A nanoparticle based on transcytosis-activating ligand, and a preparation method and application thereof

Abstract

This invention discloses nanoparticles based on transcytosis-activating ligands, their preparation method, and applications, particularly in the treatment of fundus diseases. The nanoparticles comprise an enzyme protein core and a polymer shell grown in situ on the protein surface. The polymer shell contains positively charged monomers, transcytosis-activating ligands, and neutral monomers. The nanoparticles can enhance the hydrolytic resistance of enzyme proteins (e.g., antioxidant enzymes), protecting them from degradation during transport and improving delivery stability. Simultaneously, the nanoparticles can also promote the efficient delivery of proteases (especially to the posterior segment of the eye), exhibiting good safety and showing excellent application prospects in the medical field.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a nanoparticle based on transcytosis-activating ligand (TAL), its preparation method, and its application, particularly in the treatment of fundus diseases. Background Technology

[0002] Diabetic retinopathy, glaucoma, and age-related macular degeneration are among the leading causes of irreversible vision loss worldwide. Retinal oxidative stress is widely considered a key pathogenic driver of the development and progression of these diseases. Antioxidants hold significant therapeutic potential by restoring redox homeostasis in retinal cells. However, most preclinical trials attempting to deliver small-molecule antioxidants to the posterior segment of the retina via eye drops, such as OT-551 (NCT00306488) and VisuXL® (NCT05429229), have failed to meet their study endpoints or obtain clinical approval. These failures are primarily attributed to: i) multiple ocular barriers leading to rapid clearance of the eye drops after local instillation, significantly reducing bioavailability; and ii) the complex mechanisms of action of small-molecule drugs, which may induce unpredictable toxicities in addition to the intended effects during use.

[0003] Enzymes are large molecular biocatalysts with high substrate specificity and catalytic efficiency. For example, catalase (CAT) specifically catalyzes the decomposition of hydrogen peroxide (H2O2), while superoxide dismutase (SOD) catalyzes the decomposition of superoxide anion radicals (O2). - These antioxidant enzymes exhibit promising potential in regulating oxidative stress in retinal diseases without causing unpredictable toxicity. However, their large molecular size and fragility pose challenges in crossing the eye barrier while maintaining catalytic activity.

[0004] Therefore, providing a drug delivery method and product that can improve the bioavailability of eye drops and achieve precise regulation of redox homeostasis is crucial for the safe and effective treatment of fundus diseases. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention provides nanoparticles based on transcytosis-activated ligand (TAL), their preparation method and applications, particularly in the treatment of fundus diseases.

[0006] In a first aspect of the invention, a compound or a salt thereof is provided, said compound being usable as a functional monomer (transcytosis-activating ligand monomer), having the following structure: (I) in, L is a linking group; R1 is an end group containing unsaturated chemical bonds (such as carbon-carbon double bonds) that can undergo polymerization.

[0007] In some embodiments of the present invention, L is... , where n is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0008] Furthermore, n is an integer from 1 to 6, such as 1, 2, 3, 4, 5, 6.

[0009] In some embodiments of the present invention, R1 is -C(CH3)=CH2 or -CH=CH2.

[0010] In some embodiments of the present invention, the compound has the following structure: .

[0011] In a second aspect of the invention, a nanoparticle with a core-shell structure is provided, comprising a protein as a core and a polymer shell coating the surface of the protein. The monomers of the polymer include functional monomers, positively charged monomers, and neutral monomers, wherein the functional monomers are the compounds described in the first aspect of the present invention.

[0012] Furthermore, the protein is an enzyme protein, such as an antioxidant enzyme.

[0013] In some embodiments of the present invention, the antioxidant enzyme is selected from one or more of the following: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione reductase (GR).

[0014] Furthermore, the protein may also be loaded with one or more small molecule active ingredients, such as glycerol, trehalose, panthenol, hyaluronic acid oligosaccharide, fluocinolone, bromfenac sodium, olopatadine, rifelast, cyclosporine A, taurine, vitamin B6, B12, lutein, anthocyanins, aspartic acid, levofloxacin, acyclovir, asiaticoside, menthol, borneol, etc.

[0015] Furthermore, the protein has been modified with double bonds, as described in the third aspect of the invention.

[0016] In some embodiments of the present invention, the functional unit is .

[0017] Further, the positively charged monomer is selected from one or more of N-(3-aminopropyl)acrylamide, dimethylaminoethyl methacrylate (DMAEMA), and acrylamide propyltrimethylammonium chloride (APTAC). In some embodiments of the present invention, the positively charged monomer is N-(3-aminopropyl)acrylamide.

[0018] Further, the neutral monomer can be, for example, acrylamides, acrylates, vinyl esters, etc. Even further, the neutral monomer is selected from one or more of the following: acrylamide, N,N-dimethylacrylamide, N-isopropylacrylamide, N-ethylacrylamide, N,N-diethylacrylamide, hydroxyethyl acrylate, hydroxyethyl methacrylate, hydroxypropyl acrylate, vinyl acetate, etc. In some embodiments of the present invention, the neutral monomer is acrylamide.

[0019] Furthermore, the molar ratio of the protein to the polymer monomer is 1:1000-10000 (e.g., 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000), particularly 1:4000.

[0020] Furthermore, the functional monomer accounts for 1%-45% of the sum of the molar amounts of the functional monomer, the positively charged monomer, and the neutral monomer (e.g., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%), particularly 10%-30%, 15%-25%, and 20%.

[0021] Furthermore, the proportion of the positively charged monomer to the sum of the molar amounts of the functional monomer, the positively charged monomer, and the neutral monomer is 1%-20% (e.g., 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%), particularly 1%-15%, 2.5%-10%, and 5%.

[0022] Furthermore, the proportion of the neutral monomer to the sum of the molar amounts of the functional monomer, the positively charged monomer, and the neutral monomer is 50%-95% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%).

[0023] In some preferred embodiments of the present invention, the functional monomer accounts for 20% and the positively charged monomer accounts for 5% of the total molar amount of the functional monomer, the positively charged monomer, and the neutral monomer.

[0024] Furthermore, the raw materials for the polymer further include a crosslinking agent; further, the crosslinking agent is a compound containing multiple unsaturated double bonds, such as polyisocyanates, polyamines, or acrylates, for example: one or more of N,N-methylenebisacrylamide, ethylene glycol diacrylate, polyethylene glycol diacrylate, glycerol dimethacrylate, diisocyanate, propylenediamine, divinylbenzene, and vinyltriethoxysilane. In some embodiments of the present invention, the crosslinking agent is N,N-methylenebisacrylamide (BIS).

[0025] In some embodiments of the present invention, the protein is superoxide dismutase (SOD), and the polymer forms a shell on the protein surface (“in-situ growth”) through in-situ polymerization; wherein, the raw materials for in-situ polymerization include functional monomers, positively charged monomers, neutral monomers, and crosslinking agents, wherein the functional monomers are… The positively charged monomer is N-(3-aminopropyl)acrylamide, the neutral monomer is acrylamide, the crosslinking agent is N,N-methylenebisacrylamide, and the molar ratio of protein to functional monomer, positively charged monomer and neutral monomer is 1:800:200:3000 (AT-n(SOD)).

