Self-assembling polypeptides for modulating retinal pigment epithelium permeability and methods of making and using the same

Abstract

This invention relates to a self-assembling polypeptide for regulating the permeability of retinal pigment epithelium (RPE), its preparation method, and its applications. The self-assembling polypeptide comprises an integrin receptor-targeting peptide, a self-assembling polypeptide, and a hydrophobic molecule sequentially linked. The self-assembling polypeptide designed in this invention can self-assemble into nanoparticles and specifically target RPE cells. After interacting with receptors on the cell membrane, the nanoparticles transform into nanofibers, achieving rapid targeted enrichment of RPE tissue. Furthermore, by regulating the permeability of RPE tissue, it can enhance the transcellular drug transport capacity, providing a new clinical application strategy for the treatment of macular neovascularization, especially polypoid choroidal vascular lesions.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a self-assembled polypeptide for regulating the permeability of retinal pigment epithelium, its preparation method and application. Background Technology

[0002] Macular neovascularization (MNV), caused by a variety of factors including age, genetics, and environment, carries a high risk of blindness. Polypoid choroidal vascular lesion (PCV), a typical variant of type I MNV, primarily occurs beneath the retinal pigment epithelium (RPE). The abnormal branching vascular network in the focal area leads to choroidal thickening accompanied by dilated and highly permeable choroidal neovascularization and lipid exudate accumulation, resulting in retinal pigment epithelium detachment and significant vision loss or even complete visual impairment.

[0003] The incidence of retinal neovascularization (PCV) is relatively high in Asian populations. Currently, the primary first-line treatment in clinical practice involves intravitreal injection of anti-vascular endothelial growth factor (VEGF) to inhibit the proliferation and migration of vascular endothelial cells, thereby reducing the growth of abnormal choroidal vascular lesions and the risk of leakage. However, compared to patients with neovascularization of the fundus without PCV, PCV patients respond poorly to anti-VEGF drugs. The neatly arranged and tightly connected retinopathy layer (RPE) in the fundus plays a major role in the blood-retinal outer barrier. The RPE strictly regulates the exchange of substances and restricts the transport of macromolecules to maintain the basic function of the outer retina. While the barrier function of the RPE maintains the normal physiological balance of the retina and choroid, it also hinders the entry of monoclonal antibody drugs into the lesions. Therefore, taking measures to improve drug permeability to the RPE to enhance efficacy and salvage vision before severe visual loss is achieved is extremely important.

[0004] Self-assembled peptides, as molecules with biological homology, can spontaneously form ordered and stable structures and exert specific biological functions under non-covalent interactions, and have become an innovative therapeutic approach to improve the treatment effects of various eye diseases. Due to their superior biological and chemical properties, peptides can be used to generate a variety of peptides with different structures and functions through targeted molecular design and synthesis methods. Therefore, it would be of great significance to develop a peptide material that can reversibly regulate the drug permeability of refractive polyethylene (RPE). Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a self-assembling polypeptide for regulating the permeability of retinal pigment epithelium, its preparation method, and its application.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a self-assembling polypeptide for regulating the permeability of retinal pigment epithelium, the self-assembling polypeptide comprising an integrin receptor-targeting peptide, a self-assembling polypeptide, and a hydrophobic molecule connected in sequence.

[0008] Patients with polypoid choroidal vascular disease (PCV) respond poorly to existing first-line drugs (such as VEGF monoclonal antibodies). The well-organized and tightly connected retinal pigment epithelium (RPE) in the fundus tissue is a major obstacle to the delivery of hydrophilic protein drugs and macromolecules, meaning that addressing the drug permeability problem of RPE holds promise for further improving the clinical efficacy of PCV treatment. The self-assembling peptide designed in this invention self-assembles into nanoparticles through hydrophilic-hydrophobic interactions. These nanoparticles can specifically target retinal pigment epithelium cells. After interacting with receptors on the cell membrane, they transform from nanoparticles into nanofibers, achieving rapid targeted enrichment of RPE. Furthermore, by regulating the permeability of RPE, it can enhance the transcellular transport capacity of drugs, providing a new clinical application strategy for the treatment of PCV and other pathological macular neovascularization.

[0009] Preferably, the amino acid sequence of the integrin receptor targeting peptide is HSDVHK or GGRGDS.

