A protac nanocomplex, a preparation method and application thereof in a product for treating ophthalmic diseases

Abstract

The application discloses a PROTAC nanocomposite, a preparation method and application thereof in products for treating ophthalmic diseases, relates to the technical field of biological medicine, and the PROTAC nanocomposite comprises a lipid layer and a PROTAC chimera for targeted degradation of ACSL4 wrapped in the lipid layer; wherein the lipid layer comprises an ionizable cationic lipid, cholesterol, DOPE and DSPE-PEG2000; the mass ratio of the ionizable cationic lipid, cholesterol, DOPE, DSPE-PEG2000 and the PROTAC chimera for targeted degradation of ACSL4 is (1.5-2.5):(0.8-1.2):(0.3-0.7):(1.5-2.5):1, based on the mass of the PROTAC nanocomposite; and the PROTAC nanocomposite can be applied to products for treating ophthalmic diseases, in particular, refractory corneal diseases, such as relieving and treating corneal alkali burn.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a PROTAC nanocomposite, its preparation method, and its application in products for treating ophthalmic diseases. Background Technology

[0002] Chemical eye injuries (CEIs) account for 10%–22% of all ocular trauma, with alkali burns, such as those caused by sodium hydroxide and ammonia, being the most severe. An epidemiological study reported that 67.9% of patients included in the study had alkali burns. These injuries trigger a range of pathological changes, including corneal opacity, fibrotic scarring, edema, and neovascularization, severely impairing corneal transparency and visual recovery; 92.5% of such cases ultimately result in visual impairment. The tissue necrosis and protein denaturation caused by alkali exposure trigger an inflammatory response characterized by immune cell infiltration and the release of pro-inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Although phagocytes clear debris to aid corneal repair, activated macrophages produce reactive oxygen species (ROS), upregulate vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs), further exacerbating inflammation, damaging surrounding tissues, and promoting neovascularization. This can lead to corneal edema, vision loss, and a vicious cycle of pathological changes. Therefore, excessive inflammation is a key obstacle to the healing of alkali burns and an important therapeutic target. Currently, glucocorticoids are the main means of controlling the early inflammatory response. However, the use of glucocorticoids in the early repair phase (7-21 days after injury) may accelerate corneal dissolution or even perforation, limiting their long-term use.

[0003] Ferroprelation is an iron-dependent form of cell death driven by lipid peroxidation and impaired antioxidant defense. Following alkali burns, the accumulation of reactive oxygen species (ROS) induces oxidative stress, promotes membrane lipid peroxidation, and ultimately triggers ferroptosis. Inhibiting ferroptosis in corneal alkali burns can alleviate oxidative stress, exert anti-inflammatory effects, promote epithelial repair, and reduce angiogenesis. Current interventions against ferroptosis primarily rely on ferroptosis inhibitors, such as Ferrostatin-1, which act as free radical scavenging antioxidants. However, these compounds often suffer from poor stability, low solubility, insufficient targeting, safety concerns, toxicity, poor pharmacokinetics, and limited efficacy, making them ineffective in treating corneal alkali burns.

[0004] From a nanoparticle design perspective, PROTAC molecules consist of three parts: a target protein ligand, an E3 ubiquitin ligase ligand, and a covalent linker connecting the three. This molecule can simultaneously bind to the target protein and the E3 ubiquitin ligase, forming a ternary complex that triggers the polyubiquitination of the target protein, ultimately leading to its degradation via the ubiquitin-proteasome system (UPS). As a novel targeted protein degradation strategy, PROTACs offer several advantages over traditional small-molecule inhibitors. Zhang et al. (PROTAC based targeted degradation of LRG1 for mitigating cornealneovascularization) utilized PROTACs to degrade LRG1 (an angiogenesis factor upregulated after alkali burns) to treat corneal neovascularization; however, this approach only targets neovascularization and does not address the repair of epithelial damage.

[0005] Therefore, there is an urgent need for a PROTAC nanocomposite that can be used to treat corneal alkali burns. Summary of the Invention

[0006] The purpose of this invention is to provide a PROTAC nanocomposite that can be used in products for treating ophthalmic diseases. Products containing the PROTAC nanocomposite can effectively alleviate and treat ophthalmic diseases, such as corneal alkali burns.

[0007] To achieve the above objectives, the first aspect of the present invention provides a PROTAC nanocomposite, wherein the PROTAC nanocomposite comprises a lipid layer and a PROTAC chimera that targets the degradation of ACSL4 encapsulated in the lipid layer; wherein the lipid layer comprises ionizable cationic lipids, cholesterol, DOPE and DSPE-PEG2000. Based on the mass of the PROTAC nanocomposite, the mass ratio of the ionizable cationic lipid, cholesterol, DOPE, DSPE-PEG2000 and the PROTAC chimera that targets ACSL4 degradation is (1.5-2.5):(0.8-1.2):(0.3-0.7):(1.5-2.5):1.

[0008] Compared with the prior art, the PROTAC nanocomposite provided by the present invention comprises a lipid layer and a PROTAC chimera targeting ACSL4 degradation encapsulated in the lipid layer. The lipid layer comprises ionizable cationic lipids, cholesterol, DOPE, and DSPE-PEG2000, making the lipid layer of the present invention a lipid bilayer structure. The ionizable cationic lipids, cholesterol, and DOPE constitute the main lipid phase, which can effectively enhance membrane stability, regulate fluidity, promote membrane fusion and endosome escape, and release the PROTAC chimera targeting ACSL4 degradation into the cell. DSPE-PEG2000 is distributed on the surface of the PROTAC nanocomposite in an anchored form, with its hydrophobic end (DSPE) embedded in the lipid bilayer and the hydrophilic PEG chain extending outward to form a hydration layer, thereby prolonging the blood circulation time of the PROTAC nanocomposite and thus improving the treatment effect of corneal alkali burn.

[0009] Furthermore, based on the mass of the PROTAC nanocomposite, the mass ratio of the ionizable cationic lipid, cholesterol, DOPE, DSPE-PEG2000, and the PROTAC chimera targeting ACSL4 degradation is (1.5-2.5):(0.8-1.2):(0.3-0.7):(1.5-2.5):1; this gives the PROTAC nanocomposite of the present invention excellent stability while also ensuring sufficient encapsulation of the PROTAC chimera targeting ACSL4 degradation, thereby improving the therapeutic effect of corneal alkali burns.

[0010] Furthermore, the PROTAC nanocomposite of this invention can effectively degrade ACSL4, upregulate glutathione peroxidase 4 (GPX4), inhibit lipid peroxidation and the accumulation of reactive oxygen species (ROS), and inhibit pathological activation driven by the upregulation of vascular endothelial growth factor (VEGF) without affecting cell viability. In a mouse corneal alkali burn model, topical application of the product containing the PROTAC nanocomposite of this invention can accelerate corneal healing and improve repair quality, alleviate corneal edema and inhibit inflammatory response, reduce stromal edema and opacity, significantly inhibit fibrosis and neovascularization, and demonstrate good safety in healthy corneas.

[0011] Furthermore, the structure of the PROTAC chimera that targets ACSL4 degradation is shown in Formula I; Formula I.

[0012] In this invention, when the PROTAC nanocomposite contains a PROTAC chimera that targets and degrades ACSL4 with the above-mentioned structure, the PROTAC chimera that targets and degrades ACSL4 specifically recruits the E3 ubiquitin ligase CRBN, efficiently inducing ubiquitination and proteasome degradation of the key ferroptosis protein ACSL4, thereby significantly inhibiting the ferroptosis pathway and alleviating oxidative damage to corneal epithelial cells.

[0013] Furthermore, the structural formula of the ionizable cationic lipid is shown in Formula II; Formula II.

[0014] In this invention, when the lipid layer contains ionizable cationic lipids with the structure shown in Formula II, it can efficiently encapsulate PROTAC chimeras, improve membrane stability, regulate the fluidity of PROTAC nanocomplexes, promote membrane fusion and endosome escape, thereby improving the therapeutic effect of corneal alkali burns.

[0015] Furthermore, when the aforementioned PROTAC chimera targeting ACSL4 degradation is incorporated into ROS-responsive ionizable cationic lipid nanoparticles, structural breakage occurs in the highly reactive oxygen species (ROS) microenvironment at the site of corneal alkali burn, triggering the disassembly and reassembly of the PROTAC nanocomposite and accelerating the release of the PROTAC chimera. Animal experiments have demonstrated that eye drops containing the PROTAC nanocomposite of this invention can significantly reduce corneal edema and inflammatory infiltration, promote epithelial repair, and show no obvious ocular irritation, exhibiting good biocompatibility and therapeutic potential.

[0016] Furthermore, the average hydrodynamic diameter of the PROTAC nanocomposite in water is 195 nm-204 nm; the polydispersity index of the PROTAC nanocomposite is less than or equal to 0.2; and the zeta potential of the PROTAC nanocomposite mixed with water to 0.1 mg / mL at 25 °C is 30 mV-40 mV.

[0017] In this invention, when the average hydrodynamic diameter and polydispersity index of the PROTAC nanocomposite meet the above-mentioned ranges, it indicates that the PROTAC nanocomposite has a highly uniform particle size, making it easier to penetrate through the corneal epithelial space or via intracellular pathways, thereby improving the therapeutic effect of corneal alkali burns. When the zeta potential of the PROTAC nanocomposite meets the above-mentioned ranges, it indicates that the PROTAC nanocomposite is stable, has good dispersibility, is not prone to aggregation, and is not likely to interact with cell membranes to produce cytotoxicity.