[0026] In some embodiments of the present invention, the protein is catalase (CAT), and the polymer forms a shell on the protein surface (“in-situ growth”) through in-situ polymerization; wherein the raw materials for in-situ polymerization include functional monomers, positively charged monomers, neutral monomers, and crosslinking agents, and the functional monomers are… The positively charged monomer is N-(3-aminopropyl)acrylamide, the neutral monomer is acrylamide, the crosslinking agent is N,N-methylenebisacrylamide, and the molar ratio of protein to functional monomer, positively charged monomer and neutral monomer is 1:800:200:3000 (AT-n(CAT)).

[0027] Furthermore, the average diameter of the nanoparticles is 5-50 nm (e.g., 5, 10, 11, 12, 13, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, 50 nm), particularly 10-20 nm and 10-15 nm.

[0028] Furthermore, the zeta potential of the nanoparticles is 1-5 mV (e.g., 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 mV), particularly 2.5-3.0 mV.

[0029] Furthermore, the nanoparticles are prepared by the method described in the third aspect of the present invention.

[0030] In a third aspect of the present invention, a method for preparing nanoparticles (as described in the second aspect of the present invention) is provided, comprising the following steps: (1) Double bond modification on the protein surface: The protein is mixed with a double bond modifier and reacted to obtain a protein with surface-modified double bonds; (2) In-situ polymerization: The protein with surface-modified double bonds obtained in step (1) is mixed with functional monomers, positively charged monomers, neutral monomers, cross-linking agents and initiators, and reacted; The functional monomer is the compound described in the first aspect of this invention.

[0031] Furthermore, the protein is an enzyme protein, such as an antioxidant enzyme, including, but not limited to, one or more of: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione reductase (GR).

[0032] Further, the double bond modifier is selected from one or more of acrylic acid, acrylates, and acrylate salts; even further, the double bond modifier is selected from one or more of N-acryloyloxysuccinimide, acrylamide, sodium acrylate, and N-(3-aminopropyl)acrylamide. In some embodiments of the present invention, the double bond modifier is N-acryloyloxysuccinimide. In some embodiments of the present invention, the double bond modifier is N-(3-aminopropyl)acrylamide.

[0033] Further, the molar ratio of the protein to the double bond modifier in step (1) is 1:5-200 (e.g., 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:120, 1:150), particularly 1:5-100, 1:10-50, 1:20.

[0034] Further, the reaction described in step (1) is carried out in solution; even further, the solution is a buffer solution with a pH of 6-9 (e.g., 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0), such as 2-(N-morpholino)ethanesulfonic acid (MES) buffer with a pH of 6.

[0035] Further, the reaction temperature in step (1) is 4-30℃ (e.g., 4, 5, 6, 8, 10, 15, 20, 25, 30℃).

[0036] Further, the reaction time described in step (1) is 1-16 hours (e.g., 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16 hours).

[0037] Furthermore, step (1) may also include a step of first activating the protein with carboxyl groups (e.g., adding EDC and NHS).

[0038] Furthermore, step (1) may also include a post-processing step (e.g., ultrafiltration centrifugation to remove small molecules).

[0039] In some embodiments of the present invention, the functional unit is .

[0040] Further, the positively charged monomer is selected from one or more of N-(3-aminopropyl)acrylamide, dimethylaminoethyl methacrylate (DMAEMA), and acrylamide propyltrimethylammonium chloride (APTAC). In some embodiments of the present invention, the positively charged monomer is N-(3-aminopropyl)acrylamide.

[0041] Further, the molar ratio of the protein to the functional monomer in step (2) is 1:50-2000 (e.g., 1:100, 1:200, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:1100, 1:1200, 1:1400, 1:1500, 1:1600, 1:1800, 1:2000), especially 1:100-1000, 1:400-1200, 1:800.

[0042] Further, the molar ratio of the protein to the positively charged monomer in step (2) is 1:50-1000 (e.g., 1:50, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:600, 1:800, 1:1000), especially 1:100-400, 1:200.

[0043] Further, the neutral monomer is an acrylamide, acrylate, vinyl ester, etc. Even further, the neutral monomer is selected from one or more of the following: acrylamide, N,N-dimethylacrylamide, N-isopropylacrylamide, N-ethylacrylamide, N,N-diethylacrylamide, hydroxyethyl acrylate, hydroxyethyl methacrylate, hydroxypropyl acrylate, vinyl acetate, etc. In some embodiments of the present invention, the neutral monomer is acrylamide.

[0044] Further, in step (2), the molar ratio of protein to neutral monomer is 1:1000-5000 (e.g., 1:1000, 1:1500, 1:2000, 1:2400, 1:2500, 1:2600, 1:2800, 1:2900, 1:3000, 1:3100, 1:3200, 1:3400, 1:3500, 1:3600, 1:3800, 1:4000, 1:4200, 1:4400, 1:4500, 1:4600, 1:4800, 1:5000), especially 1:2000-4000, 1:3000.

[0045] Further, the crosslinking agent is a polyisocyanate, polyamine, or acrylate, such as one or more of N,N-methylenebisacrylamide, ethylene glycol diacrylate, polyethylene glycol diacrylate, glycerol dimethacrylate, diisocyanate, propylenediamine, divinylbenzene, and vinyltriethoxysilane. In some embodiments of the present invention, the crosslinking agent is N,N-methylenebisacrylamide (BIS).

[0046] Further, in step (2), the molar ratio of protein to crosslinking agent is 1:50-1000 (e.g., 1:50, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:800, 1:1000), especially 1:100-500, 1:300.

[0047] Furthermore, the initiator is a water-soluble initiator, such as one or more of ammonium persulfate, sodium persulfate, and potassium persulfate. In some embodiments of the present invention, the initiator is ammonium persulfate.

[0048] Furthermore, in step (2), the molar ratio of protein to initiator is 1:50-1000 (e.g., 1:50, 1:100, 1:200, 1:300, 1:400, 1:450, 1:500, 1:600, 1:800, 1:1000), especially 1:300-600, 1:450.

[0049] In some embodiments of the present invention, step (2) further includes adding a co-initiator, such as sodium bisulfite or tetramethylethylenediamine.

[0050] Furthermore, in step (2), the mass ratio of the initiator to the co-initiator is 1:1-5 (e.g., 1:1, 1:2, 1:3, 1:4, 1:5).

[0051] In some embodiments of the present invention, the molar ratio of protein to functional monomer, positively charged monomer, and neutral monomer in step (2) is 1:800:200:3000. Furthermore, the molar ratio of protein to functional monomer, positively charged monomer, neutral monomer, crosslinking agent, and initiator in step (2) is 1:800:200:3000:300:450.

