NANO eye drop capable of delivering drug to posterior eye segment, and preparation method therefor and use thereof

Abstract

A nano eye drop capable of delivering a drug to the posterior eye segment, and a preparation method therefor and the use thereof. The nano eye drop comprises a liposome core and a chitosan shell, wherein the liposome core can load a hydrophilic and / or hydrophobic drug, and prolong the retention time of the drug on the ocular surface and enhance the ocular permeability of the drug.

Description

Nano eye drops capable of delivering drugs to the posterior segment of the eye, and preparation method and application thereof Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a nano eye drop capable of delivering drugs to the posterior segment of the eye, and a preparation method and application thereof. Background Art

[0002] A series of acute and chronic diseases affecting the posterior segment of the eye, including diabetic retinopathy, age-related macular degeneration, infection and non-infectious ocular inflammation, can seriously affect the patient's visual quality of life. Intravitreal injection and intravitreal implantation are currently the main means of delivering drugs to the posterior segment of the eye, but the above invasive treatment methods may cause adverse reactions such as pain, infectious endophthalmitis, and retinal detachment, thereby reducing patient compliance. Eye drops are widely used in ophthalmic diseases such as dry eye, inflammation, infection, allergy, and glaucoma due to their advantages such as convenient administration, non-invasiveness, and relative economy. However, due to the existence of multiple physiological barriers in the eye, it is difficult for traditional eye drops to deliver active ingredients to the posterior segment of the eye. Therefore, innovation and change of drug delivery methods and medication strategies are crucial to the development of safe and effective new eye drops.

[0003] Liposomes are amphiphilic and can encapsulate both hydrophilic and lipophilic drug molecules. They are a widely used class of nanomaterials that make it possible for drugs to enter the posterior segment of the eye. However, the surface of traditional liposomes is negatively charged, and their adhesion to the negatively charged surface of the eye is poor, making them easily cleared. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of existing eye drops in effectively delivering drugs to the posterior segment of the eye, and to provide a nano-eye drop capable of delivering drugs to the posterior segment of the eye, as well as its preparation method and application. By utilizing chitosan-modified liposomes with excellent biosafety, adhesion, and cationic properties, the surface charge of the liposomes is improved, enhancing the interaction between the particles and the ocular surface. This improves corneal penetration by opening tight junctions between corneal epithelial cells, thereby increasing adhesion time and thus bioavailability.

[0005] The technical solution adopted by the present invention to solve its technical problem is:

[0006] In a first aspect, the present invention provides a chitosan-modified liposome. The chitosan-modified liposome comprises a liposome core and a chitosan shell. The liposome core comprises a lipid bilayer membrane.

[0007] The lipid bilayer membrane was obtained by rotary evaporation of soybean lecithin, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol.

[0008] Preferably, the mass ratio of soybean lecithin, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol is 2-10:1-2:1-2, preferably (6-10):1-2:1-2, further preferably (6-10):1:2 or (6-10):2:1; for example, 2:1:1, 3:1:1, 4:1:1, 5:1:1, 6:1:1, 7:1:1, 8:1:1, 2:1:2, 3:1:2, 4:1:2, 5:1:2, 6:1:2, 7:1:2, 8:1:2, 2:2:1, 3:2:1, 4:2:1, 5:2:1, 6:2:1, 7:2:1, 8:2:1, 3:1:1, 4:1:1, 5:1:1, 6:1:1, 7:1:1, 8:1:1, etc.

[0009] The temperature of the rotary evaporation is 30 to 50°C, preferably 25 to 45°C, for example, 30, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50°C, etc.

[0010] The time for rotary evaporation is 10 to 20 minutes, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 minutes, etc.

[0011] Preferably, the mass ratio of the liposome core to the chitosan shell is 1:0.04-0.24, for example, 1:0.04, 1:0.05, 1:0.1, 1:0.15, 1:0.16, 1:0.17, 1:0.2 or 1:0.24, and preferably 1:0.1-0.2.

[0012] The chitosan-modified liposomes are positively charged, for example, greater than +20mV, greater than +25mV, greater than +30mV, greater than +35mV, preferably +30mV to +45mV, for example, +30, +31, +32, +33, +34, +35, +36, +37, +38, +39, +40, +41, +42, +43, +44 or +45mV, etc.

[0013] The liposome core can be loaded with drugs.

[0014] The mass ratio of chitosan to drug can be 1 to 6:4, for example, 1:4, 1:2, 3:4, 1:1, 5:4, 3:2, etc., preferably 3 to 5:4.

[0015] Preferably, the drug is a hydrophilic drug and / or a hydrophobic drug.

[0016] In some embodiments, the hydrophilic drug is loaded into the cavity of the lipid bilayer membrane.

[0017] In some embodiments, the hydrophobic drug is loaded on a lipid bilayer membrane.

[0018] In some embodiments, the hydrophilic drug includes but is not limited to ganciclovir, timolol, ciprofloxacin, ofloxacin, tobramycin, gentamicin, dexamethasone acetate, pilocarpine, hydrocortisone, fluorometholone, azalastine hydrochloride, tropicamide, naphazoline hydrochloride, naphazoline hydrochloride or pranoprofen.

[0019] In some embodiments, the hydrophobic drug includes but is not limited to curcumin, brinzolamide, sparfloxacin, ofloxacin, gatifloxacin, dexamethasone, pilocarpine, naphazoline hydrochloride or tropicamide.

[0020] The drug includes but is not limited to at least one of nucleic acid, polypeptide, protein or small molecule compound.

[0021] The drugs are ophthalmic drugs, including drugs that can be used as eye drops in the prior art, including but not limited to anti-inflammatory drugs, anti-glaucoma drugs, anti-allergic drugs, hormone drugs or anti-cataract drugs, anti-AMD drugs or drugs for treating dry eye.

[0022] The chitosan-modified liposomes can deliver drugs to the posterior segment of the eye.

[0023] The drug loading rate is greater than 10%, preferably 10%-50%.

[0024] The second aspect of the present invention provides a use of the chitosan-modified liposomes in the preparation of nano eye drops capable of delivering drugs to the posterior segment of the eye.

[0025] The third aspect of the present invention provides a nano eye drop, which comprises a liposome core and a chitosan shell, wherein the liposome core comprises a lipid bilayer membrane.

[0026] Preferably, the liposome comprises the chitosan-modified liposome according to the first aspect and a drug loaded in the inner core of the liposome.

[0027] The nano eye drops also include common auxiliary materials for eye drops.

[0028] Preferably, common excipients for eye drops include preservatives, artificial tears, buffer solutions, substances that relieve visual fatigue, cooling agents, and the like.

[0029] The mass percentage of chitosan-modified liposomes in the nano eye drops can be 0.01% to 99.5% (specifically, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, etc.).

[0030] The mass percentage of the drug in the nano eye drops is 0.1% to 0.36%, more preferably 0.15% to 0.33%, and most preferably 0.2%.

[0031] The nano eye drops can deliver drugs to the posterior segment of the eye.

[0032] A fourth aspect of the present invention provides a method for preparing chitosan-modified liposomes, the method comprising:

[0033] a) dissolving soybean lecithin, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol in an organic solvent to obtain a mixed solution, and rotary evaporating the mixed solution to obtain a lipid film;

[0034] b) adding a buffer solution to the lipid film obtained in step a) to hydrate the film to obtain a hydrated solution;

[0035] c) further adding chitosan, stirring and mixing, and then incubating to obtain chitosan-modified liposomes.

