A siRNA eye drop penetrating mucus barrier and cell barrier and a preparation method thereof

Abstract

The present application relates to the technical field of medicine, in particular to a kind of siRNA eye drop for penetrating mucus barrier and cell barrier and preparation method thereof.Innovative development side chain guanidino group-containing polydisulfide delivery carrier is used in the present application, through the quick dynamic disulfide exchange reaction between polydisulfide and sulfhydryl on mucin, tear film retention and efficient penetration are realized, and cationic polydisulfide is quickly adsorbed on the surface of negative charged cell membrane by electrostatic interaction, the quick dynamic disulfide exchange reaction between polydisulfide and sulfhydryl on cell membrane is used, and endocytosis is used to quickly penetrate tight junction cell layer.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to an siRNA eye drop that can penetrate the mucus barrier and cell barrier, and its preparation method. Background Technology

[0002] Choroidal neovascularization (CNV), also known as subretinal neovascularization, refers to the pathological formation and growth of new blood vessels originating from the choroid, extending beneath the retina, or breaching Bruch's membrane to enter the spaces between the retinal pigment epithelium. CNV is commonly seen in age-related macular degeneration (AMD), and the proliferating vascular structures can lead to visual impairment, even blindness in severe cases. A key driver of this pathological process is the excessive production of vascular endothelial growth factor (VEGF), primarily VEGFA, by retinal pigment epithelial (RPE) cells, which accelerates disease progression by increasing vascular permeability and promoting angiogenesis.

[0003] Drug treatment for CNV primarily focuses on inhibiting the growth of new blood vessels under the retina and reducing or eliminating inflammatory responses; there is currently no specific drug for treatment. Treatment effects can be achieved through photodynamic therapy and intravitreal injection of anti-VEGF drugs.

[0004] Photodynamic therapy (PDT) primarily involves intravenously injecting photosensitizing drugs into patients with idiopathic choroidal neovascularization (ICD). These photosensitizing drugs have a high affinity for choroidal neovascularization, accumulating locally within the neovascularized vessels. Low-intensity laser irradiation of the affected area then activates the photosensitizing drugs, promoting a local reaction that leads to the coagulation and necrosis of the neovascularized vessels. PDT can be applied to various subfoveal choroidal neovascularization diseases, but it also has drawbacks, including high treatment costs, high repeat treatment rates, short-term vision loss, and insufficient long-term efficacy stability.

[0005] With the discovery of VEGF's important role in neovascularization, intravitreal injection of anti-VEGF drugs such as conbercept, aflibercept, and ranibizumab has become the main clinical treatment for CNV. However, these drugs are expensive and prone to drug resistance. Frequent retinal injections can also cause serious complications such as ocular inflammation, subconjunctival hemorrhage, lens damage, and retinal detachment.

[0006] In contrast, small interfering RNA (SRNA) silences disease-related proteins at the mRNA level through RNA interference mechanisms. Due to its high specificity, long-lasting effect, and ability to circumvent traditional antibody resistance, it shows great therapeutic potential in clinical translation. Intravitreal injection is currently the most common method of drug administration for treating posterior segment ocular diseases, but repeated injections may lead to risks such as intraocular infection and retinal detachment. In comparison, non-invasive eye drops are an ideal choice due to high patient compliance and low infection risk, but their efficacy is limited by the difficulty of effectively overcoming the complex ocular barrier with existing delivery systems.

[0007] For non-invasive topical eye drops, the tear film, composed of the outermost lipid layer, the middle aqueous layer, and the deep mucous layer, becomes the primary barrier to drop penetration. This is mainly because tears dilute the drug concentration and lead to rapid drug clearance, while negatively charged mucins further hinder drug penetration through electrostatic interactions. Furthermore, the tight junctions between corneal epithelial cells and the dense collagen layer make the cornea one of the most difficult ocular barriers to penetrate. In contrast, the conjunctiva has a larger surface area and higher permeability, making the conjunctiva-sclera-choroid-RPE pathway the primary route for posterior segment delivery. However, in this pathway, lysosomal retention further impedes drug delivery to the RPE due to the epithelial barrier. Therefore, transforming the siRNA delivery method to achieve efficient CNV treatment has significant research and application value. Summary of the Invention

[0008] Therefore, the technical problem to be solved by the present invention is to overcome the problems of high price, large side effects and easy recurrence in the prior art, thereby providing siRNA eye drops.