[0052] Further, step (2) may also include the preparation of solutions of functional monomer, positively charged monomer, crosslinking agent, and initiator. Further, the solvent for the functional monomer solution, positively charged monomer solution, and initiator solution is water. Further, the concentrations of the functional monomer solution, positively charged monomer solution, and initiator solution are each 5%-20% (e.g., 5%, 8%, 10%, 12%, 15%, 18%, 20%, w / v). Further, the solvent for the crosslinking agent solution is selected from one or more of dimethyl sulfoxide, ethanol, propanol, and ethylene glycol, for example, dimethyl sulfoxide. Further, the concentration of the crosslinking agent solution is 5%-20% (e.g., 5%, 8%, 10%, 12%, 15%, 18%, 20%, w / v).

[0053] Further, the concentration of protein in the reaction system described in step (2) is 1-10 mg / mL (e.g., 1, 2, 3, 4, 5, 6, 8, 10 mg / mL).

[0054] Further, the reaction in step (2) is carried out in solution; even further, the solution is a buffer solution with a pH of 6-9 (e.g., 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0), such as sodium bicarbonate buffer solution with a pH of 8.5.

[0055] Furthermore, the reaction temperature described in step (2) is 4-30℃ (e.g., 4, 5, 6, 8, 10, 15, 20, 25, 30℃).

[0056] Furthermore, the reaction time described in step (2) is 1-12 hours (e.g., 1, 2, 3, 4, 5, 6, 8, 10, 12 hours).

[0057] Furthermore, step (2) may also include a post-processing step (e.g., ultrafiltration centrifugation to remove small molecules).

[0058] In a fourth aspect of the invention, a composition is provided comprising the compound described in the first aspect or a salt thereof, and a positively charged monomer.

[0059] In some embodiments of the present invention, the compound is The positively charged monomer is N-(3-aminopropyl)acrylamide.

[0060] In some embodiments of the invention, the composition further comprises a double bond modifier, such as that described in the third aspect of the invention, for example, N-acryloyloxysuccinimide (NAS).

[0061] In some embodiments of the invention, the composition further comprises a crosslinking agent, such as that described in the third aspect of the invention, for example N,N-methylenebisacrylamide (BIS).

[0062] In some embodiments of the invention, the composition further comprises an initiator, such as that described in the third aspect of the invention, for example, ammonium persulfate (APS).

[0063] In some embodiments of the invention, the composition further comprises a co-initiator, such as that described in the third aspect of the invention, for example tetramethylethylenediamine (TEMED).

[0064] Furthermore, the composition may also contain a carboxyl activating agent, such as EDC / NHS.

[0065] Furthermore, the composition may also contain a solvent, such as one or more of water, dimethyl sulfoxide, ethanol, propanol, ethylene glycol, etc., as described in the third aspect of the invention.

[0066] Furthermore, the composition may also contain a buffer solution, such as that described in the third aspect of the invention, for example, sodium bicarbonate buffer or 2-(N-morpholino)ethanesulfonic acid (MES) buffer.

[0067] In a fifth aspect of the invention, a pharmaceutical composition is provided comprising the nanoparticles described in the second aspect, and one or more pharmaceutically acceptable excipients.

[0068] Furthermore, the pharmaceutically acceptable excipients include, for example, one or more of the following: carriers, diluents, wetting agents, fillers, binders, lubricants, disintegrants, antioxidants, buffers, suspending agents, solubilizers, thickeners, stabilizers, flavoring agents, emulsifiers, skin moisturizers, penetration enhancers, surfactants, preservatives, preservative synergists, solvents, fragrances, colorants, pH adjusters, etc.

[0069] Furthermore, the pharmaceutical composition can be administered via any suitable route of administration, such as gastrointestinal administration (e.g., oral administration) or non-gastrointestinal administration (e.g., intraocular, intravenous, intramuscular, transdermal, subcutaneous, intradermal, mucosal administration, etc.).

[0070] In some embodiments of the present invention, the pharmaceutical composition is an ophthalmic pharmaceutical composition, which may be in the form of eye drops, ophthalmic gels, ointments, injections, etc. Further, the pharmaceutically acceptable excipients may be selected from one or more of the following: solubilizers, stabilizers, viscosity modifiers, solvents, buffers, pH adjusters, isotonic adjusters, humectants, penetration enhancers, suspending agents, preservatives, etc. In some preferred embodiments of the present invention, the pharmaceutical composition is an eye drop.

[0071] Furthermore, in the pharmaceutical composition, the nanoparticles described in the second aspect can be used alone or in combination with other types of active ingredients.

[0072] Furthermore, the pharmaceutical composition may be a unit dosage form. In this form, the formulation is further divided into unit doses containing an appropriate amount of the active ingredient.

[0073] Furthermore, the amount of the active ingredient in the unit dose formulation may be varied or adjusted from 0.1 mg to 1000 mg (e.g., 0.1, 1, 5, 10, 20, 40, 50, 100, 200, 400, 500, 1000 mg), depending on the specific application and potency of the active ingredient. If desired, the composition may also contain other suitable therapeutic agents.

[0074] Further, the amount (mass percentage) of the active component (nanoparticles as described in the second aspect) in the pharmaceutical composition can be 0.1-99.5% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%).

[0075] In a sixth aspect of the invention, the use of the nanoparticles described in the second aspect and the pharmaceutical composition described in the fifth aspect in the preparation of medicaments for the prevention and / or treatment of eye diseases is provided.

[0076] Furthermore, the diseases mentioned are selected from: age-related macular degeneration, diabetic retinopathy, glaucoma, retinitis pigmentosa, high myopia fundus disease, retinal ischemia-reperfusion injury, retinal blue light damage, dry eye syndrome, corneal injury, corneal blue light damage, keratitis, cataract, eye strain, blue light damage syndrome, etc.

[0077] In some embodiments of the present invention, the disease is a fundus disease, such as age-related macular degeneration, diabetic retinopathy, glaucoma, retinitis pigmentosa, fundus disease of high myopia, retinal ischemia-reperfusion injury, retinal blue light damage, etc.

[0078] In a seventh aspect of the invention, a method for preventing and / or treating eye diseases is provided, comprising the step of administering the nanoparticles described in the second aspect or the pharmaceutical composition described in the fifth aspect to a subject in need of such treatment.

[0079] Furthermore, the diseases mentioned are as described in the sixth aspect of the present invention, particularly age-related macular degeneration, diabetic retinopathy, glaucoma, retinitis pigmentosa, high myopia fundus disease, retinal ischemia-reperfusion injury, and retinal blue light damage.

[0080] Furthermore, the subjects are mammals, such as humans.

[0081] Furthermore, the method of administration is local ocular administration, such as ocular surface instillation, ocular gel / ointment application, subconjunctival injection, intravitreal injection, suprachoroidal administration, etc., especially ocular surface instillation.

[0082] In an eighth aspect of the invention, the use of the nanoparticles described in the second aspect and the pharmaceutical composition described in the fifth aspect in the preparation of medicaments for the prevention and / or treatment of oxidative stress diseases is provided.

[0083] Furthermore, the diseases are selected from: neurodegenerative diseases, diabetes and its complications, cardiovascular diseases, inflammatory and immune diseases, liver and kidney damage, photoaging of the skin, and eye diseases (such as those described in the sixth aspect of the present invention).

