, the solution was freeze-dried under reduced pressure to obtain P1Cy5.5.

[0074] Comparative Example 1

[0075] Compared to Example 1, the polymer carrier structure was adjusted, and the different preparation steps are as follows:

[0076] Synthesis of P2

[0077] M2 (0.50 mmol, 251.4 mg) and CHTA (0.50 mmol, 112.1 mg) were dissolved in 3 mL of anhydrous DMF. After magnetic stirring at 50 °C for 24 hours, mPEG5k-OH (363 mg) was added to the reaction system, and the reaction was allowed to proceed for another 24 hours. Subsequently, the mixture was placed in a dialysis bag (MWCO: 8000 Da) and dialyzed against deionized water for 48 hours. After 48 hours, the solution was freeze-dried under reduced pressure to obtain P2, which was analyzed by 1H NMR. Figure 2 ).

[0078] Comparative Example 2

[0079] Compared to Example 1, the polymer carrier structure was adjusted, and the different preparation steps are as follows:

[0080] P3 Synthesis

[0081] DSB (0.25 mmol, 48.1 mg), DTD (0.25 mmol, 38.1 mg), and CHTA (0.52 mmol, 117.6 mg) were dissolved in 5 mL of anhydrous DMF. After magnetic stirring at 50 °C for 24 hours, mPEG5k-OH (199 mg) was added to the reaction system, and the reaction was continued for another 24 hours. Subsequently, the mixture was placed in a dialysis bag (MWCO: 8000 Da) and dialyzed against deionized water for 48 hours. After 48 hours, the solution was freeze-dried under reduced pressure to obtain P3, which was analyzed by 1H NMR. Figure 3 ).

[0082] P3 Cy5.5 Synthesis

[0083] DSB (0.25 mmol, 48.1 mg), DTD (0.25 mmol, 38.1 mg), N-Boc-serine (0.005 mmol, 1 mg), and CHTA (0.52 mmol, 117.6 mg) were dissolved in 5 mL of anhydrous DMF. Similarly, P3Cy5.5 was obtained and purified as described above.

[0084] Example 3

[0085] Preparation of NP1:

[0086] A solution of necrostatin-1 (4 mg) and P1 (40 mg) in DMSO (1 mL) was added dropwise to deionized water (10 mL) with stirring. The mixture was dialyzed in a dialysis bag (MWCO: 8000 Da) for 12 hours to obtain NP1 (transmission electron microscopy image and zeta potential diagram are shown in [reference]). Figure 4 ).

[0087] Comparative Example 4

[0088] Compared with Example 3, the only difference is that P2 is used instead of P1 to obtain NP2.

[0089] Comparative Example 5

[0090] Compared with Example 3, the only difference is that P3 is used to replace P1 to obtain NP3.

[0091] II. Effect Measurement Experiment and Data Analysis

[0092] Example 4 - In vitro ROS determination

[0093] In vitro ROS test data;

[0094] The scavenging ability of NP1 for H2O2 was determined using the hydrogen peroxide method. First, NP1 (25 μM) and 2 mL of PBS containing 50 mM hydrogen peroxide were incubated at room temperature for 5 min, 1 h, 2 h, 4 h, and 8 h, or different concentrations of NP1 (5 μM, 10 μM, 25 μM) and 2 mL of PBS containing 50 mM hydrogen peroxide were incubated at room temperature for 2 h. Then, 50 μL of sample or standard solution (1, 2, 5, 10, 20, 50, 100 μM) was added to a 96-well plate. Next, 100 μL of hydrogen peroxide detection reagent was added to each well. After incubation at room temperature for 30 min, the absorbance at 560 nm was measured using a multi-plate reader to determine the concentration of remaining H2O2 and calculate the H2O2 scavenging ability.

[0095] In the ABTS assay, NP1 sample (25 μM) was mixed with ABTS˙+ solution (7 mM) and incubated in the dark for 1 minute, 30 minutes, 1, 2, and 4 hours, respectively. Subsequently, the absorbance of the mixture at 734 nm was measured using a multi-plate reader. The ABTS˙+ scavenging activity of NP1 was calculated using the following formula: ABTS˙+ scavenging activity (%) = (A0 - Ai) / A0 × 100%. Where A0 and Ai represent the absorbance of the ABTS˙+ solution before and after the addition of the NP1 sample, respectively.

