ITGB1-siRNA lipid nanoparticle and application of ITGB1-siRNA lipid nanoparticle in anti-angiogenesis treatment

Abstract

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to ITGB1-siRNA lipid nanoparticles and application of the ITGB1-siRNA lipid nanoparticles in anti-angiogenesis treatment. Specifically, the ITGB1-siRNA lipid nanoparticles are successfully prepared, the lipid nanoparticles are loaded with integrin beta1 small interfering RNA (ITGB1-siRNA), are modified with triptolide (TP) and are coupled with anti-Flt1 peptide, so that the lipid nanoparticles can target vascular endothelial cells (VEC) and pericytes of CNV, and a good treatment effect on the CNV is achieved. Therefore, the lipid nanoparticles researched and developed by the invention are expected to become a new choice for treating the CNV-related eye diseases, and have good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomedicine and molecular biology, and particularly relates to an ITGB1-siRNA lipid nanoparticle and its application in anti-angiogenesis therapy. Background Art

[0002] The information disclosed in the background art of the present invention is only intended to increase the understanding of the overall background of the present invention, and does not necessarily be regarded as an admission or imply in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] The cornea is a transparent and avascular tissue on the ocular surface, and maintaining its transparency is crucial for normal visual function. Under physiological conditions, the cornea is in a special state of "angiogenic immune privilege", where anti-angiogenic factors interact with pro-angiogenic factors to maintain a dynamic balance. However, in various pathological conditions, such as infection, trauma, after corneal transplantation, and long-term wearing of contact lenses, etc., the avascular balance state of the cornea is disrupted, triggering the occurrence of corneal neovascularization (CNV). Corneal neovascularization is a disease that seriously threatens vision. Corticosteroids have become the main drugs for CNV treatment due to their low cost and significant efficacy. However, long-term application can lead to secondary infections, increased intraocular pressure, and cataracts. Vascular endothelial growth factor (VEGF) and its receptors play important roles in the process of angiogenesis. Anti-VEGF drugs such as ranibizumab, bevacizumab, and aflibercept have been used for the treatment of CNV to improve the vision of patients. In addition, animal model experiments and clinical trials have both confirmed the therapeutic effects of these drugs on CNV. However, due to drug resistance or re-injury, some patients have a poor response to this type of treatment, or the situation of CNV recurrence occurs. Nucleic acid drugs such as small interfering RNA (siRNA) and antisense oligonucleotides can prevent angiogenesis by selectively inhibiting the expression of angiogenesis-related genes, which may represent a new strategy for treating CNV.

[0004] However, delivering oligonucleotides to target cells in vivo remains extremely challenging. Lipid nanoparticles (LNPs) are currently the most effective nucleic acid delivery carriers and are also considered a promising ocular drug delivery carrier because they can enhance drug solubility, improve bioavailability, and achieve sustained drug release. Ionizable cationic lipid nanoparticles (icLNPs) have a net neutral charge on their surface, avoiding the problems of rapid clearance and toxicity faced by permanent cationic lipid nanoparticles in vivo. They perform well in nucleic acid delivery, with high encapsulation efficiency, good cell uptake ability, and effective endosomal escape characteristics. icLNPs have been approved by the FDA for use in Opatrio (a therapeutic siRNA molecule developed by Alnylam Pharmaceuticals) and COVID-19 mRNA vaccines (developed by Moderna and Pfizer-BioNTech). Despite the powerful efficacy of icLNPs in nucleic acid delivery, the inflammatory side effects they cause have raised concerns. Ionizable lipids can induce the expression of interleukin-1 (IL-1) and inflammatory responses, promoting the formation of CNV, thus limiting the application of icLNPs in the treatment of CNV. At the same time, how to achieve targeted drug delivery to ensure that the drug effectively binds to a specific tissue microenvironment or specific receptors on the cell surface, thereby achieving a more effective therapeutic effect has also become an urgent problem to be solved in the treatment of corneal neovascularization. Summary of the Invention

[0005] Aiming at the deficiencies in the prior art, the purpose of the present invention is to provide an ITGB1-siRNA lipid nanoparticle and its application in anti-neovascularization treatment. Specifically, the present invention successfully prepared an ITGB1-siRNA lipid nanoparticle, which, in addition to loading integrin β1 small interfering RNA (ITGB1-siRNA), is modified with triptolide (TP) and conjugated with an anti-Flt1 peptide, enabling it to target vascular endothelial cells (VECs) and pericytes of CNV, thereby achieving a good therapeutic effect on CNV. At the same time, the lipid nanoparticles prepared by the present invention do not cause an increase in intraocular pressure and an increased risk of local infection, and can be administered in the form of eye drops. It not only avoids the side effects of existing drugs for the treatment of corneal neovascularization, but also is simple and convenient to use. Based on the above research results, the present invention is completed.

[0006] In order to achieve the above technical objectives, the technical solutions provided by the present invention are as follows:

[0007] In the first aspect of the present invention, there is provided an ITGB1-siRNA lipid nanoparticle, which loads ITGB1-siRNA and is modified with triptolide and an anti-Flt1 peptide; further, the lipid nanoparticle is an ionizable cationic lipid nanoparticle.

[0008] Among them, ITGB1-siRNA is integrin β1 small interfering RNA. Through research, the present invention finds that integrin β1 (Itgb1) plays a key role in CNV. Single-cell sequencing shows that the expression of Itgb1 is significantly increased in the corneas of mice with corneal neovascularization. Knocking out Itgb1 can significantly reduce the occurrence of CNV. Therefore, ITGB1-siRNA is selected for the treatment of CNV. Further, the concentration of the ITGB1-siRNA is 50-100 nM, and it is safer and more effective within this concentration range.

[0009] The triptolide is one of the main active components of Tripterygium wilfordii, and has powerful immunosuppressive and anti-inflammatory effects. It has also been reported that it has the activity of anti-tumor angiogenesis. In addition, triptolide can significantly inhibit the degradation of collagen in corneal fibroblasts induced by interleukin-1β (IL-1β), and the triptolide-modified LNPs exhibit good anti-inflammatory activity. However, due to the characteristics of triptolide such as high toxicity, extremely poor water solubility, and narrow therapeutic window, the concentration of triptolide has been screened and optimized. Through research, it is found that triptolide with a concentration of 10-15 ng / mL is suitable for LNPs modification to achieve anti-inflammatory and inhibit the proliferation of VEC; among them, triptolide with a concentration of 10 ng / mL is the most preferred.

[0010] The anti-Flt1 peptide is a specific antagonist of Flt1 (the gene name of VEGFR1), and its amino acid sequence is GNQWFI. The anti-Flt1 peptide can specifically bind to VEGFR1 and block the interaction between VEGFR1 and various ligands (such as VEGFA, VEGFB, and placental growth factor), thereby inhibiting the migration of vascular endothelial cells (VEC) and the formation of vascular lumens induced by VEGF. In the present invention, single-cell sequencing shows that the expression of Flt1 is significantly increased in the vascular endothelial cells and pericytes of mouse CNV. Therefore, using the anti-Flt1 peptide can effectively target CNV and enhance the therapeutic effect of drugs at the same time.

[0011] The particle size of the ITGB1-siRNA lipid nanoparticles is ∼110 nm, and PDI < 0.5.

[0012] Further, the cationic lipid in the lipid nanoparticles can be any one of SM-102, ALC-0315, and D-Lin-MC3-DMA. Since SM-102, as an ionizable cationic lipid for LNPs, shows the highest cell uptake rate, SM-102 is preferred.

[0013] In the second aspect of the present invention, a preparation method of the above ITGB1-siRNA lipid nanoparticles is provided, and the preparation method includes preparing the ITGB1-siRNA lipid nanoparticles by using a microfluidic technology;

[0014] Specifically, the preparation method includes:

[0015] S1. Dissolve ionizable cationic lipid, co-lipid, cholesterol and polyethylene glycolylated lipid in ethanol to obtain a phospholipid solution;

[0016] S2. Dissolve triptolide in ethanol, and then mix it with the phospholipid solution prepared in step S1 to obtain a phospholipid solution containing triptolide;

[0017] S3. Dissolve ITGB1-siRNA in sodium citrate buffer solution, and mix it with the phospholipid solution containing triptolide prepared in step S2 based on microfluidics method. After dilution, concentration and centrifugation with PBS buffer solution, the triptolide-modified lipid nanoparticles loaded with integrin β1 siRNA are obtained;

[0018] S4. Incubate and couple the anti-Flt1 peptide with the triptolide-modified lipid nanoparticles loaded with integrin β1 siRNA prepared in step S3 to obtain the product.