[0084] Furthermore, the subjects are mammals, such as humans.

[0085] Furthermore, the administration route can be any suitable route of administration, such as gastrointestinal administration (e.g., oral administration) or non-gastrointestinal administration (e.g., intraocular, intravenous, intramuscular, transdermal, subcutaneous, intradermal, mucosal administration, etc.).

[0086] In an eighth aspect of the invention, the use of the compound described in the first aspect or its salt, or the composition described in the fourth aspect, in the preparation of nanoparticles targeting the eye (as described in the second aspect and examples of the invention, or other nanoparticles with a protein core and a polymer shell) is provided.

[0087] This invention presents a nanoparticle prepared based on transcytosis-activated ligands. This nanoparticle can serve as an actively transported nanocapsule (ATNC), enhancing the hydrolytic resistance of proteins (e.g., antioxidant enzymes), protecting them from degradation during transport, and improving delivery stability. Simultaneously, the nanoparticle can also promote efficient protein delivery (especially to the posterior segment of the eye), exhibiting good safety profiles and demonstrating excellent application prospects in the pharmaceutical field (particularly in the treatment of retinal diseases). Attached Figure Description

[0088] Figure 1 The diagram shows the structure and function of actively transported nanocapsules (ATNC).

[0089] Figure 2 The figure shows the 1H NMR characterization results of the transcytotoxic activation ligand (TAL) prepared in Example 1.

[0090] Figure 3 The mass spectrometry characterization results of the transcytotoxic activation ligand (TAL) prepared in Example 1 are shown.

[0091] Figure 4 The figure shows the molar ratio of each monomer in 25 different formulations of ATNCs.

[0092] Figure 5 The images show the preparation and characterization of ATNCs. a) shows 25 ATNCs libraries with different ratios; b) shows the zeta potential thermograms of ATNCs with different ratios; c) shows the mean fluorescence intensity (MFI) thermograms of ATNCs with different ratios co-incubated with HCEC cells for two hours; d) shows the toxicity thermogram of ATNCs co-incubated with HCEC cells for 24 hours; e) shows a schematic diagram of ATNCs transepithelial transport; and f) shows the quantitative analysis of fluorescence intensity on the basal side of the Transwell chamber.

[0093] Figure 6 The figure shows the molar ratios of each monomer in the synthesis of n(CAT) and AT-n(CAT).

[0094] Figure 7 The figure shows the molar ratios of each monomer in the synthesis of n(SOD) and AT-n(SOD).

[0095] Figure 8 The image shows the characterization results and TEM images of the size and zeta potential of natural CAT, SOD and ATNCs.

[0096] Figure 9The results show the enzyme activity of ATNCs. In this paper, a shows the enzyme activities of natural CAT, SOD and ATNCs; b shows the changes in enzyme activity of ATNCs over 4 weeks; c and d show the relative enzyme activities of natural CAT, SOD and ATNCs after co-incubation with trypsin.

[0097] Figure 10 The figure shows the fluorescence intensity in the posterior segment of the mouse eye after a single infusion of different FITC markers (natural CAT, n(CAT), AT-n(CAT), and AT-n(CAT)+GS).

[0098] Figure 11 The images show the results of fluorescein corneal staining, fundus photographs, and representative optical coherence tomography (OCT) images of mice after 14 consecutive days of ATNCs eye drops.

[0099] Figure 12 The figure shows the efficacy of AT-n(CAT) in a mouse model of retinal ischemia-reperfusion. Figure 12 Figure a shows the TUNEL staining results of a frozen section of the retina; Figure 12 b shows the H&E staining results; Figure 12 Figure c shows a representative waveform of ERG.

[0100] Figure 13 and Figure 14 The figure shows the therapeutic effects of instilling natural SOD and ATNCs on a mouse model of retinal blue light damage. Figure 13 Figure a shows the TUNEL staining results of a frozen section of the retina; Figure 13 b shows the H&E staining results; Figure 14 a-14c shows representative OCT images and statistical results; Figure 14 Figure d-14f shows representative ERG waveforms and statistical results. Detailed Implementation

[0101] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0102] All publications, patents, and published patent specifications cited in this article are incorporated herein in their entirety through citation.

[0103] This invention utilizes actively transported nanocapsules (ATNCs) to overcome multiple ocular barriers through transcytosis, thereby precisely regulating redox homeostasis through enzymatic processes. Encapsulating the enzyme within the ATNC solves the enzyme stability problem. Specifically, this invention synthesizes a transcytosis-activating ligand derived from glycylsarcosine (N-(3-(2-(2-amino-N-methylacetamido)acetamido)propyl)methacrylamide, TAL). Glycylsarcosine is a classic substrate for peptide transporters (PEPTs), which are highly expressed in corneal and conjunctival epithelial cells. TAL is integrated into a polymer shell to construct the ATNC. After topical ocular administration, the ATNC efficiently targets PEPTs, triggering transcytosis in corneal and conjunctival epithelial cells and promoting enzyme penetration of the epithelial barrier. More importantly, the polymer shell effectively prevents enzyme degradation by proteases and hydrolases during transcytosis. Therefore, this ATNC platform can efficiently deliver antioxidant enzymes to the posterior segment of the eye, regulating oxidative stress in fundus diseases through enzymatic processes.

[0104] As a proof of concept, this invention encapsulates two representative antioxidant enzymes, CAT and SOD, into ATNCs, forming two types of enzyme nanocapsules, named AT-n(CAT) and AT-n(SOD), respectively. After ophthalmic administration, both AT-n(CAT) and AT-n(SOD) successfully overcome the eye barrier, effectively delivering CAT and SOD to the posterior segment of the eye and exerting potent antioxidant effects. In mouse models of retinal ischemia-reperfusion injury (RIRI) and light-induced retinal damage (LIRD), AT-n(CAT) and AT-n(SOD) catalyze the decomposition of H2O2 and O2. - This significantly reduced damage to retinal structure and function. Furthermore, no significant adverse effects on intraocular pressure, corneal and retinal structure, or visual function were observed in mice treated with AT-n(CAT) and AT-n(SOD). Moreover, given the versatility of this protein encapsulation method, ATNC is expected to serve as a platform strategy for the local, non-invasive delivery of therapeutic proteins to the posterior segment of the fundus, thereby achieving effective treatment of fundus diseases.

[0105] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0106] Example 1: Synthesis of transcytosis-activated ligand TAL 1. Experimental materials Reagents and their sources: Glycylsarcosine (GS) and N-(3-aminopropyl)acrylamide (APm) were purchased from Aladdin Company; dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS) and 4-dimethylaminopyridine (DMAP) were purchased from Bide Pharmaceutical Company.