[0036] The mass ratio of soybean lecithin, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol is 2-10:1-2:1-2, preferably (6-10):1-2:1-2, more preferably (6-10):1:2 or (6-10):2:1; for example, 2:1:1, 3:1:1, 4:1:1, 5:1:1, 6:1:1, 7:1:1, 8:1:1, 2:1:2, 3:1:2, 4:1:2, 5:1:2, 6:1:2, 7:1:2, 8:1:2, 2:2:1, 3:2:1, 4:2:1, 5:2:1, 6:2:1, 7:2:1, 8:2:1, 3:1:1, 4:1:1, 5:1:1, 6:1:1, 7:1:1, 8:1:1, etc.

[0037] The organic solvent may be dichloromethane, methanol, ethanol, acetone, ethyl acetate, benzene, toluene, chloroform, dimethyl sulfoxide (DMSO), isopropanol, butanone, ether, acetic acid or tetrahydrofuran.

[0038] The temperature of the rotary evaporation is 30 to 50°C, preferably 25 to 45°C, for example, 30, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50°C, etc.

[0039] The time for rotary evaporation is 10 to 20 minutes, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 minutes, etc.

[0040] The buffer includes but is not limited to phosphates (such as sodium dihydrogen phosphate, disodium hydrogen phosphate), borates (such as borax), boric acid, etc. Preferably, it is PBS.

[0041] The hydration temperature is 20-30°C, for example 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30°C.

[0042] The hydration time is 30 to 100 min, preferably 40 to 70 min; for example, 30, 40, 45, 50, 55, 60, 65, 70, 80, 90 or 100 min.

[0043] The hydrated liquid obtained after the hydration is ultrasonicated and filtered.

[0044] Preferably, the power of the ultrasound is 50 to 150 W, more preferably 80 to 120 W; for example, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 W.

[0045] Preferably, the ultrasonication time is 10 to 30 min, more preferably 15 to 25 min; for example, 10, 15, 20, 25 or 30 min.

[0046] The stirring temperature is 10 to 40°C, preferably 20 to 30°C; for example, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35 or 40°C.

[0047] The stirring and mixing time is 10 to 50 minutes, preferably 20 to 40 minutes; for example, 10, 15, 20, 25, 30, 35, 40, 45 or 50 minutes.

[0048] The incubation temperature is 1-10°C, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10°C.

[0049] The incubation time is 10 to 48 hours, for example 10, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45 or 48 hours; preferably, the incubation is overnight.

[0050] After the incubation, the method further comprises the steps of filtering and centrifuging to obtain the upper layer solution.

[0051] In some specific embodiments, the preparation method comprises:

[0052] a) dissolving soybean lecithin, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol in a mass ratio of 2-10:1-2:1-2 in dichloromethane to obtain a mixed solution, and subjecting the mixed solution to rotary evaporation at 30-50° C. for 10-20 minutes to obtain a lipid film;

[0053] b) adding phosphate buffer to the lipid film obtained in step a), hydrating at 20-30° C. for 30-100 minutes to obtain a hydrated solution, and further sonicating at 50-150W for 10-30 minutes;

[0054] c) further adding chitosan solution, stirring at 10-40° C. for 10-50 min, and incubating at 1-10° C. overnight to obtain chitosan-modified liposomes.

[0055] The chitosan content of the chitosan solution is 0.1-0.3% by mass, and the solvent is a glacial acetic acid solution with a volume concentration of 0.1% (v / v).

[0056] The fifth aspect of the present invention provides a method for preparing the nano eye drops, the method comprising:

[0057] The mixed solution in step a) of the method for preparing chitosan-modified liposomes further comprises a hydrophobic drug; and / or the buffer solution in step b) of the method for preparing chitosan-modified liposomes contains a hydrophilic drug.

[0058] The mass ratio of soybean lecithin, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol is 2-10:1-2:1-2, preferably (6-10):1-2:1-2, more preferably (6-10):1:2 or (6-10):2:1; for example, 2:1:1, 3:1:1, 4:1:1, 5:1:1, 6:1:1, 7:1:1, 8:1:1, 2:1:2, 3:1:2, 4:1:2, 5:1:2, 6:1:2, 7:1:2, 8:1:2, 2:2:1, 3:2:1, 4:2:1, 5:2:1, 6:2:1, 7:2:1, 8:2:1, 3:1:1, 4:1:1, 5:1:1, 6:1:1, 7:1:1, 8:1:1, etc.

[0059] The organic solvent may be dichloromethane, methanol, ethanol, acetone, ethyl acetate, benzene, toluene, chloroform, dimethyl sulfoxide (DMSO), isopropanol, butanone, ether, acetic acid or tetrahydrofuran.

[0060] The temperature of the rotary evaporation is 30 to 50°C, preferably 25 to 45°C, for example, 30, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50°C, etc.

[0061] The time for rotary evaporation is 10 to 20 minutes, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 minutes, etc.

[0062] The buffer includes but is not limited to phosphates (such as sodium dihydrogen phosphate, disodium hydrogen phosphate), borates (such as borax), boric acid, etc. Preferably, it is PBS.

[0063] The hydration temperature is 20-30°C, for example 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30°C.

[0064] The hydration time is 30 to 100 min, preferably 40 to 70 min; for example, 30, 40, 45, 50, 55, 60, 65, 70, 80, 90 or 100 min.

[0065] The hydrated liquid obtained after the hydration is ultrasonicated and filtered.

[0066] Preferably, the power of the ultrasound is 50 to 150 W, more preferably 80 to 120 W; for example, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 W.

[0067] Preferably, the ultrasonication time is 10 to 30 min, more preferably 15 to 25 min; for example, 10, 15, 20, 25 or 30 min.

[0068] The stirring temperature is 10 to 40°C, preferably 20 to 30°C; for example, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35 or 40°C.

[0069] The stirring and mixing time is 10 to 50 minutes, preferably 20 to 40 minutes; for example, 10, 15, 20, 25, 30, 35, 40, 45 or 50 minutes.

[0070] The incubation temperature is 1-10°C, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10°C.

[0071] The incubation time is 10 to 48 hours, for example 10, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45 or 48 hours; preferably, the incubation is overnight.

[0072] After the incubation, the method further comprises the steps of filtering and centrifuging to obtain the upper layer solution.

[0073] Preferably, the preparation method comprises:

[0074] a) dissolving soybean lecithin, cholesterol, distearoyl phosphatidylethanolamine-polyethylene glycol, and a hydrophobic drug in an organic solvent to obtain a mixed solution, and rotary evaporating the mixed solution to obtain a lipid film;

[0075] b) adding a buffer solution to the lipid film obtained in step a) to hydrate the film to obtain a hydrated solution;

[0076] c) further adding chitosan, stirring and mixing, and then incubating to obtain nano eye drops.

[0077] In some specific embodiments, the preparation method comprises:

[0078] a) dissolving soybean lecithin, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol, and a hydrophobic drug in a mass ratio of 2-10:1-2:1-2 in dichloromethane to obtain a mixed solution, and subjecting the mixed solution to rotary evaporation at 30-50° C. for 10-20 minutes to obtain a lipid film;

[0079] b) adding phosphate buffer to the lipid film obtained in step a), hydrating at 20-30° C. for 30-100 minutes to obtain a hydrated solution, and further sonicating at 50-150W for 10-30 minutes;

[0080] c) adding chitosan solution, stirring at 10-40° C. for 10-50 min, and incubating at 1-10° C. overnight to obtain nano eye drops.