[0009] To address the aforementioned technical problems, this invention provides a method for preparing siRNA eye drops that can penetrate mucus and cell barriers, comprising the following steps: S11: Under a protective atmosphere, guanidinyl-modified lipoic acid (SG) and 2-mercaptoethanol were added to a buffer solution and mixed to obtain polymer PSG; wherein the mixing reaction was as follows: Figure 2 As shown, the chemical formula of polymer PSG is as follows, n=100-800, preferably 400;

[0010] S12: The polymer PSG and VEGFA siRNA are mixed in DEPC water and incubated to obtain the siRNA eye drops that penetrate the mucus barrier and cell barrier.

[0011] Preferably, the buffer solution is a triethanolamine buffer solution; the concentration of the triethanolamine is 0.8-1.2 M, and the pH is 6.8-7.2.

[0012] Furthermore, the concentration of the triethanolamine is 1 M, and the pH is 7.

[0013] Preferably, the guanidinyl-modified thioctic acid is prepared as follows: S21: Under a protective atmosphere, thioctic acid, 1,1'-carbonyldiimidazole, L -Arginine methyl ester dihydrochloride and N,N -Diisopropylethylamine N,N The reaction solution was obtained by reacting the product in dimethylformamide (DMF) for 3-5 h. S22: Add diethyl ether to the reaction solution, separate the solid and liquid phases, and remove impurities to obtain the guanidine-modified thioctic acid.

[0014] Furthermore, the protective atmosphere is always a nitrogen atmosphere.

[0015] This invention designs a polydisulfide (PSG) with a guanidine group in its side chain and uses it to encapsulate siRNA to prepare nanocomposites (PSG NCs) for the treatment of CNV. PSG NCs can efficiently penetrate the ocular mucosa and cellular barrier to reach RPE cells in the fundus, silencing VEGFA mRNA, reducing ocular VEGFA levels, inhibiting angiogenesis in a non-invasive manner, reducing laser spot area, and thus achieving CNV treatment.

[0016] Preferably, in step S11, the mixing reaction time is 2-4 hours.

[0017] Preferably, in step S11, after the mixing reaction, the mixture is dialyzed with water for 64-80 hours and then freeze-dried; the molecular weight cutoff for the dialysis is 3500 Da. This ensures that small molecule impurities are fully dialyzed while retaining the target product. Long-term dialysis thoroughly removes residual small molecules, improving product purity; subsequent freeze-drying yields a stable solid product, facilitating storage and subsequent use.

[0018] Preferably, in step S11, the molar ratio of guanidinyl-modified thioctic acid to 2-mercaptoethanol is 100-800:1.

[0019] Furthermore, in step S11, the molar ratio of guanidinyl-modified thioctic acid to 2-mercaptoethanol is 400:1.

[0020] Preferably, in step S12, the mass ratio of polymer PSG to VEGFA siRNA is 2-20:1. VEGFA siRNA is a small interfering RNA that targets vascular endothelial growth factor A (VEGFA). It can specifically degrade VEGFA mRNA through RNA interference mechanisms, inhibiting its protein expression. It can reduce angiogenesis and vascular leakage and is often used in studies on inhibiting tumor angiogenesis and improving pathological retinal angiogenesis. It is a commonly used tool for regulating the VEGFA pathway at the gene level.

[0021] Furthermore, in step S12, the mass ratio of polymer PSG to VEGFA siRNA is 15:1.

[0022] Preferably, in step S12, the incubation temperature is 36-38℃ and the incubation time is 20-40 min.

[0023] This invention also provides an siRNA eye drop prepared by the above-described method that penetrates both the mucus barrier and the cell barrier. The tear film consists of an outer lipid layer, a middle aqueous layer, and an inner mucus layer. The efficient retention and rapid penetration of drugs within the tear film present conflicting requirements for the design of the delivery carrier. First, the tear film secretes tears to dilute the drug, reducing its retention time, and then removes the drug as the tear film is renewed. Second, the lipid and aqueous layers impose different requirements on the hydrophilicity / hydrophobicity of the delivery carrier. Finally, the non-specific adsorption and penetration of drugs by mucins in the mucus layer present conflicting requirements on the positive and negative charges of the delivery carrier.

[0024] Preferably, the siRNA eye drops contain a nanocomposite with a particle size of 90-110 nm. This avoids excessively small particles being cleared too quickly, while also preventing excessively large particles from failing to penetrate the ocular surface barrier. This size facilitates penetration of the ocular barrier, improving siRNA delivery efficiency; simultaneously, it exhibits good stability and biocompatibility, reducing ocular irritation and ensuring the drug effectively enters the eye to exert its effect.