[0107] 2. Experimental Methods The synthesis route is shown below:

[0108] The specific steps are as follows: (1) Sodium bicarbonate (NaHCO3, 2.7 g) was dissolved in a mixed solvent of water and acetone (13 mL : 10 mL). Glycylsarcosine (GS, 2.0 g) was then added, and the mixture was stirred at room temperature for 5 minutes. Next, a solution of fluorene methoxycarbonyl chloride (Fmoc-Cl, 2.0 g) in acetone was added dropwise at 0°C, and the reaction mixture was stirred for 30 minutes. The reaction was then continued to be stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography (TLC).

[0109] (2) After the reaction was complete, the solvent was removed by rotary evaporation, and the residue was acidified to pH 3 with 1 M hydrochloric acid. Subsequently, the mixture was extracted three times with ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate. After removing the solvent by rotary evaporation, the crude product was further purified by silica gel column chromatography to obtain compound 1.

[0110] (3) Compound 1 (2.0 g) was dissolved in anhydrous dichloromethane, followed by the addition of dicyclohexylcarbodiimide (DCC, 1.5 g), N-hydroxysuccinimide (HNS, 0.7 g), and 4-dimethylaminopyridine (DMAP). The mixture was stirred at room temperature for 20 minutes to activate the carboxyl group. Then, N-(3-aminopropyl)methacrylic acid (APm, 1.0 g) dissolved in dichloromethane was added dropwise, and the mixture was allowed to react overnight at 35°C. The reaction progress was monitored by TLC. After the reaction was complete, the precipitated byproducts were removed by filtration, and the crude product was purified by silica gel column chromatography to obtain compound 2.

[0111] (4) Finally, compound 2 was dissolved in a 30% (v / v) piperidine / DMF solution and stirred at 0°C for 20 minutes to remove the Fmoc protecting group. After the reaction was complete (monitored by TLC), the solvent was removed by rotary evaporation, and the residue was further purified by silica gel column chromatography to obtain a polymerizable transcytosis-activated ligand monomer (N-(3-(2-(2-amino-N-methylacetamido)acetamido)propyl)methacrylamide, TAL).

[0112] 1H NMR (400 MHz, Deuterium Oxide) δ 5.60 (s, 1H), 5.36 (s, 1H), 4.04 (s, 2H), 3.94 (s, 2H), 3.21-3.18 (m, 2H), 3.11-3.04 (m, 2H), 2.98 (s, 3H),1.84 (s, 3H), 1.68 (dd, J = 6.5, 4.7 Hz, 2H) MS: [M+H]: m / z: calcd. for (C12H22N4O3):270.3, found 271.2 Example 2: Synthesis of ATNC 1. Experimental materials Reagents and their sources: Acrylamide (AAm), N-(3-aminopropyl)acrylamide (APm), and tetramethylethylenediamine (TEMED) were purchased from Aladdin Company; N-acryloyloxysuccinimide (NAS), ammonium persulfate (APS), and N,N-methylenebisacrylamide (BIS) were purchased from Bide Pharmaceutical Company; and bovine serum albumin (BSA) was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0113] 2. Experimental Methods First, BSA was modified with NAS to provide sites for in-situ polymerization (molar ratio 20:1). After the reaction, small molecules were removed by ultrafiltration and centrifugation. TAL, AAm, APm, and APS were dissolved in ultrapure water to prepare 10% (w / v) stock solutions, and N,N-methylenebisacrylamide (BIS) was dissolved in ultradry DMSO to prepare a 10% stock solution. The polymerization system was then prepared according to the following steps: Figure 4 Add BSA, AAm, APm, TAL, and BIS to the indicated ratio (molar ratio). Adjust the concentration of the reaction system to 1 mg / mL (BSA concentration) with sodium bicarbonate buffer (50 mM, pH 8.5). After vortexing and standing for 5 minutes, add initiator APS and TEMED (APS:TEMED = 1:2, mass ratio). React at 4°C for 2 hours. After the reaction is complete, remove small molecules by ultrafiltration and centrifugation.

[0114] 3. Experimental Results like Figure 4 , Figure 5 As shown in Figure a, a total of 25 ATNCs with different surface ratios were obtained for subsequent research.

[0115] like Figure 5 As shown in Figure b, the Zeta potential characterization results show that the ATNC surface without APm carries a weak negative charge, and its surface potential gradually increases with the increase of APm content.

[0116] Example 3: Cellular uptake assay of ATNC 1. Experimental materials Testing tool: BD FACSCelesta flow cytometer.

[0117] 2. Experimental Methods (1) 1 mL of human corneal epithelial cells (HCEC) were added at a concentration of 10 × 10⁻⁶. 4 Inoculate the cells into 12-well plates at a density of cells / well and incubate for 24 hours.

[0118] (2) Remove the old culture medium, wash with PBS, and add 1 mL of culture medium containing different ATNCs to each well. After incubation for 2 h, remove the old culture medium, wash with PBS, digest with trypsin, collect the cells, and centrifuge at 2000 rpm for 5 min.

[0119] (3) Wash twice with PBS, filter with a 200-mesh copper grid to obtain a single-cell suspension and transfer it into a flow cytometer to detect the fluorescence intensity.

[0120] 3. Experimental Results like Figure 5 As shown in Figure c, only a weak fluorescence signal is observed when the APm content is low; when the APm content (APm molar amount / (AAm+APm+TAL) molar amount) increases to 5%, 10%, and 20%, the average fluorescence intensity (MFI) increases significantly.

[0121] Example 4: Cytotoxicity test of ATNC 1. Experimental materials Reagents and their sources: Fetal bovine serum (FBS), DMEM medium, and penicillin and streptomycin were purchased from Thermo Fisher Scientific, Inc., USA; CCK-8 was purchased from Beyotime Biotechnology Co., Ltd., Shanghai. Testing tool: Tecan Spark multi-functional microplate reader.

[0122] 2. Experimental Methods (1) Use DMEM / F-12 medium containing 10% FBS, 1% penicillin-streptomycin, 0.001% EGF, and 0.0006% human insulin solution. 100 μL of well-grown human corneal epithelial cells (HCEC) were cultured at 1×10⁻⁶ ppm. 4 Cells were seeded at a density of 10 cells / well in 96-well plates, with the edge wells of the plates filled with sterile PBS and incubated overnight in a cell culture incubator.

[0123] (2) After 24 h, the old culture medium was removed, and after washing with PBS, the control group was set up with 100 μL of culture medium added to each well, and the experimental group was set up with 100 μL of culture medium containing ATNC (50 μg / mL, BSA concentration) added to each well. The cells were co-cultured with the experimental group at 37℃ and 5% CO2 for 24 h.

[0124] (3) Carefully aspirate the culture medium from the wells, add 100 μL of freshly prepared CCK-8 working solution (1 / 9, v / v) to each well, and continue culturing for 2 h.

[0125] (4) Terminate the culture. Measure the absorbance of each well at OD 450 nm using a microplate reader. Cell viability is calculated using the following formula: Cell viability = OD450(samples) / OD450(control) × 100%.

[0126] 3. Experimental Results from Figure 5 The cytotoxicity results of ATNC showed that excessive APm (20%) led to significant cytotoxicity, while ATNC showed no significant cytotoxicity when the APm content was 0%, 2.5%, 5% and 10%.

