Ophthalmic liposome capable of penetrating cornea and targeting retina as well as preparation method and application of ophthalmic liposome

Abstract

The invention relates to an ophthalmic liposome capable of penetrating a cornea and targeting a retina as well as a preparation method and application of the ophthalmic liposome. The ophthalmic liposome is formed by a liposome, composed of a lipid material with good biocompatibility, entrapping an anti-VEGF drug, and the surface of which is connected with cell penetrating peptide (CPP) and hyaluronic acid (HA). The anti-VEGF drug is loaded inside the liposome, so that the defect of poor stability of the anti-VEGF drug is effectively improved; meanwhile, the amphipathy of the liposome is utilized to enhance the cornea penetrating effect; then the CPP with the penetrating effect and the HA targeting the retina are covalently connected to the surface; and it is found that the prepared liposome can obviously improve the corneal penetrability of the drug and the aggregation to the fundus part in an eye drop administration mode, so that the drug concentration of a lesion part is increased, and the treatment effect is obviously improved.

Description

technical field

[0001] The invention belongs to the technical field of medicine, and in particular relates to an ophthalmic liposome co-modified with a cell penetrating peptide CPP and hyaluronic acid HA capable of penetrating the cornea and targeting the retina, and a preparation method and application thereof. Background technique

[0002] The eye is an extremely complex and precise organ, 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 (Diabetic retinopathy, DR) and age-related macular degeneration (Age-related macular degeneration, AMD), It is a serious blinding eye disease. Fundus neovascular diseases include chor...

Examples

Embodiment 1

[0047] 1. Preparation of liposomes

[0048] (1) Link DSPE-PEG-Mal with cell penetrating peptide CPP by addition reaction.

[0049] The cell penetrating peptide CPP was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd., and CPP was thiolated. CPP is specifically

[0050] Penetratin: RQIKIWFQNRRMKWKK (SEQ ID NO: 1), wherein C is an amino acid-cysteine ​​C added during the sequence synthesis, and this amino acid contains a sulfhydryl group, that is, the sulfhydrylation of CPP is completed.

[0051] 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 the 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 shaken gently. The mixed solution was reacted at room temperature and protected from light for 24 h under the condition of nitrogen filling. The org...

Embodiment 2

[0076] During 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.

[0077] Human corneal epithelial cells HCEC were divided into 5 × 10 per well 6 The density of cells was seeded into the upper chamber of the collagen-coated Transwell chamber to construct a simulated in vitro corneal barrier. Retinal pigment epithelial cells ARPE-19 per well 2 × 10 5 The density of cells was plated in the lower chamber of the Transwell to simulate the fundus retinal environment. In order to investigate the efficiency of liposomes modified with different ratios of CPP and HA (the preparation method is the same as in Example 1) to penetrate the corneal barrier and be taken up by retinal cells, the fluorescently labeled drug-loaded liposomes modified with different ratios of CPP and HA were added to Go to the upper chamber of the Transwell chamber. After b...

Embodiment 3

[0083] Comparison of intraocular fluorescence intensity of different vectors.

[0084] PenHA-Lip: Penetratin, HA double modified liposome set; prepared according to the method of Example 1.

[0085] PenHA-PAMAM: Penetratin, HA double-modified PAMAM group; the preparation method is: first thiolate Penetratin, then take the thiolated Penetratin (16.8mg) and NHS-PEG-Mal (8.7mg) and dissolve them in 3mL of phosphate buffer and vortex After spinning for 1 min, it was added dropwise to 6 mL of PAMAM (10 mg) in phosphate buffer, and Pen-PAMAM was obtained after reaction. HA (22mg), EDC (22mg), and NHS (22mg) were dissolved in acetate buffer, and preactivated at 37°C for 2h. The activated HA was then added to Pen-PAMAM (35.5mg), adjusted to a final pH of 8 with borate buffer, and incubated at 37°C for 12h, stirring with a magnetic stirrer to couple HA to the liposome surface , forming PenHA-PAMAM.

[0086] PenHA-PLGA: Penetratin, HA double-modified PLGA group; the preparation metho...

technical field

[0001] The invention belongs to the technical field of medicine, and in particular relates to an ophthalmic liposome co-modified with a cell penetrating peptide CPP and hyaluronic acid HA capable of penetrating the cornea and targeting the retina, and a preparation method and application thereof. Background technique

[0002] The eye is an extremely complex and precise organ, 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 (Diabetic retinopathy, DR) and age-related macular degeneration (Age-related macular degeneration, AMD), It is a serious blinding eye disease. Fundus neovascular diseases include chor...

Examples

Embodiment 1

[0047] 1. Preparation of liposomes

[0048] (1) Link DSPE-PEG-Mal with cell penetrating peptide CPP by addition reaction.

[0049] The cell penetrating peptide CPP was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd., and CPP was thiolated. CPP is specifically

[0050] Penetratin: RQIKIWFQNRRMKWKK (SEQ ID NO: 1), wherein C is an amino acid-cysteine ​​C added during the sequence synthesis, and this amino acid contains a sulfhydryl group, that is, the sulfhydrylation of CPP is completed.

[0051] 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 the 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 shaken gently. The mixed solution was reacted at room temperature and protected from light for 24 h under the condition of nitrogen filling. The org...

Embodiment 2

[0076] During 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.

[0077] Human corneal epithelial cells HCEC were divided into 5 × 10 per well 6 The density of cells was seeded into the upper chamber of the collagen-coated Transwell chamber to construct a simulated in vitro corneal barrier. Retinal pigment epithelial cells ARPE-19 per well 2 × 10 5 The density of cells was plated in the lower chamber of the Transwell to simulate the fundus retinal environment. In order to investigate the efficiency of liposomes modified with different ratios of CPP and HA (the preparation method is the same as in Example 1) to penetrate the corneal barrier and be taken up by retinal cells, the fluorescently labeled drug-loaded liposomes modified with different ratios of CPP and HA were added to Go to the upper chamber of the Transwell chamber. After b...

Embodiment 3

[0083] Comparison of intraocular fluorescence intensity of different vectors.

[0084] PenHA-Lip: Penetratin, HA double modified liposome set; prepared according to the method of Example 1.

[0085] PenHA-PAMAM: Penetratin, HA double-modified PAMAM group; the preparation method is: first thiolate Penetratin, then take the thiolated Penetratin (16.8mg) and NHS-PEG-Mal (8.7mg) and dissolve them in 3mL of phosphate buffer and vortex After spinning for 1 min, it was added dropwise to 6 mL of PAMAM (10 mg) in phosphate buffer, and Pen-PAMAM was obtained after reaction. HA (22mg), EDC (22mg), and NHS (22mg) were dissolved in acetate buffer, and preactivated at 37°C for 2h. The activated HA was then added to Pen-PAMAM (35.5mg), adjusted to a final pH of 8 with borate buffer, and incubated at 37°C for 12h, stirring with a magnetic stirrer to couple HA to the liposome surface , forming PenHA-PAMAM.

[0086] PenHA-PLGA: Penetratin, HA double-modified PLGA group; the preparation metho...

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