[0025] This invention also provides a therapeutic drug for choroidal neovascularization, comprising siRNA eye drops that penetrate the aforementioned mucus and cellular barriers. The tight junctions of corneal and conjunctival epithelial cells constitute the main physical static barriers of the anterior segment of the eye, significantly limiting drug penetration into the retina. Furthermore, the negative charge and efflux pumps on the cell membranes make it even more difficult for hydrophilic macromolecular drugs and negatively charged nanomedicines to penetrate.

[0026] Compared with the prior art, the above-described technical solution of the present invention has the following advantages: This invention innovatively develops a polydisulfide delivery carrier with guanidine groups in the side chain, which achieves tear film retention and efficient penetration through a rapid dynamic disulfide exchange reaction between the polydisulfide and the thiol groups on the mucin.

[0027] This invention utilizes cationic polydisulfide to rapidly adsorb onto the negatively charged cell membrane surface through electrostatic interactions, and leverages the rapid dynamic disulfide exchange reaction between the polydisulfide and the thiol groups on the cell membrane, as well as transcytosis to rapidly penetrate the tightly connected cell layers.

[0028] This invention innovates the administration route of macromolecular drugs, enabling non-invasive treatment of CNV through eye drops, which greatly improves patient compliance. Attached Figure Description

[0029] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0030] Figure 1 This is the synthesis route of SG in Example 1.

[0031] Figure 2 This is the synthesis route of PSG in Example 1.

[0032] Figure 3 The synthetic route for PGA in Comparative Example 1 is shown.

[0033] Figure 4 The figures show the particle size and potential of the nanocomposites in Example 1 and Comparative Example 1.

[0034] Figure 5 The image shows the fluorescence change after the nanocomposite in Example 2 was incubated with the di-eosin-glutathione disulfide (Di-E-GSSG) probe.

[0035] Figure 6 This is a graph showing the viscosity change of PSG NCs after incubation with mucin in Example 2.

[0036] Figure 7 This is a diagram showing the trajectory of the nanocomposite in the mucin solution in Example 2.

[0037] Figure 8 This is an experimental diagram showing the results of the nanocomposite's penetration of the in vitro mucosal barrier in Example 2.

[0038] Figure 9 This is a graph showing the cellular uptake levels of PSG NCs on human conjunctival epithelial cells (HConEpiCs) pretreated with different endocytosis inhibitors in Example 3.

[0039] Figure 10 The graph shows the cell uptake levels after PSG NCs and HConEpiCs were incubated for 4 hours in fresh culture medium for different times in Example 3.

[0040] Figure 11 This is a graph showing the cell uptake levels after PSG NCs and HConEpiCs were incubated for 4 hours in Example 3, followed by incubation for another 4 hours in fresh culture medium containing different intracytoplasmic transport inhibitors.

[0041] Figure 12This is an experimental diagram showing the results of the nanocomposite's penetration of the conjunctival barrier in Example 3.

[0042] Figure 13 This is an experimental diagram showing the in vitro conjunctival barrier penetration results of PSG NCs in Example 3. The HConEpiCs monolayer was pretreated with different inhibitors.

[0043] Figure 14 This is an experimental diagram showing the penetration results of the nanocomposite on the scleral barrier in vitro in Example 3.

[0044] Figure 15 This is a graph showing the cellular uptake level of the nanocomposite in ARPE-19 cells in Example 4.

[0045] Figure 16 The image shows the PCR results of the nanocomposite in ARPE-19 cells in Example 4.

[0046] Figure 17 The image shows the results of the enzyme-linked immunosorbent assay (ELISA) experiment on ARPE-19 cells in Example 4 of the nanocomposite.

[0047] Figure 18 The diagram shows the tube formation and quantification of HRMECs in Example 4. The scale bar in the diagram is 200 μm.

[0048] Figure 19 The figure shows the experimental results of VEGFA mRNA expression level in the RPE-choroid-sclera complex of CNV mice in Example 5 (n=6).

[0049] Figure 20 The figure shows the experimental results of VEGFA expression level in the RPE-choroid-sclera complex of mice in Example 5 (n=6).

[0050] Figure 21 The images shown are representative fundus fluorescein angiography (FFA) images obtained 10 days after topical eye drops were applied to mice with choroidal neovascularization in Example 5; the circles indicate the locations of vascular leakage, pointing to the CNV locations.