[0010] When the integrin receptor targeting peptide is HSDVHK, the self-assembled peptide can enhance the paracellular permeation pathway of drugs by mediating the rearrangement of cytoskeleton actin initiated by αvβ3 on the RPE, thereby promoting the disintegration of intercellular adhesion junctions. When the integrin receptor targeting peptide is GGRGDS, the self-assembled peptide can mediate the upregulation of the cytoskeleton by αvβ3 and αvβ5 and activate multiple signaling networks, mainly the sphingolipid signaling pathway, thereby promoting transcellular drug transport by promoting cell transocytosis.

[0011] Preferably, the amino acid sequence of the self-assembled polypeptide is KLVFF.

[0012] Preferably, the hydrophobic molecule is a C12-C18 saturated fatty acid molecule (e.g., C12 saturated fatty acid molecule, C13 saturated fatty acid molecule, C14 saturated fatty acid molecule, C15 saturated fatty acid molecule, C16 saturated fatty acid molecule, C17 saturated fatty acid molecule, C18 saturated fatty acid molecule).

[0013] Preferably, the integrin receptor targeting peptide is linked to the C-terminus of the self-assembled polypeptide via an amide bond, and the hydrophobic molecule is linked to the N-terminus of the self-assembled polypeptide via an amide bond.

[0014] In a second aspect, the present invention provides a method for preparing a self-assembled polypeptide according to the first aspect, the method comprising:

[0015] Using resin as a carrier, and integrin receptor-targeting peptides, amino acids constituting self-assembled peptides, and hydrophobic molecules as raw materials, the self-assembled peptides were prepared by sequentially linking amino acids and hydrophobic molecules from the C-terminus to the N-terminus using a solid-phase synthesis method.

[0016] Thirdly, the present invention provides the use of the self-assembled polypeptide according to the first aspect in the preparation of products that improve the drug permeability of the retinal pigment epithelial cell layer.

[0017] Fourthly, the present invention provides a combination pharmaceutical composition comprising the self-assembled polypeptide and the drug for treating macular neovascularization as described in the first aspect.

[0018] The combined pharmaceutical composition involved in this invention creatively combines the aforementioned self-assembled peptides with a drug for treating macular neovascularization, wherein the self-assembled peptides can significantly enhance the permeability of the drug in retinal pigment epithelium, thereby improving the bioavailability and efficacy of the drug.

[0019] Preferably, the combined pharmaceutical composition further contains pharmaceutically acceptable excipients.

[0020] The combined drug composition of the present invention can be administered alone or in combination with excipients to form an appropriate dosage form for administration.

[0021] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, antibacterial agents, or buffers.

[0022] Preferably, the combined pharmaceutical composition is a single compound preparation or a combination of two separate preparations.

[0023] Preferably, the formulation is any pharmaceutically acceptable dosage form.

[0024] Fifthly, the present invention provides the use of the self-assembled polypeptide according to the first aspect or the combined pharmaceutical composition according to the fourth aspect in the preparation of a medicament for the prevention or treatment of polypoid choroidal vascular disease.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The self-assembling peptides designed in this invention self-assemble into nanoparticles under hydrophilic-hydrophobic interaction, which can specifically target retinal pigment epithelial cells. After interacting with receptors on the cell membrane, the nanoparticles transform into nanofibers, achieving rapid targeted enrichment of retinal pigment epithelial tissue (RPE). Furthermore, by regulating the permeability of retinal pigment epithelial tissue, the transcellular drug transport capacity can be enhanced, providing a new clinical application strategy for the treatment of PCV and other pathological macular neovascularization. Attached Figure Description

[0027] Figure 1 This is a mass spectrometry characterization of the self-assembled polypeptide obtained in Example 1;

[0028] Figure 2 This is a liquid chromatography characterization chromatogram of the self-assembled polypeptide obtained in Example 1;

[0029] Figure 3 This is a mass spectrometry characterization of the self-assembled polypeptide obtained in Example 2;

[0030] Figure 4 This is a liquid chromatography characterization chromatogram of the self-assembled polypeptide obtained in Example 2;

[0031] Figure 5 This is a schematic diagram of the self-assembly and morphology transformation process of the self-assembly peptide of the present invention;

[0032] Figure 6A The self-assembled polypeptide CK has / does not have Ca at 0h and 4h. 2+ Induced CD chart;

[0033] Figure 6B The self-assembled polypeptide CS is present / absent with Ca at 0h and 4h. 2+ Induced CD chart;

[0034] Figure 6C The self-assembled peptides CK and CS were present / absent with Ca at 0h and 4h. 2+ Graph showing the percentage composition of secondary structures under induced catalysis;

[0035] Figure 6D The self-assembled peptides CK and CS were in 0h and 2h Ca 2+ Transmission electron microscopy image under induced conditions;

[0036] Figure 7 This is a scanning electron microscope image of self-assembled peptides binding to ARPE-19 cells 4 h later.