[0081] In some specific embodiments, the preparation method comprises:

[0082] 1) Soy lecithin, cholesterol, distearoyl phosphatidylethanolamine-polyethylene glycol, and a hydrophobic drug are dissolved in dichloromethane to obtain a mixture, and the mixture is rotary evaporated at 40° C. for 15 minutes to form a lipid film;

[0083] 2) adding phosphate buffer to the lipid film obtained in step 1), and hydrating at 25° C. for 60 minutes to obtain a hydrated mixed solution;

[0084] 3) The hydrated solution obtained in step 2) was subjected to 90W water bath ultrasonic treatment for 15 minutes, and then extruded through a 0.45 μm filter membrane to obtain a liposome solution loaded with a hydrophobic drug;

[0085] 4) adding chitosan solution to the liposome solution obtained in step 3); mixing under magnetic stirring at 25° C. for 30 minutes, and then incubating at 4° C. overnight to obtain nano eye drops.

[0086] Preferably, the preparation method comprises:

[0087] a) dissolving soybean lecithin, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol in an organic solvent to obtain a mixed solution, and rotary evaporating the mixed solution to obtain a lipid film;

[0088] b) adding a buffer containing a hydrophilic drug to the lipid film obtained in step a) to hydrate the film to obtain a hydrated solution;

[0089] c) further adding chitosan, stirring and mixing, and then incubating to obtain nano eye drops.

[0090] In some specific embodiments, the preparation method comprises:

[0091] a) dissolving soybean lecithin, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol in a mass ratio of 2-10:1-2:1-2 in dichloromethane to obtain a mixed solution, and subjecting the mixed solution to rotary evaporation at 30-50° C. for 10-20 minutes to obtain a lipid film;

[0092] b) adding a phosphate buffer containing a hydrophilic drug to the lipid film obtained in step a), hydrating at 20-30° C. for 30-100 minutes to obtain a hydrated solution, and further sonicating at 50-150W for 10-30 minutes;

[0093] c) adding chitosan solution, stirring at 10-40° C. for 10-50 min, and incubating at 1-10° C. overnight to obtain nano eye drops.

[0094] The chitosan content of the chitosan solution is 0.1-0.3% by mass, and the solvent is a glacial acetic acid solution with a volume concentration of 0.1% (v / v).

[0095] In some specific embodiments, the preparation method comprises:

[0096] 1) Soy lecithin, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) were dissolved in dichloromethane to obtain a mixture, and the mixture was rotary evaporated at 40°C for 15 minutes to form a lipid film;

[0097] 2) adding phosphate buffer containing ganciclovir to the lipid film obtained in step 1), and hydrating at 25° C. for 60 minutes to obtain a hydrated mixed solution;

[0098] 3) The hydrated solution obtained in step 2) was subjected to 90W water bath ultrasonic treatment for 15 minutes, and then extruded through a 0.45 μm filter membrane to obtain a liposome solution loaded with ganciclovir;

[0099] 4) adding chitosan solution to the liposome solution obtained in step 3); mixing under magnetic stirring at 25° C. for 30 minutes, and then incubating at 4° C. overnight to obtain nano eye drops.

[0100] Furthermore, in step 2), the ratio of the hydrophilic drug to the phosphate buffer in mg:mL is 16-24:6-14, more preferably 18-22:8-12, and most preferably 20:10.

[0101] Furthermore, in step 4), the chitosan solution contains chitosan at a mass percentage of 0.1-0.3%, and the solvent is a 0.1% (v / v) glacial acetic acid solution. More preferably, the chitosan solution contains chitosan at a mass percentage of 0.15-0.25%, and most preferably 0.2%.

[0102] Furthermore, the mass percentage of the drug in the nano eye drops is 0.1% to 0.36%, more preferably 0.15% to 0.33%, and most preferably 0.2%.

[0103] The sixth aspect of the present invention provides a method for delivering a drug to the posterior segment of the eye, which comprises dripping an effective amount of the chitosan-modified liposomes or the nano eye drops onto the ocular surface of a subject in need.

[0104] The seventh aspect of the present invention provides a use of the chitosan-modified liposome or the nano eye drops as and / or in the preparation of ophthalmic drugs.

[0105] The ophthalmic drug is a drug for treating or preventing diseases related to the posterior segment of the eye, such as retinal disease, vitreous disease, choroidal disease or scleral disease.

[0106] Preferably, the ophthalmic drug is a drug for treating or preventing eye inflammation, allergy, glaucoma, cataract; for example, a drug for diabetic retinopathy and / or age-related macular degeneration and / or retinal light damage and / or infectious and non-infectious eye inflammation (such as cytomegalovirus retinitis).

[0107] The eighth aspect of the present invention provides a method for treating ophthalmic diseases, which comprises dripping an effective amount of the chitosan-modified liposomes or the nano eye drops onto the ocular surface of a subject in need.

[0108] The ophthalmic diseases include diseases related to the posterior segment of the eye, such as drugs for retinal diseases, vitreous diseases, choroidal diseases or scleral diseases.

[0109] Preferably, the ophthalmic disease includes drugs for ocular inflammation, allergies, glaucoma, and cataracts; for example, diabetic retinopathy and / or age-related macular degeneration and / or retinal light damage and / or infection and non-infectious ocular inflammation (e.g., cytomegalovirus retinitis). The infection can be a bacterial infection, a viral infection, or a fungal infection. Bacteria include, but are not limited to, Staphylococcus aureus, Bacillus mutans, Escherichia coli, Staphylococcus epidermidis, Pseudomonas aeruginosa, Bacillus cereus, or Pseudomonas spp. The virus includes, but is not limited to, cytomegalovirus, Epstein-Barr virus, adenovirus, and Ebola virus. The fungus includes, but is not limited to, Candida, Fusarium, or Aspergillus spp.

[0110] As used herein, "treating" means slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-associated signs, symptoms, conditions, or disorders.

[0111] The "effective amount" of the present invention refers to the amount or dosage of the drug of the present invention that provides the desired treatment or prevention after administration to an individual or organ in a single or multiple doses.

[0112] The term "prevention" used in the present invention refers to a method implemented to prevent or delay the occurrence of a disease, disorder or symptom in the body.

[0113] The "subject" described in the present invention can be a human or non-human mammal, or a cell, tissue, or organ of a human or non-human mammal. The non-human mammal can be a wild animal, zoo animal, commercial animal, pet, laboratory animal, etc. Preferably, the non-human mammal includes, but is not limited to, pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels), monkeys, etc.

[0114] The advantages and positive effects achieved by the present invention are:

[0115] 1. The nano-eye drops of the present invention comprise a liposome core and a chitosan shell. These nano-eye drops can effectively load both hydrophilic and hydrophobic drug molecules, prolong the drug's ocular surface retention time, and enhance its ocular penetration. Results from the examples of the present invention showed that after ocular administration of ganciclovir-loaded nano-eye drops, drug levels exceeding the effective concentration were detected in the retina, choroid, and other posterior ocular structures of rats and rabbits. The nano-eye drops' efficient posterior ocular drug delivery capability provides a new strategy for promoting the clinical application of new ophthalmic drugs.