[0019] Among them, in step S1, the molar ratio of ionizable cationic lipid, co-lipid, cholesterol and polyethylene glycolylated lipid is 45-55:35-40:5-15:0.5-5, preferably 50:38.5:10:1.5;

[0020] In step S2, the concentration of triptolide in the ethanol solution is 50-150 μg / mL, preferably 100 μg / mL.

[0021] In step S3, the concentration of ITGB1-siRNA in sodium citrate buffer solution is 0.01-0.2 mg / mL, preferably 0.087 mg / mL;

[0022] The concentration is carried out using a 50-200KD ultrafiltration tube, and among them, the 100KD ultrafiltration tube concentration is preferred.

[0023] The specific centrifugation conditions are: centrifuge at a rotation speed of 3000-8000 revolutions per minute for 1-30 minutes, preferably centrifuge at a rotation speed of 5000 revolutions per minute for 10 minutes to effectively remove free triptolide.

[0024] In step S4, the anti-Flt1 peptide contains a linker peptide, so it is easier to couple with the lipid nanoparticles, and the amino acid sequence of the linker peptide can be GGGSC.

[0025] In a third aspect of the present invention, there is provided the use of the above ITGB1-siRNA lipid nanoparticles in the preparation of a drug for anti-corneal neovascularization. The present invention has demonstrated through experiments that ITGB1-siRNA lipid nanoparticles can effectively inhibit CNV by blocking the activation of the PI3K / AKT and NF-κB pathways and reducing the expression of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α.

[0026] In a fourth aspect of the present invention, there is provided a drug for anti-corneal neovascularization, and the active ingredient of the drug for corneal neovascularization comprises the above ITGB1-siRNA lipid nanoparticles.

[0027] According to the present invention, the drug further comprises at least one pharmaceutically inactive ingredient.

[0028] The pharmaceutically inactive ingredient may be a carrier, excipient, diluent, etc. commonly used in pharmacy. Moreover, according to common methods, it can be formulated into oral preparations such as powders, granules, suspensions, emulsions, syrups, sprays, etc., topical preparations, and dosage forms in the form of sterile injection solutions for use.

[0029] The carrier, excipient, diluent, etc. that may be included as non-pharmaceutical active ingredients are well-known in the art, and those of ordinary skill in the art can determine that they meet clinical standards.

[0030] In yet another specific embodiment of the present invention, the carrier, excipient, and diluent include but are not limited to lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl paraben, propyl paraben, talc, magnesium stearate, and mineral oil, etc.

[0031] In yet another specific embodiment of the present invention, the drug of the present invention can be administered into the body by known means. For example, it can be delivered systemically via intravenous injection or locally by instillation (such as ocular instillation) into the tissue of interest (such as the cornea). Such administration can be carried out via a single dose or multiple doses. Those skilled in the art understand that the actual dose to be administered in the present invention can vary to a large extent depending on various factors such as the target cell, biological type or its tissue, the general condition of the subject to be treated, the route of administration, the mode of administration, and so on.

[0032] In yet another specific embodiment of the present invention, the subjects to which the drug is administered can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, chimpanzees, etc. Among them, humans are preferred.

[0033] In particular, the drug of the present invention can be used as a drug dosage form of eye drops, which has the advantages of non-invasiveness, convenience, and effectiveness.

[0034] The fifth aspect of the present invention provides a method for treating corneal neovascularization and its related diseases, the method comprising administering to a subject a therapeutically effective dose of the above-mentioned ITGB1-siRNA lipid nanoparticles or an anti-corneal neovascularization drug.

[0035] The subject refers to an animal that has been the subject of treatment, observation or experiment, preferably a mammal, and most preferably a human. The "therapeutically effective amount" refers to the amount of an active compound or agent, including the compounds of the present invention, which amount can elicit a biological or medical response in a tissue system, animal or human that is sought by a researcher, veterinarian, physician or other medical personnel, which includes alleviating or partially alleviating the symptoms of the disease, syndrome, disorder or condition being treated. It must be recognized that the optimal dosage and dosing interval of the active ingredient of the present invention are determined by its nature and external conditions such as the form, route and site of administration and the particular mammal being treated, and this optimal dosage can be determined by conventional techniques. It must also be recognized that the optimal course of treatment, i.e., the daily dose of the compound over a specified period of time, can be determined by methods well known in the art.

[0036] In the present invention, the corneal neovascularization-related diseases include but are not limited to keratitis (such as bacterial keratitis, fungal keratitis, viral keratitis, etc.), corneal ulcer, scleritis / uveitis, allergic eye diseases, diabetic corneal diseases, etc., and are not specifically limited herein.

[0037] Advantageous technical effects of the above one or more technical solutions:

[0038] The above technical solution provides a novel, CNV-targeted icLNPs, which are loaded with Itgb1-siRNA and modified with TP. The lipid nanoparticles effectively inhibit CNV by blocking the activation of the PI3K / AKT and NF-κB pathways and reducing the expression of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α.

[0039] In addition, the lipid nanoparticles prepared above can be used as eye drops, which have been proven to be a non-invasive, convenient and effective treatment means with extremely low cytotoxicity and high biosafety in vivo; at the same time, it shows an effective therapeutic inhibitory effect on CNV in vivo. Therefore, the lipid nanoparticles developed by the present invention are expected to become a new option for treating CNV-related ocular diseases, and thus have good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0041] Figure 1 Characterization of Itgb1-siRNA@TPL in the embodiments of the present invention

[0042] A. Schematic diagram of the preparation process of Itgb1-siRNA@TPL. B-E. Representative transmission electron microscopy (TEM) images, particle size and polydispersity of Itgb1-siRNA@TPL (siRNA@TPL) and SM-102LNPs loaded with Itgb1-siRNA (siRNA@SM). TEM scale bar: 100 nm. F. Agarose gel electrophoresis shows that Itgb1-siRNA was successfully encapsulated in TPL and SM-102LNPs. 2% agarose gel was electrophoresed in 1× Tris-acetate-ethylenediaminetetraacetic acid buffer at 120 V for 40 min. G, H. Zeta potential of representative siRNA@TPL and siRNA@SM LNPs, indicating that they are nearly electrically neutral. I. Release curve of TP of siRNA@TPL in PBS, 1% triton X-100 solution and cytoplasmic solution containing 1% triton X-100 solution. The cytoplasmic solution containing 1% triton X-100 was used to simulate the TP release of siRNA@TPL in cells. J, K. Fluorescence microscopy images and flow cytometry analysis show the cellular uptake of siRNA@TPL (siRNA-cy3@TPL) and siRNA@SM-102LNPs (siRNA-cy3@SM) by C166 cells. The cellular uptake of siRNA@TPL was much higher than that of siRNA@SM or siRNA-cy3. The incubation time was 24 hours. ns: no significant difference; *p<0.05; ***p<0.001.

[0043] Figure 2 Single-cell sequencing analysis of mouse CNV in the embodiments of the present invention

[0044] A. UMAP plot of the single-cell transcriptome dataset (n = 20098 cells), showing 19 different cell clusters. B. Results of clustering analysis using the clusterree R package with resolution parameters ranging from 0 to 0.5 at intervals of 0.1. C. Determination of cell types for each cell cluster based on the expression levels of known representative cell markers. D, E. Distribution (D) and proportion (E) of each cell type in the corneas of CNV mice and NC mice. F. Differences in cell-cell communication in the corneas of CNV group and NC group mice indicate that integrin plays an important role in CNV. G. Comparison of significant ligand-receptor pairs related to Itga1, Itgb1, Itga6 and Itgb4 in the CNV group and NC group. The color of the dots reflects the communication probability, and the size of the dots represents the calculated p-value.

[0045] Figure 3 In the examples of the present invention, to evaluate the anti-angiogenic effects of Itbg1, Itgb4, Itga1, and Itga6 gene knockdown. A, B. Results of Transwell assay (A) and scratch assay (B) showed that knockdown of Itgb1, Itgb4, Itga1, and Itga6 genes inhibited the migration of HUVECs. C, D. CCK8 (C) and EdU staining (D) were used to evaluate the inhibitory effect of knocking down Itgb1, Itgb4, Itga1, and Itga6 genes on the proliferation of HUVECs. E. Representative images of tube formation assay of HUVECs in the NC group and the groups with knockdown of Itgb1, Itgb4, Itga1, or Itga6 genes. F-H. Clinical evaluation of the effect of Itgb1 gene knockdown on suture-induced CNV in mice. Representative ocular surface images before and on the 3rd, 7th, and 11th days after modeling (F). Vessel length (G) and CNV area (H) were measured on the 7th and 11th days. Results are presented as mean ± standard error of the mean (SEM; n = 10). Between each designated group: *p < 0.05, **p < 0.01, ***p < 0.001.