[0018] A second aspect of the present invention provides a method for preparing a PROTAC nanocomposite, comprising the following steps: PROTAC chimeras targeting ACSL4 degradation were prepared to generate ionizable cationic lipids; The ionizable cationic lipid, cholesterol, DOPE and the PROTAC chimera that targets ACSL4 degradation were mixed in chloroform, and the resulting first mixture was dried to obtain a lipid membrane. The lipid membrane was dissolved in a C1-C3 monohydric alcohol to obtain a second mixture; DSPE-PEG2000 was dispersed in a buffer solution with a pH of 7-7.4; the second mixture was mixed with a buffer solution containing DSPE-PEG2000; and after dialysis, a PROTAC nanocomposite was obtained.

[0019] Compared with existing technologies, this invention mixes ionizable cationic lipids, cholesterol, DOPE, and a PROTAC chimera that targets and degrades ACSL4 in chloroform. The resulting first mixture is dried to obtain a lipid membrane. Chloroform can completely dissolve the ionizable cationic lipids, cholesterol, and DOPE, ensuring uniform dispersion of the raw materials. The organic solvent is thoroughly removed through drying to form the membrane, avoiding toxicity. The lipid membrane is dissolved in a C1-C3 monohydric alcohol to obtain a second mixture. DSPE-PEG2000 is dispersed in a buffer solution with a pH of 7-7.4. The mixture is combined with a buffer solution containing DSPE-PEG2000. By rapidly dissolving the lipid membrane with a C1-C3 monohydric alcohol and then mixing it with a buffer solution containing DSPE-PEG2000, the self-assembly of the PROTAC nanocomplex can be promoted, and the encapsulation efficiency of the PROTAC chimera targeting ACSL4 degradation can be improved. This facilitates the acquisition of a PROTAC nanocomplex with high stability and good fluidity, promotes membrane fusion and endosome escape, and allows the PROTAC chimera targeting ACSL4 degradation to be released into the cells, thereby improving the therapeutic effect of corneal alkali burns.

[0020] Furthermore, the ratio of the ionizable cationic lipid to the amount of chloroform added is (150-250) μg: 2mL.

[0021] In this invention, when the amounts of ionizable cationic lipids and chloroform meet the above-mentioned range, ionizable cationic lipids, cholesterol, and DOPE are more easily dissolved in chloroform, thereby improving the efficiency of lipid membrane preparation.

[0022] Furthermore, the temperature at which the mixture is dried is 30-40°C.

[0023] Furthermore, when the lipid membrane is dissolved in a C1-C3 monohydric alcohol, the ratio of the amount of ionizable cationic lipid in the lipid membrane to the amount of the monohydric alcohol added is (150-250) μg: 50 μL.

[0024] In this invention, when the ratio of ionizable cationic lipids to monohydric alcohols in the lipid membrane meets the above-mentioned range, it not only ensures complete lipid dissolution and prevents precipitation due to incomplete dissolution when the second mixture is added to the buffer containing DSPE-PEG2000, thus avoiding a widening of the particle size distribution of the PROTAC nanocomposite; it also optimizes the formation efficiency of the lipid layer, ensuring the stability and consistency of the PROTAC nanocomposite; simplifies subsequent processes, and avoids damage to the structure of the lipid layer or the PROTAC chimera due to solvent residue.

[0025] Furthermore, the C1-C3 monohydric alcohol is ethanol.

[0026] In this invention, ethanol is used to rapidly dissolve the lipid membrane. The resulting second mixture is then mixed with a buffer solution, which promotes the self-assembly of ionized cationic lipids, cholesterol, and DOPE, improves the encapsulation rate and amount of PROTAC chimera, enhances the stability of the PROTAC nanocomposite, and promotes membrane fusion and endosome escape; thereby improving the therapeutic effect of corneal alkali burns.

[0027] Further, in the buffer solution containing DSPE-PEG2000, the ratio of the amount of DSPE-PEG2000 to the amount of buffer solution added is (150-250) μg:1mL; wherein, the buffer solution is a phosphate buffer, the concentration of the phosphate buffer solution is 5-20mM, and the pH value is 7.2-7.4.

[0028] In this invention, when the buffer solution is a phosphate buffer, and the concentration, pH value, and the relationship between DSPE-PEG2000 and the amount of buffer solution satisfy the above-mentioned ranges, the concentration of the phosphate buffer solution is maintained in the range of 5-20 mM. This provides sufficient buffering capacity to resist environmental pH fluctuations, ensuring the structural stability of the PROTAC nanocomplex (LNP), while also avoiding excessive compression of the electric double layer on the surface of the PROTAC nanocomplex due to high ionic strength. This maintains the zeta potential in the ideal range of +30 mV to +40 mV, endowing the particles with good electrostatic repulsion and colloidal stability. At the same time, maintaining the pH value in a weakly alkaline environment of 7.0-7.6 promotes the appropriate protonation of ionizable cationic lipids, ensuring sufficient positive charge for efficient binding of negatively charged PROTAC molecules, while avoiding cytotoxicity or non-specific protein adsorption caused by excessive positive charge. In addition, optimizing the relationship between the amount of DSPE-PEG2000 and the buffer solution can further stabilize the surface of the PROTAC nanocomplex, reduce the risk of aggregation, and ultimately achieve a PROTAC nanocomplex with high encapsulation efficiency, low cytotoxicity, and long-term storage stability.

[0029] Furthermore, the preparation of the PROTAC chimera targeting ACSL4 degradation includes the following steps: Pomalidomide, 1,2-bis(2-bromoethoxy)ethane, potassium carbonate and potassium iodide were dissolved in acetonitrile and refluxed at 80-85℃ for 12-24 h to prepare pomalidomide-bromopolyethylene glycol intermediate. Troglitazone, the pomalidomide-bromopolyethylene glycol intermediate, potassium carbonate, and potassium iodide were dissolved in acetonitrile and reacted under reflux conditions in the presence of a protective gas for 12-24 hours. The resulting reaction product was post-processed to obtain the PROTAC chimera that targets the degradation of ACSL4.

[0030] Furthermore, the preparation of the ionizable cationic lipid includes the following steps: The fatty amine with the structure shown in Formula A and the compound with the structure shown in Formula B are mixed in a molar ratio of 1:(3-3.5) and reacted at 75-85°C. The reaction product is purified and concentrated to dryness to obtain the ionizable cationic lipid. Formula A; Formula B.

[0031] Furthermore, the steps of purifying and concentrating the reaction product to dryness include: purifying the reaction product by silica gel column chromatography, then eluting with dichloromethane or methanol as the eluent, and concentrating the resulting eluent to dryness under reduced pressure to obtain ionized cationic lipids.

[0032] A third aspect of the present invention provides the application of the above-mentioned PROTAC nanocomposite in a product for treating ophthalmic diseases; wherein the treatment of ophthalmic diseases includes the treatment of corneal alkali burns.

[0033] Furthermore, the treatment of corneal alkali burns includes: Inhibits ferroptosis, reduces oxidative stress, and inhibits vascular endothelial cell proliferation; and / or, Accelerate corneal healing and improve repair quality; and / or, Relieves corneal edema and inhibits inflammatory response; and / or, Inhibits corneal neovascularization. Attached Figure Description

[0034] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a transmission electron microscope image of dACSL4@401-TK12 from Example 1; Figure 2 The image shows the anterior segment of the cornea of ​​the mouse after alkali burn in Test Example 2 and a fluorescein sodium staining image. Figure 3 This is a statistical chart in Test Example 2 that quantifies the corneal epithelial defect area (%) based on the fluorescein staining results on days 1, 2, and 3; Figure 4 This is a statistical chart showing the corneal opacity scores assessed on days 7, 14, 21, and 28 in Test Example 2. Figure 5 This is an immunofluorescence staining image of α-smooth muscle actin (α-SMA, a marker of corneal fibrosis) in test example 2; Figure 6 This is a quantitative statistical graph of the relative fluorescence intensity of α-SMA in Test Example 2; Figure 7 This is an optical coherence tomography (OCT) image of the mouse cornea in Test Example 2, with the red dashed line representing corneal thickness; Figure 8 This is a quantitative analysis diagram of the corneal thickness of mice in Test Example 2; Figure 9 This is an image of hematoxylin-eosin (H&E) staining of a mouse corneal tissue section from Test Example 2; Figure 10 In test example 2, a lateral view of the anterior segment of the mouse cornea is shown, with white dashed lines marking the corneal neovascularization area and white arrows pointing to the neovascularization. Figure 11 This is a statistical chart showing the quantitative analysis of the length from the limbus to the neovascularization on days 7 and 14 in test example 2. Figure 12 This is a statistical chart of the area of ​​new blood vessels on days 21 and 28 in test case 2; Figure 13 In test example 2, the immunofluorescence staining of CD31 (a vascular marker) is shown, with the white arrow indicating a CD31-positive vascular lumen; Figure 14 This is a quantitative analysis graph of the relative fluorescence intensity of CD31 in Test Example 2; Figure 15 This is an immunofluorescence staining image of ACSL4 in corneal tissue from test example 2; Figure 16 This is a quantitative analysis graph of the relative fluorescence intensity of ACSL4 in Test Example 2; Figure 17 This refers to the immunofluorescence staining of GPX4 in corneal tissue in test example 2; Figure 18 This is the quantitative analysis of the relative fluorescence intensity of GPX4 in Test Example 2; Figure 19 This is a dihydroethidium (DHE) staining image of corneal tissue from test example 2; Figure 20This is a quantitative analysis graph of the relative fluorescence intensity of DHE in Test Example 2; Figure 21 These are anterior segment photographs and sodium fluorescein staining images of the corneas of healthy control mice in Test Example 2; Figure 22 This is a hematoxylin-eosin (H&E) staining image of corneal tissue sections from healthy control mice in test example 2; Figure 23 This is the immunoblot band of ACSL4 protein in HUVECs in test example 3; Figure 24 This is a quantitative analysis graph of ACSL4 protein levels in HUVECs in test example 3; Figure 25 This is the immunoblot band of GPX4 protein in HUVECs in test example 3; Figure 26 This is a quantitative analysis graph of GPX4 protein levels in HUVECs in Test Example 3; Figure 27 This is a statistical chart of malondialdehyde (MDA) content in HUVECs in Test Example 3; Figure 28 This is a graph of intracellular ROS fluorescence intensity obtained from CM-H2DCFDA staining in Test Example 3; Figure 29 This is a quantitative analysis diagram of the relative fluorescence intensity of intracellular ROS in test example 3; Figure 30 This is an image of the scratch wound area in test example 4, with the dashed lines representing cell boundaries; Figure 31 This is a statistical graph showing the percentage of migration area of ​​VEGF-induced HUVECs cells after treatment in Test Example 4. Detailed Implementation