[0051] Figure 22 This is a representative OCT image obtained 10 days after topical eye drops were applied to mice with choroidal neovascularization in Example 4; the arrow points to the CNV location. Detailed Implementation

[0052] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0053] Example 1: Guanidino-modified lipoic acid (SG) is synthesized according to the following steps: Lipoic acid (412 mg, 2.0 mmol) was dissolved in anhydrous water. N,N The mixture was added to dimethylformamide (DMF, 4 mL), followed by 1,1'-carbonyldiimidazole (324 mg, 2.0 mmol). After stirring at room temperature for 1 hour under nitrogen protection, a solution dissolved in anhydrous DMF (4 mL) was added. L Arginine methyl ester dihydrochloride (261 mg, 1.0 mmol) and N,N-diisopropylethylamine (174 μL, 1.0 mmol) were mixed and stirred at room temperature for 3.5 hours under nitrogen protection. The reaction mixture was then added dropwise to diethyl ether (40 mL) and centrifuged at 4000 rpm for 3 minutes. The precipitate was collected and washed with a dichloromethane / diethyl ether mixture (1:2, v / v, 5 × 10 mL). After solvent removal under vacuum, a pale yellow viscous solid was obtained, which was guanidine-modified lipoic acid (SG).

[0054] See synthesis path Figure 1 .

[0055] 2-Mercaptoethanol (0.12 mM) was dissolved in triethanolamine buffer (1.0 M, 2 mL, pH 7.0), and SG (2.4 mM) in triethanolamine buffer solution (1.0 M, 10 mL, pH 7.0) was added. The mixture was stirred vigorously for 3 hours under nitrogen protection. The reaction mixture was then collected, dialyzed against deionized water (molecular weight cutoff 3500 Da) for three days, and lyophilized to give a white solid polymer PSG (41.2 kDa).

[0056] See synthesis path Figure 2 .

[0057] A VEGFA siRNA (siVEGFA, purchased from Sangon Biotech) solution (0.1 mg / mL, 65 μg, dissolved in DEPC water) and a PSG solution (1 mg / mL, 975 μg, dissolved in DEPC water) were mixed at a PSG / siVEGFA weight ratio of 15:1. The mixture was vortexed for 5 seconds and incubated at 37°C for 30 minutes to form PSG / siVEGFA nanocomposites (PSG NCs).

[0058] The particle size and zeta potential of PSG NCs were determined using a Malvern particle size analyzer (Zetasizer Nano-ZS, Malvern). Using the same method as for VEGFA siRNA, PSG / FAM-siVEGFA nanocomposites (PSG) were prepared by replacing siVEGFA with FAM-siVEGFA and Cy5-siVEGFA (purchased from Sangon Biotech), respectively. FAM NCs) and PSG / Cy5-siVEGFA nanocomposite (PSG Cy5 NCs).

[0059] Comparative Example 1: PBLG was synthesized via ring-opening polymerization of γ-benzyl-L-glutamate N-carboxycyclic intracyclic anhydride (BLG-NCA) using ethanolamine as an initiator, as follows: γ-Benzyl-L-glutamate N-carboxycyclic anhydride (BLG-NCA, 634 mg, 2 mmol) was dissolved in anhydrous DMF (5 mL) under nitrogen protection, and ethanolamine (1.1 μL, 0.018 mmol) was added. The mixture was stirred at room temperature for 72 hours. The product PBLG was precipitated with diethyl ether (60 mL), washed with diethyl ether (5 × 10 mL), and lyophilized to give a white solid PBLG.

[0060] Dissolve PBLG (200 mg) in acetic acid (HOAc, 4 mL), then add hydrobromic acid (HBr) / acetic acid solution (1:3). v / v The reaction mixture was stirred at 40°C for 4 hours. The product was precipitated with diethyl ether (60 mL), washed with diethyl ether (5 × 10 mL), and lyophilized to obtain a white solid PP.

[0061] For the synthesis of PGA, PP (136 mg, 10 μmol), EDC (5.8 mg, 30 μmol), and NHS (1.9 mg, 15 μmol) were premixed in DMF (2 mL) and stirred at room temperature for 2 hours. Subsequently, this mixture was added to a solution of 1-(4-aminobutyl)guanidine (145 mg, 1.1 mmol), and stirring was continued at room temperature for 24 hours. The product PGA was precipitated with diethyl ether (60 mL), washed with diethyl ether (5 × 10 mL), and lyophilized to obtain PGA (23.5 kDa).

[0062] See synthesis path Figure 3 .