[0046] Figure 4 In the examples of the present invention, to evaluate the vec targeting ability of LNPs conjugated with anti-flt1 peptide

[0047] A. Single-cell sequencing analysis showed high expression of Flt1 in VECs and pericytes of CNV mice. B, C. Immunofluorescence and western blot assays showed a significant upregulation of the expression of FLT1 (VEGFR1) in CNV. Scale bar: 50 μm. D-F. Fluorescence microscopy images and flow cytometry analysis showed that anti-Flt1 peptide significantly enhanced the uptake of LNPs by C166 cells. Scale bar: 20 μm. G. The knockdown efficiency of siRNA against Itgb1 at different concentrations (10, 20, 50, and 100 nM) was evaluated using qRT-PCR. H. Comparison of the knockdown efficiency of Itgb1 siRNA@SM (siRNA@SM) loaded with and without anti-Flt1 peptide. I-L. Inhibitory effects of LNPs loaded with Itgb1-siRNA at different concentrations (10, 20, 50, and 100 nM) on the proliferation and migration of C166 cells. Scale bar for EdU staining: 100 μm; scale bar for scratch assay: 200 μm; scale bar for Transwell assay: 50 μm. M. Cytotoxicity of LNPs loaded with Itgb1-siRNA at different concentrations (20, 50, 100, and 200 nM) against C166 cells. *p < 0.05.

[0048] Figure 5 In the examples of the present invention, to screen the optimal concentration of TP-modified LNPs

[0049] A. Unsupervised subclustering analysis of VECs using UMAP. B. Heatmap of the top three differentially expressed genes in each subgroup, showing distinguishable transcriptomic features among the seven cell subgroups. C. Proportion of cells in each subcluster of the CNV group and the NC group. D-F. Single-cell sequencing and western blot experiments showed that the expression levels of inflammatory cytokines IL-1β, IL-6, and TNF-α in the CNV group were significantly higher than those in the NC group. G, H. qRT-PCR and ELISA assays showed that SM-102 LNPs could upregulate the expression of IL-1β, IL-6, and TNF-α in the mouse ocular surface. I. Evaluation of the cytotoxicity of TP at different concentrations (5, 10, 15, and 20 ng / mL) on C166 cells using flow cytometry. J-L. Effects of TP at different concentrations (5, 10, and 15 ng / mL) on the proliferation (J and K) and migration (L) of C166 cells. M. Evaluation of the inhibitory effect of TP at different concentrations (5, 10, and 15 ng / mL) on the production of inflammatory cytokines IL-1β, IL-6, and TNF-α using qRT-PCR and ELISA. *p < 0.05, **p < 0.01. Figure 6 Biological safety and efficacy evaluation of Itgb1-siRNA@TPL in the treatment of CNV in the embodiments of the present invention A, B. Evaluation of the cytotoxicity of Itgb1-siRNA@TPL modified with 10 ng / mL [TPL(10)] or 15 ng / mL [TPL(15)] TP on C166 cells using calcein-AM / propidium iodide staining and flow cytometry. C. Slit lamp and AS-OCTA images of CNV in each group before and on the 3rd, 7th, 11th, and 14th days after modeling. D. Vascular length and area of CNV in each group on the 7th and 14th days after modeling. E, F. Changes in corneal epithelial injury area in the corneal epithelial injury models of different groups. G. Hematoxylin and eosin (H&E) staining images of the major organs and cornea of normal mice and mice treated with siRNA@TPL(10) for 14 days. *p < 0.05, **p < 0.01, ***p < 0.001.

[0050] Figure 7In the embodiments of the present invention, Itgb1-siRNA@TPL inhibits CNV through the PI3K / AKT and NF-κB signaling pathways. Heatmap of the top 20 differentially expressed genes in VECs of the CNV group and the control group. B. KEGG analysis of differentially expressed genes between VECs of the CNV group and the control group. C. Protein levels of p-AKT, p-PI3K, p-NF-κB p65 (p-p65), AKT, PI3K, NF-κB, IL-1IL-1β, IL-6, and TNF-α in the CNV group, the Itgb1-siRNA@TPL group, and the NC group. D. Western blot shows the inhibitory effect of Itgb1-siRNA@TPL on AKT activation induced by UCL-TRO-1938 (UCL). E. Representative immunofluorescence images of p-AKT in the UCL group and the UCL+siRNA@TPL group. Scale bar: 50 μm. F, G. CCK8 and EdU staining assays show the inhibitory effect of siRNA@TPL on the proliferation of C166 cells induced by UCL. H. Transwell assay and scratch assay show that siRNA@TPL significantly inhibits the migration of C166 cells induced by UCL. Scale bar for Transwell assay: 50 μm; scale bar for scratch assay: 200 μm. I. Western blot shows the inhibitory effect of Itgb1-siRNA@TPL on NF-κB activation induced by LPS. J. Representative immunofluorescence images of p-NF-κB p65 (p-p65) in the LPS group and the LPS+siRNA@TPL group. Scale bar: 50 μm. K, L. qRT-PCR and ELISA are used to detect the inhibitory effect of siRNA@TPL on the expression of inflammatory cytokines IL-1β, IL-6, and TNF-α induced by LPS. *p<0.05, **p<0.01, ***p<0.001. Detailed Description of the Invention

[0051] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the present invention belongs.

[0052] It should be noted that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the exemplary embodiments of the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprises" and / or "includes" are used in this specification, they specify the presence of the described features, steps, operations, devices, components, and / or combinations thereof.

[0053] The present invention will be further described below in conjunction with specific examples. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not specified in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0054] The present invention will be further explained and illustrated below by way of examples, but this does not constitute a limitation to the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The test methods for which specific conditions are not indicated in the following examples are usually carried out under conventional conditions.

[0055] Example

[0056] 1. Materials and Methods

[0057] 1.1. Materials

[0058] SM-102 (HY-134541), ALC-0315 (HY-138170) and D-Lin-MC3-DMA (HY-112251) were purchased from MCE. DSPC (D130429) and DSPE-PEG-MAL (D163618) were purchased from Aladdin. DMSO (D8418), collagenase IV (C4-BIOC) and collagenase I (SCR103) were purchased from Sigma. Human umbilical vein endothelial cells (HUVECs) and mouse vascular endothelial cell line (C166) were purchased from ATCC. The anti-Flt1 peptide (GGNQWFI) with GGGSC linker was synthesized by Sangon Biotech. Other chemical reagents were purchased from Sinopharm Group. Anti-ITGB1 antibody (34971T), anti-phosphorylated PI3K antibody, anti-AKT antibody (9272S) and anti-phosphorylated AKT antibody (4060T) were purchased from Cell Signaling Technology; anti-NF-κB antibody (ab207297) and anti-phosphorylated NF-κB p65 antibody (ab239882) were purchased from Abcam; anti-CD31 antibody (GB12063) was purchased from Wuhan Sevier Biotechnology Co., Ltd.; anti-PI3K antibody (A19742), anti-IL-6 antibody (A22222), anti-IL-1β antibody (A16288), anti-TNFα antibody (A23264) and anti-β-actin antibody (AC026) were purchased from Abcam. Secondary antibodies, including FITC-labeled donkey anti-rabbit IgG (H+L) (AS042) and Cy3-labeled goat anti-mouse IgG (H+L) (AS008), were both purchased from Abcam; DMEM medium (11965092), DMEM / F12 medium (11320033) and FBS (10099141C) were purchased from Thermo Fisher Scientific. Cell Counting Kit-8 (G4103) and fluorescein isothiocyanate-phalloidin (G1248) were purchased from Wuhan Sevier Biotechnology Co., Ltd.; triptolide (TP, B20709) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. RNeasy Mini Kit and Prime Script TM RT Master Mix Kit were purchased from Qiagen and Takara Biotechnology (Beijing) Co., Ltd., respectively.