[0116] 2. The nano eye drops of the present invention are composed of a liposome core and a chitosan shell. The liposome core contains a lipid bilayer membrane and an aqueous cavity, which enables the nano eye drops to effectively load lipophilic and hydrophilic drug molecules. The chitosan shell gives the nano eye drops a positively charged surface, which can enhance mucin binding and prolong the residence time in front of the eye. In addition, chitosan can temporarily open the tight junctions in the corneal and junctional epithelial cells, thereby promoting the ocular permeability of the drug. In this study, we used ganciclovir as a drug model. Our research results show that the nano eye drops exhibit excellent ocular surface retention and ocular penetration capabilities, and can effectively deliver ganciclovir to the posterior segment of rat and rabbit eyes. Through these in vitro and in vivo verifications, the nano eye drops can efficiently and non-invasively deliver drug molecules to the posterior segment of the eye, and this method provides a new strategy for promoting the clinical application of new ophthalmic drugs.

[0117] 3. The nano eye drops of the present invention are composed of a liposome core and a chitosan shell. The liposome components and chitosan have been approved by the FDA (U.S. Food and Drug Administration) for medical use and have good biosafety. The results of the examples of the present invention show that the nano eye drops have no significant toxicity to corneal epithelial cells, and continuous eye application does not cause corneal damage or retinal structural abnormalities. The good biocompatibility and ocular tolerance of the nano eye drops provide a new strategy for promoting the clinical application of new ophthalmic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figure 1: Schematic diagram of the synthesis of ganciclovir-loaded nano-eye drops;

[0119] Figure 2: Transmission electron microscopy (TEM) of liposomes (a, upper row) and nano-eye drops (a, lower row), and zeta potential (b) characterization data, where LP is liposomes and CMLP is chitosan-modified liposome nano-eye drops;

[0120] Figure 3: Effects of different concentrations of nano-eye drops on HCEC cell viability in a CCK-8 cytotoxicity assay, where LP represents liposomes and CMLP represents chitosan-modified liposome nano-eye drops.

[0121] Figure 4: Effects of nano eye drops on the corneal structure of mice in the ocular surface sodium fluorescein staining experiment. In the figure, the first row is the photos of the eye surface of each group of mice under white light after continuous eye drops for 1 week; the second row is the photos of the cornea of ​​each group of mice under cobalt blue light after continuous eye drops for 1 week; the third row is the photos of the eye surface of each group of mice under white light after continuous eye drops for 2 weeks; the fourth row is the photos of the cornea of ​​each group of mice under cobalt blue light after continuous eye drops for 2 weeks.

[0122] Figure 5: Effects of nano-eye drops on mouse retinal structure in retinal H&E staining experiment;

[0123] Figure 6: HCEC uptake test of nano-eye drops; Figures a and b are flow cytometric uptake results of HCEC cells at 1 hour and 2 hours; Figure c is a quantitative analysis of the mean fluorescence intensity corresponding to the flow cytometric uptake results;

[0124] Figure 7: Effects of nano-eye drops on tight junctions between HCEC cells in the phalloidin labeling experiment;

[0125] Figure 8: Photographs of the anterior segment of the mouse eye at different time points after a single instillation of free FITC, LP-FITC, and CMLP-FITC;

[0126] Figure 9: Fluorescence distribution in the posterior segment of mouse eyes 3 hours and 6 hours after a single instillation of free RhB, LP-RhB, and CMLP-RhB;

[0127] Figure 10: Pharmacokinetic graphs of nano eye drops in the posterior segment tissues of rats and rabbits; Figure a is a graph showing the ganciclovir concentration detected in the rat retina at different time points; Figure b is a graph showing the ganciclovir concentration detected in the rat sclera-choroid at different time points; Figure c is a graph showing the ganciclovir concentration detected in the retina, sclera and choroid of the posterior segment of the rat eye at different time points; and Figure d is a graph showing the drug content of ganciclovir in the posterior segment tissue of the rabbit eye 6 hours after a single instillation.

[0128] Figure 11: Schematic diagram of the synthesis of curcumin-loaded nano-eye drops.

[0129] Figure 12: Schematic diagram of the construction of the mouse retinal light damage model.

[0130] Figure 13: Curcumin-loaded nano-eye drops for the treatment of retinal light damage. DETAILED DESCRIPTION

[0131] The present invention will be further described below with reference to the following examples. The following examples are descriptive and not restrictive, and the scope of protection of the present invention cannot be limited by the following examples.

[0132] The various experimental operations involved in the specific embodiments are all routine techniques in the field. For parts not specifically annotated in this document, ordinary technicians in this field can refer to various commonly used reference books, scientific literature or related instructions, manuals, etc. before the filing date of this invention to implement them.

[0133] Sources of reagents used in the examples:

[0134] Ganciclovir (GCV) and cholesterol were purchased from Shanghai TCI Chemical Industry Development Co., Ltd.

[0135] Soy lecithin (CAS: 8002-43-5), DSPE-PEG and chitosan (product number: C-35700, viscosity value about 155 mPa.s) were purchased from Tianjin Xiens Biochemical Technology Co., Ltd.

[0136] Loading rate = (mass of loaded drug / mass of fed drug)*100%.

[0137] Example 1: Preparation of nano eye drops loaded with ganciclovir

[0138] Instruments and equipment: Rotary evaporator.

[0139] 8 mg of soy lecithin, 1 mg of cholesterol, and 1 mg of DSPE-PEG were dissolved in dichloromethane, mixed thoroughly, and then placed on a rotary evaporator and evaporated at 40°C for 15 minutes to form a lipid film. 8 mL of a PBS solution containing 0.2% GCV was then added and hydrated at 25°C for 60 minutes. The resulting hydrated solution was sonicated in a 90W waterbath for 15 minutes and then extruded through a 0.45 μm filter membrane to obtain a GCV-loaded liposome solution. An equal volume of a 0.05%-0.3% chitosan solution (solvent: 0.1% v / v glacial acetic acid) was then added to the resulting GCV liposome solution. The mixture was mixed with magnetic stirring at 25°C for 30 minutes and incubated at 4°C overnight. After overnight incubation, the solution was sterile-filtered through a 0.45 μm microporous filter membrane and ultrafiltration centrifuged. The supernatant solution was collected as the nano-eye drop solution and stored at 4°C until further use. A schematic diagram of the synthesis is shown in Figure 1.

[0140] The prepared nano-eye drops were characterized using a zeta potential analyzer and transmission electron microscopy. The characterization and subsequent examples were conducted using a 0.2% chitosan solution as an example. The characterization results, shown in Figure 2, show a particle size of approximately 80 nm, a surface charge of approximately +38 mV, and a loading of 46%.

[0141] Example 2: Toxicity test of nano eye drops

[0142] Test method: CCK-8 method.

[0143] Testing tool: Tecan Spark multifunctional microplate reader

[0144] Reagents and their sources:

[0145] Fetal bovine serum (FBS), DMEM culture medium and penicillin-streptomycin were purchased from Thermo Fisher Scientific, USA.

[0146] CCK-8 was purchased from Tongren Company in Japan.

[0147] Experimental steps:

[0148] 1. Use DMEM / F-12 culture medium containing 10% FBS, 1% penicillin-streptomycin, 0.001% EGF, and 0.0006% human insulin solution. Cells of 100 μL well-grown human corneal epithelial cells (HCEC) were plated at 1×10 4 The cells were seeded at a density of 100 cells / well in a 96-well plate, and the edge wells of the plate were filled with sterile PBS and incubated in a cell culture incubator overnight.