[0063] VEGFA siRNA (siVEGFA) solution (0.1 mg / mL, dissolved in DEPC water) and PGA solution (1 mg / mL, dissolved in DEPC water) were mixed at a PGA / siVEGFA weight ratio of 15:1. The mixture was vortexed for 5 seconds and incubated at 37°C for 30 minutes to form PGA / siVEGFA nanocomposites (PGA NCs). The particle size and zeta potential of PGA NCs were determined using a Malvern particle size analyzer (Zetasizer Nano-ZS, Malvern). Using the same method, PGA / FAM-siVEGFA nanocomposites (PGA FAMNCs) and PGA / Cy5-siVEGFA nanocomposites (PGA) were prepared by replacing siVEGFA with FAM-siVEGFA and Cy5-siVEGFA, respectively. Cy5 NCs).

[0064] Figure 4 The figures show the particle size and potential of the nanocomposites in Example 1 and Comparative Example 1. This invention can synthesize siRNA nanocomposites with controllable size, a particle size of approximately 100 nm, and a positive surface charge.

[0065] Example 2:

[0066] Di-E-GSSG (purchased from Cayman) was dissolved in PBS (0.1 M, containing 1 mM EDTA, pH 7.4, 200 μL), followed by the addition of PSG NCs from Example 1 or PGA NCs from Comparative Example 1 (1 μg siVEGFA / mL). Fluorescence spectrophotometry was used to analyze the NCs. λ ex =510 nm, λ em The fluorescence intensity was measured every 15 seconds at a wavelength of 560 nm.

[0067] To determine the viscosity of mucin, PSG NCs (1 μg siVEGFA / mL) from Example 1 were added to different concentrations (0.1%, 0.3%, and 0.5%). w / v The mixture was then placed in a mucin solution (1 mL). Rheological characterization of the mixture was subsequently performed at a constant shear rate of 93 1 / s, and the viscosity was measured at 120 seconds.

[0068] Brownian motion of NCs in mucin solution was studied using particle tracking. PSG was then used. FAM NCs and PGA FAM NCs (1 μg FAM-siVEGFA / mL) were respectively mixed with mucin solution (0.3%, dissolved in PBS). w / vMix 200 μL of the mixture and then transfer the mixture to a glass-bottomed cell culture dish. Ф The nanocomposite was equilibrated at 37 °C for 30 minutes in a 15 mm (x=15 mm) medium. The motion trajectory of the nanocomposite was recorded at a rate of 3 frames / second using a confocal laser scanning microscope.

[0069] Add 1% PBS solution containing mucin to the upper chamber of the Transwell (0.4 μm pore size). w / v 100 μL of PBS (pH 7.4, 1 mL) was added to the lower chamber to construct an in vitro mucosal barrier model. During the assay, PSG was added to the upper chamber. FAM NCs and PGA FAM NCs (5 μg FAM-siVEGFA / mL, 200 μL). The lower chamber mixture (100 μL) was collected at specific time points and the same volume of fresh PBS was added. The fluorescence intensity of FAM-siVEGFA in the lower chamber mixture was determined by fluorescence spectrophotometry. λ ex =488nm, λ em =525 nm). The permeation efficiency (%) of the comparative example and the embodiment on the mucosal barrier = (cumulative content of FAM-siVEGFA in the lower chamber × 100) / content of added FAM-siVEGFA.

[0070] The Di-E-GSSG probe is a glutathione dimer composed of two eosin fluorescein molecules linked by a disulfide bond. Fluorescence self-quenching occurs due to the proximity of the eosin group, but the fluorescence signal is restored after a disulfide exchange reaction with the disulfide bond. Figure 5 As shown, the fluorescence intensity of Di-E-GSSG did not change significantly after co-incubation with the comparative example for one hour, while the fluorescence intensity increased significantly after incubation with the example, proving that disulfide exchange occurred between the example and Di-E-GSSG. During mucus penetration, this dynamic covalent cross-linking mechanism may further affect the macroscopic physical properties of the mucus layer. Further rheological evaluation revealed (…). Figure 6 In the example, after co-incubation with a mucin solution, the viscosity of the mucin increased significantly, and this increase was further observed with increasing mucin concentration. This phenomenon can be attributed to the formation of dynamic covalent cross-linking points between the nanoparticles and the mucin through disulfide exchange, thereby translating the chemical bonding into a change in the macroscopic viscosity of the solution. The dynamic covalent interaction between this nanocomposite and the mucin in the mucus was then evaluated to assess its diffusion and permeation properties in the mucus. Brownian motion of the nanocomposite in the mucin solution was assessed using microparticle tracking assays, such as... Figure 7As shown, the embodiment exhibits a wider motion trajectory, with a significantly greater travel distance than the comparative example. Subsequently, the transmucosal delivery efficiency of the NCs was determined using the Transwell chamber system. Figure 8 The transport efficiency of the examples was significantly higher than that of the comparative examples. Notably, the thiol blocker 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) significantly inhibited the transport efficiency of the examples. These results collectively demonstrate that the examples can efficiently penetrate the mucus layer via thiol-disulfide bond exchange reactions.