[0035] To make the technical problems, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0036] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined with "first" or "second" may explicitly or implicitly include one or more of that feature.

[0037] The term "PROTAC chimera targeting the degradation of ACSL4" is abbreviated as dACSL4.

[0038] The first aspect of the present invention provides a PROTAC nanocomposite, wherein the PROTAC nanocomposite comprises a lipid layer and a PROTAC chimera targeting the degradation of ACSL4 encapsulated in the lipid layer; wherein the lipid layer comprises ionizable cationic lipids, cholesterol, DOPE and DSPE-PEG2000. Based on the mass of the PROTAC nanocomposite, the mass ratio of the ionizable cationic lipid, cholesterol, DOPE, DSPE-PEG2000 and the PROTAC chimera that targets ACSL4 degradation is (1.5-2.5):(0.8-1.2):(0.3-0.7):(1.5-2.5):1.

[0039] When the above technical solution is adopted, the PROTAC nanocomposite comprises a lipid layer and a PROTAC chimera targeting ACSL4 degradation encapsulated in the lipid layer. The lipid layer comprises ionizable cationic lipids, cholesterol, DOPE, and DSPE-PEG2000, making the lipid layer of the present invention a lipid bilayer structure. The ionizable cationic lipids, cholesterol, and DOPE constitute the main lipid phase, which can effectively enhance membrane stability, regulate fluidity, promote membrane fusion and endosome escape, and release the PROTAC chimera targeting ACSL4 degradation into the cell. DSPE-PEG2000 is distributed on the surface of the PROTAC nanocomposite in an anchored form, with its hydrophobic end (DSPE) embedded in the lipid bilayer and the hydrophilic PEG chain extending outward to form a hydration layer, thereby prolonging the blood circulation time of the PROTAC nanocomposite and thus improving the treatment effect of corneal alkali burn.

[0040] Furthermore, based on the mass of the PROTAC nanocomposite, the mass ratio of the ionizable cationic lipid, cholesterol, DOPE, DSPE-PEG2000, and the PROTAC chimera targeting ACSL4 degradation is (1.5-2.5):(0.8-1.2):(0.3-0.7):(1.5-2.5):1; this gives the PROTAC nanocomposite of the present invention excellent stability while also ensuring sufficient encapsulation of the PROTAC chimera targeting ACSL4 degradation, thereby improving the therapeutic effect of corneal alkali burns.

[0041] Furthermore, the PROTAC nanocomposite of this invention can effectively degrade ACSL4, upregulate glutathione peroxidase 4 (GPX4), inhibit lipid peroxidation and the accumulation of reactive oxygen species (ROS), and inhibit pathological activation driven by the upregulation of vascular endothelial growth factor (VEGF) without affecting cell viability. In a mouse corneal alkali burn model, topical application of the product containing the PROTAC nanocomposite of this invention can accelerate corneal healing and improve repair quality, alleviate corneal edema and inhibit inflammatory response, reduce stromal edema and opacity, significantly inhibit fibrosis and neovascularization, and demonstrate good safety in healthy corneas.

[0042] For example, the mass ratio of the ionizable cationic lipid, cholesterol, DOPE, DSPE-PEG2000 and the PROTAC chimera that targets ACSL4 degradation can be 1.5 : 0.8 : 0.3 : 1.5 : 1, 2 : 1 : 0.5 : 2 : 1 or 2.5 : 1.2 : 0.7 : 2.5 : 1.

[0043] Preferably, the mass ratio of the ionizable cationic lipid, cholesterol, DOPE, DSPE-PEG2000 and the PROTAC chimera that targets ACSL4 degradation is (1.8-2.2):(0.9-1.1):(0.4-0.6):(1.8-2.2):1; more preferably, it is 2:1:0.5:2:1.

[0044] In some embodiments, the structure of the PROTAC chimera that targets ACSL4 degradation is shown in Formula I; Formula I.

[0045] Using the above technical solution, when the PROTAC nanocomposite contains a PROTAC chimera that targets and degrades ACSL4, the PROTAC chimera specifically recruits the E3 ubiquitin ligase CRBN, efficiently inducing ubiquitination and proteasome degradation of the key ferroptosis protein ACSL4, thereby significantly inhibiting the ferroptosis pathway and alleviating oxidative damage to corneal epithelial cells.

[0046] In some embodiments, the ionizable cationic lipid is a ROS-responsive ionizable cationic lipid.

[0047] In some embodiments, the ionizable cationic lipid has the structural formula shown in Formula II; Formula II.

[0048] Using the above technical solution, when the lipid layer contains ionizable cationic lipids with the structure shown in Formula II, it can efficiently encapsulate PROTAC chimeras, improve membrane stability, regulate the fluidity of PROTAC nanocomplexes, promote membrane fusion and endosome escape, thereby improving the treatment effect of corneal alkali burns.

[0049] Furthermore, when the aforementioned PROTAC chimera targeting ACSL4 degradation is incorporated into ROS-responsive ionizable cationic lipid nanoparticles, structural breakage occurs in the highly reactive oxygen species (ROS) microenvironment at the site of corneal alkali burn, triggering the disassembly and reassembly of the PROTAC nanocomposite and accelerating the release of the PROTAC chimera. Animal experiments have demonstrated that eye drops containing the PROTAC nanocomposite of this invention can significantly reduce corneal edema and inflammatory infiltration, promote epithelial repair, and show no obvious ocular irritation, exhibiting good biocompatibility and therapeutic potential.

[0050] In some embodiments, the average hydrodynamic diameter of the PROTAC nanocomposite in water is 195 nm-204 nm; the polydispersity index of the PROTAC nanocomposite is less than or equal to 0.2; and the zeta potential of the PROTAC nanocomposite mixed with water to 0.1 mg / mL at 25 °C is 30 mV-40 mV.

[0051] Using the above technical solution, when the average hydrodynamic diameter and polydispersity index of the PROTAC nanocomposite meet the aforementioned ranges, it indicates that the PROTAC nanocomposite has a highly uniform particle size, making it easier to penetrate through the corneal epithelial space or via intracellular pathways, thereby improving the treatment effect of corneal alkali burns. When the zeta potential of the PROTAC nanocomposite meets the aforementioned ranges, it indicates that the PROTAC nanocomposite is stable, well-dispersible, not prone to aggregation, and not likely to interact with cell membranes to produce cytotoxicity.

[0052] A second aspect of the present invention provides a method for preparing a PROTAC nanocomposite, comprising the following steps: PROTAC chimeras targeting ACSL4 degradation were prepared to generate ionizable cationic lipids; The ionizable cationic lipid, cholesterol, DOPE and the PROTAC chimera that targets ACSL4 degradation were mixed in chloroform, and the resulting first mixture was dried to obtain a lipid membrane. The lipid membrane was dissolved in a C1-C3 monohydric alcohol to obtain a second mixture; DSPE-PEG2000 was dispersed in a buffer solution with a pH of 7-7.4; the second mixture was mixed with a buffer solution containing DSPE-PEG2000; and after dialysis, a PROTAC nanocomposite was obtained.

[0053] When using the above technical solution, ionizable cationic lipids, cholesterol, DOPE, and a PROTAC chimera targeting ACSL4 degradation are mixed in chloroform. The resulting first mixture is dried to obtain a lipid membrane. Chloroform can completely dissolve ionizable cationic lipids, cholesterol, and DOPE, ensuring uniform dispersion of all raw materials. The organic solvent is thoroughly removed through drying to form a film, avoiding toxicity. The lipid membrane is dissolved in a C1-C3 monohydric alcohol to obtain a second mixture. DSPE-PEG2000 is dispersed in a buffer solution with a pH of 7-7.4. The second mixture is then dissolved in the buffer solution. The mixture is combined with a buffer solution containing DSPE-PEG2000. By rapidly dissolving the lipid membrane with a C1-C3 monohydric alcohol and then mixing it with a buffer solution containing DSPE-PEG2000, the self-assembly of the PROTAC nanocomplex can be promoted, and the encapsulation efficiency of the PROTAC chimera targeting ACSL4 degradation can be improved. This facilitates the acquisition of a PROTAC nanocomplex with high stability and good fluidity, promotes membrane fusion and endosome escape, and allows the PROTAC chimera targeting ACSL4 degradation to be released into the cells, thereby improving the therapeutic effect of corneal alkali burns.