[0059] 1.2. Synthesis and characterization of tgb1-siRNA@TPL

[0060] icLNPs (ionizable cationic lipids) were prepared using microfluidic technology. The raw materials were weighed according to the molar ratio of ionizable cationic lipids (including SM-102, ALC-0315, and D-Lin-MC3-DMA): cholesterol: DSPC: DSPE-PEG2000 = 50:38.5:10:1.5, and dissolved in absolute ethanol to make the final concentration of icLNPs reach 12 mmol / L, forming a colorless and transparent solution for standby. Next, 5 mg of TP was weighed and dissolved in absolute ethanol to prepare a solution with a concentration of 100 μg / mL. This TP solution was mixed with 510 μL of phospholipid solution, and then absolute ethanol was added to make the final phospholipid concentration reach 1 μmoL / L. The siRNA was diluted to 0.087 mg / mL with 50 μmoL / L sodium citrate buffer at pH 4.0. Using a microfluidic device, the nucleic acid sodium citrate buffer and the phospholipid solution containing TP were mixed at different volume ratios. The mixed solution was diluted 30 times with 10 mmol / L PBS (pH 7.4), and then concentrated with a 100KD ultrafiltration tube and centrifuged at a speed of 5000 revolutions per minute for 10 minutes to remove free triptolide (TP). The finally concentrated solution was the triptolide-modified lipid nanoparticles loaded with integrin β1 siRNA. Finally, 50 μg of anti-Flt1 peptide (containing a linker, sequence: GNQWFI-GGGSC) was added to the nanoparticle complex suspension and incubated with rotation at room temperature for 2 hours to obtain the nanoparticle complex Itgb1-siRNA@TPL. The lipid nanoparticle siRNA@SM was prepared from the ionizable cationic lipid SM-102 and was not modified with TP and anti-Flt1 peptide.

[0061] After resuspending the nanoparticles in deionized water, the average particle size and polydispersity index (PDI) were measured using a Zetasizer Lab instrument from Malvern Panalytical. The structure of Itgb1-siRNA@TPL was observed using a transmission electron microscope (JEOL JEM-2100 type from JEOL Ltd.) with an accelerating voltage of 200 keV.

[0062] 1.3. Animals

[0063] All animal experiment operations were carried out in accordance with the principles of the "Statement on the Use of Animals in Ophthalmic and Vision Research" issued by the Association for Research in Vision and Ophthalmology (ARVO) in the United States and were approved by the Animal Care and Use Committee of the author's institution. 8-week-old male C57BL6 / J mice were purchased from Jinan Qinglongshan Experimental Animal Center in China, and the breeding environment was maintained with a cycle of 12 hours of light and 12 hours of darkness. Before the experiment, slit lamp microscopy examinations were performed on all of them to exclude corneal abnormalities, lens damage and dislocation, vitreous hemorrhage, and iris hemorrhage and adhesions, etc.

[0064] 1.4. Establishment of the CNV model

[0065] Refer to the literature to construct a suture-induced CNV mouse model. The mice were anesthetized by intraperitoneal injection of sodium pentobarbital injection (0.2 mL / 100 g) and topical application of 0.5% proxymetacaine hydrochloride eye drops. Three 10-0 nylon sutures were placed into the corneal stroma approximately 1 mm from the pupil center without burying the knots. Levofloxacin eye drops (4 times a day) were used to prevent infection 1 week after surgery. CNV evaluation was performed according to the literature report. Slit lamp examination was performed on days 3, 7, 11, and 14 after modeling to detect the position of the corneal suture, corneal infection, and the growth of CNV. An effective experimental model was selected based on the growth and stability of CNV. Image J software was used to measure the length, diameter, and area of CNV in the animal model.

[0066] 1.5 Evaluation of CNV

[0067] Before treatment and on days 3, 7, 11, and 14 after treatment, the corneal neovascularization of the mice was evaluated by slit lamp and optical coherence tomography (DREAM OCT of China Interlight Co., Ltd.). TM ) The area of CNV and the length of the longest neovascularization were quantified with reference to the literature report. The area of CNV was calculated according to the following formula: Area of CNV = C / 12 × 3.14 × [R 2 - (R - L) 2 , where C is the number of clock hours occupied by corneal neovascularization, L is the length of the longest blood vessel from the corneal limbus, and R is the radius of the cornea.

[0068] 1.6 Single-cell RNA sequencing

[0069] 1.6.1 Isolation of single cells from mouse anterior corneal limbus tissue

[0070] We observed that dense neovascularization formed on the corneas of mice 7 days after modeling. Therefore, on the 7th day after modeling, the mice were euthanized by overdose anesthesia, and the limbal tissues (within 1 mm inside and outside the limbus) were immediately removed under a microscope. This tissue included the peripheral cornea and the conjunctiva and scleral tissues adjacent to the limbus. Then, the posterior one-third of the obtained tissues (including corneal endothelium, Descemet's membrane, and the posterior parts of corneal stroma and sclera) was removed. Each remaining limbal tissue was evenly cut into 4 pieces and incubated in DMEM / F12 medium containing 3% collagenase IV and 2% collagenase I at 37°C for 30 minutes. Subsequently, it was digested 3 times with 2 mL of 0.25% trypsin / 35 mmol / L EDTA at 37°C for 5 minutes each time. The cell suspension after each digestion was transferred to another centrifuge tube, and the digestion was terminated with medium containing 10% fetal bovine serum. The single-cell suspension was centrifuged at 1000 revolutions per minute and resuspended in PBS at 4°C. The proportion and viability of single cells were detected using a fluorescence cell counter (Beijing EkoTec Biotechnology Co., Ltd.). Normal mouse corneas treated in the same way were used as controls.

[0071] 1.6.2. Single-cell RNA sequencing and analysis

[0072] Single cells from the CNV group and the control group were respectively processed for library construction on the 10×Genomics system. After constructing the cDNA library, quality inspection (using FragmentAnalyzer 2100 from Agilent Technologies) and sequencing were performed (sequencing platform: Illumina NovaSeq 6000; read length: 150 bp, paired-end sequencing). The raw data was processed with default parameters using Cell Ranger (version 7.1.0, 10xGenomics) software and aligned with the mouse mm10 genome. Subsequently, we used the R package Seurat (version 4.3.2) for subsequent quality control, normalization, dimensionality reduction, clustering, and downstream analysis. The data was normalized using the NormalizaData function in the R package Harmony (version 4.3.2). By the variance-stabilizing transformation method, the top 2000 variable genes were selected as the input for the "FindVariableFeatures" function. The gene expression values were scaled using the "ScaleData" function. For cell clustering, principal component analysis (PCA) was performed on the highly variable genes. Through the "FindClusters" function, the top 15 principal components (PCs) were clustered at a resolution of 0.5. Next, the UMAP function was used to visually display the clustering results of the samples. The "FindAllMarkers" function combined with the Wilcoxon rank-sum test was used for single-cell gene expression analysis to identify marker genes.

[0073] Cell - cell communication analysis was performed using the R package Cellchat (version 4.3.2). The CellChatDB database was applied to analyze the secreted signals, extracellular matrix - receptor interactions, and cell - cell communication among different cell clusters. In these cell clusters, overexpressed ligands and receptors were identified. The netVisual_bubble tool was used to visually display the significant ligand - receptor interactions between target cells.

[0074] 1.7. Cell transfection

[0075] Short hairpin RNAs (shRNAs) targeting integrin β1 (Itgb1), integrin α1 (Itga1), integrin α6 (Itga6), and integrin β4 (Itgb4) were cloned into the plko - 1 plasmid. A scrambled shRNA provided by Addgene that does not target any known genes in the human and mouse genomes was used as a control. Two target - specific shRNAs were used for knockdown of each gene, and each shRNA was used alone. When the cell confluence reached approximately 80%, the cells were transfected with lentiviral particles encoding the specific shRNA for 24 hours. For small interfering RNA (siRNA) transfection, Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) was used according to the manufacturer's protocol, and the incubation time was 24 hours.

[0076] After cell transfection, positive cells were selected with 2 μg / mL puromycin. Then total RNA was extracted, and real - time quantitative PCR was performed to detect the expression of the target gene. The gene expression levels were normalized to the expression level of β - actin. The shRNAs, siRNAs, and sequence information used in this invention are shown in Table 1.

