WO2022037063A1

WO2022037063A1 - Ophthalmic liposome capable of penetrating cornea and targeting retina, preparation method therefor and application thereof

Info

Publication number: WO2022037063A1
Application number: PCT/CN2021/082922
Authority: WO
Inventors: 丁雪鹰; 徐楠; 张姝月; 刘堃; 高申; 丁宝月; 王卓; 何美; 台宗光
Original assignee: 上海市第一人民医院
Priority date: 2020-08-18
Filing date: 2021-03-25
Publication date: 2022-02-24
Also published as: CN111888484A; CN111888484B

Abstract

The present disclosure relates to an ophthalmic liposome capable of penetrating a cornea and targeting a retina, a preparation method therefore and an application thereof. The ophthalmic liposome is a liposome which is made of biocompatible lipid material and encapsulates an anti-VEGF drug, and whose surface is connected to cell-penetrating peptide (CPP) and hyaluronic acid (HA). According to the present disclosure, the anti-VEGF drug is encapsulated by the liposome, thereby effectively mitigating the instability of the anti-VEGF drug. Corneal penetration is enhanced through the amphiphilicity of the liposome, and the surface of the liposome is covalently linked to the cell-penetrating peptide (CPP) which has penetration effects and the hyaluronic acid (HA) which targets the retina. It is found that the liposome prepared can significantly increase corneal penetration and accumulation in the ocular fundus of the drug by means of eye drop instillation, thereby enhancing drug concentration at a lesion site and significantly improving the treatment effect.

Description

An ophthalmic liposome capable of penetrating the cornea and targeting the retina and its preparation method and application

technical field

The invention belongs to the technical field of medicine, in particular to an ophthalmic liposome co-modified with a cell-penetrating peptide CPP and hyaluronic acid HA, which can penetrate the cornea and target the retina, and a preparation method and application thereof.

Background technique

The eye is an extremely complex organ, which can be anatomically divided into two parts: the anterior segment and the posterior segment. The anterior segment mainly includes the cornea, conjunctiva, aqueous humor, and lens, and the posterior segment mainly includes the vitreous, sclera, choroid, and retina. Intraocular neovascular diseases are mainly located in the posterior segment of the eye, such as retinopathy of prematurity (ROP), diabetic retinopathy (DR) and age-related macular degeneration (AMD). It is a serious blinding eye disease. Fundus neovascular disease includes choroidal neovascularization and retinal neovascularization. The lesions mainly exist in the retina. Vascular endothelial growth factor (VEGF) is the main pathogenic factor. It inhibits the activity of VEGF and can effectively control neovascularization. generation.

The main method of clinical treatment of fundus neovascularization is repeated intravitreal injection of biological drugs, such as conbercept and aflibercept. Repeated injection into the vitreous cavity is an invasive method of administration, which inevitably leads to some serious complications, such as cataract formation, intraocular hypertension, retinal detachment, vitreous hemorrhage, intraocular inflammation, and tissue damage. Frequent visits to the hospital and related nursing costs also put patients under greater financial pressure and have poor patient compliance. There is an urgent need for a safe, effective, easy-to-use and high patient compliance dosing regimen for the treatment of fundus neovascular disease.

Eye drop administration can be used as an alternative to intravitreal injection for the treatment of ocular diseases. It is convenient to use and has good patient compliance. It is the most commonly used administration method for the clinical treatment of ocular diseases. When administering eye drops, drug absorption is the main limiting factor, and amphiphilic small-molecule drugs are most likely to enter the eye through the corneal barrier. Compared with small-molecule drugs, biopharmaceuticals have large molecular weight and strong hydrophilicity, and it is difficult to enter the eye through eye drops, resulting in low drug bioavailability. Improving the permeability of eye drop administration to the ocular barrier is an urgent problem that needs to be overcome.

Liposomes are composed of phospholipid bilayers wrapped around the aqueous phase. The composition is similar to cell membranes and has amphiphilicity. Encapsulating drugs in liposomes can promote the penetration of drugs into the corneal barrier, and liposomes are loaded with drugs. With excellent performance, high biocompatibility, easy surface modification, and strong clinical transformation, it is a promising ocular drug delivery carrier.

Cell-penetrating peptides (CPP) are short peptides composed of several or more than a dozen amino acids, which have the ability to deliver a variety of macromolecular substances into cells, such as dendrimers, liposomes, glues Bundles and proteins, etc., show excellent performance in ocular permeability and biocompatibility. However, CPP has poor targeting ability and cannot specifically accumulate the drug in the retina after penetrating the cornea, resulting in low drug concentration at the lesion site.

Hyaluronic acid (HA) is a glycosaminoglycan composed of a disaccharide structure and is the main component of the vitreous body. After HA modification, it can greatly improve the fluidity of the carrier in the vitreous body and make it easier to reach the retina. The study found that human retinal pigment epithelial cells (ARPE-19) highly expressed the CD44 receptor under disease conditions, and HA could specifically bind to the CD44 receptor, and the carrier modified HA to achieve targeting to the retina. Therefore, hyaluronic acid modification may be an effective strategy to improve retinal targeting of drugs.

Studies have shown that high expression of integrin αvβ3 can be detected in retinal or choroidal neovascular endothelial cells of CNV, AMD, and DR patients. RGD peptide is a kind of arginine-glycine-aspartic acid (Arg-Gly-Asp). ), the peptide containing the RGD sequence has a specific binding function to the integrin αvβ3 receptor. Patent CN201510749931.6, literature (Yongchao C, Ning C, Huajun Y, et al. Topical ocular delivery to laser-induced choroidal neovascularization by dual internalizing RGD and TAT peptide-modified nanoparticles [J]. International Journal of Nanomedicine, 2017,12 :1353-1368.doi:10.2147/IJN.S126865), (Yang X, Wang L, Li L, et al. A novel dendrimer-based complex co-modified with cyclic RGD hexapeptide and penetration for noninvasive targeting and penetration of the ocular posterior segment[J].Drug Delivery,2019,26(1):989-1001.doi:10.1080/10717544.2019.1667455)(Liu C,Jiang K,Tai L,et al.Facile Noninvasive Retinal Gene Delivery Enabled by Penetratin. ACS Appl Mater Interfaces.2016;8(30):19256-19267.doi:10.1021/acsami.6b04551) Co-modification of RGD and cell penetrating peptide CPP on dendrimer PAMAM and PLGA nanoparticles for the treatment of posterior ocular neovascularization However, the dendritic polymer PAMAM is highly irritating to the eyes and has high toxicity, so it is not suitable for ocular administration, and the drug delivery system does not improve the problem of poor fluidity of the carrier in the vitreous body. Compared with the free drug, the double-modified drug-loaded nanoparticles The solution only increased the drug concentration by a factor of about 4.