[0096] Test results are available Figure 5 ,pass Figure 5 It was found that the scavenging rate of H2O2 by NP1 increased significantly with increasing NP1 treatment time. Specifically, after 8 hours of NP1 treatment, the scavenging rate of H2O2 reached over 60%. Furthermore, increasing the concentration of NP1 also increased the scavenging rate of H2O2. The ABTS results also showed that the scavenging rate of ABTS˙+ increased significantly with prolonged NP1 treatment time; the absorbance at 734 nm decreased by approximately tenfold with increasing time, and the scavenging of ABTS˙+ by NP1 also exhibited a dose-dependent effect.

[0097] Example 5 - Cell Experiment

[0098] Cell culture and modeling

[0099] R28 cells are retinal progenitor cells with differentiation potential that express the RGC-specific antigen Thy1.1 and are commonly used in vitro to study neuronal function and neuroprotection. R28 cells were cultured in DMEM low-glucose medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. The construction of the oxygen-glucose deprivation (OGD) model, in short, involves replacing the cell culture medium with serum-free and glucose-free medium, culturing in an anoxic incubator for 4 hours, then replacing it with normal culture medium and culturing under standard conditions until the experiment is conducted.

[0100] Cell membrane targeted detection

[0101] Cells were seeded at a density of 1 × 10⁵ cells / well in 24-well plates. In NP1 Cy5.5 After intervening in cells for 15 min, 30 min, and 1 h respectively, the cell culture medium was discarded, and the cells were fixed in 4% paraformaldehyde at 37°C for 10 min. Cell membrane probes were then incubated at 37°C for 5 min, and the slides were mounted for confocal microscopy examination. NP1 Cy5.5 Compared to NP3 Cy5.5 After 15 and 30 minutes of cell intervention, it clearly colocalized with the cell membrane and was taken up by endocytosis. Figure 6 .

[0102] CCK8

[0103] Cells were seeded at a density of 5000 cells / well in 96-well plates. OGD modeling was performed 4 hours after drug intervention. 24 hours after OGD modeling, 10 μl of CCK8 assay reagent was added to 100 μl of culture medium per well, and the plates were incubated at 37°C in the dark for 1 hour. Od values ​​were read at 450 nm using a microplate reader. NP1 showed the most significant protective effect at 100 μmol, and was more effective than small molecule inhibitors NP2 and NP3. Figure 7 .

[0104] ROS Flow Cytometry

[0105] Cells were arranged at a ratio of 1×10 6 Cells were seeded at a density of cells / well in 6-well plates. Twelve hours after OGD modeling, cells from each group were processed into single-cell suspensions and incubated with DCFH-DA at 37°C in the dark for 30 minutes. After staining, the cells were washed twice with PBS and then subjected to quantitative real-time fluorescence detection. NP1 significantly reduced the level of ROS production in OGD-modeled cells compared to NP2. Figure 8 .

[0106] Example 6

[0107] Animal breeding and modeling

[0108] This study used 7-week-old (20-25g) male C57BL / 6 mice without specific pathogens. The IR model (MiceIR) was established according to standard procedures. After puncturing the anterior chamber of the mice with a 30G needle, physiological saline was perfused at a constant rate of 120mmHg for 40 minutes, and drugs were injected intravitreally. The intervention was performed 2 days before model establishment.

[0109] Distribution and residence time of NP1 in the retina

[0110] NP1 Cy5.5Immediately after intravitreal injection in mice at 1, 2, and 14 days, the eyeballs were enucleated and placed in ice-cold PBS solution for careful dissection and rinsing of the retina. The retina was then immersed in 4% paraformaldehyde (pH 7.4) for 1 hour, followed by two 5-minute dips in fresh PBS. Subsequently, at room temperature, the retina was carefully placed on a slide with a drop of antifluorescence quencher and covered with a coverslip. Three equidistant frame samples (center, middle, and periphery) were photographed in each retinal quadrant. NP1 Cy5.5 It can accumulate significantly within the retina, and even two weeks after injection, it still exhibits strong fluorescence expression and distribution. For example... Figure 9 .