[0077] Table 1

[0078]

[0079] 1.8. Toxicity test

[0080] The cytotoxicity of Itgb1 - siRNA@TPL and TP was evaluated by CCK - 8 assay and flow cytometry. For the CCK - 8 assay, HUVECs or C166 were seeded at 1×10 per well 4Cells were seeded at a density of [X] cells per well in a 96-well plate, and 100 μL of medium was added to each well and incubated for 24 hours. Then, the cells were treated with different concentrations of Itgb1-siRNA@TPL or TP for 24 hours. Untreated cells were used as controls. Subsequently, 10 μL of CCK-8 solution was added to each well. After incubation in the incubator for 2 hours, the absorbance at 450 nm was measured using an absorption spectrophotometer (Thermo Fisher Scientific). For flow cytometry, cells were collected by trypsin digestion according to the standard cell culture protocol. After washing and centrifuging at 1000 rpm for 5 minutes with PBS, the cells were resuspended in 250 μL of PBS containing 2% bovine serum albumin (BSA) and 1 mmol / L EDTA. [X] cells were collected and the data obtained were analyzed using a Beckman flow cytometer (Beckman Coulter Life Sciences). 4 cells, and the data obtained were analyzed using a Beckman flow cytometer (Beckman Coulter Life Sciences).

[0081] For in vivo toxicity assays, a mouse corneal epithelial injury model was established. Half of the corneal surface and adjacent limbus were scraped using a corneal rust ring remover (product of Alger, Lago Vista, Texas, USA). Then, Itgb1-siRNA@TPL eye drops were instilled into the scratched corneal epithelium eyes 4 times a day. Dexamethasone (1 mg / mL) (DEX) and PBS were used as controls. Corneal fluorescein staining and slit lamp photography were performed to detect the degree of corneal epithelial defects. 1.9. Transwell assay

[0082] Cell migration analysis was performed using a Transwell chamber (product of Corning, Corning, NY, USA). After suspending the cells in serum-free medium, 3×10 4 cells were placed in the upper chamber of the Transwell chamber, and DMEM medium containing 10% serum was added to the lower chamber. After incubation for 48 hours, the cells on the lower surface of the membrane were fixed with 4% paraformaldehyde and stained with crystal violet at room temperature for 20 minutes. Subsequently, the number of cells on the lower side of the filter membrane was counted under a microscope (Olympus). Each experiment was repeated three times.

[0083] 1.10. Scratch assay

[0084] Cells were seeded in a 6-well plate and cultured until the cells covered the entire well. A scratch was made on the cell layer using a 200 μL pipette tip. After washing three times with PBS, the cells were treated with medium supplemented with Itgb1-siRNA@TPL or TP, and then the culture plate was placed in an incubator at 37°C and 5% CO2. Cells cultured in normal medium were used as controls. Images were collected at 0 hours and 24 hours after scratching.

[0085] 1.11. Western blot assay

[0086] Total proteins were extracted from mouse corneal tissues and vascular endothelial cells, and the protein concentration was measured using a BCA kit (Beyotime). 30 μg of protein samples were taken and separated by 6%-15% SDS-PAGE, and then transferred onto a PVDF membrane (Beyotime). Next, the PVDF membrane was blocked in a 5% milk solution and then bound to the primary antibody. Then, the membrane was bound to a secondary antibody labeled with horseradish peroxidase (HRP). Finally, a molecular imaging system (Bio-Rad) was used to detect the bands.

[0087] 1.12. Real-time quantitative polymerase chain reaction (qRT-PCR)

[0088] qRT-PCR was used to measure the mRNA expression levels in cells or limbal tissues. According to the manufacturer's instructions, RNA was extracted using an RNeasy Mini kit. The RNA concentration was measured using a Nanodrop 2000 system (Thermo Fisher Scientific), and then 1 μg of RNA was taken and cDNA synthesis was performed using HiScript II reverse transcriptase (Vazyme). qRT-PCR was carried out using a SYBR Green PCR premix in an ABIStepOnePlus system (Applied Biosystems). The RNA expression levels of the target genes were normalized to GAPDH and analyzed using the 2-ΔΔCt method. Each experiment was independently performed three times.

[0089] 1.13. H&E staining and immunofluorescence (IF)

[0090] Mouse eyeballs were removed and fixed overnight in 4% paraformaldehyde at 4°C, and then dehydrated and paraffin-embedded. Then, the tissues were sectioned into 5-μm sections using a microtome. H&E staining: After deparaffinization of tissue sections, they were stained with hematoxylin and eosin, and then images were captured using a microscope imaging system (Olympus). Immunofluorescence (IF) staining: After deparaffinization of paraffin sections, they were first incubated in 5% goat serum for 1 hour to block non-specific binding sites, and then incubated with the primary antibody overnight at 4°C. The next day, the sections were washed with PBS and incubated with a fluorescein-labeled secondary antibody at 37°C for 1 hour. Subsequently, the nuclei were counterstained with DAPI for 5 minutes. Then, a Zeiss Cell discoverer 7 confocal laser scanning microscope was used to analyze the fluorescence intensity.

[0091] For cell samples, they were fixed in 4% paraformaldehyde at room temperature for 15 minutes and then permeabilized with a PBS solution containing 0.3% Triton X-100 for 20 minutes. The subsequent steps were the same as those for tissue immunofluorescence staining.

[0092] 1.14. Enzyme-linked immunosorbent assay (ELISA)

[0093] According to the manufacturer's instructions (eBioscience), the levels of inflammatory cytokines IL-1β, TNF-α, and IL-6 in corneal extracts or cell supernatants were measured by ELISA using an absorption spectrophotometer (Thermo Fisher Scientific). The levels of inflammatory cytokines were then normalized relative to the total protein level, and a standard curve was plotted based on the measured values of the diluted standard solution. By comparing with this curve, the levels of inflammatory cytokines in each sample were determined.

[0094] 1.15. Statistical analysis

[0095] Data are representative results of three independent experiments and are expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS version 19 software (IBM Corporation, Armonk, NY, USA), and p < 0.05 was considered statistically significant.

[0096] 2. Results and discussion

[0097] 2.1 Synthesis and characterization of siRNA@TPL

[0098] The emergence of LNPs (lipid nanoparticles) has provided new therapeutic directions for modern medicine. LNPs are usually composed of four components with different functions: cationic lipids, helper lipids, cholesterol, and polyethylene glycolylated lipids. Among these four components, cationic lipids are the most important for achieving in vivo gene transfection efficiency. So far, icLNPs such as the cationic lipids SM-102 and ALC-0315 have been approved by the FDA for clinical use due to their excellent nucleic acid delivery performance and biosafety. Therefore, we developed an icLNP modified with TP and anti-Flt1 peptide to reduce inflammation and improve the targeting to CNV. In this study, this icLNP was used to deliver integrin β1 small interfering RNA (Itgb1-siRNA) to target cells for the treatment of CNV ( Figure 1 A). To prepare Itgb1-siRNA@TPL, we investigated three representative ionizable cationic lipids; the uptake rates of these lipids by HUVECs and C166 cells were detected by flow cytometry and fluorescence microscopy. Among these three lipids, we found that SM-102, as an ionizable cationic lipid for LNPs, showed the highest cell uptake rate. Therefore, SM-102 was used as the ionizable cationic lipid to modify the nanoparticles.

[0099] Nanoparticle delivery systems with particle sizes in the range of 20 - 200 nm are considered to enter target cells more effectively than those with other particle sizes. Additionally, a narrow particle size distribution (polydispersity index [PDI] ≤ 0.5) is required to prepare a safe, stable, and effective formulation. Itgb1-siRNA@TPL prepared in this study showed a uniformly dispersed and regular spherical conformation in the dry state under transmission electron microscopy ( Figure 1 B), with a particle size of 110 ± 5.897 nm detected by dynamic light scattering ( Figure 1 C and D), and a PDI of approximately 0.3 ( Figure 1 E). In summary, these findings indicate that although the particle size and polydispersity index of Itgb1-siRNA@TPL are slightly larger than those of the SM102 lipid nanoparticles loaded with Itgb1-siRNA (Itgb1-siRNA@SM), it may have good stability and a relatively high cellular uptake rate. The siRNA encapsulation efficiency detected by a Qubit 4 fluorometer was 99.9%, and agarose gel electrophoresis also confirmed the successful complexation of LNPs with siRNA ( Figure 1 F). Compared with cationic lipids, ionizable cationic lipids with a neutral charge at physiological pH can minimize cytotoxicity and may be protonated and positively charged in the acidic environment in vivo to facilitate endosomal escape. Therefore, we measured the zeta potential of siRNA@TPL, which showed no significant difference from that of Itgb1-siRNA@SM ( Figure 1 G and H). This finding indicates that Itgb1-siRNA@TPL is safe for in vivo use. The next key step is to ensure the effective release of TP and the delivery of siRNA to the cytoplasm to exert its therapeutic effect. First, the release of TP from Itgb1-siRNA@TPL was evaluated. In PBS, almost no TP was released within 4 days; however, rapid release of TP was observed in the cytoplasm extracted from C166 cells after demulsification, and more than 60% of the TP in Itgb1-siRNA@TPL was released within 4 hours ( Figure 1 I). This release pattern suggests that Itgb1-siRNA@TPL may have good therapeutic effects in target cells and reduce the risk of TP release at unintended sites. Effective delivery of siRNA to target cells is crucial for achieving therapeutic goals, and VECs are the main effector cells in CNV. Therefore, the cellular uptake rate of Itgb1-siRNA@TPL was evaluated in C166 cells. Fluorescence microscopy and flow cytometry analysis showed that the uptake rate of Itgb1-siRNA@TPL in C166 cells was much higher than that of Itgb1-siRNA@SM ( Figure 1(J-K). This may be due to the binding of anti-Flt1 peptide in siRNA@TPL, indicating that Itgb1-siRNA@TPL may be more effective in treating CNV.