[0054] For example, the DSPE-PEG2000 is dispersed in a buffer solution, the pH of which can be 7, 7.1, 7.2, 7.3 or 7.4, or a range consisting of any two of the above values.

[0055] In some embodiments, the ratio of the ionizable cationic lipid to the amount of chloroform added is (150-250) μg: 2 mL.

[0056] Using the above technical solution, when the amounts of ionizable cationic lipids and chloroform meet the above range, ionizable cationic lipids, cholesterol and DOPE are more easily dissolved in chloroform, thus improving the efficiency of lipid membrane preparation.

[0057] For example, the ratio of the ionizable cationic lipid to the amount of chloroform added can be 150 μg: 2 mL, 160 μg: 2 mL, 180 μg: 2 mL, 200 μg: 2 mL, 220 μg: 2 mL, 240 μg: 2 mL, or 250 μg: 2 mL, or any range consisting of any two of the above values.

[0058] Preferably, the ratio of the ionizable cationic lipid to the amount of chloroform added is (180-220) μg: 2 mL.

[0059] In some embodiments, the mixture is dried at a temperature of 30-40°C.

[0060] In some embodiments, when the lipid membrane is dissolved in a C1-C3 monohydric alcohol, the ratio of the amount of ionizable cationic lipid in the lipid membrane to the amount of the monohydric alcohol added is (150-250) μg: 50 μL.

[0061] By employing the above technical solution, when the ratio of ionizable cationic lipids to monohydric alcohols in the lipid membrane meets the aforementioned range, it not only ensures complete lipid dissolution and prevents precipitation due to incomplete dissolution when the second mixture is added to a buffer containing DSPE-PEG2000, thus avoiding a widening of the PROTAC nanocomposite particle size distribution, but also optimizes the formation efficiency of the lipid layer, ensuring the stability and consistency of the PROTAC nanocomposite; simplifies subsequent processes, and avoids damage to the structure of the lipid layer or the PROTAC chimera due to solvent residue.

[0062] For example, the ratio of the amount of ionizable cationic lipid to the amount of monohydric alcohol added in the lipid membrane can be 150 μg: 50 μL, 160 μg: 50 μL, 180 μg: 50 μL, 200 μg: 50 μL, 220 μg: 50 μL, 240 μg: 50 μL, or 250 μg: 50 μL, or a range consisting of any two of the above values.

[0063] Preferably, the C1-C3 monohydric alcohol is ethanol; preferably, the ratio of the amount of ionizable cationic lipid to the amount of monohydric alcohol added in the lipid membrane is (180-220) μg: 50 μL.

[0064] Using the above technical solution, ethanol can rapidly dissolve the lipid membrane. The resulting second mixture, when mixed with buffer, can promote the self-assembly of ionized cationic lipids, cholesterol, and DOPE, improve the encapsulation rate and amount of PROTAC chimera, enhance the stability of PROTAC nanocomplex, and promote membrane fusion and endosome escape; thereby improving the treatment effect of corneal alkali burns.

[0065] In some embodiments, the ratio of DSPE-PEG2000 to the buffer solution in the buffer solution is (150-250) μg:1mL; wherein the buffer solution is a phosphate buffer solution with a concentration of 5-20 mM and a pH of 7.2-7.4.

[0066] Using the above technical solution, when the buffer solution is phosphate buffer, and the concentration, pH value, and the relationship between DSPE-PEG2000 and the amount of buffer solution meet the above-mentioned ranges, the concentration of phosphate buffer solution is maintained in the range of 5-20 mM. This provides sufficient buffering capacity to resist environmental pH fluctuations, ensuring the structural stability of PROTAC nanocomplexes (LNPs), while avoiding excessive compression of the electric double layer on the surface of PROTAC nanocomplexes due to high ionic strength. This maintains the zeta potential in the ideal range of +30 mV to +40 mV, endowing the particles with good electrostatic repulsion and colloidal stability. At the same time, maintaining a weakly alkaline environment of 7.0-7.6 promotes the appropriate protonation of ionizable cationic lipids, ensuring sufficient positive charge for efficient binding of negatively charged PROTAC molecules, while avoiding cytotoxicity or non-specific protein adsorption caused by excessive positive charge. In addition, optimizing the relationship between the amount of DSPE-PEG2000 and the amount of buffer solution can further stabilize the surface of PROTAC nanocomplexes, reduce the risk of aggregation, and ultimately achieve PROTAC nanocomplexes with high encapsulation efficiency, low cytotoxicity, and long-term storage stability.

[0067] For example, the ratio of DSPE-PEG2000 to the added buffer solution can be 150 μg:1 mL, 160 μg:1 mL, 180 μg:1 mL, 200 μg:1 mL, 220 μg:1 mL, 240 μg:1 mL, or 250 μg:1 mL, or any range formed by any two of the above values. The concentration of the phosphate buffer solution can be 5 mM, 10 mM, 15 mM, or 20 mM, or any range formed by any two of the above values; the pH value of the phosphate buffer solution is 7.2, 7.3, or 7.4, or any range formed by any two of the above values.

[0068] Preferably, the ratio of the amount of DSPE-PEG2000 to the amount of buffer solution added is (180-220) μg:1mL.

[0069] In some embodiments, the second mixture is mixed with the buffer solution containing DSPE-PEG2000 by ultrasonic mixing, with the probe ultrasonic power being 90-110W, the ultrasonic mode being a cyclic mode of turning on for 2-4 seconds and turning off for 1-3 seconds, and the ultrasonic mixing time being 1.5-3 minutes.

[0070] Using the above technical solution, when the probe's ultrasonic power, ultrasonic mode, and ultrasonic mixing time meet the above range, it can effectively promote the self-assembly of PROTAC chimeras containing ionizable cationic lipids, cholesterol, DOPE, and targeted degradation of ACSL4, thereby improving the encapsulation efficiency and encapsulation amount; at the same time, it can promote the embedding of the hydrophobic end (DSPE) of DSPE-PEG2000 into the lipid bilayer, and the hydrophilic PEG chains extend outward to form a hydration layer, thereby prolonging the blood circulation time of the PROTAC nanocomplex.

[0071] In some embodiments, the second mixture is mixed with a buffer containing DSPE-PEG2000 and then subjected to dialysis, which includes: mixing the second mixture with a buffer containing DSPE-PEG2000, transferring the resulting mixture to a dialysis bag with a molecular weight cutoff of 9kDa-11kDa, and dialyzing in 1-1.5L of phosphate buffer (concentration of 9-11mM, pH of 7.2-7.4) at 3-5°C for 20-30 hours, changing the buffer at least 5 times during dialysis; after dialysis, filtering with a 0.22μm filter membrane, and the particles in the resulting suspended particulate solution are the PROTAC nanocomposite.

[0072] According to the present invention, the PROTAC chimera that targets and degrades ACSL4 can be prepared according to existing methods.

[0073] In some embodiments, the preparation of the PROTAC chimera targeting ACSL4 degradation includes the following steps: Pomalidomide, 1,2-bis(2-bromoethoxy)ethane, potassium carbonate and potassium iodide were dissolved in acetonitrile and refluxed at 80-85℃ for 12-24 h to prepare pomalidomide-bromopolyethylene glycol intermediate. Troglitazone, the pomalidomide-bromopolyethylene glycol intermediate, potassium carbonate, and potassium iodide were dissolved in acetonitrile and reacted under reflux conditions in the presence of a protective gas for 12-24 hours. The resulting reaction product was post-processed to obtain the PROTAC chimera that targets the degradation of ACSL4.

[0074] According to the present invention, the pomalidomide-bromopolyethylene glycol intermediate refers to 4-((2-(2-(2-bromoethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione.

[0075] In some embodiments, the mass ratio of pomalidomide, 1,2-bis(2-bromoethoxy)ethane, potassium carbonate, and potassium iodide is 1:(1.3-1.5):(1-1.3):(0.1-0.2).

[0076] According to the present invention, the method of dissolving pomalidomide, 1,2-bis(2-bromoethoxy)ethane, potassium carbonate and potassium iodide in acetonitrile is not particularly limited in the amount of acetonitrile used.

[0077] In some embodiments, the acetonitrile is anhydrous acetonitrile.

[0078] In some embodiments, the preparation method of the pomalidomide-bromopolyethylene glycol intermediate includes: dissolving pomalidomide, 1,2-bis(2-bromoethoxy)ethane, potassium carbonate, and potassium iodide in acetonitrile, refluxing at 80-85°C for 12-24 h, filtering to remove insoluble matter after the reaction, concentrating the filtrate to dryness under reduced pressure at 30-40°C, purifying the crude product by silica gel column chromatography using a petroleum ether solution containing 55-65 vol% ethyl acetate as the eluent, collecting the eluent, and concentrating it to dryness under reduced pressure at 30-40°C to obtain the pomalidomide-bromopolyethylene glycol intermediate. In the preparation of the pomalidomide-bromopolyethylene glycol intermediate, there is no particular limitation on the pressure of the reduced pressure concentration; those skilled in the art can adjust it according to actual conditions. The silica gel column chromatography purification and elution are commonly used chromatographic purification and elution process conditions in the prior art and are not particularly limited; those skilled in the art can adjust them according to actual conditions.