In addition, literature (Tai L, Liu C, Jiang K, et al. A novel penetratin-modified complex for noninvasive intraocular delivery of antisense oligonucleotides[J]. International Journal of Pharmaceutics, 2017:347-356.doi:10.1016/j. ijpharm.2017.06.090) Coated hyaluronic acid and Penetratin on the surface of PAMAM successively by electrostatic adsorption to improve the ocular biocompatibility and permeability of PAMAM, and found that the distribution and retention time of the complex in the posterior segment of the eye increased, but the transparent The acid is coated inside Penetratin and cannot effectively improve the fluidity of the vitreous and retinal targeting.

At present, there is no report of a liposome encapsulated with anti-VEGF drug and attached with cell penetrating peptide CPP and hyaluronic acid HA on the surface for topical eye drops for the treatment of posterior ocular diseases.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an ophthalmic liposome that can penetrate the cornea and target the retina and its preparation method and application in view of the deficiencies in the prior art.

In the first aspect, the present invention provides an ophthalmic liposome co-modified with a cell-penetrating peptide and hyaluronic acid that can penetrate the cornea and target the retina, which is a lipid material with good biocompatibility. The constructed liposomes are loaded with anti-VEGF drugs and have cell penetrating peptide CPP and hyaluronic acid HA attached to the surface.

In some embodiments of the present invention, the lipid materials with good biocompatibility include, but are not limited to, egg yolk lecithin (EPC), soybean lecithin (SPC), hydrogenated soybean lecithin (HSPC), dipalmitoyl phosphatidyl Choline (DPPC), cholesterol (CHOL), dimyristoyl phosphatidyl ethanolamine (DMPE) or its pegylated derivatives, distearoyl phosphatidyl ethanolamine (DSPE) or its pegylated derivatives, di One or more of palmitoyl phosphatidyl ethanolamine (DPPE) or its PEGylated derivatives, and dioleoyl phosphatidyl ethanolamine (DOPE) or its PEGylated derivatives.

In some embodiments of the present invention, the cell-penetrating peptide CPP includes, but is not limited to, the hexadeceptide Penetratin (RQIKIWFQNRRMKWKK, SEQ ID NO: 1) derived from the homology domain of the Drosophila antennal gene, human immunodeficiency virus protein Transduction peptide TAT (GRKKRRQRRRPPQ, SEQ ID NO: 2), octamer arginine R8 (RRRRRRRR, SEQ ID NO: 3), low molecular weight protamine LMWP (VSRRRRRRGGRRRR, SEQ ID NO: 4).

In some embodiments of the invention, the anti-VEGF drug includes, but is not limited to, bevacizumab, ranibizumab, conbercept, aflibercept.

In some embodiments of the present invention, the mass ratio of the hyaluronic acid HA to the total lipid is 0.1-10, more preferably 1-5; and/or the ratio of the cell penetrating peptide CPP to the total lipid The molar ratio is 1% to 50%, more preferably 10% to 50%.

In some embodiments of the invention, the hyaluronic acid HA has a molecular weight of 6.4 kDa-1500 kDa.

In some embodiments of the invention, the cell penetrating peptide CPP is chemically bonded to the exterior of the liposome; and/or the hyaluronic acid HA is chemically bonded to the exterior of the liposome.

In a second aspect, the present invention provides a method for preparing a retina-targeting cell-penetrating peptide and hyaluronic acid co-modified ophthalmic liposome, comprising the following steps:

(1) using an addition reaction to connect the phospholipid containing the Mal group with the cell penetrating peptide CPP;

(2) The cell-penetrating peptide CPP-modified liposomes encapsulating anti-VEGF drugs were prepared by thin film dispersion method;

(3) The hyaluronic acid HA was connected with the liposome modified by the cell penetrating peptide CPP encapsulating the anti-VEGF drug by chemical coupling method.

In some embodiments of the present invention, the specific operation of the addition reaction is to dissolve DSPE-PEG-Mal (all phospholipids containing Mal group) and thiol-modified CPP (all thiolated CPP) in chloroform In the solution, the reaction was performed in the dark for 24 h under nitrogen-filled conditions, and then evaporated to dryness under reduced pressure to obtain DSPE-PEG-CPP.

In some embodiments of the present invention, the specific operation of the film dispersion method is to dissolve EPC, CHOL, DPPE, DSPE-PEG, DSPE-PEG-CPP in a chloroform solution, evaporate under reduced pressure to obtain a lipid film, and use a Liposomes were obtained by hydration of the anti-VEGF drug in PBS.

In some embodiments of the present invention, the specific operation of the chemical coupling method is to dissolve HA, EDC, and NHS in acetate buffer, incubate at 37° C. for 2 hours for pre-activation, and add the HA carboxyl group to the step after activation. (2) In the obtained liposomes, chemical coupling with amino groups on the surface of DPPE is carried out to obtain liposomes loaded with anti-VEGF drugs and connected with cell penetrating peptide CPP and hyaluronic acid HA on the surface.

In a third aspect, the present invention provides an application of the ophthalmic liposome as described above in the preparation of a medicament for treating fundus neovascular disease.

The advantages of the present invention are:

1. Ophthalmic drug delivery is one of the main drug delivery methods for the treatment of ocular diseases. Ophthalmic preparations include eye drops, eye ointments, injections, and the like. For the treatment of fundus diseases, injections cause great pain to patients and have many complications, while the current eye drops and eye ointments have defects such as poor corneal penetration ability and low local bioavailability, resulting in poor treatment effect.

There are many carriers for ocular administration, such as PAMAM, PLGA, HSA, etc. In order to promote the penetration of drugs, there are also many kinds of ocular penetration enhancers, such as cyclodextrins, chelating agents, crown ethers, bile acids and Bile salts, surfactants, cell-penetrating peptides, etc.; in order to improve targeting, known ligands targeting the fundus include RGD, YSA, folic acid, HA, and the like. Based on rich research experience, the inventors of the present application chose to use liposomes as drug carriers to load anti-VEGF drugs into liposomes, which can effectively improve the shortcoming of poor stability of anti-VEGF drugs, and utilize the amphipathic properties of liposomes. Enhance the corneal penetration effect, and then covalently connect the penetrating cell-penetrating peptide CPP and retina-targeting hyaluronic acid HA to the surface to further enhance the corneal penetration effect. Penetration and aggregation to the fundus of the eye, thereby increasing the drug concentration at the lesion site and significantly improving the treatment effect. And the effect of the ophthalmic liposome of the present invention is significantly better than that of the ophthalmic drugs disclosed in the prior art.

2. Since the administration route of the medicine of the present invention is eye drop administration, compared with the administration route of injection, the patient's medication compliance will be greatly improved.