[0127] Example 5: Detection of ATNC's in vitro transcytosis capacity 1. Experimental materials Testing tools: Tecan Spark multi-functional microplate reader, resistance meter.

[0128] 2. Experimental Methods Transwell chambers were used to construct an in vitro epithelial barrier model.

[0129] like Figure 5 As shown in Figure e, in the upper layer of the Transwell cell, at a depth of 3×10 4 HCECs were seeded at a density of cells / well, and resistance changes were monitored using a resistance meter. Once the resistance reached the plateau phase, indicating the formation of tight junctions, the old culture medium was removed, and the cells were washed with PBS. 500 μL of fresh culture medium containing ATNC was added to the upper layer of the chamber, and the cells were co-cultured for 3 hours. The lower layer of culture was then collected to detect its fluorescence intensity.

[0130] 3. Experimental Results like Figure 5 As shown in Figure f, when the APm content is constant, the transcytosis efficiency of ATNCs increases with increasing TAL content, which is consistent with the TAL-mediated transporter protein involvement mechanism. At the same TAL ratio, ATNCs containing 5% APm exhibit higher transcytosis efficiency than those containing 10% APm. Based on these results, ATNCs with a surface APm content of 5% and a TAL content of 20% were selected for subsequent studies.

[0131] Example 6: Synthesis of AT-n (CAT) and AT-n (SOD) 1. Experimental materials Reagents and their sources: Acrylamide (AAm), N-(3-aminopropyl)acrylamide (APm), catalase (CAT), and TEMED were purchased from Aladdin Company; N-acryloyloxysuccinimide (NAS), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), ammonium persulfate (APS), and N,N-methylenebisacrylamide (BIS) were purchased from Bide Pharmaceutical Company; and superoxide dismutase (SOD) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0132] 2. Experimental Methods (1) Synthesis of AT-n(CAT): First, CAT was modified with NAS to provide sites for in-situ polymerization (molar ratio 20:1). After the reaction, small molecules were removed by ultrafiltration and centrifugation. TAL, AAm, APm, and APS were dissolved in ultrapure water to prepare 10% (w / v) stock solutions, and N,N-methylenebisacrylamide (BIS) was dissolved in ultradry DMSO to prepare a 10% stock solution. The polymerization system was prepared according to the following steps: Figure 6 Add BSA, AAM, APm, TAL, and BIS to the indicated ratio, and adjust the reaction system concentration to 1 mg / mL (CAT concentration) with sodium bicarbonate buffer (50 mM, pH 8.5). After vortexing and standing for 5 minutes, add the initiator APS and TEMED, and react at 4°C for 2 hours. After the reaction is complete, remove small molecules by ultrafiltration and centrifugation, and store at 4°C for later use after determining the protein concentration.

[0133] (2) Synthesis of AT-n(SOD): SOD was dissolved in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (20 mM, pH 6.0), and EDC was added to activate the SOD carboxyl group (molar ratio 35:1). The reaction was carried out at 4°C for 15 minutes, and then N-hydroxysuccinimide (NHS, molar ratio 3.5:1) was added. After reacting at 4°C for 2 hours, the mixture was transferred to sodium bicarbonate buffer (50 mM, pH 8.5) by ultrafiltration and centrifugation. APm (molar ratio to SOD 100:1) was added, and the reaction was carried out at 4°C overnight (double bond modification). Then, according to... Figure 7 Add AAM, APm, TAL and BIS to the indicated ratio, and adjust the reaction solution concentration to 1 mg / mL (SOD concentration) with sodium bicarbonate buffer (50 mM, pH 8.5). After vortexing and standing for 5 minutes, add initiator APS and TEMED, and react at 4°C for 2 hours. After the reaction is completed, remove small molecules by ultrafiltration and centrifugation, and store at 4°C for later use after determining the protein concentration.

[0134] 3. Experimental Results The prepared AT-n(CAT) and AT-n(SOD) were characterized by dynamic light scattering (DLS) and zeta potential, and the results are as follows: Figure 8 As shown, its average diameter is 11-13 nm and its zeta potential is 2.5-3.0 mV.

[0135] Example 7: Enzyme activity and stability detection 1. Experimental materials Reagents and their sources: Hydrogen peroxide and total superoxide dismutase assay kits were purchased from Shanghai Beyotime Biotechnology Co., Ltd. Testing tools: Tecan Spark multi-functional microplate reader, ultraviolet spectrophotometer.

[0136] 2. Experimental Methods CAT activity was detected by ultraviolet spectrophotometry. 20 μL of 0.1 mg / mL (CAT concentration) sample was mixed with 2 mL of hydrogen peroxide solution in a quartz cuvette, and the absorbance at a wavelength of 240 nm was monitored by an ultraviolet spectrophotometer (Shimadzu).

[0137] SOD activity was detected using the total SOD activity assay kit (WST-8 method) manufactured by Beyotime Corporation, following the instructions.

[0138] (1) The activities of CAT, n(CAT), AT-n(CAT) and SOD, n(SOD) and AT-n(SOD) before and after in situ polymerization were measured to evaluate the effect of polymer shell loading on enzyme activity.

[0139] (2) AT-n(CAT) and AT-n(SOD) samples were stored at 4°C for 4 weeks, and the activity changes of CAT and SOD during the period were detected to evaluate their stability.

[0140] (3) Dilute each sample to 100 μg / mL (CAT or SOD concentration), add trypsin (0.025 mg / mL), and incubate at 37˚C. Take the solution at the set time points (0, 10 min, 20 min, 30 min, 60 min, 120 min) and measure the corresponding enzyme residual activity.

[0141] 3. Experimental Results like Figure 9 As shown, compared with natural CAT and SOD, the polymerized n(CAT), AT-n(CAT), n(SOD), and AT-n(SOD) can better maintain enzyme activity (e.g., Figure 9 (As shown in Figure a); the enzyme activity did not change significantly after 4 weeks of storage (e.g., Figure 9 As shown in Figure b), AT-n(CAT) and AT-n(SOD) exhibit good stability; natural CAT and SOD rapidly lose their activity during incubation with trypsin, while the polymerized n(CAT), AT-n(CAT), n(SOD), and AT-n(SOD) retain more than 70% of their activity even after 120 minutes (e.g., ...). Figure 9 As shown in c and d), this indicates that ATNC encapsulation enhances resistance to protein hydrolysis and improves the stability of postocular delivery.

[0142] Example 8: ATNC mouse eye tissue distribution experiment 1. Experimental materials Testing tool: Zeiss LSM800 laser confocal microscope (Germany); Animals and their source: Female 6-8 week old C57BL / 6J mice were purchased from Beijing Spefol Biotechnology Co., Ltd. The following reagents were administered to the experimental animals in each group: control group: PBS; CAT group: FITC-labeled CAT (concentration: 1 mg / mL, based on enzyme protein); n(CAT) group: FITC-labeled n(CAT) (concentration: 1 mg / mL); AT-n(CAT) group: FITC-labeled AT-n(CAT) (concentration: 1 mg / mL); AT-n(CAT)+GS group: glycylsarcosine (GS) (2 mg / mL) was administered first, followed by FITC-labeled AT-n(CAT) (concentration: 1 mg / mL) 5 minutes later; PBS was used as the dispersion solvent for each group's solution.