[0149] 2. After 24 h, the old culture medium was removed and the cells were washed with PBS. 100 μL of culture medium was added to each well of the control group and 100 μL of culture medium was added to each well of the experimental group. The cells were co-cultured with the GCV-loaded nano-eye drops (0-1 mg / mL) prepared in Example 1 at a concentration gradient, and the cells were incubated in a 37°C, 5% CO2 incubator for 24 h.

[0150] 3. Carefully aspirate the culture medium in the wells, add 100 μL of freshly prepared CCK-8 working solution (1 / 9, v / v) to each well, and continue incubation for 2 hours.

[0151] 4. Terminate the culture. Measure the absorbance of each well at OD 450 nm using a microplate reader. Calculate cell viability using the following formula: Cell viability = OD 450 (samples) / OD 450 (control) × 100%.

[0152] From the cytotoxicity results of the CCK-8 method in Figure 3 , it can be seen that the nano eye drops have no obvious cytotoxicity.

[0153] Example 3: Ocular surface biosafety test of nano eye drops

[0154] Test method: Sodium fluorescein staining

[0155] Test tool: slit lamp biological microscope

[0156] Reagents and sources: Fluorescein sodium was purchased from Shanghai Sigma-Aldrich Trading Co., Ltd.

[0157] Animals and their sources: Female 6-8 week old C57BL mice were purchased from Beijing Weitonglihua Company.

[0158] Experimental steps:

[0159] 1. Healthy 18-22 g C57BL mice were divided into three groups, with three mice in each group. Each group of mice was treated with eye drops containing different nanoparticles: PBS, LP (GCV-loaded liposomes, prepared in Example 1), and CMLP (GCV-loaded chitosan-modified liposomes, prepared in Example 1) (concentration: 1 mg / mL, 5 μL per injection, twice daily).

[0160] 2. The integrity of the mouse corneal epithelium was observed under cobalt blue light by slit lamp biomicroscopy and sodium fluorescein staining at week 1 and week 2.

[0161] As shown in Figure 4, after 1 week and 2 weeks of eye drops containing different nanoparticles, there was no obvious damage to the corneal epithelium by sodium fluorescein staining, and the corneal epithelium had good integrity, indicating that the prepared nano eye drops had good tolerance and high biocompatibility.

[0162] Example 4: Biosafety Assessment of Nano-Eye Drops in Ocular Tissue

[0163] Test method: Hematoxylin / eosin (H&E) staining

[0164] Testing tools: microscope

[0165] Animals and their sources: Female 6-8 week old C57BL mice were purchased from Beijing Weitonglihua Company.

[0166] Experimental steps:

[0167] 1. Healthy 18-22 g C57BL mice were divided into three groups, with three mice in each group. Each group of mice was treated with eye drops containing different nanoparticles: PBS, LP (GCV-loaded liposomes, prepared in Example 1), and CMLP (GCV-loaded chitosan-modified liposomes, prepared in Example 1) (concentration: 1 mg / mL, 5 μL per injection, twice daily).

[0168] 2. After two weeks of continuous eye drops, mice were sacrificed on the 14th day. Eyeballs were removed and fixed in 4% paraformaldehyde for 24 hours before being sent to the pathology laboratory for paraffin sectioning and H&E staining. Retinal structural changes were then observed under a microscope and photographed.

[0169] As shown in Figure 5, after two weeks of continuous eye drops, no obvious pathological changes occurred in the retinal tissue of mice, indicating that the prepared nano eye drops have good eye tissue safety.

[0170] Example 5: Preparation of fluorescent molecule-loaded nano-eye drops

[0171] Instruments and equipment: Rotary evaporator.

[0172] Reagents and their sources:

[0173] Shanghai TCI Chemical Industry Development Co., Ltd.

[0174] Fluorescein isothiocyanate (FITC), rhodamine B (RhB), soybean lecithin, DSPE-PEG and chitosan were purchased from Tianjin Xiens Biochemical Technology Co., Ltd.

[0175] 8 mg of soybean lecithin, 1 mg of cholesterol, and 1 mg of DSPE-PEG were dissolved in 4 mL of dichloromethane, mixed thoroughly, and then placed on a rotary evaporator and evaporated at 40°C for 15 minutes to form a lipid film. Subsequently, 8 mL of a 0.5 mg / mL FITC solution in PBS was added and the mixture was hydrated at 25°C for 60 minutes. The resulting hydrated solution was sonicated in a 90W waterbath for 15 minutes and then extruded through a 0.45 μm filter membrane to obtain a FITC-loaded liposome solution. An equal volume of a 0.2% chitosan solution (solvent: 0.1% v / v glacial acetic acid solution) was then added to the resulting FITC-loaded liposome solution. The mixture was mixed with magnetic stirring at 25°C for 30 minutes and incubated at 4°C overnight. After sterile filtration through a 0.45 μm microporous filter membrane, the supernatant solution was collected and stored at 4°C until further use.

[0176] The same method was used to prepare RhB-loaded liposomes and RhB-loaded chitosan-modified liposomes.

[0177] Example 6: HCEC uptake test of nano eye drops

[0178] Test method: flow cytometry

[0179] Test tool: BD FACSCelesta flow cytometer

[0180] Experimental steps:

[0181] 1. Use Dulbecco's Modified Eagle's Medium / Ham's F-12 (DF-12) culture medium containing 10% fetal bovine serum (FBS, v / v), 7 μg / mL insulin, 100 U / mL penicillin and 100 mg / mL streptomycin to culture 1 mL of HCEC cells at a rate of 10×10 4 The cells were seeded into 12-well plates at a density of 1000 cells / well and incubated for 24 hours.

[0182] 2. Remove the old culture medium, wash with PBS, replace with fresh culture medium, add free FITC (fluorescein isothiocyanate) solution, LP-FITC (fluorescein isothiocyanate-loaded liposomes, prepared in Example 5), and CMLP-FITC (fluorescein isothiocyanate-loaded chitosan-modified liposomes, prepared in Example 5), respectively, incubate for 1 h and 2 h, respectively, remove the culture medium, wash with PBS, collect the cells after trypsin digestion, and centrifuge at 1500 rpm for 5 min.

[0183] 3. Repeat washing with PBS twice, filter through a 200-mesh copper mesh to obtain a single-cell suspension and transfer it into a flow tube. Detect the fluorescence intensity by flow cytometry.

[0184] As shown in Figure 6, the chitosan-modified liposome group detected significantly higher mean fluorescence intensities than the other groups at both 1 and 2 hours, indicating that the nano-eye drops can enhance the uptake of active ingredients by HCEC and facilitate enhanced ocular tissue penetration through the intracellular pathway.

[0185] Example 7: Detection of tight junctions between HCEC cells

[0186] Test method: Phalloidin labeling method

[0187] Test tool: German Zeiss LSM800 laser confocal microscope

[0188] Test steps:

[0189] 1. Place circular adhesive coverslips in a 12-well plate using Dulbecco's Modified Eagle's Medium / Ham's F-12 (DF 12) medium containing 10% fetal bovine serum (FBS, v / v), 7 μg / mL insulin, 100 U / mL penicillin, and 100 mg / mL streptomycin. Then, plate 1 mL of HCEC cells at a rate of 5 × 10 4 The cells were seeded into 12-well plates at a density of 1000 cells / well and incubated for 24 h.