3. In order to ensure that the drug can reach the therapeutic concentration at the lesion site, the inventors of the present application obtained a better modification ratio of the cell-penetrating peptides CPP and HA on the liposome through screening experiments, which allows the anti-VEGF drug to penetrate the eye. After the external barrier reaches the fundus, it can reach the therapeutic concentration and have a better therapeutic effect.

Description of drawings

Figure 1 is a schematic diagram of the preparation of liposomes.

Figure 2 is a characterization diagram of liposomes. In the figure: A is the particle size distribution of liposomes determined by dynamic light scattering particle size analyzer; B is the morphological image of liposomes observed by transmission electron microscope.

Figure 3 is the cytotoxicity of different liposomes.

Figure 4 shows the apparent permeability coefficients of different liposomes. Free drug: free drug group; Lip: unmodified drug-loaded liposome group; HA-Lip: HA single-modified drug-loaded liposome group; Pen-Lip: Penetratin single-modified drug-loaded liposome group; PenHA-Lip: Double-modified drug-loaded liposome group.

Figure 5 shows the cellular uptake of different liposomes after co-incubation with ARPE-19 cells.

Figure 6 shows the distribution of liposomes in mouse eyes.

Figure 7 is a graph showing the results of in vivo efficacy.

Figure 8 is a comparison of intraocular fluorescence intensity of different carriers.

Figure 9 is a comparison of intraocular fluorescence intensity of liposomes modified with different penetration enhancers.

Figure 10 is a comparison of retinal fluorescence intensity of liposomes modified with different targeting ligands.

Figure 11 is the mean fluorescence intensity of ARPE-19 cells after the liposomes of Example 6 penetrated the corneal barrier in vitro.

12 is the mean fluorescence intensity of ARPE-19 cells after the liposomes of Example 7 penetrated the corneal barrier in vitro.

13 is the mean fluorescence intensity of ARPE-19 cells after the liposomes of Example 8 penetrated the corneal barrier in vitro.

detailed description

The specific embodiments provided by the present invention will be described in detail below with reference to the accompanying drawings.

Example 1

1. Preparation of liposomes

(1) DSPE-PEG-Mal was linked to the cell penetrating peptide CPP by an addition reaction.

The cell penetrating peptide CPP was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd., and CPP was thiolated. CPP is specifically Penetratin: RQIKIWFQNRRMKWKK (SEQ ID NO: 1), wherein C is an amino acid-cysteine C added during the synthesis of the sequence, and the amino acid contains a sulfhydryl group, that is, the sulfhydrylation of CPP is completed.

DSPE-PEG-CPP is obtained by 1,4 addition reaction between the maleimide group of DSPE-PEG-Mal and the sulfhydryl group on the cysteine residue of cell penetrating peptide CPP. DSPE-PEG-Mal (11.6 mg) and thiolated Penetratin (16.8 mg) were dissolved in chloroform solution, triethylamine was added dropwise as a catalyst, and the mixture was shaken gently. The mixed solution was reacted in the dark at room temperature for 24 h under nitrogen-filled conditions. The organic solvent is filtered, and the obtained filtrate is evaporated by a rotary evaporator to obtain a solid crystal, which is freeze-dried to obtain DSPE-PEG-CPP.

(2) The cell-penetrating peptide CPP-modified liposomes containing Conbercept were prepared by thin film dispersion method.

Weigh egg yolk lecithin 10 mg (EPC), cholesterol 2.5 mg (CHOL), DPPE 5 mg, DSPE-PEG 1 mg, DSPE-PEG-CPP 17 mg and dissolve them in an eggplant bottle containing 10 mL of chloroform solution. The organic solvent was removed by evaporation under reduced pressure at 35°C by a rotary evaporator, and a uniform lipid film was formed on the bottle wall. Vacuum drying was continued to remove residual organic solvent. 5ml of PBS solution with a concentration of 5mg/ml Conbercept (pH 7.4, preparation method: take a certain volume of Conbercept injection with a concentration of 10mg/ml, add PBS to dilute to the desired concentration) into the eggplant type In the bottle, shake quickly to hydrate the solution with the lipid film. After hydration, the lipid film was completely detached from the bottle wall by water bath ultrasound on ice, and stirred for 1 h in the dark. The obtained milky white liquid was passed through 0.45 μm and 0.22 μm microporous filter membranes three times to obtain liposomes with suitable particle size, which were stored at 4° C. in the dark for future use.

(3) The hyaluronic acid HA was connected to the liposome modified with the cell penetrating peptide CPP encapsulating Conbercept by chemical coupling method.

HA (22 mg), EDC (22 mg), NHS (22 mg) were dissolved in acetate buffer and preactivated at 37°C for 2 h. Activated HA was then added to Penetratin-modified liposomes loaded with conbercept (total lipid (mass of all lipid material added) was 35.5 mg) and the final pH was adjusted to 8 with borate buffer , and incubated at 37 °C for 12 h, and stirred with a magnetic stirrer to couple HA to the surface of liposomes to form conbercept-loaded liposomes co-modified with HA and Penetratin.

A schematic diagram of the preparation of liposomes is shown in Figure 1.

2. Characterization of liposomes

Take 100 μL of freshly prepared liposome solution, dilute it 10 times, and analyze and measure the average particle size of liposomes with dynamic light scattering particle size analyzer. After staining the liposome solution with 2% phosphotungstic acid, the morphology of the liposome was observed by transmission electron microscope. The particle size distribution and electron microscope images of liposomes are shown in Figure 2.

3. Cytotoxicity study

The cytotoxicity of unloaded liposomes was investigated by CCK-8 method. HCEC and ARPE-19 cells were seeded in a 96-well cell culture plate at a density of 5 × 10 ³ per well, and after culturing in an incubator for 24 h, the liposomes were diluted with serum-free medium to a concentration of 12.5, 25, 50, 125, 250, 500, 1000 μg·mL ^-1 solutions were added to the 96-well plate. After 24 hours of incubation, the cells were washed three times with PBS, added with 100 μL of medium containing 10% CCK-8 reagent, and placed in an incubator for 1 hour of incubation. The absorbance of each well at 450nm was measured with a microplate reader and the cell viability under the treatment of each concentration was calculated.

The results are shown in Figure 3, when the liposome concentration was in the range of 12.5 μg·mL ^-1 and 1000 μg·mL ^-1 , the human corneal epithelial cell HCEC and human retinal pigment epithelial cell ARPE-19 were more than 90% higher than those of the human corneal epithelium. The cell viability had no significant effect on the growth of the two types of cells, indicating that the liposomes before and after penetrating peptide and hyaluronic acid modification had low cytotoxicity and high biocompatibility.