[0100] 2.2 Single-cell RNA sequencing shows that integrin is involved in the CNV process

[0101] In recent years, the molecular mechanism of CNV has been widely and deeply studied. Many signaling molecules related to angiogenesis and inflammatory processes, such as VEGF, basic fibroblast growth factor (bFGF), matrix metalloproteinases, hypoxia-inducible factor (HIF), and IL-1, have been identified as pro-angiogenic factors in this process. However, the regulatory network involved in CNV remains not fully clear. Single-cell RNA sequencing technology can characterize different cell populations, functional transcriptome maps, and cell-cell communication. This technology has been widely used in the study of various ophthalmic diseases, including glaucoma, diabetic retinopathy, and age-related macular degeneration, to explore potential pathogenic factors and therapeutic targets.

[0102] To deeply explore the regulatory network in the CNV process, we performed single-cell RNA sequencing on a mouse CNV model. The Seurat software package was used for data preprocessing, and the UMAP method was used for non-linear dimensionality reduction. Through this unbiased analysis, we identified 19 different cell clusters ( Figure 2 A). The clusterree R package was used to group similar cell clusters and clarify their lineage directions, providing more information for the identification of cell types ( Figure 2 B). According to the curated classical markers, these cells were divided into 10 cell lines, including corneal epithelial cell line (Krt12 + , cell clusters 1, 2, 3, 7, and 15), conjunctival epithelial cell line (Krt13 + and Krt4 + , cell cluster 6), corneal stromal cell line (Lum + , cell clusters 5, 8, and 11), monocytes (Itgam + , Tnf + and Mrc1 + , cell cluster 9), T cells (Cd3e + and Cd4 + , cell cluster 10), neutrophils (S100a8 + , S100a9 + and CD14 + , cell clusters 0 and 17), dendritic cells (Itgax + , Cd83 + and Cd80 + , cell cluster 13), vascular endothelial cells (Eng+ , Tek + and Vwf + , cell cluster 12), pericytes (Rgs + and Mcam + , cell cluster 16) and lymphatic endothelial cells (Lyve1 + , cell cluster 18)( Figure 2 C). Cell clusters 1, 3, 7, and 15 expressed the limbal stem cell marker Trp63 and were identified as epithelial stem cells. Cell clusters 1 and 3 with high expression of Ck14 were identified as corneal epithelial basal cells, while cell clusters 7 and 15 were identified as limbal stem cells. Cell clusters 4 and 14 co-expressed Krt13 and Krt12, and we speculated that they might be corneal or conjunctival epithelial cells, but the specific cell types have not been fully determined. Cell cluster 5 expressing Kera was identified as corneal stromal cells. Cell clusters 8 and 11 did not express Kera and were defined as corneal stromal fibroblasts. Subsequently, we analyzed the differences in cell populations between the CNV group and the normal control group (NC group) and found that inflammatory cells such as neutrophils, monocytes, and T cells were significantly increased in the CNV group. This indicates that inflammation plays an important role in the CNV process ( Figure 2 D and E).

[0103] Intercellular communication plays a crucial role in the activation of VEC and the CNV process. During the occurrence of CNV, corneal epithelial cells, inflammatory cells, and fibroblasts produce a large amount of pro-angiogenic vascular endothelial growth factor (VEGF), and the expression of VEGF receptors in VEC is significantly increased. In addition, in patients with corneal alkali burns or those who have received corneal transplants, basic fibroblast growth factor (bFGF) derived from the corneal epithelium, stromal layer, and endothelium directly activates corneal VEC and promotes the occurrence of CNV. Therefore, we used the CellChat R software package to perform intercellular communication analysis to detect changes in intercellular signals during the CNV process. We set VEC as the target cell and visualized the intercellular communication between each cluster of cells and the target cell. The results showed that there were significant differences in integrin-related signals between the CNV group and the control group ( Figure 2 F). It has been reported in the literature that integrin regulates angiogenesis by interacting with various extracellular molecules, transmits signals in a bidirectional manner, and acts as a "hub" during angiogenesis to coordinate the behavior of endothelial cells and vascular smooth muscle cells. The binding of integrin to growth factors (such as VEGF, FGFs, and angiopoietin-1) or their receptors (VEGF receptor 2 and FGF receptor) can stimulate blood vessel growth. Subsequently, we found that in the CNV group, the intercellular signals related to four integrins (Itga1, Itgb1, Itga6, and Itgb4) were significantly upregulated; among them, the signal changes involving Itgb1 were the most significant ( Figure 2 G).

[0104] To explore the functions of these four integrins in angiogenesis, we performed gene knockdown experiments in C166 cells. Transwell assays and scratch assays showed that knockdown of these four integrin genes significantly inhibited the migration of HUVECs ( Figure 3 A and B). Subsequently, we used the CCK-8 assay and EdU to detect the effects of these four integrins on the proliferation of HUVECs. Knockdown of these integrin genes slowed down cell proliferation; however, in the CCK-8 assay, there was no significant difference between the ITGA6 or integrin ITGB4 group and the control group (p>0.05)( Figure 3 C and D). In addition, we observed that the tube formation ability of HUVECs was also impaired after knockdown of these integrins ( Figure 3 E). Notably, knockdown of ITGB1 had the most obvious inhibitory effects on the proliferation, migration, and tube formation ability of HUVECs. The inhibitory effects of knockdown of Itgb1 on proliferation and migration were also confirmed in C166 cells. Similarly, in the Itgb1 knockdown group, the proliferation and migration abilities of C166 cells were significantly reduced. Abnormal proliferation, migration, and tube formation of vascular endothelial cells (VECs) are key links in the angiogenic effect. Knockdown of Itgb1 significantly inhibited the proliferation, migration, and tube formation of VECs. Therefore, Itgb1 may be a suitable target for treating CNV. To further confirm the role of Itgb1 in in vivo CNV, we injected lentivirus loaded with Itgb1-shRNA plasmid subconjunctivally in mice. Knockdown of Itgb1 significantly inhibited CoNV; on the 7th and 11th days after modeling, the length and area values of the neovessels were much lower than those of the control group ( Figure 3 F-H). Previous studies have also confirmed that Itgb1 mediates angiogenesis in cancer and diabetic retinopathy. Based on these results, we used Itgb1 as the target for subsequent research on anti-CoNV therapeutic drugs.

[0105] 2.3. Evaluation of the targeting ability of LNPs conjugated with anti-Flt1 peptide to VECs

[0106] FLT1 is a VEGF receptor encoded by the FLT1 gene. Anti-Flt1 peptide is an antagonistic peptide of FLT1, which can specifically bind to FLT1 and prevent it from binding to ligands. In previous studies, anti-Flt1 peptide has been applied to anti-angiogenic therapy for tumors, diabetic retinopathy, and CNV. In this study, we showed by single-cell sequencing that compared with the NC group, the expression of Flt1 in vascular endothelial cells (VECs) and pericytes in the CNV group was significantly increased ( Figure 4 A). Immunofluorescence and western blot also confirmed this result ( Figure 4B and C). Therefore, we prepared lipid nanoparticles (LNPs) conjugated with anti-Flt1 peptide for targeted therapy of CNV. Fluorescence microscopy and flow cytometry analysis confirmed that conjugation with anti-Flt1 peptide enhanced the internalization ability of LNPs in C166 cells ( Figure 4 D-F). Next, we used qRT-PCR to detect the gene silencing efficiency of Itgb1-siRNA@SM102 LNPs (Itgb1-siRNA@SM). After transfecting C166 cells with Itgb1-siRNA@SM conjugated with anti-Flt1 peptide for 48 hours, the expression of Itgb1 mRNA was detected. We found that there was no significant difference in the knockdown efficiency between 50 nM siRNA and 100 nM siRNA ( Figure 4 G), so 50 nM Itgb1-siRNA was encapsulated in LNPs. We further found that anti-FLT1 peptide could improve the knockdown efficiency of LNPs loaded with Itgb1-siRNA. When transfecting cells with 50 nM siRNA, the average knockdown efficiency of LNPs conjugated with anti-Flt1 peptide was increased by 1.48-fold compared with LNPs without conjugated anti-Flt1 peptide ( Figure 4 H).