[0071] Example 3:

[0072] HConEpiCs were used at 1×10 per orifice. 4 Cells were seeded at a density of 100 mcg / well in 96-well plates and cultured for 24 hours. Cells were pretreated for 30 minutes with a combination of endocytosis inhibitors, including DTNB (1 mM), methyl-β-cyclodextrin (5 mM), genistein (100 μg / mL), wollamic acid (10 μg / mL), and chlorpromazine (10 μg / mL). After replacing the medium with fresh serum-free medium, PSG was added. FAM NCs (1 μg FAM-siVEGFA / mL) were incubated with cells at 37°C for 4 hours. Simultaneously, cells not pretreated with inhibitors were incubated with PSG. FAM NCs were incubated at 4°C for 4 hours. Subsequently, the cells were washed three times with cold PBS containing heparin (20 U / mL) to lyse them. Cells were then analyzed by fluorescence spectrophotometry (λ). ex =488 nm,λ em The amount of PSG NCs taken up by cells was determined by (535 nm). The uptake level was expressed as micrograms of FAM-siVEGFA per milligram of cellular protein.

[0073] To evaluate the exocytosis kinetics of PSG NCs, HConEpiCs were loaded at a density of 1 × 10⁻⁶ per well. 4 Cells were seeded at a density of [number] cells per well in 96-well plates and cultured for 24 hours. After replacing the medium with serum-free medium, PSG was added. FAM NCs (1 μg FAM-siVEGFA / mL) were added. After incubation at 37°C for 4 hours, the medium was replaced with fresh medium, and the cells were cultured for another 1, 2, 4, or 8 hours. The amount of NCs taken up by the cells was determined by fluorescence spectrophotometry using the method described above. Simultaneously, PSG... FAM After 4 hours of incubation with NCs, the culture medium was replaced with a medium containing different cell transport inhibitors (including brefidobacterium A (90 μM), monensin (50 μM) and N-ethylmaleimide (50 μM, all purchased from Beyotime), and the cells were cultured for another 4 hours. The amount of PSG NCs taken up by the cells was then determined by fluorescence spectrophotometry as described above.

[0074] HConEpiCs were set at 1.0-1.5×10 5 cells / cm 2 The HConEpiCs were inoculated into the upper chamber of a Transwell substrate at a density of 0.4 μm, and the culture medium was changed every 2 days for 23-25 ​​days. The transmembrane resistance of the HConEpiCs was measured using a transmembrane resistivity meter. When the transmembrane resistance reached 1000 Ω·cm... 2 At this time, it indicates that the HConEpiCs monolayer has formed, and the in vitro conjunctival barrier model has been successfully constructed. During the assay, the Transwell medium was replaced with fresh serum-free Duchenne modified Eagle medium (DMEM), and PSG was added to the upper chamber. FAM NCs and PGA FAM NCs (5 μg FAM-siVEGFA / mL, 200 μL) were incubated at 37°C. At specific time points, 100 μL of the lower chamber medium was collected and the same volume of fresh serum-free DMEM was added. The fluorescence intensity (λ) of FAM-siVEGFA in the medium was determined by fluorescence spectrophotometry. ex =488 nm,λ em =535 nm). The permeation efficiency (%) of the comparative example and the example on the HConEpiCs monolayer = (cumulative content of FAM-siVEGFA in the lower chamber × 100) / initial content of added FAM-siVEGFA.

[0075] Pig eyeballs were obtained from the slaughterhouse. After collection, the periorbital tissue was removed and the sclera was separated, then washed three times with PBS. The sclera was fixed between the supply and receiving chambers of a Franz transdermal diffusion cell (Φ=9 mm), with its outer surface facing the supply chamber, to construct an in vitro scleral barrier model. During measurement, PSG was added to the supply chamber. FAM NCs and PGA FAM NCs (5 μg FAM-siVEGFA / mL, 200 μL). Simultaneously, the lower receiving chamber was filled with PBS and incubated at 37°C. At specific time points, the receiving chamber mixture (200 μL) was collected and the same volume of fresh PBS was added. The content of Aflibercept in the receiving chamber mixture (λ) was determined by fluorescence spectrophotometry. ex =488 nm,λ em =535 nm). The penetration efficiency (%) of the comparative example and the embodiment to the scleral barrier = (cumulative content of FAM-siVEGFA in the receiving chamber × 100) / content of FAM-siVEGFA added in the supply chamber.