4. Penetration of liposomes in isolated rabbit cornea

Penetration of different liposomes through isolated rabbit corneas was determined using a diffusion cell apparatus. The rabbits were sacrificed by injection of a lethal dose of sodium pentobarbital via the ear vein, the eyeball was enucleated, the cornea (along with the surrounding sclera of approximately 2 mm width) was carefully removed from the eyeball and rinsed gently with PBS. The cornea was placed horizontally between the donor and recipient chambers with the corneal epithelial surface facing the donor solution and secured with clips. The donor fluid is the sample solution, and the recipient fluid is PBS solution. Exhaust air bubbles to make the corneal tissue fully contact with the recipient fluid. During 4 hours, an equal amount of 100 μL sample was drawn from the receptor chamber every 0.5 h, and then immediately replenished with an equal amount of PBS. The concentration of the extracted samples was measured using an ELISA detection kit, and the apparent permeability coefficient was calculated.

The results are shown in Figure 4. The apparent permeability coefficients of the five groups were (1.215±0.081), (2.422±0.178), (3.35±0.368), (5.815±0.177), and (9.33±0.50), respectively. The apparent permeability coefficient was 7.7 times that of the free drug group, and PenHA-Lip had the strongest ability to penetrate rabbit cornea. The apparent permeability coefficient of PenHA-Lip is 2.8 times and 1.6 times higher than that of HA-Lip group and Pen-Lip group, respectively, and the data are significantly different. The double-modified group is better than the single-modified group in promoting the ability to penetrate the cornea. The Q value calculated by the mean method was greater than 1.15, which indicated that the modification of Penetratin and HA on liposomes could synergistically promote corneal penetration.

5. The cellular uptake ability of ARPE-19 cells to different liposomes

Sterile round coverslips were pre-plated in 24-well plates and ARPE-19 cells were seeded on the coverslips at a density of ⁵ x 10 per well in a 37°C, 5% _CO incubator After 24 h of culture, cells were serum starved overnight. Rinse twice with PBS, and add the serum-free medium containing Lip, Pen-Lip, PenHA-Lip to the 24-well plate. After cells were incubated with liposomes at 37°C for 3 h, they were washed three times on ice with PBS containing 1000 IU·mL ^-1 heparin to remove extracellular liposomes. Fixed with pre-cooled 4% paraformaldehyde for 30 min and washed 3 times with PBS. Pipette 10 μL of DAPI-containing anti-fluorescence quenching mounting slides onto the slide, carefully remove the coverslip, stick the side with cells on the slide with mounting fluid, and place in the dark for 30 min. The uptake of liposomes by cells was observed using a laser scanning confocal microscope. All operations were performed under dark conditions.

The results are shown in Fig. 5, blue represents DAPI-stained nuclei, red represents Nile red-labeled liposomes, the control group has no red fluorescence, the Lip group has only weak red fluorescence, the Pen-Lip group has medium red fluorescence, and PenHA The -Lip group had the strongest fluorescence. The fluorescence intensity was semi-quantitatively analyzed by ImageJ. The mean fluorescence intensity of the Lip group, Pen-Lip group and PenHA-Lip group were (21.61±3.69), (29.32±2.26) and (47.37±3.99), respectively. The uptake efficiency of Lip was 2.2 and 1.6 times higher than that of Lip and Pen-Lip, respectively. The results showed that liposomes did not cause cell damage after co-incubating with cells for 3 hours. PenHA-Lip had both the cell penetration ability of Penetratin and the targeting ability of HA, and the most prominent performance in ARPE-19 cell uptake.

6. Intraocular distribution of liposomes

The upper and lower eyelids of the mouse were gently opened with a cotton swab, PenHA-Lip was instilled into the conjunctival sac of the mouse, and the eyelids were slowly closed after waiting for a few seconds. Mice were sacrificed 30min, 1h, 3h, 6h, 12h, and 24h after administration, and the eyeballs were immediately removed, rinsed gently with PBS, fixed with eye fixative overnight, and dehydrated in 30% sucrose solution for 12h. The eyeballs were then made into 10 μm-thick cryosections, stained with DAPI, and the sections were scanned under an inverted fluorescence microscope.

The results are shown in Figure 6. It can be observed that the retinal ganglion cell layer (GCL), inner plexiform layer (IPL) and outer plexiform layer (OPL) exhibit moderate fluorescence, while the inner nuclear layer (INL) and outer nuclear layer (ONL) ) showed weak fluorescence, the RPE layer had strong fluorescence, and the RPE layer was the target position of drug administration. Only 30 minutes after eye drop administration, PenHA-Lip could reach the retinal area at the back of the eye, and the fluorescence intensity was high in the first 6 hours, and then gradually decreased with the passage of time, but still visible at 24 hours. Fluorescence.

7. In vivo efficacy test

Male C57/BL6J mice were anesthetized by intraperitoneal injection of 1% pentobarbital solution (25 mg/kg body weight), compound tropicamide was used to dilate the pupils, and ofloxacin eye ointment was applied to the cover glass and contacted the center of the cornea. The 532 multi-wavelength laser treatment machine was used for photocoagulation at 4 points equidistant from the optic disc (laser parameters: wavelength of 532 nm, power of 360 mW, and exposure time of 100 ms). During photocoagulation, pay attention to avoid the position of the retinal vessels. If bubbles are generated after photocoagulation, it means that Bruch's membrane is broken, and the operation is successful.

Immediately after laser photocoagulation, C57BL/6 mice were randomly divided into four groups and given the following treatments respectively: (1) eye drops, 5 μL PBS, 3 times/d, for 7 days; (2) eye drops, 5 μL Kang Bercept, 3 times/d, administered for 7 days; (3) eye drop administration, 5 μL PenHA-Lip/Conb, 3 times/d, administered for 7 days; (4) intravitreal injection of 1 μl Conbercept. The living status and behavior of mice in each group were closely observed. After 7 days of administration, 3 mice were taken from each group, choroid slices were made and immunofluorescence staining was performed, observed and photographed under a fluorescence microscope, and the CNV area within the same fluorescence intensity range was measured by ImageJ software.

The results are shown in FIG. 7 , the CNV areas of the four groups were (20590±1107) μm ² , (20279±1596) μm ² , (14109±1540) μm ² , and (21676±1065) μm ² , respectively. Compared with the PBS eye drop administration group, the double-modified drug-loaded liposome eye drop administration group and the intravitreal Conbercept group significantly reduced the area of CNV, but the free drug eye drop administration group had no significant difference difference. In addition, there was no significant difference in CNV area between the double-modified drug-loaded liposome eye drop administration group and the intravitreal injection conbercept group.