[0190] 2. After removing the old culture medium and washing with PBS, 1 mL of culture medium containing 10 μg / mL chitosan-modified liposome nano-eye drops (prepared in Example 5) was added to the well plates and incubated with HCEC cells for 2 h.

[0191] 3. Remove the culture medium, wash with PBS three times, and then fix with 4% paraformaldehyde for 15 minutes.

[0192] 4. Add 300 μL of FITC phalloidin containing 1% BSA (1:300 dilution) to each well and incubate at room temperature in the dark for 15 minutes.

[0193] 5. Wash with PBS three times, add 3-4 μL of anti-fluorescence quenching mounting medium containing DAPI to each well, and leave at room temperature for 10 minutes to stain the cell nucleus.

[0194] 6. Use CLSM at a wavelength of 488 nm to observe the changes in tight junctions between HCEC cells after co-incubation with different nanoparticles.

[0195] F-actin is a major protein in the cell cortex and plays a key role in regulating epithelial permeability. FITC-labeled phalloidin can stain F-actin, revealing the presence of tight junctions between cells. As shown in Figure 7, F-actin in the PBS and liposome-treated groups is distributed along the cell membrane with clear outlines. However, after treatment with the chitosan-modified liposome nanoparticle eye drops, F-actin exhibits a fibrous appearance with blurred outlines, indicating that tight junctions between cells are open and permeability is enhanced.

[0196] Example 8: Nano eye drops prolong the ocular surface retention time of drugs

[0197] Testing tools: Slit lamp

[0198] Animals and their sources: Female 6-8 week old C57BL mice were purchased from Beijing Weitonglihua Company.

[0199] Experimental steps:

[0200] 1. Mice were randomly divided into three groups, with three mice in each group. Each group received 7 μL of free FITC (fluorescein isothiocyanate) solution, LP-FITC (fluorescein isothiocyanate-loaded liposomes, prepared in Example 5), and CMLP-FITC (fluorescein isothiocyanate-loaded chitosan-modified liposomes, prepared in Example 5) eye drops, respectively.

[0201] 2. Use a slit lamp biomicroscope to take pictures of the mouse eyes under white light at different time intervals (0, 1, 4, 8, 12, and 16 minutes) to compare the fluorescence intensity of the ocular surface between different groups at the same time interval.

[0202] As shown in Figure 8, 4 minutes after eye application, the green fluorescence of FITC could no longer be observed on the ocular surface of the free FITC solution group and the LP-FITC group; while at 16 minutes, strong green fluorescence could still be observed on the ocular surface and orbital margin of the mice in the CMLP-FITC group, indicating that the nano eye drops can effectively prolong the retention time on the ocular surface.

[0203] Example 9: Mouse eye tissue distribution experiment of nano eye drops

[0204] Test tool: German Zeiss LSM800 laser confocal microscope

[0205] Animals and their sources: Female 6-8 week old C57BL mice were purchased from Beijing Weitonglihua Company.

[0206] Experimental steps:

[0207] 1. Mice were randomly divided into 3 groups, with 3 mice in each group at each time point, for a total of 18 mice. 7 μL of free RhB (rhodamine B), LP-RhB (liposomes loaded with rhodamine B, prepared in Example 5), and CMLP-RhB (chitosan-modified liposomes loaded with rhodamine B, prepared in Example 5) were instilled into the eyes of the mice, respectively.

[0208] 2. The mice were killed 3 hours and 6 hours after eye drop, and the eyeballs were removed and embedded in OCT frozen section embedding medium and sliced.

[0209] 3. Remove frozen sections and fix with 4% paraformaldehyde at room temperature for 20 minutes. Wash three times with PBS. Add DAPI-containing anti-fluorescence quenching mounting medium to stain cell nuclei and mount the sections. Use CLSM to observe differences in fluorescence intensity in the posterior segment of the mouse eye, including the retina and choroid, between the different groups.

[0210] As shown in Figure 9, the fluorescence intensity in the retina and choroid of mice in the chitosan-modified liposome nano-eye drops group was significantly stronger than that in the other two groups, indicating that the chitosan-modified liposome nano-eye drops can deliver the active ingredients to the posterior segment structure of the mouse eye.

[0211] Example 10: Pharmacokinetics of Nano-Eye Drops in Rat Eye Tissue

[0212] Test tool: Thermo Fisher UltiMate 3000 high performance liquid chromatograph

[0213] Animals and their sources: Female 6-8 week old SD rats were purchased from Beijing Weitonglihua Company.

[0214] Experimental steps:

[0215] 1. Rats were randomly divided into three groups, and 30 μL of GCV solution containing 0.1% GCV (ganciclovir), LP-GCV (liposomes loaded with ganciclovir), and CMLP-GCV (chitosan liposomes loaded with ganciclovir) (prepared as in Example 1) were instilled into the conjunctival sac of the rats respectively;

[0216] 2. At predetermined time points (1, 3, 6, and 12 hours after a single eye drop), rats were humanely euthanized by intraperitoneal overdose of anesthetic. The eyeballs were then removed, rinsed with PBS, and the retina and choroid were isolated. The samples were then transferred to 1.5 mL EP tubes and weighed.

[0217] 3. Add 100 μL PBS and 3 zirconium beads to the EP tube containing the sample and grind it using a tissue grinder (frequency 60 Hz, time 10 min).

[0218] 4. Add 200 μL PBS and an equal volume of methanol to the ground EP tube, mix thoroughly, and centrifuge at 13,000 rpm for 15 minutes.

[0219] 5. Take 100 μL of the supernatant and use high performance liquid chromatography (HPLC) to determine the GCV content in rat eye tissue. Chromatographic conditions: mobile phase: methanol: water = 5:95; chromatographic column: Hypersil TM ODS-2C18 (100×4.6 mm, 5 μm), column temperature: 25°C, detection wavelength: 254 nm, flow rate: 1.0 mL / min, injection volume: 20 μL, run time: 17 min.

[0220] As shown in Figure 10a-c, compared with the control group, after a single instillation of the nano eye drops, significantly increased GCV content was detected in the posterior segment of the rat eye tissues, including the retina and choroid, indicating that the nano eye drops can efficiently deliver the drug to the posterior segment of the rat eye tissue. Among them, the drug concentrations in the retina and sclera-choroid reached 31.59 ng / mg and 32.53 ng / mg at 3 hours, and the drug concentrations in the retina and sclera-choroid reached 36.43 ng / mg and 93.22 ng / mg at 6 hours, respectively. These concentrations are higher than the IC50 of ganciclovir against cytomegalovirus (0.02 μg / mL-3.5 μg / mL), indicating that the nano eye drops can achieve effective drug concentrations in the posterior segment of the eye by eye administration.

[0221] Example 11: Detection of drug concentration in rabbit eye tissue of nano eye drops

[0222] Test tool: Thermo Fisher UltiMate 3000 high performance liquid chromatograph

[0223] Animals and their sources: Female Dutch rabbits were purchased from Beijing Weitonglihua Company

[0224] Experimental steps:

[0225] 1. The rabbits were randomly divided into three groups, and 50 μL of GCV solution containing 0.1% GCV, LP-GCV, and CMLP-GCV (prepared according to the method of Example 1) were instilled into the conjunctival sac of the rats respectively;

[0226] Six hours after the single eye drop, air was injected into the marginal ear vein to kill the Dutch rabbit. The eyeball was then removed and rinsed with PBS. The cornea, aqueous humor, retina, and choroid were then separated. The samples were then transferred to 1.5 mL EP tubes and weighed.