[0079] In some embodiments, the mass ratio of the troglitazone, the pomalidomide-bromopolyethylene glycol intermediate, potassium carbonate and potassium iodide is 50:(100-140):(70-90):(8-12).

[0080] In some embodiments, the ratio of troglitazone to acetonitrile is 50 mg: (4-6) mL.

[0081] In some embodiments, the protective gas is nitrogen.

[0082] In some embodiments, the post-processing of the reaction product includes: cooling the reaction product to room temperature, filtering to remove solids, concentrating the filtrate to dryness under reduced pressure at 30-40°C, purifying the crude product by silica gel column chromatography, collecting the eluent, and concentrating it to dryness under reduced pressure at 30-40°C; wherein, when purifying the crude product by silica gel column chromatography, a mixed solvent of dichloromethane (DCM) and methanol (MeOH) at a volume ratio of 15:1 is used as the eluent. During the post-processing of the reaction product, there are no special limitations on the pressure of the reduced pressure concentration; those skilled in the art can adjust it according to actual conditions. The silica gel column chromatography purification and elution are commonly used chromatographic purification and elution process conditions in the prior art and are not particularly limited; those skilled in the art can adjust them according to actual conditions.

[0083] According to the present invention, ionizable cationic lipids can be prepared according to existing methods.

[0084] In some embodiments, the preparation of ionizable cationic lipids includes the following steps: The fatty amine with the structure shown in Formula A and the compound with the structure shown in Formula B are mixed in a molar ratio of 1:(3-3.5) and reacted at 75-85°C. The reaction product is purified and concentrated to dryness to obtain the ionizable cationic lipid. Formula A; Formula B.

[0085] In some embodiments, the step of purifying and concentrating the reaction product to dryness includes: purifying the reaction product by silica gel column chromatography, then eluting with dichloromethane or methanol as the eluent, and concentrating the resulting eluent to dryness under reduced pressure to obtain ionized cationic lipids. According to the present invention, in the process of purifying and concentrating the reaction product to dryness, the silica gel column chromatography purification and elution are commonly used chromatographic purification and elution process conditions in the prior art, without special limitations, and can be adjusted by those skilled in the art according to actual conditions.

[0086] In some embodiments, during the process of concentrating the eluent to dryness under reduced pressure, the temperature of the reduced pressure concentration is 30-40°C. According to the present invention, there is no particular limitation on the pressure of the reduced pressure concentration, and those skilled in the art can adjust it according to actual conditions.

[0087] A third aspect of the present invention provides the application of the above-mentioned PROTAC nanocomposite in a product for treating ophthalmic diseases; wherein the treatment of ophthalmic diseases includes the treatment of corneal alkali burns.

[0088] In some embodiments, the treatment of corneal alkali burns includes: Inhibits ferroptosis, reduces oxidative stress, and inhibits vascular endothelial cell proliferation; and / or, Accelerate corneal healing and improve repair quality; and / or, Relieves corneal edema and inhibits inflammatory response; and / or, Inhibits corneal neovascularization.

[0089] In some embodiments, the product for treating corneal alkali burns includes eye drops and / or rinsing solutions.

[0090] In some embodiments, the effective amount of PROTAC nanocomposite added to the product is 0.005%-0.2% based on the product weight, for the treatment of corneal alkali burns.

[0091] To better illustrate the technical solution of the present invention, the following specific embodiments are also provided.

[0092] Unless otherwise specified, all raw materials used in the examples and comparative examples were obtained commercially.

[0093] Example 1 - Preparation of PROTAC nanocomposites Example 1 provides a PROTAC nanocomposite comprising a lipid layer and a PROTAC chimera (dACSL4) of Formula I encapsulated within the lipid layer; wherein the lipid layer comprises an ionizable cationic lipid, cholesterol, DOPE, and DSPE-PEG2000 of Formula II, and the mass ratio of the ionizable cationic lipid, cholesterol, DOPE, DSPE-PEG2000, and the PROTAC chimera of Formula II to the PROTAC nanocomposite is 2:1:0.5:2:1. Formula I; Formula II; The preparation method of the above-mentioned PROTAC nanocomposite includes the following steps: (1) Preparation of PROTAC chimera targeting ACSL4 degradation (dACSL4 for short): Pomalidomide, 1,2-bis(2-bromoethoxy)ethane, potassium carbonate, and potassium iodide were dissolved in 20 mL of anhydrous acetonitrile and refluxed at 80 °C for 16 h. After the reaction was completed, insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure at 40 °C to dryness. The crude product was purified by silica gel column chromatography using a petroleum ether solution containing 60 vol% ethyl acetate as the eluent. The eluent was collected and concentrated under reduced pressure at 40 °C and a vacuum of 5 Pa to dryness to obtain a pomalidomide-bromopolyethylene glycol intermediate (4-((2-(2-(2-bromoethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione). The mass ratio of pomalidomide, 1,2-bis(2-bromoethoxy)ethane, potassium carbonate, and potassium iodide was 1:1.4:1.2:0.1. Troglitazone (50 mg), the above-mentioned pomalidomide-bromopolyethylene glycol intermediate (120 mg), potassium carbonate (80 mg), and potassium iodide (10 mg) were dissolved in 5 mL of anhydrous acetonitrile and reacted under reflux under nitrogen protection for 18 h. After the reaction product was cooled to room temperature, the solid was removed by filtration. The filtrate was concentrated to dryness under reduced pressure at 40 °C. The crude product was purified by silica gel column chromatography using a mixed solvent of dichloromethane (DCM) and methanol (MeOH) in a volume ratio of 15:1 as the eluent. The eluent was collected and concentrated to dryness at 40 °C and a vacuum of 5 Pa to obtain the PROTAC chimera that targets the degradation of ACSL4.

[0094] (2) Preparation of ionizable cationic lipid (abbreviated as 401-TK12) includes the following steps: The aliphatic amine with the structure shown in Formula A and the compound with the structure shown in Formula B were mixed in a molar ratio of 1:3 and reacted at 80°C.

[0095] Formula A; Formula B.

[0096] The above reaction product was purified by silica gel column chromatography, and then eluted with dichloromethane. The resulting eluent was concentrated to dryness under reduced pressure at 40°C and 5 Pa to obtain an ionized cationic lipid, abbreviated as 401-TK12.

[0097] (3) Mix the above proportions of ionizable cationic lipids, cholesterol, DOPE and the above-mentioned PROTAC chimera (dACSL4) that targets and degrades ACSL4 in chloroform, and the resulting first mixture is dried (35°C) to obtain a lipid membrane.

[0098] (4) The above lipid membrane was dissolved in ethanol to obtain a second mixture; the above-prepared DSPE-PEG2000 was dispersed in phosphate buffer (10 mM) with a pH of 7.2; the second mixture was ultrasonically mixed with the buffer containing DSPE-PEG2000, the ultrasonic power of the probe was 100W, the ultrasonic mode was a cyclic mode of 3s on and 2s off, and the ultrasonic mixing time was 2min; the resulting mixture was transferred to a dialysis bag with a molecular weight cutoff of 9kDa-11kDa, and dialyzed in 1L of phosphate buffer (concentration of 10 mM, pH of 7.4) at 4℃ for 24h, with the buffer changed 5 times during dialysis; after dialysis, the mixture was filtered with a 0.22 μm filter membrane, and the particles in the resulting suspended particle solution were the PROTAC nanocomplex dACSL4@401-TK12; The mass ratio of ionizable cationic lipids, cholesterol, DOPE, DSPE-PEG2000, and dACSL4 is 2:1:0.5:2:1; the ratio of ionizable cationic lipids to chloroform is 200 μg:2 mL; the ratio of ionizable cationic lipids to ethanol in the lipid membrane is 200 μg:50 μL; and the ratio of DSPE-PEG2000 to the buffer solution is 200 μg:1 mL.

[0099] Comparative Example 1 - Blank Carrier 401-TK12 A blank carrier 401-TK12 was prepared according to Example 1. The difference between the preparation method of Example 1 and Example 1 is that steps (1)-(3) are not performed in Comparative Example 1, and the raw materials in steps (4)-(6) do not contain dACSL4. The amount of other raw materials remains unchanged. The blank carrier 401-TK12 was prepared.

[0100] Test Example 1 - Physicochemical Characterization After mixing dACSL4@401-TK12 from Example 1 and blank carrier 401-TK12 from Comparative Example 1 with water to a concentration of 0.1 mg / mL, the zeta potential and hydrodynamic diameter of dACSL4@401-TK12 from Example 1 and blank carrier 401-TK12 from Comparative Example 1 were determined using a Zetasizer Nano ZS (25 °C, Malvern, UK). The results are shown in Table 1.

[0101] The morphology of dACSL4@401-TK12 was observed using a transmission electron microscope (TEM, HT770, Hitachi, Japan) at 100 kV and stained with 1% phosphotungstic acid solution. The TEM images of dACSL4@401-TK12 are shown below. Figure 1 .

[0102] Table 1 from Figure 1 As can be seen, the PROTAC nanocomposite of Example 1 of the present invention has a uniform spherical morphology and a uniform particle size distribution.