[0037] Figure 8 This is a graph showing the results of an in vitro RPE barrier transepithelial resistance assay for self-assembled peptides.

[0038] Figure 9This is a fluorescence confocal microscope image of the self-assembled peptide interacting with ARPE-19 cells;

[0039] Figure 10A This is an immunofluorescence staining image of tight junction protein 1 after the self-assembled peptide interacts with ARPE-19;

[0040] Figure 10B This is an immunofluorescence staining image of actin after the self-assembled peptide interacts with ARPE-19;

[0041] Figure 10C yes Figure 10A The fluorescence intensity quantification results of each stained image;

[0042] Figure 10D yes Figure 10B The fluorescence intensity quantification results of each stained image;

[0043] Figure 11 This is a graph showing the effect of self-assembled peptides on drug penetration.

[0044] Figure 12 This is a biological transmission electron microscope image of the reaction between self-assembled peptides and iPSC-RPE.

[0045] Figure 13 This is a stained section image of a tissue section after a self-assembled polypeptide was injected into the vitreous cavity. Detailed Implementation

[0046] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0047] Example 1

[0048] This embodiment provides a self-assembly polypeptide (abbreviated as CK in this invention), the chemical structure of which is shown below:

[0049]

[0050] Its preparation method is as follows:

[0051] (1) First, weigh 300 mg of Fmoc-Lys-Wang Resin into a polypeptide synthesis tube, add 10 mL of anhydrous DMF, and place the tube on a shaker for 6 h to swell the resin.

[0052] (2) After swelling the resin, remove the DMF solution. First, add DMF solvent to rinse the resin and remove it, then add DCM solvent to rinse and remove it. Use DMF / DCM to rinse three times alternately, then add deprotecting agent (DMF solution containing 2% DBU and 5% anhydrous piperazine by volume) and act for 20 min to remove the protecting group Fmoc so that the amino group at the reaction end is exposed.

[0053] (3) After the deprotection step is completed, remove the deprotecting agent and rinse the resin three times alternately with DMF / DCM solvent. Add a small amount of resin particles to the ninhydrin detection reagent and heat in a boiling water bath for 30 seconds. If the resin particles turn blue-purple, the deprotection step is complete. If the color is still transparent white, the deprotection has failed and step (2) needs to be repeated.

[0054] (4) After confirming successful deprotection, weigh an amount of the next amino acid containing the Fmoc protecting group and HBTU catalyst powder, which is 10 times the amount of amino acid relative to the resin loading, into a 15 mL centrifuge tube. Add 10 mL of coupling agent (DMF solution containing 5% N-methylmorpholine by volume) for pre-activation for 15 min. Then add the peptide synthesis tube and shake on a shaker for 45 min.

[0055] (5) After the coupling step is completed, remove the coupling agent and rinse the resin three times with DMF / DCM solvent alternately. Add a small amount of resin particles to the ninhydrin detection reagent and heat in a boiling water bath for 30 seconds. If the resin particles do not change color, the coupling is successful. If the resin particles change color, the coupling is incomplete and step (4) needs to be repeated.

[0056] (6) After all amino acids in the polypeptide sequence have been successfully coupled and deprotected, add palmitic acid (C16) containing the Fmoc protecting group at a concentration 10 times the amount of amino acids relative to the resin loading and react overnight on a shaker. After confirming successful coupling again, remove the Fmoc protecting group using a deprotecting agent, and then rinse three times alternately with DMF / DCM solvent. After confirming successful deprotection, add 10 mL of methanol solution to the polypeptide synthesis tube and soak for 15 min. Then remove the methanol and transfer the resin to a clean serum bottle.