[0076] like Figure 9As shown, DTNB and chlorpromazine reduced cellular uptake by approximately 71.2% and 53.4%, respectively, indicating that the samples entered cells primarily via thiol-disulfide bond exchange and clathrin-mediated endocytosis. Furthermore, with increasing incubation time, the uptaken PSG FAMNCs were gradually expelled from the cells. Figure 10 Intracellular transport inhibitors such as N-ethylmaleimide (thiol-disulfide bond exchange inhibitor), Brefield's styrosinase A (endoplasmic reticulum to Golgi pathway inhibitor), and monensin (Golgi to plasma membrane pathway inhibitor) significantly reduced PSG. FAM extrinsic levels of NCs ( Figure 11 This demonstrates that the disulfide exchange pathway and exocytosis (first reaching the Golgi apparatus via the endoplasmic reticulum, then being transported to the cell membrane via the Golgi apparatus) are involved in cell transport in the examples. A transwell system was used to culture a conjunctival cell monolayer to simulate the conjunctival barrier; the permeability efficiency of the examples was increased by 4 times compared to the comparative example. Figure 12 Furthermore, treatment with disulfide exchange inhibitors, endocytosis inhibitors, and exocytosis inhibitors effectively reduced the total amount of transport in the examples, demonstrating that the disulfide exchange process, endocytosis, intracellular transport, and exocytosis pathways discussed above all participate in the transport of the product of Example 1 in the cell monolayer. Figure 13 In the scleral transdermal penetration experiment, the penetration volume of the example was increased by 13 times compared with the control example. Figure 14 This indicates that the product of Example 1 also has good penetration efficiency through the scleral barrier and has a greater chance of accumulating in the eye.

[0077] Example 4:

[0078] ARPE-19 cells (purchased from ATCC) were added at a rate of 1 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 12-well plates and cultured for 24 hours. After replacing the medium with serum-free medium, PSG was added. FAM NCs and PGA FAM NCs (1 μg FAM-siVEGFA / mL). In parallel experiments, PSG was added... FAM Before NCs, ARPE-19 cells were pretreated with DTNB (1 mM) for 30 minutes. After incubation at 37°C for 4 hours, the cells were washed three times with cold PBS containing heparin (20 U / mL, purchased from Solarbio) and then analyzed by flow cytometry.

[0079] ARPE-19 cells were loaded at 2×10 5Cells were seeded at a density of 100 cells / well in 6-well plates and cultured for 24 hours. Fresh serum-free DMEM was then added, along with an aqueous solution of nanoparticles (1 μg FAM-siVEGFA / mL) from Comparative Example 1 and Example 1, and incubated at 37°C for 24 hours. Cells were collected, RNA was extracted, and intracellular VEGFA mRNA levels were determined by reverse transcription-polymerase chain reaction (RT-PCR). The concentration of VEGFA protein in the culture medium was determined using an ELISA kit.

[0080] ARPE-19 cells were loaded at 2×10 5 Cells were seeded at a density of 100 cells / well in 6-well plates and cultured for 24 hours. Fresh serum-free DMEM was then added, and nanoparticles (1 μg FAM-siVEGFA / mL) from the comparative and example studies were added. The plates were incubated at 37°C for 24 hours, and the cell culture medium was collected. HRMECs were then seeded at a density of 1 × 10⁶ cells / well. 4 Cells were seeded at a density of 5 μg / mL in 50 μL pre-coated matrix gel and treated with ARPE-19 cell culture medium for 8 hours. Tube formation was recorded using an inverted microscope, and the number of lumens and nodes was quantitatively analyzed using ImageJ software.

[0081] Flow cytometry analysis revealed that RPE cells exhibited significantly higher uptake of the sample compared to the control group. After DTNB was used to block thiol groups on the cell surface, the uptake efficiency of RPE cells decreased significantly, becoming comparable to the control group, demonstrating the role of disulfide exchange in promoting cell internalization. Figure 15 Furthermore, after cellular uptake, the example effectively silenced VEGFA mRNA and inhibited VEGFA secretion, reducing VEGFA mRNA and protein levels by 75.6% and 48.9%, respectively, which was superior to the comparative example (…). Figure 16 and Figure 17 Since endothelial cell tube formation is a key step in angiogenesis, ARPE-19 cells with silenced VEGFA mRNA were co-incubated with vascular endothelial cells. In tube formation experiments, the product from Example 1 effectively reduced the tube-forming ability of endothelial cells, thereby inhibiting angiogenesis. Figure 18 ).