[0103] Based on the results in Table 1, the average hydrodynamic diameter of the blank support 401-TK12 was (166.4 ± 35.28) nm, while that of the dACSL4@401-TK12 encapsulated with dACSL4 increased slightly to (199.14 ± 4.13) nm. The polydispersity index (PDI) of both dACSL4@401-TK12 and the blank support 401-TK12 was less than 0.2, indicating that the PROTAC nanocomposite dACSL4@401-TK12 of this invention possesses excellent particle size uniformity and colloidal stability. The zeta potential of the blank support 401-TK12 was (38.2 ± 2.9) mV, while that of the dACSL4@401-TK12 loaded with dACSL4 was (35.6 ± 3.7) mV, demonstrating the good colloidal stability of the dACSL4@401-TK12 of this invention.

[0104] Test Example 2 - Mouse Corneal Alkali Burn Experiment Ninety-nine C57BL / 6 mice (6-8 weeks old) were selected for corneal alkali burn experiments. They were first anesthetized with tribromoethanol (0.3 mL / 20 g), then mydriasis was achieved with compound tropicamide eye drops, and finally local anesthesia was performed with promecaine hydrochloride. A 2 mm diameter filter paper soaked in 2.5 μL of 0.5 mol / L NaOH solution was placed in the center of the right cornea for 30 seconds, and then immediately rinsed with 20 mL of sterile physiological saline. The mice treated with corneal alkali burn were randomly divided into three experimental groups (n=3 at each time point, that is, 3 mice were randomly selected from the three groups at the same time point of the experiment): (1) untreated control group, (2) blank vector 401-TK12 group, (3) dACSL4@401-TK12 group. In this study, mice in the untreated control group received no medication or treatment. Mice in the blank vector 401-TK12 group were given 10 μL of 0.1 mg / mL 401-TK12 phosphate buffer (10 mM buffer concentration), and mice in the dACSL4@401-TK12 group were given 10 μL of 0.1 mg / mL dACSL4@401-TK12 phosphate buffer (10 mM buffer concentration). Mice in both the blank vector 401-TK12 and dACSL4@401-TK12 groups received the medication four times daily for 14 consecutive days via eye drops.

[0105] At days 0, 1, 3, 7, 14, 21, and 28 post-injury, three mice were randomly selected from each of the three groups. Each mouse underwent anterior segment photography, corneal opacity scoring, optical coherence tomography, and hematoxylin and eosin (H&E) staining. Mice selected on days 3, 7, 14, 21, and 28 also underwent corneal neovascularization assessment. The remaining mice were randomly selected from the three groups at days 7, 14, 21, and 28 post-injury for immunofluorescence staining and dihydroethidium (DHE) staining.

[0106] Twelve C57BL / 6 mice (6-8 weeks old) were selected as a healthy control group. The healthy control mice were administered the same drug as the dACSL4@401-TK12 group in terms of administration method and frequency. On days 0, 3, 7, and 14, three mice were randomly selected each day for anterior segment photography, corneal neovascularization assessment, and hematoxylin-eosin (H&E) staining experiments.

[0107] 2.1 Pre-section Photography Mice in the untreated control group, the blank vector 401-TK12 group, and the dACSL4@401-TK12 group underwent anterior segment photography and cobalt blue fluorescein staining on days 0, 1, 3, 7, 14, 21, and 28 to assess corneal epithelial healing. The fluorescein staining area was recorded and quantified. Healthy control mice underwent anterior segment photography and cobalt blue fluorescein staining on days 0, 3, 7, and 14 to assess the safety of dACSL4@401-TK12.

[0108] 2.2 Assessment of corneal neovascularization Neovascularization was assessed using lateral radiography. Lateral radiography was performed on healthy control mice on day 0 (recorded as day 0). Lateral radiography was performed on untreated mice, mice in the blank vector 401-TK12 group, and mice in the dACSL4@401-TK12 group on days 3, 7, 14, 21, and 28, respectively. The length of the longest vessel extending from the limbus was measured on days 7 and 14. The neovascularization area (S) was calculated on days 21 and 28 using the following formula: S = 1 / 2C × π[r² − (r − l)²] Where C is the number of clock hour sectors occupied by the blood vessel (12 in total), l is the maximum length of the blood vessel (maximum 1.5 mm), and r is the estimated corneal radius (1.5 mm).

[0109] 2.3 Corneal Opacity Score Corneal opacity was scored on mice in the untreated control group, the blank vector 401-TK12 group, and the dACSL4@401-TK12 group at days 0, 1, 3, 7, 14, 21, and 28, respectively. The degree of corneal opacity is graded from 0 to 4: 0 = Clear cornea; 1 = Mild matrix turbidity; 2 = Moderate matrix turbidity; 3 = Severe smog, iris details are clearly visible; 4 = The cornea is opaque, and the iris cannot be seen.

[0110] Quantitative analysis of images was performed using ImageJ software.

[0111] 2.4 Optical Coherence Tomography (OCT) Corneal thickness was measured using OCT in mice in the untreated control group, the blank vector 401-TK12 group, and the dACSL4@401-TK12 group at days 0, 1, 3, 7, 14, 21, and 28 to assess the severity of edema. Mice were anesthetized by intraperitoneal injection of tribromoethanol and then fixed to a specially designed mouse scaffold. High-resolution spectral-domain optical coherence tomography (OCT) was used to acquire cross-sectional images of the cornea, with at least three repeated scans centered on the corneal apex for each eye. Central corneal thickness was quantified using ImageJ software, and the average value of the repeated scans was used for statistical analysis.

[0112] 2.5 Hematoxylin-eosin (H&E) staining Corneal tissues from mice in the untreated control group, the blank vector 401-TK12 group, and the dACSL4@401-TK12 group were paraffin-embedded and sectioned on days 0, 1, 3, 7, 14, 21, and 28, respectively; corneal tissues from healthy control mice were paraffin-embedded and sectioned on days 0, 3, 7, and 14.

[0113] Paraffin sections were first dewaxed with xylene I and II (15 minutes each), then rehydrated with a gradient of ethanol solutions (100% → 100% → 75%, 5 minutes each), and finally rinsed with distilled water. Sections were stained with hematoxylin for 3 minutes, briefly differentiated (3-5 seconds), blued (3-6 seconds), dehydrated with 95% ethanol (1 minute), and counterstained with eosin for 15 seconds. Afterwards, sections were sequentially dehydrated with anhydrous ethanol (three times, 1 minute each), cleared with butanol (twice) and xylene (twice, 1 minute each), and finally mounted with neutral resin. Histological morphology was evaluated under a bright-field microscope.

[0114] 2.6 Immunofluorescence staining and dihydroethidium (DHE) staining On days 7, 14, 21, and 28 after injury, three mice were randomly selected from each of the three groups. Corneal tissues from the untreated control group, the blank vector 401-TK12 group, and the dACSL4@401-TK12 group were embedded and frozen sections were prepared.

[0115] α-SMA immunofluorescence staining: Frozen sections from the three experimental groups were fixed with 4% paraformaldehyde at room temperature for 10 minutes, washed three times with PBS, and then permeated with 0.5% Triton X-100 in PBS for 30 minutes. The sections were blocked with 10% normal goat serum for 2 hours, and then incubated overnight at 4°C with anti-α-SMA primary antibody. After washing, the sections were incubated with fluorescently labeled secondary antibody and imaged using confocal microscopy.

[0116] CD31 immunofluorescence staining: Refer to the method of α-SMA immunofluorescence staining, except that anti-α-SMA is replaced with anti-CD31.

[0117] ACSL4 / FACL4 immunofluorescence staining: Refer to the method of α-SMA immunofluorescence staining, except that anti-α-SMA is replaced with anti-ACSL4 / FACL4.

[0118] GPX4 immunofluorescence staining: The method is the same as that for α-SMA immunofluorescence staining, except that anti-α-SMA is replaced with anti-GPX4.

[0119] Dihydroethidium (DHE) staining: DHE was prepared as 10 μM phosphate buffer. Frozen sections of mice from the untreated control group, the blank vector 401-TK12 group, and the dACSL4@401-TK12 group were incubated at room temperature (25℃) in the dark for 60 min. After incubation, the tissues were washed three times with PBS.

[0120] 2.7 Statistical Methods All data were analyzed and graphs generated using GraphPad Prism (version 10.0). Results are expressed as mean ± standard deviation (SD). Differences between groups were assessed using Student's t-test or one-way ANOVA, as appropriate. Statistical significance was defined as p < 0.05, where p ≤ 0.05 (where p < 0.05 is a statistical significance level). p ≤ 0.01 p ≤ 0.001 ) and p ≤ 0.0001 ( ).

[0121] 2.8 Test Results 2.8.1 The dACSL4@401-TK12 of the present invention can accelerate corneal healing and improve repair quality. Corneal epithelial healing was assessed using anterior segment photography of the mouse cornea and cobalt blue fluorescein staining. The area stained with fluorescein was recorded and quantified. Figure 2 As can be seen, the untreated control group and the 401-TK12 group both exhibited persistent epithelial defects and significant angiogenesis, while the dACSL4@401-TK12 group showed significantly accelerated epithelial regeneration and reduced angiogenesis. Quantitative analysis confirmed this; see details below. Figure 3In mice in the dACSL4@401-TK12 group, the corneal damage area was much smaller than that in the untreated control group and the blank vector 401-TK12 group on days 1-3, indicating that the epithelial healing speed of the dACSL4@401-TK12 group was significantly faster, and the defect was almost completely closed on day 3.