Example 2

In the research process, our research group investigated the influence of multiple factors on the therapeutic effect of liposomes, including the following CPP and HA ratio screening experiments.

Human corneal epithelial cells HCEC were seeded into the upper chamber of collagen-coated Transwell chamber at a density of 5×10 ⁶ cells per well to construct a simulated in vitro corneal barrier. The retinal pigment epithelial cells ARPE-19 were plated in the lower chamber of Transwell at a density of 2×10 ⁵ cells per well to simulate the retinal environment of the fundus. In order to investigate the efficiency of liposomes modified with different proportions of CPP and HA (the preparation method is the same as that of Example 1) to penetrate the corneal barrier and be taken up by retinal cells, fluorescently labeled drug-loaded liposomes modified with different proportions of CPP and HA were added to the into the upper chamber of the Transwell chamber. After 12 hours in the incubator, the lower chamber was washed with PBS, and the cellular uptake of liposomes was observed using a fluorescence microscope and the mean fluorescence intensity was analyzed semi-quantitatively.

The effect of the ratio of CPP and HA on the mean fluorescence intensity (MFI) uptake by cells was studied, and the number of replicates was 3. Table 1 shows the MFI values of cellular uptake when the CPP ratio is 1%, 5%, 10%, 35%, and 50%, and the HA ratio is 0.1, 1, 5, and 10.

Table 1 Effects of different ratios of CPP and HA on the uptake of MFI by ARPE-19 cells

The results are shown in Table 1. When the mass ratio of HA to total lipids remained unchanged, with the increase of the CPP modification ratio, the higher the fluorescence intensity value of liposomes penetrating the corneal barrier and being taken up by retinal cells, the higher the CPP was. The MFI when the ratio is 10% to 50% is significantly different from the MFI of 1% and 5%, and there is no significant difference between the groups when the CPP ratio is 10%, 35% and 50%. When the modification ratio of CPP remained unchanged, the fluorescence intensity value that penetrated the corneal barrier and was taken up by retinal cells first increased and then decreased as the proportion of HA increased. The better ratio of CPP modification was 10%-50%, and the better ratio of HA modification was 1-5.

Example 3

Comparison of intraocular fluorescence intensity of different carriers.

PenHA-Lip: Penetratin, HA double-modified liposome group; prepared according to the method of Example 1.

PenHA-PAMAM: Penetratin, HA double-modified PAMAM group; the preparation method is as follows: firstly thiolated Penetratin, then take the thiolated Penetratin (16.8 mg) and NHS-PEG-Mal (8.7 mg) and dissolve them in 3 mL of phosphate buffer solution and vortex After spinning for 1 min, it was added dropwise to the phosphate buffer solution of 6 mL of PAMAM (10 mg), and Pen-PAMAM was obtained after the reaction. HA (22 mg), EDC (22 mg), NHS (22 mg) were dissolved in acetate buffer and preactivated at 37°C for 2 h. Activated HA was then added to Pen-PAMAM (35.5 mg), adjusted to a final pH of 8 with borate buffer, and incubated at 37 °C for 12 h with stirring using a magnetic stirrer to couple HA to the liposome surface , forming PenHA-PAMAM.

PenHA-PLGA: Penetratin, HA double-modified PLGA group; the preparation method is: PLGA-PEG-Mal (18.7mg) and thiolated Penetratin (16.8mg) are dissolved in chloroform solution, and triethylamine is added dropwise as a catalyst. Shake lightly. The mixed solution was reacted in the dark at room temperature for 24 h under nitrogen-filled conditions. The organic solvent was filtered, and the obtained filtrate was evaporated by a rotary evaporator to obtain a solid crystal, which was freeze-dried to obtain Pen-PLGA. HA (22 mg), EDC (22 mg), NHS (22 mg) were dissolved in acetate buffer and preactivated at 37°C for 2 h. Activated HA was then added to Pen-PLGA (35.5 mg), adjusted to a final pH of 8 with borate buffer, and incubated at 37 °C for 12 h with stirring using a magnetic stirrer to couple HA to the liposome surface , forming PenHA-PLGA.

PenHA-HSA: Penetratin, HA double-modified HSA group; the preparation method is as follows: firstly, thiolated Penetratin, then take the thiolated Penetratin (16.8 mg) and NHS-PEG-Mal (8.7 mg) and dissolve them in 3 mL of phosphate buffer solution and vortex After spinning for 1 min, it was added dropwise to 6 mL of HSA (10 mg) in phosphate buffer, and Pen-HSA was obtained after the reaction. HA (22 mg), EDC (22 mg), NHS (22 mg) were dissolved in acetate buffer and preactivated at 37°C for 2 h. Activated HA was then added to Pen-HSA (35.5 mg), adjusted to a final pH of 8 with borate buffer, and incubated at 37 °C for 12 h with stirring using a magnetic stirrer to couple HA to the liposome surface , forming PenHA-HSA.

The upper and lower eyelids of the mouse were gently opened with a cotton swab, PenHA-Lip, PenHA-PAMAM, PenHA-PLGA and PenHA-HSA were dropped into the conjunctival sac of the mouse, and the eyelids were slowly closed after waiting for a few seconds. Mice were sacrificed 6 h after administration, and the eyeballs were immediately removed, washed three times with PBS, and the vitreous and retina were taken out, freeze-thawed and homogenized in 110 μL sterile PBS, and the mean fluorescence intensity was measured with a microplate reader.

The results are shown in Figure 8. The average fluorescence intensities of the four groups were (7.5±0.5), (3.1±0.3), (1.8±0.2), and (2.1±0.6), respectively. The average fluorescence intensity of the PenHA-Lip group was PenHA -PAMAM, PenHA-PLGA, PenHA-HAS 2.4 times, 4.2 times, 3.6 times, the data have significant differences, indicating that the ophthalmic liposome of the present invention is more effective than other ophthalmic drugs.

Example 4

Comparison of intraocular fluorescence intensity of liposomes modified with different penetration enhancers.

CDHA-Lip: cyclodextrin, HA double-modified liposome group; preparation method: weigh 20.5 mg of egg yolk lecithin (EPC), 5 mg of cholesterol (CHOL), and 10 mg of DPPE and dissolve them in an eggplant-shaped bottle containing 10 mL of chloroform solution . The organic solvent was removed by evaporation under reduced pressure at 35°C by a rotary evaporator, and a uniform lipid film was formed on the bottle wall. Vacuum drying was continued to remove residual organic solvent. 5ml of the PBS solution with a concentration of 5mg/ml Conbercept (pH 7.4, preparation method: take a certain volume of Conbercept injection with a concentration of 10mg/ml, add PBS to dilute to the desired concentration) into the eggplant type In the bottle, shake quickly to hydrate the solution with the lipid film. After hydration, the lipid film was completely detached from the bottle wall by water bath ultrasound on ice, and stirred for 1 h in the dark. The obtained milky white liquid was passed through 0.45 μm and 0.22 μm microporous filter membranes three times to obtain liposomes with suitable particle size, which were stored at 4° C. in the dark for future use.