[0057] (7) Add 5 mL of the prepared lysis buffer (containing 2.5% TIS, 2.5% ultrapure water, and 95% TFA by volume) to a serum bottle containing resin, stir in an ice bath for 3 h, then filter to remove the resin and collect the lysis buffer. Concentrate the lysis buffer to a viscous state using nitrogen, add pre-cooled ice-cold ether to precipitate the peptide solid, transfer the suspension to a centrifuge tube and centrifuge at 8000 r / min for 3 min to collect the precipitate, add fresh ice-cold ether and repeat the washing and centrifugation twice. Place the collected precipitate in a vacuum drying oven at 37℃ to dry, obtaining a white solid powder, and finally purify it using preparative liquid chromatography.

[0058] The purified self-assembled peptides were characterized by mass spectrometry. The mass spectrometry characterization results are as follows: Figure 1 As shown, by Figure 1 As can be seen from the main peak of the mass spectrum, the molecular weight of the peptide material is basically consistent with that of the design. This indicates that the target molecule has been synthesized, demonstrating that the self-assembled material with the above structure has been successfully synthesized.

[0059] The purified product was analyzed by high-performance liquid chromatography (HPLC), and the chromatogram is shown below. Figure 2 As shown, by Figure 2 It can be seen that the purified polypeptide material has a molecular purity of 96.5%.

[0060] Example 2

[0061] This embodiment provides a self-assembling polypeptide (abbreviated as CS in this invention), the chemical structure of which is shown below:

[0062]

[0063] Its preparation method is as follows:

[0064] (1) First, weigh 300 mg of Fmoc-Ser-Wang Resin into a polypeptide synthesis tube, add 10 mL of anhydrous DMF, and place the tube on a shaker for 6 h to swell the resin.

[0065] (2) After swelling the resin, remove the DMF solution. First, add DMF solvent to rinse the resin and remove it, then add DCM solvent to rinse and remove it. Use DMF / DCM to rinse three times alternately, then add deprotecting agent (DMF solution containing 2% DBU and 5% anhydrous piperazine by volume) and act for 20 min to remove the protecting group Fmoc so that the amino group at the reaction end is exposed.

[0066] (3) After the deprotection step is completed, remove the deprotecting agent and rinse the resin three times alternately with DMF / DCM solvent. Add a small amount of resin particles to the ninhydrin detection reagent and heat in a boiling water bath for 30 seconds. If the resin particles turn blue-purple, the deprotection step is complete. If the color is still transparent white, the deprotection has failed and step (2) needs to be repeated.

[0067] (4) After confirming successful deprotection, weigh an amount of the next amino acid containing the Fmoc protecting group and HBTU catalyst powder, which is 10 times the amount of amino acid relative to the resin loading, into a 15 mL centrifuge tube. Add 10 mL of coupling agent (DMF solution containing 5% N-methylmorpholine by volume) for pre-activation for 15 min. Then add the peptide synthesis tube and shake on a shaker for 45 min.

[0068] (5) After the coupling step is completed, remove the coupling agent and rinse the resin three times with DMF / DCM solvent alternately. Add a small amount of resin particles to the ninhydrin detection reagent and heat in a boiling water bath for 30 seconds. If the resin particles do not change color, the coupling is successful. If the resin particles change color, the coupling is incomplete and step (4) needs to be repeated.

[0069] (6) After all amino acids in the polypeptide sequence have been successfully coupled and deprotected, palmitic acid (C16) containing the Fmoc protecting group, in an amount 10 times the amount of amino acids relative to the resin loading, is added and the mixture is shaken on a shaker and reacted overnight. After confirming successful coupling again, the Fmoc protecting group is removed using a deprotecting agent, and the mixture is then rinsed three times alternately with DMF / DCM solvent. After confirming successful deprotection, 10 mL of methanol solution is added to the polypeptide synthesis tube and soaked for 15 min. Subsequently, the methanol is removed and the resin is transferred to a clean serum bottle.

[0070] (7) Add 5 mL of the prepared lysis buffer (containing 2.5% TIS, 2.5% ultrapure water, and 95% TFA by volume) to a serum bottle containing resin, stir in an ice bath for 3 h, then filter to remove the resin and collect the lysis buffer. Concentrate the lysis buffer to a viscous state using nitrogen, add pre-cooled ice-cold ether to precipitate the peptide solid, transfer the suspension to a centrifuge tube and centrifuge at 8000 r / min for 3 min to collect the precipitate, add fresh ice-cold ether and repeat the washing and centrifugation twice. Place the collected precipitate in a vacuum drying oven at 37℃ to dry, obtaining a white solid powder, and finally purify it using preparative liquid chromatography.