[0227] 3. Add 100 μL PBS and 3 zirconium beads to the EP tube containing the sample and grind it using a tissue grinder (frequency 60 Hz, time 10 min).

[0228] 4. Add 200 μL PBS and an equal volume of methanol to the ground EP tube, mix thoroughly, and centrifuge at 13,000 rpm for 15 minutes.

[0229] 5. Take 100 μL of the supernatant and use high performance liquid chromatography (HPLC) to determine the content of GCV in rabbit eye tissue. Chromatographic conditions: mobile phase: methanol: water = 5:95; chromatographic column: Hypersil TM ODS-2C18 (100×4.6 mm, 5 μm), column temperature: 25°C, detection wavelength: 254 nm, flow rate: 1.0 mL / min, injection volume: 20 μL, run time: 17 min.

[0230] Considering that rabbit eyes are similar in size to human eyes and have a more complete ocular barrier system. The delivery efficiency of nano eye drops in rabbit eyes was further tested. As shown in Figure 10d, compared with the free GCV and LP-GCV groups, significantly increased GCV content was detected in the retina and choroid tissues of Dutch rabbits administered with CMLP-GCV nano eye drops. The concentrations in the retina and sclera-choroid at 6 hours can reach 27.21ng / mg and 50.85ng / mg, respectively. This concentration is also higher than the IC50 of ganciclovir against cytomegalovirus (0.02μg / mL-3.5μg / mL), indicating that nano eye drops can efficiently deliver drugs to the posterior segment tissues of Dutch rabbit eyes.

[0231] Example 12: Preparation of nano eye drops loaded with timolol

[0232] 8 mg of soy lecithin, 1 mg of cholesterol, and 1 mg of DSPE-PEG were dissolved in dichloromethane, mixed thoroughly, and placed on a rotary evaporator for 15 minutes at 40°C to form a lipid film. Subsequently, 8 mL of a PBS solution containing 0.2% timolol was added and hydrated at 25°C for 60 minutes. The resulting hydrated solution was sonicated in a 90W waterbath for 15 minutes and then extruded through a 0.45 μm filter membrane to obtain a timolol-loaded liposome solution. Subsequently, an equal volume of 0.2% chitosan solution (solvent: 0.1% v / v glacial acetic acid solution) was added to the resulting timolol liposome solution. After mixing with magnetic stirring at 25°C for 30 minutes, the mixture was incubated at 4°C overnight. After overnight incubation, the mixture was sterile filtered through a 0.45 μm microporous filter membrane and ultrafiltration centrifuged. The upper layer of the solution was collected as the nano-eye drop solution and stored at 4°C until use, with a loading rate of approximately 35%.

[0233] Example 13: Preparation of curcumin-loaded nano eye drops

[0234] 8 mg of soy lecithin, 1 mg of cholesterol, 1 mg of DSPE-PEG, and 1.6 mg of curcumin were dissolved in dichloromethane, mixed thoroughly, and placed on a rotary evaporator. Rotary evaporation was performed at 40°C for 15 minutes to form a lipid film. 8 mL of PBS was then added and hydrated at 25°C for 60 minutes. The resulting hydrated solution was sonicated in a 90W water bath for 15 minutes and then extruded through a 0.45 μm filter membrane to obtain a curcumin-loaded liposome solution. An equal volume of 0.2% chitosan solution (solvent: 0.1% v / v glacial acetic acid solution) was then added to the obtained curcumin-loaded liposome solution. After mixing under magnetic stirring at 25°C for 30 minutes, the mixture was placed at 4°C and incubated overnight. After incubation overnight, the mixture was sterile filtered through a 0.45 μm microporous filter membrane, followed by ultrafiltration and centrifugation. The upper tube solution was collected as the nano eye drops and stored at 4°C for future use. The loading rate was 32%. The synthesis schematic is shown in Figure 11.

[0235] Example 14: Preparation of Brinzolamide-loaded Nano Eye Drops

[0236] 8 mg of soy lecithin, 1 mg of cholesterol, 1 mg of DSPE-PEG, and 1.6 mg of brinzolamide were dissolved in dichloromethane, mixed thoroughly, and then placed on a rotary evaporator and rotary evaporated at 40°C for 15 minutes to form a lipid film. 8 mL of PBS was then added and hydrated at 25°C for 60 minutes. The resulting hydrated solution was sonicated in a 90W waterbath for 15 minutes and then extruded through a 0.45 μm filter membrane to obtain a brinzolamide-loaded liposome solution. An equal volume of 0.2% chitosan solution (solvent: 0.1% v / v glacial acetic acid) was then added to the resulting brinzolamide-loaded liposome solution. The mixture was magnetically stirred at 25°C for 30 minutes and incubated at 4°C overnight. After overnight incubation, the solution was sterile filtered through a 0.45 μm microporous filter membrane and ultrafiltration centrifuged. The supernatant solution was collected as the nano-eye drop solution and stored at 4°C until use. The loading rate was approximately 35%.

[0237] Example 15: Preparation of dexamethasone-loaded nano-eye drops

[0238] 8 mg of soy lecithin, 1 mg of cholesterol, 1 mg of DSPE-PEG, and 1.6 mg of dexamethasone were dissolved in dichloromethane, mixed thoroughly, and then placed on a rotary evaporator and evaporated at 40°C for 15 minutes to form a lipid film. 8 mL of PBS was then added and the mixture was hydrated at 25°C for 60 minutes. The resulting hydrated solution was sonicated in a 90W waterbath for 15 minutes and then extruded through a 0.45 μm filter membrane to obtain a dexamethasone-loaded liposome solution. An equal volume of 0.2% chitosan solution (solvent: 0.1% v / v glacial acetic acid) was then added to the dexamethasone-loaded liposome solution. The mixture was mixed with magnetic stirring at 25°C for 30 minutes and incubated at 4°C overnight. After overnight incubation, the mixture was sterile filtered through a 0.45 μm microporous filter membrane and ultrafiltration centrifuged. The supernatant solution was collected as the nano-eye drop solution and stored at 4°C until use. The loading rate was approximately 10%.

[0239] Example 16: Construction of mouse retinal light damage (LIRD) model

[0240] To establish the LIRD model, a dark box with a blue light source mounted on top and surrounded by mirrors was used. As shown in Figure 12, female BALB / c mice were first placed in the box for 16 hours of dark adaptation. A 0.5% tropicamide solution was topically applied to the mouse cornea to ensure pupil dilation, and then the mice were exposed to strong blue light for 45 minutes. The mice were then kept in darkness for 16 hours before treatment verification.

[0241] Example 17: Nano-eye drops for treating retinal light damage

[0242] Curcumin is a hydrophobic drug with anti-oxidative stress and anti-inflammatory activities, and can be used to alleviate retinal light damage. This example further verifies the effect of the curcumin-loaded nano eye drops prepared in Example 13 in the retinal light damage model prepared in Example 16.