HA (22 mg), cyclodextrin CD (16.8 mg), EDC (22 mg), NHS (22 mg) were dissolved in acetate buffer and preactivated at 37°C for 2 h. Activated HA, CD were then added to Lip (total lipid (mass of all lipid materials added) was 35.5 mg), the final pH was adjusted to 8 with borate buffer, and incubated at 37°C for 12 h, The HA and CD were coupled to the liposome surface by stirring with a magnetic stirrer to form CDHA-Lip.

CSHA-Lip: chitosan, HA double-modified liposome group; preparation method: weigh 20.5 mg of egg yolk lecithin (EPC), 5 mg of cholesterol (CHOL), and 10 mg of DPPE and dissolve them in an eggplant-shaped bottle containing 10 mL of chloroform solution . The organic solvent was removed by evaporation under reduced pressure at 35°C by a rotary evaporator, and a uniform lipid film was formed on the bottle wall. Vacuum drying was continued to remove residual organic solvent. 5ml of the PBS solution with a concentration of 5mg/ml Conbercept (pH 7.4, preparation method: take a certain volume of Conbercept injection with a concentration of 10mg/ml, add PBS to dilute to the desired concentration) into the eggplant type In the bottle, shake quickly to hydrate the solution with the lipid film. After hydration, the lipid film was completely detached from the bottle wall by water bath ultrasound on ice, and stirred for 1 h in the dark. The obtained milky white liquid was passed through 0.45 μm and 0.22 μm microporous filter membranes three times to obtain liposomes with suitable particle size, which were stored at 4° C. in the dark for future use.

HA (22 mg), EDC (22 mg), NHS (22 mg) were dissolved in acetate buffer and preactivated at 37°C for 2 h. Activated HA was then added to Lip (total lipid (mass of all lipid material added) was 35.5 mg), the final pH was adjusted to 8 with borate buffer, and incubated at 37 °C for 12 h, using a magnetic force Stirring with a stirrer couples HA to the liposome surface, forming HA-Lip. CS (16.8 mg) was dissolved in acetic acid solution, added dropwise to HA-Lip, and sonicated for 5 min to form CSHA-Lip.

BAHA-Lip: bile acid and HA double-modified liposome group; the preparation method is as follows: weigh 10 mg of egg yolk lecithin (EPC), 16.8 mg of bile acid (BA), 3.7 mg of cholesterol (CHOL), and 5 mg of DPPE, and dissolve them in 10 mL of chloroform. solution in an eggplant-shaped bottle. The organic solvent was removed by evaporation under reduced pressure at 35°C by a rotary evaporator, and a uniform lipid film was formed on the bottle wall. Vacuum drying was continued to remove residual organic solvent. 5ml of the PBS solution with a concentration of 5mg/ml Conbercept (pH 7.4, preparation method: take a certain volume of Conbercept injection with a concentration of 10mg/ml, add PBS to dilute to the desired concentration) into the eggplant type In the bottle, shake quickly to hydrate the solution with the lipid film. After hydration, the lipid film was completely detached from the bottle wall by water bath ultrasound on ice, and stirred for 1 h in the dark. The obtained milky white liquid was passed through 0.45 μm and 0.22 μm microporous filter membranes three times to obtain liposomes with suitable particle size, which were stored at 4° C. in the dark for future use.

HA (22 mg), EDC (22 mg), NHS (22 mg) were dissolved in acetate buffer and preactivated at 37°C for 2 h. Activated HA was then added to BA-Lip (total lipid (mass of all lipid material added) was 35.5 mg), the final pH was adjusted to 8 with borate buffer, and incubated at 37 °C for 12 h, Stirring with a magnetic stirrer coupled HA to the liposome surface to form BAHA-Lip.

The upper and lower eyelids of the mouse were gently opened with a cotton swab, and Penetratin, HA double-modified liposome and chitosan were dropped into the conjunctival sac of the mouse, and the eyelids were slowly closed after waiting for a few seconds. Mice were sacrificed 6 h after administration, and the eyeballs were immediately removed, washed three times with PBS, and the vitreous and retina were taken out, freeze-thawed and homogenized in 110 μL sterile PBS, and the mean fluorescence intensity was measured with a microplate reader.

The results are shown in Figure 9. The average fluorescence intensities of the four groups were (8.6 ± 0.4), (2.2 ± 0.3), (3.8 ± 0.3), and (3.7 ± 0.5), respectively, and the average fluorescence intensities of the PenHA-Lip group were CDHA -Lip, CSHA-Lip, BAHA-Lip 3.9 times, 2.3 times, 2.3 times, the data have significant differences, indicating that the ophthalmic liposome of the present invention is better than other ophthalmic drugs.

Example 5

Comparison of retinal fluorescence intensity of liposomes modified with different targeting ligands.

PenRGD-Lip: Penetratin, RGD double-modified liposome group; the preparation method is as follows: Penetratin is thiolated, DSPE-PEG-Mal (11.6 mg) and thiolated Penetratin (16.8 mg) are dissolved in chloroform solution, dropwise added Triethylamine was used as a catalyst with gentle shaking. The mixed solution was reacted in the dark at room temperature for 24 h under nitrogen-filled conditions. The organic solvent was filtered, and the obtained filtrate was evaporated by a rotary evaporator to obtain a solid crystal, which was freeze-dried to obtain DSPE-PEG-Pen.

RGD was thiolated, DSPE-PEG-Mal (11.6 mg) and thiolated RGD (22 mg) were dissolved in chloroform solution, triethylamine was added dropwise as a catalyst, and shaken gently. The mixed solution was reacted in the dark at room temperature for 24 h under nitrogen-filled conditions. The organic solvent was filtered, and the obtained filtrate was evaporated by a rotary evaporator to obtain a solid crystal, which was freeze-dried to obtain DSPE-PEG-RGD.

10 mg of egg yolk lecithin (EPC), 2.5 mg of cholesterol (CHOL), DSPE-PEG-Pen (17 mg), and DSPE-PEG-RGD (22 mg) were weighed and dissolved in an eggplant-shaped bottle containing 10 mL of chloroform solution. The organic solvent was removed by evaporation under reduced pressure at 35°C by a rotary evaporator, and a uniform lipid film was formed on the bottle wall. Vacuum drying was continued to remove residual organic solvent. 5ml of the PBS solution with a concentration of 5mg/ml Conbercept (pH 7.4, preparation method: take a certain volume of Conbercept injection with a concentration of 10mg/ml, add PBS to dilute to the desired concentration) into the eggplant type In the bottle, shake quickly to hydrate the solution with the lipid film. After hydration, the lipid film was completely detached from the bottle wall by water bath ultrasound on ice, and stirred for 1 h in the dark. The obtained milky white liquid was passed through 0.45 μm and 0.22 μm microporous membranes three times to obtain PenRGD-Lip liposomes with suitable particle size, which were stored at 4° C. in the dark for future use.