[0071] The purified self-assembled peptides were characterized by mass spectrometry. The mass spectrometry characterization results are as follows: Figure 3 As shown, by Figure 3As can be seen from the main peak of the mass spectrum, the molecular weight of the peptide material is basically consistent with that of the design. This indicates that the target molecule has been synthesized, demonstrating that the self-assembled material with the above structure has been successfully synthesized.

[0072] The purified product was analyzed by high-performance liquid chromatography (HPLC), and the chromatogram is shown below. Figure 4 As shown, by Figure 4 It can be seen that the molecular purity of the purified polypeptide material reaches 96.1%.

[0073] Test Example 1

[0074] Self-assembly behavior and morphological transformation behavior of self-assembly peptides:

[0075] The schematic diagram of the self-assembly and morphology transformation process of the self-assembly peptide involved in this invention is shown below. Figure 5 As shown, the following experiment was conducted to investigate this process:

[0076] The self-assembled polypeptides CK and CS obtained in Examples 1 and 2 were dissolved in deionized water to prepare 3×10⁻⁶ solutions. -3 For the mother liquor of M, take 10 μL of the mother liquor and add it to 990 μL of ultrapure water. After thorough mixing, the concentration of the resulting solution is 3 × 10⁻⁶. -5 M, then nanoparticles can be obtained. Additionally, 10 μL of calcium chloride solution (final concentration 3 × 10⁻⁶) is added to each of the two solutions. -5 M) (with a control group without calcium chloride solution added), the nanoparticle samples and the samples after adding calcium chloride for 4 hours were used for circular dichroism spectroscopy and transmission electron microscopy.

[0077] Self-assembled peptide CK with / without Ca at 0h and 4h 2+ Induced CD spectrum as follows Figure 6A As shown, the self-assembled peptide CS was observed with and without Ca at 0h and 4h. 2+ Induced CD spectrum as follows Figure 6B As shown, the self-assembled peptides CK and CS showed presence / absence of Ca at 0h and 4h. 2+ The percentage of secondary structure composition under induced conditions is as follows: Figure 6C As shown, the self-assembled peptides CK and CS were in the range of 0h and 2h Ca 2+ Transmission electron microscopy image under induced conditions as shown below Figure 6D (Scale bar is 200 nm).

[0078] As shown in the figure, the self-assembling peptides CK and CS can self-assemble into nanofiber structures with a typical β-sheet configuration. The addition of Ca... 2+ The β-sheet content in the nanofibers was significantly increased.

[0079] Test Example 2

[0080] Observation of the phenomenon of self-assembled peptides binding to ARPE-19 cells:

[0081] Human retinal pigment epithelial cell line ARPE-19 was cultured in MEM-Nic medium (MEM-α containing GlutaMAX), supplemented with 1% fetal bovine serum, 1% penicillin / streptomycin, 1% N1 supplement, taurine (0.25 mg / mL), hydrocortisone (20 ng / mL), triiodothyronine (0.013 ng / mL), and nicotinamide (10 mM). Cells were cultured at 37°C in a humid environment containing 5% CO2, with the medium replaced three times a week.

[0082] ARPE-19 cells were seeded on silicon wafers and co-incubated with self-assembled peptides CK and CS (30 μM) or PBS for 4 h. Cells were then dehydrated using gradient ethanol solutions (ethanol-water solutions with ethanol percentages of 10%, 30%, 50%, 70%, 90%, and 100%) for 10 minutes at each gradient. After treatment with tert-butanol for 10 minutes, the cells were dried and then sputter-coated with gold for scanning electron microscopy to observe the cell surface morphology.

[0083] Scanning electron microscopy image of self-assembled peptides binding to ARPE-19 cells 4 h later. Figure 7 As shown in the figure (the scale bars in the first and second columns are 5 μM and 1 μM, respectively), it can be seen that the polypeptide can bind to the cell surface and self-assemble to form a wrinkled fibrous network structure.

[0084] Test Example 3

[0085] In vitro transepithelial electrical resistance assay of self-assembled peptides:

[0086] The culture method for the human retinal pigment epithelial cell line ARPE-19 was the same as that for test case 2.

[0087] APRE-19 cells were seeded in Transwell chambers, and transepithelial resistance was monitored daily in monolayer cells. The RPE barrier layer was considered to have been successfully constructed when the transepithelial resistance value remained stable.