[0111] Retinal HE staining

[0112] One week after NP1 intervention in normal mice, paraffin-embedded retinal tissue sections (6 μm) were harvested and cut along the vertical meridians, parallel to the maximum circumference of the eyeball, through the optic disc. The sections were placed on microscope slides, dewaxed, and stained with hematoxylin and eosin. Microscopic images of the stained retina were taken using an inverted fluorescence microscope. It was observed that the retinal structure of the NP1-interventioned mice was similar to that of normal mice, with no retinal damage or toxicity observed. Figure 10 .

[0113] Small molecule inhibitors NEC, NP1, and NP2 were used to treat mice with retinal irritation (IR) models. HE staining of the retina showed that NP1 provided significantly better protection for the retina, especially the RGC layers, than NEC and NP2. Figure 11 .

[0114] Summarize and analyze the test results.

[0115] We designed and synthesized a biodegradable polymer material, P1. The synthesis of P1 involves the polymerization of 1,2,4,5-cyclohexanetetracarboxylic dianhydride (CHTA) and the anhydrides of dihydroxy monomers, including 2,2'-(propane-2,2-diylbis(thioalkyldiyl))bis(ethanol-1-ol) (DSB)), 1,4-dithiaran-2,5-diol (DTD), and dihydroxycholesterol (M2) via a condensation reaction. The polymer is then capped with the hydrophilic polymer mPEG5K-OH, ultimately yielding a ROS-responsive, biodegradable amphiphilic polymer. The design considerations are: 1) Cholesterol is an important component of cell and organelle membrane structures, possessing cell membrane affinity, thus endowing P1 with the ability to bind to cell membranes, making it easier for cells to take up; 2) P1 can degrade under ROS by breaking thioketal bonds, exhibiting low toxicity; 3) DTD in the molecular chain can further consume large amounts of ROS; 4) P1 has hydrophilic PEG blocks, which can self-assemble into nanoparticles in water through hydrophilic-hydrophobic interactions. Subsequently, we encapsulated NEC with P1 to form NP1. In vitro, we demonstrated that NP1 could efficiently enter and accumulate within R28 cells using an oxygen-glucose deprivation (OGD) model. Following this, under high levels of ROS in the OGD pathological model, the thioclase bond in the P1 molecule broke, leading to the release of NEC. NEC then inhibited the function of the key molecule RIPK1, suppressing necrotizing apoptosis. In animal studies, we also established a mouse model of acute glaucoma by injecting saline into the anterior chamber of the eye. IR We verified that, on the one hand, NP1 can consume Mice. IR Excessive ROS within RGCs can alleviate oxidative stress damage; on the other hand, NP1 can inhibit Mice. IR The necrosis and apoptosis of RGCs in mice protect them and restore visual function in glaucoma mice. This study provides new insights for the development of drugs for the clinical treatment of glaucoma.

[0116] The main differences from existing technologies are as follows: First, it is the first time that the classic necrosis and apoptosis inhibitor Necrostatin-1 has been constructed into a nanomedicine for the treatment of glaucoma; second, the cholesterol monomers carried by the nanomedicine have cell membrane affinity, which allows them to enter cells efficiently; third, the nanomedicine has both ROS-responsive release and ROS consumption, that is, it can release the drug in response while clearing away pathologically generated excessive ROS.

Claims

1. A drug-loaded polymer, characterized in that, It has the structural formula of Equation 1: Formula 1 The n mentioned is an integer from 1 to 250; x is an integer from 1 to 100; The y is an integer from 1 to 100; The z mentioned is an integer from 1 to 100.

2. The drug-loaded polymer as described in claim 1, characterized in that, The value of n is 80~150; The value of x is 1 to 14; The value of y is 1 to 14; The value of z is 1 to 35.