[0107] To determine the optimal concentration of Itgb1-siRNA@LNPs for anti-angiogenesis, we investigated its effects on the proliferation and migration of C166 cells by CCK8, EdU, scratch assay and Transwell assay. CCK8 and EdU assays showed that when the concentration of siRNA was lower than 50 nM, the inhibitory effect of Itgb1-siRNA@LNPs on the proliferation of C166 cells was dose-dependent. However, when C166 cells were exposed to LNPs loaded with 50 nM or 100 nM siRNA for 72 hours, there was no significant difference in cell proliferation ( Figure 4 I and J). Similarly, when the concentration of Itgb1-siRNA was <50 nM, the inhibitory effect of LNPs loaded with Itgb1-siRNA on cell migration was also dose-dependent; while no significant difference was observed in the concentration range of 50 - 100 nM ( Figure 4 K and L). Subsequently, we detected the apoptosis of C166 cells by flow cytometry to evaluate the cytotoxicity of Itgb1-siRNA@LNPs. The results showed that LNPs loaded with no more than 100 nM Itgb1-siRNA did not induce cell apoptosis; however, when cells were exposed to LNPs loaded with 200 nM Itgb1-siRNA for 48 hours, the apoptosis rate increased slightly ( Figure 4M). In summary, these results indicate that LNPs loaded with Itgb1-siRNA at concentrations between 50 nM and 100 nM may be safe and effective anti-angiogenic therapeutic agents.

[0108] 2.4 Screening for the optimal concentration of TP for LNP modification

[0109] Inflammation is considered a core pathological process during CNV formation. It has been reported that some inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, can induce CNV by promoting the proliferation, migration, and tube formation of VECs. In addition, it has been reported that ionizable lipids can induce inflammation. Glucocorticoids can inhibit CNV through their anti-inflammatory effects; however, long-term use can lead to recurrent infections, increased intraocular pressure, and cataracts. TP is the main bioactive component of the traditional Chinese medicine Tripterygium wilfordii, which has been used for the treatment of various autoimmune diseases for hundreds of years. Increasing evidence indicates that TP has anti-inflammatory, anti-proliferative, and immunosuppressive activities without the side effects caused by glucocorticoids. In this experiment, we used VECs as the research object and analyzed the changes in cell composition and inflammatory cytokines in the VEC clusters during CNV using single-cell sequencing. Unsupervised sub-clustering analysis was performed, and the VECs were divided into 7 sub-populations by UMAP analysis ( Figure 5 A). The top three differential genes of each cluster are shown as Figure 5 B. In control mice (NC group), most VECs were assigned to cluster 3 (98.48%), and this cluster hardly expressed IL-1β, IL-6, or TNF-α ( Figure 5 C-D). Most VECs derived from the CNV group highly expressed Il6 (clusters 0, 1, 2, 4, and 6), and cluster 6, which was not detected in the NC group, also highly expressed IL-1β and TNF-α ( Figure 5 D). Western blot detection showed that the expression levels of the inflammatory cytokines IL-1β, IL-6, and TNF-α in the CNV group were significantly higher than those in the NC group ( Figure 5 E and F). It has been previously reported that icLNPs have inflammatory side effects. To confirm whether icLNPs can trigger ocular surface inflammation, we detected the inflammatory factors IL-1β, IL-6, and TNF-α in the extracts of mouse ocular surface tissues by qRT-PCR and ELISA methods ( Figure 5 G and H), and the results showed that the expressions of IL-1β, IL-6, and TNF-α were significantly upregulated 14 days after using icLNPs eye drops. This indicates that long-term use of icLNPs poses a risk of exacerbating ocular surface inflammation and CNV.

[0110] TP has shown a strong anti-inflammatory effect in the treatment of various immune diseases. This indicates that modification with TP is a feasible option to mitigate the inflammatory response induced by icLNPs. However, it has been reported that TP has the characteristics of high toxicity, extremely poor water solubility, and a narrow therapeutic window. Therefore, screening for the optimal TP concentration for LNPs modification is a crucial step in this study. First, the cytotoxicity of TP was evaluated through apoptosis experiments. The results showed that when the TP concentration exceeded 20 ng / mL, the apoptosis rate of C166 cells increased significantly ( Figure 5 I). This implies that TP with a concentration below 20 ng / mL may be suitable for LNPs modification. Subsequently, the effects of TP on the proliferation and migration of C166 cells were evaluated. We found that treatment with 10 ng / mL of TP for 48 hours and 15 ng / mL of TP for 24 hours could significantly inhibit the proliferation of C166 cells ( Figure 5 J and K). In addition, the effects of TP on the migration of C166 cells were evaluated through Transwell experiments and scratch experiments. The results showed that TP with a concentration exceeding 10 ng / mL could significantly inhibit the migration of C166 cells. The anti-inflammatory effect of TP was also measured. As previously reported, we found that TP could inhibit the expression of LPS-induced inflammatory cytokines in a concentration-dependent manner. At concentrations of 10 ng / mL and 15 ng / mL, TP showed a strong inhibitory effect on the expression of IL-1β, IL-6, and TNF-α ( Figure 5 M). These results indicate that TP with a concentration of 10 - 15 ng / mL may be suitable for LNPs modification to achieve anti-inflammatory effects and inhibit the proliferation of VEC.

[0111] 2.5 Itgb1-siRNA@TPL Anti-CNV Effect and Safety Evaluation

[0112] We first detected the cytotoxicity of Itgb1-siRNA@TPL in vitro. Compared with the control group, there was no significant change in cell apoptosis after treating C166 cells with Itgb1-siRNA@TPL containing 10 and 15 ng / mL of TP for 24 hours (p > 0.05) ( Figure 6 A and B). Subsequently, a CNV model was constructed and observed continuously for 14 days after surgery. Slit lamp examination and anterior segment optical coherence tomography angiography (AS-OCTA) were performed before and on the 3rd, 7th, 11th, and 14th days after modeling ( Figure 6C). By continuously observing and comparing the length and area of CNV with the control group, the inhibitory effect of Itgb1-siRNA@TPL on CNV was evaluated. We tested Itgb1-siRNA@TPL with TP concentrations of 10 ng / mL and 15 ng / mL [denoted as siRNA@TPL(10) and siRNA@TPL(15)], respectively. The results of each group were similar from day 1 to day 3 after modeling, showing limbal vascular congestion and neovascular buds in a few areas. From day 4 to day 14, CNV growth was very slow in the groups treated with siRNA@TPL(10) and siRNA@TPL(15). In contrast, CNV growth was significant and time-dependent in the control group ( Figure 6 C). The non-invasive imaging technique AS-OCTA developed in recent years can generate high-resolution angiography images of the cornea and ocular surface. AS-OCTA showed that the blood flow signal decreased significantly after treatment with siRNA@TPL(10) and siRNA@TPL(15), indicating their role in reducing corneal neovascularization. The length and area of neovascularization were quantified on day 7 and day 14 after modeling. On day 7 and day 14, the length and area of neovascularization in the Itgb1-siRNA@TPL(10) and Itgb1-siRNA@TPL(15) groups were significantly lower than those in the control group (p < 0.001) and slightly lower than those in the dexamethasone (DEX) group, although this difference was not significant (p > 0.05) ( Figure 6 D).