[0088] Then, the self-assembled peptides CK, CS, or PBS were added to the chambers and co-incubated with the cells. After 3 hours, the peptides were removed and the resistance value was measured. After replacing the culture medium, the resistance value was measured continuously for 24 h and 48 h.

[0089] The results are as follows Figure 8 As shown in the figure, the self-assembled peptide can disrupt the tightness of the RPE barrier and recover to the baseline level after the peptide is removed.

[0090] Test Example 4

[0091] Fluorescence confocal microscopy imaging of self-assembled peptides after interaction with ARPE-19 cells:

[0092] The culture method for the human retinal pigment epithelial cell line ARPE-19 was the same as that for test case 2.

[0093] Cy5-labeled self-assembling peptides CK or CS were co-incubated with ARPE-19 cells for 2 h, 4 h, and 6 h, after which the material was removed and fluorescence confocal microscopy was performed.

[0094] The results are as follows Figure 9 As shown in the figure (where red represents Cy5-labeled self-assembled peptides, blue represents the cell nucleus, and the scale bar is 10 μM), it can be seen that the peptides first accumulate on the cell membrane surface through receptor recognition (2 h), and then enter the cell through internalization over time (4-6 h).

[0095] Test Example 5

[0096] Immunofluorescence staining after interaction of self-assembled peptides with ARPE-19 cells:

[0097] The human retinal pigment epithelial cell line ARPE-19 was cultured using the same method as in test case 2, and cultured for 12 days until tight junctions formed.

[0098] The self-assembled peptides CK, CS, or PBS were added to the culture medium and co-incubated for 3 h. After removing the culture medium and 24 h later, the cells were fixed with paraformaldehyde and then stained with tight junction-associated protein 1 (ZO-1) and F-actin for fluorescence confocal microscopy imaging. The distribution and expression levels of ZO-1 and F-actin in the cells were analyzed based on the imaging results.

[0099] Immunofluorescence staining image of tight junction protein 1 (ZO-1) after interaction of self-assembled peptides with ARPE-19 is shown below. Figure 10A As shown (green represents ZO-1, blue represents the cell nucleus, scale bar is 10 μM); Immunofluorescence staining image of actin (F-actin) after the self-assembled peptide interacts with ARPE-19 is shown below. Figure 10B As shown (red represents F-actin, blue represents the cell nucleus, scale bar is 10 μM). Figure 10C yes Figure 10A The fluorescence intensity quantification results of each stained image; Figure 10D yes Figure 10B The fluorescence intensity quantification results of each stained image are shown in the figure.

[0100] As shown in the figure, both CK and CS significantly upregulated F-actin expression. CK treatment downregulated ZO-1 expression, while CS treatment only resulted in diffuse staining of ZO-1 without significantly affecting its expression level. This indicates a significant difference in the regulation of RPE by the two peptides: CK can significantly remodel the cytoskeleton, downregulating tight junctions, while CS treatment affects ZO-1 localization by upregulating F-actin.

[0101] Test Example 6

[0102] The permeation-enhancing effect of self-assembled peptides on drugs:

[0103] The culture method for the human retinal pigment epithelial cell line ARPE-19 was the same as that for test case 2.

[0104] APRE-19 cells were seeded in Transwell chambers, and transepithelial resistance was monitored daily in monolayer cells. The RPE barrier layer was considered to have been successfully constructed when the transepithelial resistance value remained stable.

[0105] Then, the self-assembled peptides CK or CS were simultaneously added to the Transwell chamber and co-incubated with FITC-labeled fareximab (FA) (the final concentration of the drug was 20 μM, and the final concentration of the self-assembled peptide was 30 μM). The fluorescence intensity of the solution in the lower chamber was detected at different time points, and the permeability was calculated.

[0106] The results are as follows Figure 11 As shown in the figure, compared with the free FA group, the combined use of self-assembled peptides and FA increased the cumulative penetration rate of FA from about 20% to about 40%-55%, which is nearly 2-3 times higher.

[0107] Test Example 7

[0108] Transmission electron microscopy imaging of bio-assembled peptides after interaction with iPSC-RPE:

[0109] A highly similar RPE barrier to in vivo was constructed using induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE, P0-P1 generation). Specifically, iPSC-RPE cells were seeded on matrix gel-coated tissue culture medium for 24 hours before further seeding. The culture medium was DMEM / F-12 basal medium supplemented with 1% KnockOut serum substitute (KSR), 0.01 μL NEAA, 20 µM L-glutamine, and 1% penicillin / streptomycin. During the P0-P1 generation culture, fetal bovine serum (FBS) was used instead of KSR. To prevent excessive fibroblast proliferation during monolayer formation, the ROCK inhibitor Y-27632 (1:1000 dilution) was added until tight junctions formed. After the formation of a confluent differentiated monolayer, the Y-27632 concentration was maintained at a 1:2000 ratio. iPSC-RPE cells were cultured at 37°C with 5% CO2, and the culture medium was changed three times per week.