[0243] The results are shown in Figure 13. Curcumin-loaded nano-eye drops were used to treat retinal light-damaged mice twice daily. Immunofluorescence staining and immunohistochemistry were used to observe the retinal tissue of mice in different treatment groups. The results showed that compared with curcumin eye drops and curcumin liposomes, curcumin nano-eye drops significantly reduced cell apoptosis in the outer nuclear layer of retinal tissue, thereby protecting the retinal structure. This result further demonstrates the wide applicability of the nano-eye drops of the present invention in the treatment of posterior segment diseases.

[0244] Industrial Applicability:

[0245] The present invention provides a preparation method and application of nano eye drops capable of delivering drugs to the posterior segment of the eye. The chitosan-modified liposomes of the present invention can be combined with appropriate active drug molecules to prepare corresponding eye drop dosage forms, which are suitable for industrial applications.

[0246] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A chitosan-modified liposome, characterized in that: The chitosan-modified liposome comprises a liposome core and a chitosan shell, wherein the liposome core comprises a lipid bilayer membrane; The lipid bilayer membrane was obtained by rotary evaporation of soybean lecithin, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol.

2. The chitosan-modified liposome according to claim 1, characterized in that The mass ratio of soybean lecithin, cholesterol and distearoyl phosphatidylethanolamine-polyethylene glycol is 2-10:1-2:1-2.

3. The chitosan-modified liposome according to claim 1, wherein The temperature of the rotary evaporation is 30-50°C.

4. The chitosan-modified liposome according to claim 1, wherein The rotary evaporation time is 10 to 20 minutes.

5. The chitosan-modified liposome according to claim 1, characterized in that The chitosan-modified liposomes are positively charged, for example, greater than +20 mV.

6. The chitosan-modified liposome according to claim 1, characterized in that The inner core of the liposome can be loaded with drugs. Preferably, the drugs are hydrophilic drugs and / or hydrophobic drugs.

7. The chitosan-modified liposome according to claim 6, characterized in that in: The hydrophilic drug is selected from ganciclovir, timolol, ciprofloxacin, ofloxacin, tobramycin, gentamicin, dexamethasone acetate, pilocarpine, hydrocortisone, fluorometholone, azalastine hydrochloride, tropicamide, naphazoline hydrochloride, naphazoline hydrochloride or pranoprofen; The hydrophobic drug is selected from curcumin, brinzolamide, sparfloxacin, ofloxacin, gatifloxacin, dexamethasone, pilocarpine, naphazoline hydrochloride or tropicamide.

8. A nano eye drop, characterized in that: The nano eye drops comprise the chitosan-modified liposomes according to any one of claims 1 to 7 and a drug loaded in the inner core of the liposomes.

9. A method for preparing chitosan-modified liposomes according to any one of claims 1 to 7, characterized in that: The preparation method comprises: a) dissolving soybean lecithin, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol in an organic solvent to obtain a mixed solution, and rotary evaporating the mixed solution to obtain a lipid film; b) adding a buffer solution to the lipid film obtained in step a) to hydrate the film to obtain a hydrated solution; c) further adding chitosan, stirring and mixing, and then incubating to obtain chitosan-modified liposomes.

10. A method for preparing the nano eye drops according to claim 8, characterized in that: The preparation method comprises: the mixed solution in step a) of claim 9 further comprises a hydrophobic drug; and / or the buffer solution in step b) of claim 9 contains a hydrophilic drug.

11. The preparation method according to any one of claims 9-10, characterized in that The hydration temperature is 20-30°C.

12. The preparation method according to any one of claims 9-10, characterized in that The hydration time is 30 to 100 minutes.

13. The preparation method according to any one of claims 9-10, characterized in that The hydrated liquid obtained after the hydration is sonicated and filtered; Preferably, the power of the ultrasound is 50 to 150 W; Preferably, the ultrasonication time is 10 to 30 minutes.

14. The preparation method according to any one of claims 9-10, characterized in that The stirring temperature is 10-40°C.

15. The preparation method according to any one of claims 9-10, characterized in that The stirring and mixing time is 10 to 50 minutes.

16. The preparation method according to any one of claims 9-10, characterized in that The incubation temperature is 1-10°C.

17. The preparation method according to any one of claims 10 to 16, characterized in that: The preparation method comprises: a) dissolving soybean lecithin, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol in a mass ratio of 2-10:1-2:1-2 in dichloromethane to obtain a mixed solution, and subjecting the mixed solution to rotary evaporation at 30-50° C. for 10-20 minutes to obtain a lipid film; b) adding a phosphate buffer containing a hydrophilic drug to the lipid film obtained in step a), hydrating at 20-30° C. for 30-100 minutes to obtain a hydrated solution, and further sonicating at 50-150W for 10-30 minutes; c) adding chitosan solution, stirring at 10-40° C. for 10-50 min, and incubating at 1-10° C. overnight to obtain nano eye drops.

18. The preparation method according to claim 17, characterized in that: In step b), the ratio of the hydrophilic drug to the phosphate buffer in mg:mL is 16-24:6-14.

19. The preparation method according to any one of claims 10 to 16, characterized in that: The preparation method comprises: a) dissolving soybean lecithin, cholesterol, distearoyl phosphatidylethanolamine-polyethylene glycol, and a hydrophobic drug in a mass ratio of 2-10:1-2:1-2 in dichloromethane to obtain a mixed solution, and subjecting the mixed solution to rotary evaporation at 30-50° C. for 10-20 minutes to obtain a lipid film; b) adding phosphate buffer to the lipid film obtained in step a), hydrating at 20-30° C. for 30-100 minutes to obtain a hydrated solution, and further sonicating at 50-150W for 10-30 minutes; c) adding chitosan solution, stirring at 10-40° C. for 10-50 min, and incubating at 1-10° C. overnight to obtain nano eye drops.

20. The preparation method according to any one of claims 10 to 19, characterized in that: The mass percentage of the drug in the nano eye drops is 0.1% to 0.36%.

21. A method for delivering a drug to the posterior segment of the eye, characterized in that: The method comprises adding the chitosan-modified liposomes according to any one of claims 1 to 7 or the nano eye drops according to claim 8 to the ocular surface of a subject in need.

22. Use of the chitosan-modified liposome according to any one of claims 1 to 7 or the nano eye drops according to claim 8 as and / or in the preparation of an ophthalmic drug.

23. The use according to claim 22, characterized in that The ophthalmic drug is a drug for treating or preventing diseases related to the posterior segment of the eye, such as retinal disease, vitreous disease, choroidal disease or scleral disease.

24. The use according to claim 22, characterized in that The ophthalmic drugs are drugs for treating or preventing eye inflammation, allergies, glaucoma, cataracts; for example, drugs for diabetic retinopathy and / or age-related macular degeneration and / or retinal light damage and / or infectious and non-infectious eye inflammation (such as cytomegalovirus retinitis).

25. A method for treating ophthalmic diseases, characterized in that: The method comprises adding the chitosan-modified liposomes according to any one of claims 1 to 7 or the nano eye drops according to claim 8 to the ocular surface of a subject in need.

26. The method according to claim 25, characterized in that The ophthalmic diseases include diseases related to the posterior segment of the eye, such as drugs for retinal diseases, vitreous diseases, choroidal diseases or scleral diseases.

27. The method according to claim 25, characterized in that Such ophthalmic diseases include drugs for ocular inflammation, allergies, glaucoma, cataracts; for example, diabetic retinopathy and / or age-related macular degeneration and / or retinal light damage and / or infectious and non-infectious ocular inflammation (such as cytomegalovirus retinitis).

Images