PenYSA-Lip: Penetratin, YSA double-modified liposome group; the preparation method is as follows: Penetratin is thiolated, DSPE-PEG-Mal (11.6 mg) and thiolated Penetratin (16.8 mg) are dissolved in chloroform solution, dropwise added Triethylamine was used as a catalyst with gentle shaking. The mixed solution was reacted in the dark at room temperature for 24 h under nitrogen-filled conditions. The organic solvent was filtered, and the obtained filtrate was evaporated by a rotary evaporator to obtain a solid crystal, which was freeze-dried to obtain DSPE-PEG-Pen.

YSA was thiolated, DSPE-PEG-Mal (11.6 mg) and thiolated YSA (22 mg) were dissolved in chloroform solution, triethylamine was added dropwise as a catalyst, and the mixture was shaken gently. The mixed solution was reacted in the dark at room temperature for 24 h under nitrogen-filled conditions. The organic solvent was filtered, and the obtained filtrate was evaporated by a rotary evaporator to obtain a solid crystal, which was freeze-dried to obtain DSPE-PEG-YSA.

10 mg of egg yolk lecithin (EPC), 2.5 mg of cholesterol (CHOL), DSPE-PEG-Pen (17 mg), and DSPE-PEG-YSA (22 mg) were weighed and dissolved in an eggplant-shaped bottle containing 10 mL of chloroform solution. The organic solvent was removed by evaporation under reduced pressure at 35°C by a rotary evaporator, and a uniform lipid film was formed on the bottle wall. Vacuum drying was continued to remove residual organic solvent. 5ml of the PBS solution with a concentration of 5mg/ml Conbercept (pH 7.4, preparation method: take a certain volume of Conbercept injection with a concentration of 10mg/ml, add PBS to dilute to the desired concentration) into the eggplant type In the bottle, shake quickly to hydrate the solution with the lipid film. After hydration, the lipid film was completely detached from the bottle wall by water bath ultrasound on ice, and stirred for 1 h in the dark. The obtained milky white liquid was passed through 0.45 μm and 0.22 μm microporous membranes three times to obtain PenYSA-Lip liposomes with suitable particle size, which were stored at 4° C. in the dark for future use.

PenFA-Lip: Penetratin, FA double-modified liposome group; the preparation method is as follows: Penetratin is thiolated, DSPE-PEG-Mal (11.6 mg) and thiolated Penetratin (16.8 mg) are dissolved in chloroform solution, dropwise added Triethylamine was used as a catalyst with gentle shaking. The mixed solution was reacted in the dark at room temperature for 24 h under nitrogen-filled conditions. The organic solvent was filtered, and the obtained filtrate was evaporated by a rotary evaporator to obtain a solid crystal, which was freeze-dried to obtain DSPE-PEG-Pen.

FA (22 mg), EDC (22 mg), NHS (22 mg) were dissolved in acetate buffer and preactivated at 37°C for 2 h. The activated FA was reacted with NH2-PEG-COOH (15mg) and DSPE (30mg) for 48h, and DSPE-PEG-FA was obtained after lyophilization.

10 mg of egg yolk lecithin (EPC), 2.5 mg of cholesterol (CHOL), DSPE-PEG-Pen (17 mg), and DSPE-PEG-FA (22 mg) were weighed and dissolved in an eggplant-shaped bottle containing 10 mL of chloroform solution. The organic solvent was removed by evaporation under reduced pressure at 35°C by a rotary evaporator, and a uniform lipid film was formed on the bottle wall. Vacuum drying was continued to remove residual organic solvent. 5ml of the PBS solution with a concentration of 5mg/ml Conbercept (pH 7.4, preparation method: take a certain volume of Conbercept injection with a concentration of 10mg/ml, add PBS to dilute to the desired concentration) into the eggplant type In the bottle, shake quickly to hydrate the solution with the lipid film. After hydration, the lipid film was completely detached from the bottle wall by water bath ultrasound on ice, and stirred for 1 h in the dark. The obtained milky white liquid was passed through 0.45 μm and 0.22 μm microporous filter membranes three times to obtain PenFA-Lip liposomes with suitable particle size, which were stored at 4° C. in the dark for future use.

The upper and lower eyelids of the mouse were gently opened with a cotton swab, PenHA-Lip, PenRGD-Lip, PenYSA-Lip, PenFA-Lip were dropped into the conjunctival sac of the mouse, and the eyelids were slowly closed after waiting for a few seconds. Mice were sacrificed 6 h after administration, the eyeballs were immediately removed, rinsed three times with PBS, the retinas were taken out, freeze-thawed and homogenized in 110 μL sterile PBS, and the mean fluorescence intensity was measured with a microplate reader.

The results are shown in Figure 10. The average fluorescence intensities of the four groups were (5.5 ± 0.3), (2.6 ± 0.2), (2.8 ± 0.3), and (3.4 ± 0.3), respectively, and the average fluorescence intensities of the PenHA-Lip group were PenRGD -Lip, PenYSA-Lip, PenFA-Lip 2.1 times, 2.0 times, 1.6 times, the data have significant differences, indicating that the ophthalmic liposome of the present invention is better than other ophthalmic drugs.

Example 6

Ophthalmic liposomes were prepared according to the method of Example 1, except that the encapsulated drug was bevacizumab.

Human corneal epithelial cells HCEC were seeded into the upper chamber of collagen-coated Transwell chambers at a density of 5×10 ⁶ cells per well to construct a simulated in vitro corneal barrier. The retinal pigment epithelial cells ARPE-19 were plated in the lower chamber of Transwell at a density of 2×10 ⁵ cells per well to simulate the retinal environment of the fundus. To investigate the efficiency of liposomes penetrating the corneal barrier and being taken up by retinal cells, fluorescently labeled drug-loaded liposomes were added to the upper chamber of the Transwell chamber, respectively. After 12 hours in the incubator, the lower chamber was washed with PBS, and the cellular uptake of liposomes was observed using a fluorescence microscope and the mean fluorescence intensity was analyzed semi-quantitatively.

The results are shown in Figure 11. The average fluorescence intensities of the three groups were (33.7±13.3), (188.3±39.2), and (287±22.6), respectively. The average fluorescence intensity of the PenHA-Lip group was 8.5 of that of Lip and Pen-Lip, respectively. Times, 1.5 times, the data has a significant difference.