[0110] In tightly connected iPSC-RPE cells, self-assembling peptides CK or CS (30 μM) or PBS were added and co-incubated for 3 hours, followed by fixation and sectioning for transmission electron microscopy imaging.

[0111] The results are as follows Figure 12 As shown in the figure (scale bar is 1 μM), it can be seen that: after CK treatment, obvious gaps appeared at the adhesion junctions between iPSC-RPE cells, while the tight junctions at the apex of RPE cells were not completely destroyed. No significant changes were found between iPSC-RPE cells treated with CS compared to the control group, and the intercellular connections were not significantly disrupted.

[0112] Test Example 8

[0113] Observation of sections after self-assembled peptides are injected into the vitreous cavity:

[0114] Six-week-old adult male C57BL / 6J mice were injected intravitreal with Cy5-labeled self-assembled peptides CK or CS (1 μL, 30 μM). The mice were sacrificed 12 h later, and the eyeballs were harvested. The eyeballs were fixed overnight at 4°C with 4% paraformaldehyde at room temperature and then dehydrated in 30% sucrose solution at 4°C for 24 h. The eyeballs were then frozen, embedded, and sectioned. Immediately afterward, DAPI-containing anti-fluorescence quenching mounting medium was added to the slides for fluorescence confocal microscopy imaging.

[0115] The results are as follows Figure 13 As shown in the figure (scale bar is 200 μM, where red represents Cy5-labeled self-assembled peptides and blue represents cell nuclei), it can be seen from the figure that the self-assembled peptides can penetrate through the RPE and enter the choroidal blood flow 12 hours after treatment.

[0116] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0117] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0118] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A self-assembling polypeptide for regulating the permeability of retinal pigment epithelium, characterized in that, The self-assembled polypeptide comprises an integrin receptor-targeting peptide, a self-assembled polypeptide, and a hydrophobic molecule connected in sequence.

2. The self-assembling polypeptide according to claim 1, characterized in that, The amino acid sequence of the integrin receptor targeting peptide is HSDVHK or GGRGDS.

3. The self-assembling polypeptide according to claim 1 or 2, characterized in that, The amino acid sequence of the self-assembled polypeptide is KLVFF.

4. The self-assembling polypeptide according to any one of claims 1-3, characterized in that, The hydrophobic molecule is a C12-C18 saturated fatty acid molecule.

5. The self-assembling polypeptide according to any one of claims 1-4, characterized in that, The integrin receptor-targeting peptide is linked to the C-terminus of the self-assembled polypeptide via an amide bond, and the hydrophobic molecule is linked to the N-terminus of the self-assembled polypeptide via an amide bond.

6. The method for preparing a self-assembled polypeptide according to any one of claims 1-5, characterized in that, The preparation method includes: Using resin as a carrier, and integrin receptor-targeting peptides, amino acids constituting self-assembled peptides, and hydrophobic molecules as raw materials, the self-assembled peptides were prepared by sequentially linking amino acids and hydrophobic molecules from the C-terminus to the N-terminus using a solid-phase synthesis method.

7. The use of the self-assembling polypeptide according to any one of claims 1-5 in the preparation of products that improve the drug permeability of the retinal pigment epithelial cell layer.

8. A combination pharmaceutical composition, characterized in that, The combined pharmaceutical composition comprises any one of the self-assembled peptides and a treatment for macular neovascularization disease according to any one of claims 1-5.

9. The combined pharmaceutical composition according to claim 8, characterized in that, The combined drug composition also contains pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, antibacterial agents, or buffers. Preferably, the combined pharmaceutical composition is a single compound preparation or a combination of two separate preparations; Preferably, the formulation is any pharmaceutically acceptable dosage form.

10. The use of the self-assembled polypeptide according to any one of claims 1-5 or the combination pharmaceutical composition according to any one of claims 8-9 in the preparation of a medicament for the prevention or treatment of polypoid choroidal vascular disease.

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