[0143] 2. Experimental Methods (1) Mice were randomly divided into 5 groups, and 5 μL of different FITC-labeled preparations were instilled into the eyes of the mice.

[0144] (2) Three hours after eye drops, the mice were sacrificed, the eyeballs were removed and embedded in OCT frozen section embedding medium and sectioned.

[0145] (3) Remove the frozen sections, fix with 4% paraformaldehyde at room temperature for 20 min, wash three times with PBS, add DAPI-containing anti-fluorescence quenching mounting medium to stain cell nuclei and mount. Observe the differences in fluorescence intensity in the posterior segment of the mouse eye, including the retina and choroid, among different groups using a confocal microscope.

[0146] 3. Experimental Results like Figure 10 As shown, the fluorescence intensity in the retina and choroid of the AT-n(CAT) group mice was significantly stronger than that of the other two groups, indicating that ATNC can effectively deliver the enzyme to the posterior segment of the mouse eye.

[0147] Example 9: Biosafety Testing of AT-n (CAT) and AT-n (SOD) 1. Experimental materials Testing tools: slit-lamp biological microscope, small animal fundus photography, small animal optical coherence tomography scanner; Animals and their source: Female 6-8 week old C57BL / 6J mice (weight: 18-22 g) were purchased from Beijing Speford Biotechnology Co., Ltd. The experimental animals were administered the following reagents: control group, PBS; AT-n(CAT) group, AT-n(CAT) solution (dispersant was PBS, concentration: 1 mg / mL, based on enzyme protein); AT-n(SOD) group, AT-n(SOD) solution (dispersant was PBS, concentration: 1 mg / mL).

[0148] 2. Experimental Methods (1) Healthy C57BL mice were randomly divided into 3 groups, and each group of mice was given PBS, AT-n(CAT) and AT-n(SOD) (5 μL each time, twice a day).

[0149] (2) On day 14, the integrity of the corneal epithelium of mice was observed under cobalt blue light by fluorescein staining under a slit-lamp biological microscope; fundus photographs were taken using small animal fundus photography to observe retinal vessels, optic discs and overall fundus morphology; retinal OCT images were obtained using small animal optical coherence tomography (OCT) to analyze the structural integrity of each layer of the retina.

[0150] 3. Experimental Results like Figure 11 As shown, 14 days after application of the preparations to the eyes, fluorescein sodium staining showed no obvious damage to the corneal epithelium, and the corneal epithelium was intact. Fundus photography and optical coherence tomography (OCT) of small animals showed clear optic disc boundaries, with no vascular tortuosity, hemorrhage or retinal pallor. All groups showed intact retinal structures with clear boundaries between layers, and no retinal edema, detachment or changes in thickness were observed, indicating that AT-n(CAT) and AT-n(SOD) have good ocular safety.

[0151] Example 10: Efficacy of AT-n (CAT) in treating a mouse model of retinal ischemia-reperfusion 1. Experimental materials Testing tools: Zeiss LSM800 laser confocal microscope, visual electrophysiology examination instrument, paraffin microtome; Animals and their source: Female 6-8 week old C57BL / 6J mice were purchased from Beijing Spefol Biotechnology Co., Ltd.

[0152] 2. Experimental Methods (1) Establishment of a mouse retinal ischemia-reperfusion model: Mice were randomly divided into 5 groups, one of which was left untreated as a control group; the other four groups were modeled through mouse ischemia-reperfusion. After general anesthesia, mice were anesthetized with obuprocaine hydrochloride eye drops and mydriatic with compound tropicamide eye drops. Anterior chamber puncture was performed using a needle connected to a saline infusion bottle to rapidly increase intraocular pressure to 50-60 mmHg and maintain it for 60 minutes to induce ischemia, and then the pressure device was slowly removed to achieve reperfusion. To reduce the risk of infection, tobramycin ointment was applied to the ocular surface after injection. After modeling, each group was given eye drops. 5 μL of different formulations were administered to each mouse every 12 hours.

[0153] (2) TUNEL staining: At 72 hours after treatment, mice in each group were euthanized and their eyeballs were completely removed. After embedding the eyeballs with embedding agent, frozen sections with a thickness of 8 µm were prepared. The sections were fixed with 4% paraformaldehyde solution and permeabilized with 0.1% Triton X-100 solution at room temperature; then TUNEL staining was performed according to the kit instructions. After staining, the nuclei were stained with DAPI, and the slides were mounted with anti-fluorescence quenching mounting medium. Finally, the slides were observed and images were acquired using a confocal microscope.

[0154] (3) H&E staining: 72 hours after treatment, mice in each group were euthanized and their eyeballs were removed. The retina of the eyeballs was embedded in paraffin and then stained with hematoxylin and eosin (H&E). Images of the sections in each group were obtained using an inverted fluorescence microscope.

[0155] (4) Electroretinography (ERG) detection: 72 hours after treatment, mice were first anesthetized by intraperitoneal injection under dark adaptation conditions, and then mydriasis was achieved using compound tropicamide eye drops. The reference electrode was placed at the base of the tail and the forehead, and the gold electrode was fixed to the cornea. Electroretinography (ERG) was recorded after each flash stimulation.

[0156] 3. Experimental Results like Figure 12 As shown, the TUNEL staining results ( Figure 12 a) showed that, compared with the model group and other treatment groups, AT-n(CAT) treatment significantly reduced apoptosis of retinal nuclear layer cells; histopathological analysis further showed ( Figure 12 (b) AT-n(CAT) treatment can effectively alleviate inflammatory cell infiltration, structural disorder of the inner and outer nuclear layers, retinal detachment, and loss of retinal ganglion cells (RGCs) caused by retinal ischemia-reperfusion injury; ERG waveforms show that AT-n(CAT) treatment can effectively restore visual function ( Figure 12c). The above results demonstrate that AT-n(CAT) can effectively deliver CAT to retinal tissue, enhance the antioxidant capacity of the mouse retina, inhibit cell apoptosis and inflammatory response, maintain the structural integrity of the retina, and thus ultimately maintain visual function after ischemia-reperfusion injury.

[0157] Example 11: Efficacy of AT-n(SOD) in treating a mouse model of retinal blue light damage 1. Experimental materials Testing tools: Zeiss LSM800 laser confocal microscope, visual electrophysiology examination instrument, paraffin microtome, small animal optical coherence tomography scanner; Animals and their source: Female 6-8 week old BALB / c mice were purchased from Beijing Spaford Biotechnology Co., Ltd.