[0082] Example 5:

[0083] A mouse CNV model was established using laser photocoagulation. C57BL / 6 mice were anesthetized, and tropicamide eye drops were instilled onto the ocular surface to dilate the pupils. Four burn spots (50 μm in diameter) were created around the optic nerve using a laser (wavelength 532 nm, power 475 mW, 0.05 s). Successful penetration of the Bruch's membrane resulted in bubble-like burns, which were considered effective laser spots. To prevent intraocular infection, erythromycin ointment was applied to the ocular surface of the mice postoperatively.

[0084] On day 3 after laser photocoagulation, mice were randomly divided into four groups. Mice in the fourth group received daily intravitreal injections of PBS eye drops (5 μL), PGA NCs, and PSG NCs eye drops (2.5 μg siVEGFA / eye, 5 μL) once daily for ten consecutive days. On day 3, mice in the fourth group received an intravitreal injection of aflibercept (2.5 mg / kg, 5 μL). The Normal group consisted of normal, untreated mice. On day 13, mice were sacrificed, and eyeballs were collected. The retinal pigment epithelium-choroid-sclera complex was isolated. Trizol reagent (1 mL) was added to homogenize the retinal pigment epithelium-choroid-sclera complex, and total RNA was extracted. Intracellular VEGFA mRNA content was measured by RT-PCR. Additionally, the retinal pigment epithelium-choroid-sclera complex was collected, lysed using RIPA lysis buffer (purchased from Beyotime) containing protease inhibitors (200 μL) for 30 min, centrifuged (12000 g, 15 min, 4℃), and the supernatant was collected. VEGF concentration was measured using an ELISA kit. In parallel experiments, mice were anesthetized and mydriatics were dilated using tropicamide eye drops (1%). Subsequently, mice were intraperitoneally injected with sodium fluorescein (5%, w / v, 100 μL), and fundus fluorescein angiography was performed using a Micron III retinal imaging microscope (Phoenix Research Labs, California, USA). Optical coherence tomography images were acquired using a Spectralis optical coherence tomography scanner (Heidelberg Engineering, Inc., Heidelberg, Germany).

[0085] After 10 days of continuous administration, the example demonstrated effective silencing of VEGFA mRNA in RPE cells of the fundus. Figure 19 ), and inhibited the secretion of VEGFA ( Figure 20 Fundus imaging experiments revealed a significant reduction in the area of ​​the laser spot in the mouse fundus and a decrease in fluorescence leakage. Figure 21 Further analysis of the lesion along the transverse section using OCT revealed a reduction in fundus swelling and tissue proliferation after treatment. Figure 22 The results were all better than those of the control group 1.

[0086] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing siRNA eye drops that can penetrate mucus and cell barriers, characterized in that, Includes the following steps: S11: Under a protective atmosphere, guanidine-modified lipoic acid and 2-mercaptoethanol are added to a buffer solution and mixed to react and obtain polymer PSG; wherein, the chemical formula of polymer PSG is as follows, n=100-800; ； S12: The polymer PSG and VEGFA siRNA are mixed in water and incubated to obtain the siRNA eye drops that penetrate the mucus barrier and cell barrier.

2. The preparation method according to claim 1, characterized in that: The buffer solution is a triethanolamine buffer solution; the concentration of the triethanolamine is 0.8-1.2 M, and the pH is 6.8-7.

2.

3. The preparation method according to claim 1, characterized in that: In step S11, the mixing reaction takes 2-4 hours.

4. The preparation method according to claim 1, characterized in that: In step S11, after the mixture is reacted, it is dialyzed with water for 64-80 h and then freeze-dried; the molecular weight cutoff for the dialyz is 3500 Da.

5. The preparation method according to claim 1, characterized in that: In step S11, the molar ratio of guanidinyl-modified thioctic acid to 2-mercaptoethanol is 100-800:

1.

6. The preparation method according to claim 1, characterized in that: In step S12, the mass ratio of polymer PSG to VEGFAsiRNA is 2-20:

1.

7. The preparation method according to claim 1, characterized in that: In step S12, the incubation temperature is 36-38℃ and the incubation time is 20-40 min.

8. An siRNA eye drop that penetrates the mucus barrier and cell barrier, prepared by the preparation method according to any one of claims 1-7.

9. The siRNA eye drops that penetrate mucus and cell barriers according to claim 8, characterized in that, The siRNA eye drops contain a nanocomposite with a particle size of 90-110 nm.

10. A therapeutic drug for choroidal neovascularization, characterized in that: siRNA eye drops that penetrate the mucus barrier and cell barrier as described in claim 8 or 9.

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