3. The drug-loaded polymer as described in claim 1, characterized in that, The n is 100~120; The value of x is 1 to 6; The value of y is 1 to 6; The value of z is 2 to 30.

4. A method for preparing the drug-loaded polymer according to any one of claims 1 to 3, characterized in that, Formulas 2, 3, 4, and 5 are subjected to a polycondensation reaction, followed by a capping reaction with Formula 6 to obtain the ophthalmic drug-loaded polymer. 。 5. The method for preparing the drug-loaded polymer as described in claim 4, characterized in that: The molar ratios of Equations 2, 3, 4, and 5 are 2~3:1~1.2:1~1.2:0.1~0.

5.

6. The method for preparing the drug-loaded polymer as described in claim 5, characterized in that: The molar ratios of Equations 2, 3, 4, and 5 are 2.4~2.6:1~1.1:1~1.1:0.3~0.

4.

7. The method for preparing the drug-loaded polymer as described in claim 4, characterized in that: The weight ratio of Equation 2 to Equation 6 is 1:1~2.

8. The method for preparing the drug-loaded polymer as described in claim 7, characterized in that: The weight ratio of Equation 2 to Equation 6 is 1:1.6~1.

9.

9. The method for preparing the drug-loaded polymer as described in claim 4, characterized in that: The solvent for the reaction is at least one of DMF and DMSO; The reaction temperature is 30~60℃; The drug-loaded polymer was obtained by dialysis after the reaction.

10. The application of a drug-loaded polymer according to any one of claims 1 to 3 or a drug-loaded polymer prepared by the preparation method according to any one of claims 4 to 9, characterized in that, It is used as a carrier for active pharmaceutical ingredients in drug preparation.

11. The application as described in claim 10, characterized in that, It is used as a carrier for the active ingredients of ophthalmic drugs in the preparation of ophthalmic drugs.

12. The application as described in claim 11, characterized in that, It was used as a carrier for RIPK1 inhibitors to prepare ophthalmic drugs for treating progressive death of RGCs and loss of their axons.

13. The application as described in claim 12, characterized in that, The RIPK1 inhibitor is at least one of Necrostatins, RIPA-56, and RIPK1-IN-7.

14. The application as described in claim 13, characterized in that, The drug in question is for the treatment of glaucoma.

15. A drug-loaded polymer-RIPK1 inhibitor composite nanoparticle, characterized in that, This includes RIPK1 inhibitors loaded onto vectors; The carrier is the drug-loaded polymer according to any one of claims 1 to 3 or the drug-loaded polymer prepared by the preparation method according to any one of claims 4 to 9.

16. The drug-loaded polymer-RIPK1 inhibitor composite nanoparticles as described in claim 15, characterized in that, The RIPK1 inhibitor is at least one of Necrostatins, RIPA-56, and RIPK1-IN-7.

17. A method for preparing the drug-loaded polymer-RIPK1 inhibitor composite nanoparticles according to claim 15 or 16, characterized in that, The RIPK1 inhibitor and drug-loaded polymer are dispersed in an organic solvent, then water is added, and the mixture is dialyzed to obtain the final product.

18. The application of the drug-loaded polymer-RIPK1 inhibitor composite nanoparticles according to claim 15 or 16, characterized in that, It was used to prepare ophthalmic drugs for treating the progressive death of RGCs and the loss of their axons.

19. The application of the drug-loaded polymer-RIPK1 inhibitor composite nanoparticles as described in claim 18, characterized in that, It is used to prepare ophthalmic drugs for the treatment of glaucoma.

20. The application of the drug-loaded polymer-RIPK1 inhibitor composite nanoparticles as described in claim 19, characterized in that, It was prepared into an injectable drug formulation for the treatment of glaucoma.

21. A drug for treating glaucoma, characterized in that, The drug-loaded polymer-RIPK1 inhibitor composite nanoparticles of claim 15 or 16 include a pharmaceutically effective amount.

22. The medicament for treating glaucoma as described in claim 21, characterized in that, It also contains pharmaceutically acceptable excipients.

23. The medicament for treating glaucoma as described in claim 21 or 22, characterized in that, It is a locally injectable preparation.

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