Example 7

Ophthalmic liposomes were prepared according to the method of Example 1, except that the cell penetrating peptide CPP was octamer arginine R8.

The results are shown in Figure 12. The average fluorescence intensities of the three groups were (28.3±7.1), (144±15), and (203.7±24.79), respectively. The average fluorescence intensity of the R8HA-Lip group was 7.2 of that of Lip and R8-Lip, respectively. Times, 1.4 times, the data has a significant difference.

Example 8

Ophthalmic liposomes were prepared according to the method of Example 1, except that the lipid materials in step (2) were soybean lecithin (SPC), cholesterol (CHOL), dimyristoylphosphatidylethanolamine (DMPE), DPPE -PEG, DPPE-PEG-CPP.

Human corneal epithelial cells HCEC were seeded into the upper chamber of collagen-coated Transwell chamber at a density of 5×10 ⁶ cells per well to construct a simulated in vitro corneal barrier. The retinal pigment epithelial cells ARPE-19 were plated in the lower chamber of Transwell at a density of 2×10 ⁵ cells per well to simulate the retinal environment of the fundus. To investigate the efficiency of liposomes penetrating the corneal barrier and being taken up by retinal cells, fluorescently labeled drug-loaded liposomes were added to the upper chamber of the Transwell chamber, respectively. After 12 hours in the incubator, the lower chamber was washed with PBS, and the cellular uptake of liposomes was observed using a fluorescence microscope and the mean fluorescence intensity was analyzed semi-quantitatively.

The results are shown in Figure 13. The average fluorescence intensities of the three groups were (43.7±6.1), (187.3±24.0), and (312.3±21.5), respectively. The average fluorescence intensity of the PenHA-Lip group was 7.1 of that of Lip and Pen-Lip, respectively. Times, 1.7 times, the data has a significant difference.

Example 9

The ophthalmic liposomes were prepared according to the method of Example 1, except that the encapsulated drug was ranibizumab.

Example 10

Ophthalmic liposomes were prepared according to the method of Example 1, except that the encapsulated drug was aflibercept.

Example 11

Ophthalmic liposomes were prepared according to the method of Example 1, except that the encapsulated drugs were conbercept and aflibercept in a mass ratio of 1:1.

Example 12

Ophthalmic liposomes were prepared according to the method of Example 1, except that the cell penetrating peptide CPP was the human immunodeficiency virus protein transduction peptide TAT.

Example 13

Ophthalmic liposomes were prepared according to the method of Example 1, except that the cell penetrating peptide CPP was low molecular weight protamine LMWP.

Example 14

Ophthalmic liposomes were prepared according to the method of Example 1, except that the lipid materials in step (2) were hydrogenated soybean phospholipid (HSPC), cholesterol (CHOL), dipalmitoyl phosphatidylethanolamine (DPPE), DOPE- PEG, DOPE-PEG-CPP.

Example 15

The ophthalmic liposomes were prepared according to the method of Example 1, except that the lipid material in step (2) was different in that the lipid material in step (2) was dipalmitoyl phosphatidylcholine (DPPC), Cholesterol (CHOL), Dioleoylphosphatidylethanolamine (DOPE), DMPE-PEG, DMPE-PEG-CPP.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the method of the present invention, several improvements and supplements can also be made, and these improvements and supplements should also be regarded as It is the protection scope of the present invention.

Claims

An ophthalmic liposome co-modified with a cell-penetrating peptide and hyaluronic acid that can penetrate the cornea and target the retina, is characterized in that it is an encapsulated lipid material composed of a good biocompatibility. Anti-VEGF drugs and liposomes with cell penetrating peptides and hyaluronic acid attached to the surface.
The ophthalmic liposome according to claim 1, wherein the lipid material with good biocompatibility is selected from egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, dipalmitoyl phosphatidylcholine, Cholesterol, dimyristoyl phosphatidyl ethanolamine or its pegylated derivatives, distearoyl phosphatidyl ethanolamine or its pegylated derivatives, dipalmitoyl phosphatidyl ethanolamine or its pegylated derivatives, di- One or more of oleoylphosphatidylethanolamine or its PEGylated derivatives.
The ophthalmic liposome according to claim 1, wherein the cell penetrating peptide is selected from the group consisting of hexadeceptide Penetratin derived from the homology domain of Drosophila antennae gene, human immunodeficiency virus protein transduction peptide TAT , octameric arginine R8, low molecular weight protamine LMWP; and/or the anti-VEGF drug is selected from one or more of bevacizumab, ranibizumab, conbercept, and aflibercept kind.
The liposome according to claim 1, wherein the mass ratio of the hyaluronic acid to the total lipid is 0.1-10; and/or the molar ratio of the cell penetrating peptide to the total lipid is 1 %～50%.
The liposome according to claim 1, wherein the molecular weight of the hyaluronic acid is 6.4kDa-1500kDa.
The preparation method of the arbitrary described ophthalmic liposome of claim 1-5, is characterized in that, comprises the following steps:

(1) using an addition reaction to connect the phospholipid containing the Mal group to the cell penetrating peptide;

(2) The cell-penetrating peptide-modified liposomes encapsulating anti-VEGF drugs were prepared by thin film dispersion method;

(3) The hyaluronic acid was linked to the cell-penetrating peptide-modified liposomes loaded with anti-VEGF drugs by chemical coupling method.
The preparation method according to claim 6, wherein the specific operation of the addition reaction is: dissolving DSPE-PEG-Mal and the thiol-modified cell penetrating peptide in a chloroform solution, and reacting in the dark under nitrogen-filled conditions 24h, evaporated to dryness under reduced pressure to obtain DSPE-PEG-CPP.
The preparation method according to claim 6, characterized in that, the specific operation of the film dispersion method is to dissolve EPC, CHOL, DPPE, DSPE-PEG, DSPE-PEG-CPP in a chloroform solution, reduce The lipid film was obtained by pressure evaporation, and liposomes were obtained by hydration with PBS solution containing anti-VEGF drug.
The preparation method according to claim 6, wherein the specific operation of the chemical coupling method is to dissolve HA, EDC and NHS in an acetate buffer, incubate at 37°C for 2 hours for pre-activation, and the HA carboxyl group After activation, it is added to the liposome obtained in step (2), and chemically coupled with the amino group on the surface of DPPE to obtain a liposome encapsulated with an anti-VEGF drug and connected with a cell-penetrating peptide and hyaluronic acid on the surface.
Application of the ophthalmic liposome according to any one of claims 1 to 5 in the preparation of a medicine for treating fundus neovascularization diseases.