[0158] 2. Experimental Methods (1) Establishment of a mouse retinal blue light injury model: Mice were randomly divided into 5 groups: one group received no treatment and served as the blank control group; the other four groups were irradiated with blue light to establish a mouse retinal blue light injury model. Before modeling, mice underwent 16 hours of dark adaptation treatment, and then were placed in a light-proof box equipped with a top blue light source and surrounding reflectors to ensure uniform illumination. Before blue light irradiation, compound tropicamide eye drops were used to dilate the pupils. After the pupils were fully dilated, the mice were exposed to high-intensity blue light for 45 minutes. After the light exposure ended, the mice were placed back in the dark environment for another 16 hours. After modeling was completed, each group was given eye drops. Administered twice daily, with each mouse receiving 5 μL of different nano-formulations each time.

[0159] (2) TUNEL staining: On the 3rd day of treatment, mice in each group were euthanized and their eyeballs were completely removed. After embedding the eyeballs with embedding agent, frozen sections with a thickness of 8 µm were prepared. The sections were fixed with 4% paraformaldehyde solution and permeabilized with 0.1% Triton X-100 solution at room temperature; then TUNEL staining was performed according to the kit instructions. After staining, the nuclei were stained with DAPI, and the slides were mounted with anti-fluorescence quenching mounting medium. Finally, the slides were observed and images were acquired using a confocal microscope.

[0160] (3) H&E staining: On day 7 of treatment, mice in each group were euthanized and their eyeballs were removed. After the eyeballs were embedded in paraffin, 4 µm horizontal retinal sections were cut and then stained with hematoxylin and eosin (H&E). Images of each section of each eye were obtained using an inverted fluorescence microscope.

[0161] (4) Optical coherence tomography (OCT) imaging: Mice were first anesthetized and their pupils were dilated with 0.5% tropicamide in preparation for imaging. Then, an OCT camera positioned directly in front of the cornea was used to image the retina to ensure that the optic disc was centered in the image. OCT image data were captured using a connected computer, and the outer nuclear layer and total retinal thickness measurements were automatically quantified using OCT system software.

[0162] (5) Electroretinography (ERG) detection: Under dark-adapted conditions, mice were first anesthetized by intraperitoneal injection, and then mydriasis was achieved using compound tropicamide eye drops. The reference electrode was placed at the base of the tail and the forehead, and the gold electrode was fixed to the cornea. Electroretinography (ERG) was recorded after each flash stimulation.

[0163] 3. Experimental Results For example, TUNEL staining results ( Figure 13 As shown in a), compared with the model group and other treatment groups, AT-n(SOD) treatment significantly reduced apoptosis of extraretinal nuclear layer cells. This effective anti-apoptotic effect effectively maintained the integrity of the retinal structure. H&E staining results ( Figure 13 (b) The results showed that AT-n(SOD) treatment significantly reduced thinning of the outer nuclear layer of the retina. Furthermore, on day 7 post-treatment, the thickness of the outer nuclear layer and the total retinal thickness were observed and statistically analyzed using OCT. Figure 14 As shown in a-14c, compared with the healthy control group, LIRD mice treated with PBS, natural SOD, or AT-n(SOD) showed significantly reduced ONL and total retinal thickness. In contrast, AT-n(SOD) treatment effectively alleviated the thinning of the outer nuclear layer and the entire retinal layer. ERG analysis ( Figure 14 The results (d-14f) indicate that AT-n(SOD) treatment can effectively restore visual function impairment.

[0164] The above results demonstrate that AT-n(SOD) can effectively deliver SOD to retinal tissue, enhance the antioxidant capacity of the mouse retina, reduce retinal damage in the LIRD mouse model, and maintain the integrity of retinal structure and function.

[0165] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0166] The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0167] Listing the steps of the method in a certain order in this invention does not constitute any restriction on the order of the method steps.

Claims

1. A compound or a salt thereof, characterized in that, The compound has the following structure: （Ⅰ） in, L is , where n is an integer from 1 to 10; R1 is either -C(CH3)=CH2 or -CH=CH2.

2. The compound or its salt as claimed in claim 1, characterized in that, The compound is selected from the following structures: 。 3. A nanoparticle with a core-shell structure, characterized in that, The nanoparticles contain a protease as the core and a polymer shell coating the surface of the protease. The monomers of the polymer include functional monomers, positively charged monomers, and neutral monomers, wherein the functional monomers are the compounds described in claim 1; The protease is an antioxidant enzyme; The molar ratio of the protease to the functional monomer is 1:50-2000; The molar ratio of the protease to the positively charged monomer is 1:50-1000; The molar ratio of the protease to the neutral monomer is 1:1000-5000.

4. The nanoparticles as described in claim 3, characterized in that, The protease is selected from one or more of the following: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione reductase (GR); and / or, The positively charged monomer is selected from one or more of N-(3-aminopropyl)acrylamide, dimethylaminoethyl methacrylate (DMAEMA), and acrylamide propyltrimethylammonium chloride (APTAC); and / or, The neutral monomer is selected from one or more of the following: acrylamide, N,N-dimethylacrylamide, N-isopropylacrylamide, N-ethylacrylamide, N,N-diethylacrylamide, hydroxyethyl acrylate, hydroxyethyl methacrylate, hydroxypropyl acrylate, vinyl acetate, etc.

5. The nanoparticles as described in claim 3, characterized in that, The functional monomer accounts for 1%-45% of the sum of the molar amounts of the functional monomer, positively charged monomer, and neutral monomer; and / or, The positively charged monomer accounts for 1%-20% of the sum of the molar amounts of the functional monomer, the positively charged monomer, and the neutral monomer.

6. The nanoparticles as described in claim 3, characterized in that, Of the total molar amounts of the functional monomer, positively charged monomer, and neutral monomer, the functional monomer accounts for 20% and the positively charged monomer accounts for 5%.

7. The nanoparticles as described in claim 3, characterized in that, The polymer raw materials also include a crosslinking agent; the crosslinking agent is selected from one or more of the following: N,N-methylenebisacrylamide, ethylene glycol diacrylate, polyethylene glycol diacrylate, glycerol dimethacrylate, diisocyanate, propylenediamine, divinylbenzene, and vinyltriethoxysilane.

8. The nanoparticles as described in claim 5 or 6, characterized in that, The protein is superoxide dismutase (SOD), and the polymer forms a shell on the protein surface through in-situ polymerization. The raw materials for in-situ polymerization include functional monomers, positively charged monomers, neutral monomers, and a cross-linking agent. The functional monomers are... The positively charged monomer is N-(3-aminopropyl)acrylamide, the neutral monomer is acrylamide, and the crosslinking agent is N,N-methylenebisacrylamide; or, The protein is catalase (CAT), and the polymer forms a shell on the protein surface through in-situ polymerization; wherein, the raw materials for in-situ polymerization include functional monomers, positively charged monomers, neutral monomers, and cross-linking agents, and the functional monomers are... The positively charged monomer is N-(3-aminopropyl)acrylamide, the neutral monomer is acrylamide, and the crosslinking agent is N,N-methylenebisacrylamide.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the nanoparticles as described in any one of claims 3-8, and one or more pharmaceutically acceptable excipients.

10. The use of the nanoparticles according to any one of claims 3-8 in the preparation of medicaments for the prevention and / or treatment of diseases, characterized in that, The disease in question is an eye disease.

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