[0113] D). The biosafety of siRNA@TPL was further evaluated by histological examination of the mouse corneal epithelial injury model and major organ tissues. For the corneal epithelial injury model, half of the corneal epithelium was scraped off, and Itgb1-siRNA@TPL, DEX, and PBS were given to the eyes with corneal epithelial injury, respectively. Within 3 days, the corneal epithelium in the Itgb1-siRNA@TPL(10) group and the control group healed completely. However, both the Itgb1-siRNA@TPL(15) group and the DEX group delayed corneal epithelial healing, and on day 4 after modeling, some mice in the Itgb1-siRNA@TPL(15) group and the DEX group had punctate corneal epithelial defects ( Figure 6 E). When quantifying the area of corneal epithelial defects, it was found that the values in the Itgb1-siRNA@TPL(10) group were significantly lower than those in the Itgb1-siRNA@TPL(15) group and the DEX group on day 2 and day 3 ( Figure 6 F). These results indicate that Itgb1-siRNA@TPL with a TP concentration of 10 ng / mL may be the safest and most effective preparation for treating CNV.

[0114] TP has certain toxic and side effects in the body, such as hepatotoxicity, nephrotoxicity, pulmonary toxicity, and cardiotoxicity, etc. In addition, Itgb1-siRNA@TPL may enter the blood circulation through the conjunctival blood vessels or the nasolacrimal duct. To further examine the biosafety of Itgb1-siRNA@TPL(10), we used hematoxylin-eosin staining to perform histological examinations on the tissues of the main organs (heart, liver, spleen, lung, and kidney) and the cornea. In the Itgb1-siRNA@TPL(10) group, no obvious signs of damage were observed in the main organs and the cornea. This finding indicates that Itgb1-siRNA@TPL(10) has good safety, and no obvious pathological changes occurred in the main organs and the cornea after its use on the ocular surface( Figure 6 G).

[0115] 2.6 Itgb1-siRNA@TPL inhibits CoNV through the PI3K / Akt and NF-κB signaling pathways

[0116] The relaxation of the connections between vascular endothelial cells (VECs), the formation of tip cells, and migration are key steps in angiogenesis; changes in gene expression can reveal these transformations at the molecular level. Therefore, we determined the differentially expressed genes of VECs in the CNV group and the NC group( Figure 7 A), and then used the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway to perform functional analysis on these genes. Both the PI3K / AKT and NF-κB signaling pathways were significantly enriched in the CNV group( Figure 7 B). It has been reported that PI3K plays a key role in angiogenesis and is involved in stimulating hypoxia-inducible factor (HIF) in the brain. The PI3K / AKT pathway also regulates cell survival, proliferation, migration, and metabolism, and can lead to the invasion and migration of cancer. Activating the PI3K / AKT pathway can promote corneal vascular permeability and angiogenesis. NF-κB is a heterodimeric transcription factor that plays a key role in the inflammatory responses in multiple parts of the body by promoting the abnormal expression of pro-inflammatory cytokine genes, especially Il1b, Il6, and Tnfa. It has been reported that cytokines such as IL-1 and IL-6 can activate the PI3K / AKT signaling pathway. The NF-κB pathway may also indirectly promote angiogenesis by inducing the production of pro-inflammatory cytokines. In addition, TP, which is used to modify Itgb1-siRNA@TPL, has been reported to inhibit the PI3K / AKT and NF-κB signaling pathways in ovarian cancer. These results indicate that the PI3K / AKT and NF-κB pathways may be the action targets of Itgb1-siRNA@TPL for inhibiting CNV.

[0117] To further verify the regulatory effects of Itgb1-siRNA@TPL on PI3K / AKT and NF-κB, we used western blot to compare the protein levels of phosphorylated (p)-PI3K, p-AKT, and p-NF-κB p65 in the Itgb1-siRNA@TPL, CNV, and NC groups. The results showed that Itgb1-siRNA@TPL could significantly inhibit the phosphorylation of PI3K, AKT, and NF-κB p65( Figure 7 C). In addition, we explored whether Itgb1-siRNA@TPL inhibited the proliferation and migration of VECs through the PI3K / AKT pathway in vitro. UCL-TRO-1938 is a PI3K activator that can increase the level of p-AKT (S473) and promote the proliferation of mouse embryonic fibroblasts. In this study, UCL-TRO-1938 significantly promoted the proliferation and migration of C166 cells, while Itgb1-siRNA@TPL blocked these proliferation and migration effects( Figure 7 F-H). Western blot and immunofluorescence experiments showed that Itgb1-siRNA@TPL inhibited the phosphorylation and nuclear translocation of AKT induced by UCL-TRO-1938( Figure 7 D and E). These results confirmed that the PI3K / AKT pathway is the target of Itgb1-siRNA@TPL to reduce the proliferation and migration of VECs.

[0118] Subsequently, we explored the inhibitory effect of siRNA@TPL on the activation of the NF-κB signaling pathway. Western blot results showed that Itgb1-siRNA@TPL significantly reduced the phosphorylation level of NF-κB induced by LPS. Immunofluorescence experiments also confirmed that it inhibited the activation of the NF-κB signaling pathway by preventing the nuclear translocation of NF-κB( Figure 7 I and J). Then, we used qRT-PCR and ELISA to evaluate the gene and protein expression levels of pro-inflammatory cytokines. The results showed that Itgb1-siRNA@TPL significantly reduced the expression levels of IL-1β, IL-6, and TNF-α( Figure 7 K and L). These findings indicated that the anti-inflammatory effect of Itgb1-siRNA@TPL was achieved by blocking the activation of the NF-κB signaling pathway.

[0119] Matters not covered by this invention are well-known technologies.

[0120] The above embodiments are only for illustrating the technical concept and features of the present invention, and the purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. It is not intended to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. An ITGB1-siRNA lipid nanoparticle, characterized in that: The lipid nanoparticles are loaded with ITGB1-siRNA and modified with triptolide and anti-Flt1 peptide; and the lipid nanoparticles are ionizable cationic lipid nanoparticles.

2. The lipid nanoparticle according to claim 1, wherein The concentration of the ITGB1-siRNA is 50-100 nM; the concentration of triptolide is 10-15 ng / mL.

3. The lipid nanoparticle according to claim 1, characterized in that The particle size of the ITGB1-siRNA lipid nanoparticles is ∼110 nm, and the PDI is <0.5; The cationic lipid in the lipid nanoparticles is any one of SM-102, ALC-0315 and D-Lin-MC3-DMA, preferably SM-102.

4. The method for preparing the ITGB1-siRNA lipid nanoparticles according to any one of claims 1 to 3, characterized in that: The preparation method comprises preparing the ITGB1-siRNA lipid nanoparticles by adopting microfluidics technology; Furthermore, the preparation method comprises: S1. dissolving an ionizable cationic lipid, an auxiliary lipid, cholesterol and a PEGylated lipid in ethanol to obtain a phospholipid solution; S2, dissolving triptolide in ethanol, and then mixing with the phospholipid solution prepared in step S1 to obtain a phospholipid solution containing triptolide; S3, dissolving ITGB1-siRNA in sodium citrate buffer, mixing it with the phospholipid solution containing triptolide prepared in step S2 based on a microfluidic method, and diluting, concentrating and centrifuging with PBS buffer to obtain triptolide-modified lipid nanoparticles loaded with integrin β1 siRNA; S4, incubate and couple the anti-Flt1 peptide with the triptolide-modified lipid nanoparticles loaded with integrin β1 siRNA prepared in step S3.

5. The preparation method according to claim 4, characterized in that: In step S1, the molar ratio of the ionizable cationic lipid, the auxiliary lipid, the cholesterol and the pegylated lipid is 45-55:35-40:5-15:0.5-5.

6. The preparation method according to claim 4, characterized in that: In the step S2, the concentration of triptolide in the ethanol solution is 50-150 μg / mL.

7. The preparation method according to claim 4, characterized in that: In step S3, the concentration of ITGB1-siRNA in sodium citrate buffer is 0.01-0.2 mg / mL; The concentration is carried out using a 50-200KD ultrafiltration tube; The specific conditions of the centrifugation are: centrifugation at a speed of 3000-8000 rpm for 1-30 minutes.

8. The preparation method according to claim 4, characterized in that: In the step S4, the anti-Flt1 peptide contains a linker peptide, and the amino acid sequence of the linker peptide is GGGSC.

9. Use of the ITGB1-siRNA lipid nanoparticles according to any one of claims 1 to 3 in the preparation of an anti-corneal neovascularization drug.

10. An anti-corneal neovascularization drug, characterized in that: The active ingredient of the corneal neovascularization drug comprises the ITGB1-siRNA lipid nanoparticles; Furthermore, the anti-corneal neovascularization drug also includes at least one drug inactive ingredient; Furthermore, the dosage form of the anti-corneal neovascularization drug is eye drops.

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