[0122] Corneal opacity assessment results: The corneal opacity of mice was scored according to the established grading system. The scoring results are shown below. Figure 4 At all examination time points (days 7, 14, 21, and 28), the turbidity scores of the dACSL4@401-TK12 group were significantly lower than those of the untreated control group and the blank carrier 401-TK12 group, indicating that treatment with dACSL4@401-TK12 can improve corneal transparency.

[0123] Immunofluorescence staining results: Figure 5 , Figure 6 During the study, on days 14 and 21, α-smooth muscle actin (α-SMA), a marker of corneal fibrosis, was mainly expressed in the stroma in both the untreated control group and the 401-TK12 group (white arrows indicate α-SMA fluorescence in the corneal stroma). In contrast, the α-SMA fluorescence intensity was significantly reduced in the dACSL4@401-TK12 group, indicating that the inhibitory effect of dACSL4@401-TK12 on ferroptosis can alleviate corneal scarring and fibrosis.

[0124] The above-mentioned anterior segment photographs, cobalt blue fluorescein sodium staining, corneal opacity assessment results, and immunofluorescence staining results indicate that dACSL4@401-TK12 can significantly promote corneal epithelial regeneration, accelerate corneal healing, reduce tissue opacity and scar formation, and improve the quality of corneal repair.

[0125] 2.8.2 The dACSL4@401-TK12 of the present invention can relieve corneal edema and inhibit inflammatory response. Corneal alkali burns are often accompanied by complications such as corneal edema and inflammation. The dACSL4@401-TK12 of this invention can relieve corneal edema and inhibit inflammatory response.

[0126] Optical coherence tomography (OCT) results: Optical coherence tomography was used to monitor corneal thickness in live mice. Figure 7 , Figure 8 The results showed that the dACSL4@401-TK12 group significantly reduced corneal edema, with statistically significant differences observed on days 1, 3, 7, and 14 post-injury compared to the untreated control group.

[0127] Hematoxylin-eosin (H&E) staining results: From Figure 9As can be seen, the corneas of the untreated control group exhibited epithelial defects, stromal edema, and extensive infiltration of inflammatory cells into the stroma. In contrast, the corneas treated with dACSL4@401-TK12 showed continuous epithelium, well-preserved tissue structure, and a significant reduction in inflammatory cells.

[0128] The above optical coherence tomography (OCT) hematoxylin-eosin (H&E) staining results show that dACSL4@401-TK12 can not only promote corneal epithelial repair, but also effectively inhibit corneal edema caused by alkali burns and reduce inflammatory response.

[0129] 2.8.3 The dACSL4@401-TK12 of the present invention can inhibit corneal neovascularization. Corneal neovascularization assessment results: From Figure 10 This is a lateral view of the anterior segment of the mouse cornea, with white dashed lines marking the corneal neovascularization area. From the lateral view of the mouse eye, it can be seen that in the healthy control group (day 0), the cornea is transparent, with no blood vessels extending from the limbus, and the iris is clearly visible. In the untreated control group, neovascularization began to emerge from the limbus on day 3 after alkali burn; by day 7, the neovascularization radiated towards the center of the cornea; by day 14, the neovascularization had spread to the pupillary area; and by day 21, the neovascularization had expanded to the center of the cornea and could not be defined by area (white arrows point to the neovascularization). The corneal neovascularization area in the dACSL4@401-TK12 group was less than that in the untreated control group and the blank vector 401-TK12 group. Figure 11 The results show that the neovascularization length in the dACSL4@401-TK12 group was significantly lower than that in the untreated control group and the blank vector 401-TK12 group on both days 7 and 14. Figure 12 It can be seen that on days 21 and 28, the neovascular network in the dACSL4@401-TK12 group was sparse and gradually degenerated.

[0130] Immunofluorescence staining was performed on CD31 (a vascular endothelial-specific marker) in corneal tissue sections to obtain... Figure 13 , Figure 14 It can be seen that the CD31 positive signal was significantly reduced in the dACSL4@401-TK12 group. Figure 13 The white arrow in the middle indicates CD31 positive vascular cavities, indicating a significant reduction in pathological angiogenesis.

[0131] The above-mentioned corneal neovascularization assessment and immunofluorescence staining results of CD31 in corneal tissue sections indicate that the dACSL4@401-TK12 of the present invention can inhibit corneal neovascularization.

[0132] 2.8.4 The dACSL4@401-TK12 of the present invention can downregulate ACSL4 and inhibit ferroptosis and oxidative stress in corneal tissue.

[0133] The expression of key ferroptosis-related proteins in corneal tissue was detected using immunofluorescence. Figure 15 , Figure 16 It can be seen that the ACSL4 protein level was significantly reduced in the dACSL4@401-TK12 group; from Figure 17 , Figure 18 It can be seen that GPX4 expression was upregulated in the dACSL4@401-TK12 group, indicating that dACSL4@401-TK12 can effectively inhibit ferroptosis in the cornea of ​​mice with alkali damage.

[0134] Dihydroethidium (DHE) staining showed a significant decrease in fluorescence intensity in the dACSL4@401-TK12 group at days 14 and 21 (see details). Figure 19 , Figure 20 This indicates that oxidative stress in the body has been reduced.

[0135] 2.8.5 The dACSL4@401-TK12 of the present invention exhibits good safety. The results of prostaglandic photography of healthy control mice show (see details) Figure 21 The dACSL4@401-TK12 of this invention does not cause epithelial defects in the cornea of ​​healthy mice; hematoxylin-eosin (H&E) staining results of healthy control mice show (see details) Figure 22 Tissue sections from healthy mice showed no signs of inflammatory infiltration, indicating that the mice tolerated dACSL4@401-TK12 well and could be safely used for topical ocular treatment.

[0136] Test Example 3 - Cell Culture Assay of Human Umbilical Vein Endothelial Cells (HUVECs) Human umbilical vein endothelial cells (HUVECs) were cultured in a complete medium consisting of high-glucose DMEM, 10% fetal bovine serum (FBS), and 1% penicillin / streptomycin. Cells were cultured at 37°C and 5% CO2 for 24 hours to allow them to adhere.

[0137] The cultured cells were randomly divided into four experimental groups: (1) Control group; (2) Erastin group; (3) Erastin and 401-TK12 combination group; (4) Erastin and dACSL4@401-TK12 combination group; The control group received no treatment; the Erastin group was treated with Erastin-induced ferroptosis and cultured with 10 μM Erastin; the Erastin and 401-TK12 combination group was treated with cultured with 10 μM Erastin and 1 μM blank vector 401-TK12; and the Erastin and dACSL4@401-TK12 combination group was treated with cultured with 10 μM Erastin and 1 μM PROTAC nanocomposite dACSL4@401-TK12. All cultures were prepared using complete culture medium and incubated at 37°C for 24 h.

[0138] 3.1 Western blot analysis Total protein was extracted from cells in the four experimental groups using RIPA lysis buffer containing protease inhibitors, and protein concentration was determined by the BCA method. Equal amounts of protein were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% skim milk at room temperature for 2 hours. Cells in the four experimental groups were incubated overnight at 4°C with primary antibodies against rabbit anti-ACSL4 / FACL4 and rabbit anti-GPX4, respectively. After washing, the cells were incubated with HRP-labeled secondary antibody at room temperature for 2 hours. Colorimetric analysis was performed using Super ECL Plus chemiluminescent substrate, and quantification was performed using ImageJ software.

[0139] 3.2 Determination of malondialdehyde (MDA) content Cellular proteins from the four experimental groups were extracted using RIPA lysis buffer containing protease inhibitors, and protein concentrations were determined by the BCA method. Cellular proteins extracted with RIPA lysis buffer were used as samples for malondialdehyde (MDA) content determination. Intracellular lipid peroxidation was detected using a malondialdehyde (MDA) assay kit (TBA method) developed by Nanjing Jiancheng, following the method described in Part 5 of the kit's instructions. The cells were incubated at 95°C for 40 minutes, centrifuged at 4000 rpm for 10 minutes, and the supernatant was collected. OD values ​​were measured at 532 nm using a microplate reader and statistical analysis was performed.

[0140] 3.3 CM-H2DCFDA fluorescent staining HUVECs from four experimental groups were seeded in 24-well plates with a confluence of 70%-80% and treated for 24 h. Thirty min before the end of treatment, 10 μM CM-H2DCFDA probe was added, and the plates were incubated at 37°C in the dark. After washing with PBS buffer, green fluorescence images were captured under an inverted fluorescence microscope, and the average fluorescence intensity was quantified using ImageJ.

[0141] 3.4 Statistical Methods All data were analyzed and graphs generated using GraphPad Prism (version 10.0). Results are expressed as mean ± standard deviation (SD). Differences between groups were assessed using Student's t-test or one-way ANOVA, as appropriate. Statistical significance was defined as p < 0.05, where p ≤ 0.05 (where p < 0.05 is a statistical significance level). p ≤ 0.01 p ≤ 0.001 ) and p ≤ 0.0001 ( ).

[0142] 3.5 Test Results Western blot analysis showed (see details) Figure 23 , Figure 24 Treatment with the ferroptosis inducer Erastin significantly upregulated ACSL4 protein levels. Conversely, the combination of Erastin and dACSL4@401-TK12 significantly reduced ACSL4 expression, confirming that dACSL4@401-TK12 effectively inhibited ACSL4 accumulation during ferroptosis.

[0143] Western blot results (see details) Figure 25 , Figure 26 Erastin-induced ferroptosis resulted in a significant decrease in GPX4 levels, while GPX4 expression recovered 24 hours after treatment in the combination of Erastin and dACSL4@401-TK12. By detecting the expression of GPX4 (a key antiferroptosis protective factor), the antiferroptosis effect of dACSL4@401-TK12 of this invention was verified to be mediated by ACSL4 degradation.

[0144] Malondialdehyde (MDA) content determination results: Since lipid peroxidation is a hallmark of ferroptosis, MDA content can assess the level of lipid peroxidation. Figure 27 The statistical results show that Erastin treatment significantly increased MDA levels, while the combination of Erastin and dACSL4@401-TK12 significantly reduced this increase, indicating that dACSL4@401-TK12 can effectively reduce lipid peroxidation and inhibit ferroptosis.

[0145] CM-H2DCFDA fluorescent staining can assess intracellular oxidative stress. Figure 28 , Figure 29It can be seen that Erastin treatment significantly enhanced the fluorescence signal of intracellular reactive oxygen species (ROS), while the ROS fluorescence was significantly reduced after treatment with dACSL4@401-TK12 in the Erastin and dACSL4@401-TK12 combination group, indicating that dACSL4@401-TK12 of the present invention can alleviate oxidative stress in cells.

[0146] In summary, the dACSL4@401-TK12 of the present invention can inhibit ferroptosis and reduce oxidative stress.

[0147] Test Example 4 - Scratch Healing Experiment 5×10 5 Personal umbilical vein endothelial cells (HUVECs) were seeded in six-well plates and cultured overnight at 37°C and 5% CO2. The next day, when cell density reached 80%-90%, cells were streaked vertically through the wells using a 200 μL sterile pipette tip and washed twice with PBS buffer to remove residual cells. The cells were then divided into four experimental groups. (1) Control group: Add 2 mL of low serum culture medium containing 2% fetal bovine serum (FBS); (2) VEGF group: Add to 2 mL of low serum culture medium containing 40 ng / mL rh-VEGF and 2% fetal bovine serum (FBS); (3) VEGF and 401-TK12 combination group: added to 2 mL of low serum culture medium containing 40 ng / mL rh-VEGF, 1 μM blank vector 401-TK12 and 2% fetal bovine serum (FBS); (4) VEGF and dACSL4@401-TK12 combination group: added to 2 mL of low serum culture medium containing 40 ng / mL rh-VEGF, 1 μM PROTAC nanocomplex dACSL4@401-TK12 and 2% fetal bovine serum (FBS); Cell growth was observed and photographed in each experimental group at 0 and 12 hours, and the percentage of scar area was statistically analyzed using ImageJ software. The healing rate was calculated according to the following formula: Healing rate (%) = [(0h scratch area - 12h scratch area) / 0h scratch area] × 100%.

[0148] Test results: In corneal alkali burns, pathological neovascularization is the primary cause of corneal blindness and impaired functional recovery. Neovascularization is driven by multiple factors, including reactive oxygen species (ROS) and inflammatory cytokines, which promote the production of vascular endothelial growth factor (VEGF).

[0149] Scratch healing experiments show, see details Figure 30 , Figure 31 In the VEGF group, the wound healing rate of human umbilical vein endothelial cells treated with VEGF was approximately 70%. However, in the VEGF and dACSL4@401-TK12 combination group, treatment with dACSL4@401-TK12 reduced the wound healing rate to 45.5%, indicating that the dACSL4@401-TK12 of this invention can effectively inhibit VEGF-induced HUVEC proliferation and vascular endothelial cell proliferation. Notably, in the VEGF and 401-TK12 combination group, treatment with the blank 401-TK12 vector did not show a significant difference in cell migration and wound healing rate compared to the VEGF group, suggesting that the inhibitory effect of dACSL4@401-TK12 originates from its effective payload (dACSL4) rather than the vector itself.

[0150] Test Example 5 - Cytotoxicity Assay Cytotoxicity was assessed using the CCK-8 assay. Human umbilical vein endothelial cells (HUVECs) were cultured in complete medium consisting of high-glucose DMEM, 10% fetal bovine serum (FBS), and 1% penicillin / streptomycin. Cells were cultured at 37°C under humidified conditions of 5% CO2 for 24 hours. The cultured HUVECs were then treated for 24 hours in 0.5 μM, 1 μM, and 1.5 μM dACSL4@401-TK12 media, respectively.

[0151] The cell survival rate of the different concentration treatment groups all exceeded 95%, indicating that the PROTAC nanocomposite of the present invention has negligible cytotoxicity at treatment-related doses.

[0152] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A PROTAC nanocomposite, characterized in that, The PROTAC nanocomposite comprises a lipid layer and a PROTAC chimera encapsulated within the lipid layer that targets the degradation of ACSL4; wherein the lipid layer comprises ionizable cationic lipids, cholesterol, DOPE, and DSPE-PEG2000. Based on the mass of the PROTAC nanocomposite, the mass ratio of the ionizable cationic lipid, cholesterol, DOPE, DSPE-PEG2000 and the PROTAC chimera that targets ACSL4 degradation is (1.5-2.5):(0.8-1.2):(0.3-0.7):(1.5-2.5):

1.

2. The PROTAC nanocomposite according to claim 1, characterized in that, The structural formula of the PROTAC chimera that targets ACSL4 degradation is shown in Formula I. Equation I.

3. The PROTAC nanocomposite according to claim 1 or 2, characterized in that, The structural formula of the ionizable cationic lipid is shown in Formula II; Formula II; and / or, The average hydrodynamic diameter of the PROTAC nanocomposite in water is 195 nm-204 nm; the polydispersity index of the PROTAC nanocomposite is less than or equal to 0.2; and the zeta potential of the PROTAC nanocomposite mixed with water to 0.1 mg / mL at 25 °C is 30 mV-40 mV.

4. A method for preparing the PROTAC nanocomposite according to any one of claims 1-3, characterized in that, Includes the following steps: PROTAC chimeras targeting ACSL4 degradation were prepared to generate ionizable cationic lipids; The ionizable cationic lipid, cholesterol, DOPE and the PROTAC chimera that targets ACSL4 degradation were mixed in chloroform, and the resulting first mixture was dried to obtain a lipid membrane. The lipid membrane was dissolved in a C1-C3 monohydric alcohol to obtain a second mixture; DSPE-PEG2000 was dispersed in an atmosphere with a pH of 7- In a buffer solution of 7.4, the second mixture was mixed with a buffer solution containing DSPE-PEG2000, and after dialysis, the PROTAC nanocomposite was obtained.

5. The method for preparing the PROTAC nanocomposite according to claim 4, characterized in that, The ratio of the ionizable cationic lipid to the amount of chloroform added is (150-250) μg: 2 mL; and / or, The mixture is dried at a temperature of 30-40℃.

6. The method for preparing the PROTAC nanocomposite according to claim 4 or 5, wherein when the lipid membrane is dissolved in a C1-C3 monohydric alcohol, the ratio of the amount of ionizable cationic lipid in the lipid membrane to the amount of the monohydric alcohol added is (150-250) μg: 50 μL; and / or, The C1-C3 monohydric alcohol is ethanol; and / or, In the buffer solution containing DSPE-PEG2000, the ratio of DSPE-PEG2000 to the amount of buffer solution added is (150-250) μg:1mL; wherein, The buffer solution is a phosphate buffer solution with a concentration of 5-20 mM and a pH value of 7.2-7.

4.

7. The method for preparing the PROTAC nanocomposite according to claim 4 or 5, characterized in that, The preparation of the PROTAC chimera that targets ACSL4 degradation includes the following steps: Pomalidomide, 1,2-bis(2-bromoethoxy)ethane, potassium carbonate and potassium iodide were dissolved in acetonitrile and refluxed at 80-85℃ for 12-24 h to prepare pomalidomide-bromopolyethylene glycol intermediate. Troglitazone, the pomalidomide-bromopolyethylene glycol intermediate, potassium carbonate, and potassium iodide were dissolved in acetonitrile and reacted under reflux conditions in the presence of a protective gas for 12-24 hours. The resulting reaction product was post-processed to obtain the PROTAC chimera that targets the degradation of ACSL4.

8. The method for preparing the PROTAC nanocomposite according to claim 4 or 5, characterized in that, The preparation of the ionizable cationic lipid includes the following steps: The fatty amine with the structure shown in Formula A and the compound with the structure shown in Formula B are mixed in a molar ratio of 1:(3-3.5) and reacted at 75-85°C. The reaction product is purified and concentrated to dryness to obtain the ionizable cationic lipid. Formula A; Formula B; The steps of purifying and concentrating the reaction product to dryness include: purifying the reaction product by silica gel column chromatography, then eluting with dichloromethane or methanol as the eluent, and concentrating the eluent under reduced pressure to dryness to obtain ionized cationic lipids.

9. The use of the PROTAC nanocomposite according to any one of claims 1-3 in a product for treating ophthalmic diseases; wherein, The treatment of ophthalmic diseases includes the treatment of corneal alkali burns.

10. The application according to claim 9, characterized in that, The treatment for corneal alkali burns includes: Inhibits ferroptosis, reduces oxidative stress, and inhibits vascular endothelial cell proliferation; and / or, Accelerate corneal healing and improve repair quality; and / or, Relieves corneal edema and inhibits inflammatory response; and / or, Inhibits corneal neovascularization.

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