A complex based on neutrophil nanovesicles exhibiting both anti-inflammatory and anti-angiogenic bidirectional trapping combined with autoluminescent dynamics, its preparation method, and its application.

Abstract

This invention provides a complex based on neutrophil nanovesicles that combines anti-inflammatory and anti-angiogenic bidirectional trapping with autoluminescent dynamics, along with its preparation method and applications, belonging to the field of biopharmaceutical manufacturing technology. The complex uses neutrophil nanovesicles as a carrier and also encapsulates bis(2,4,5-trichlorosalicylate n-amyl)oxalate (CPPO) and the photosensitizer Ce6. Simultaneously, the surface is modified with ranibizumab via ROS-responsive ketothiols. The complex possesses the characteristics of trapping anti-inflammatory factors and spontaneously targeting corneal neovascularization lesions. Based on ROS-responsive release of ranibizumab to bind vascular endothelial growth factor, it exerts an anti-angiogenic function. Simultaneously, the chemical energy generated by CPPO under hyperoxia conditions excites Ce6 to produce singlet oxygen, thereby inhibiting the tubular formation ability of vascular endothelial cells. This invention provides a new approach for the treatment of eye-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical manufacturing technology, specifically relating to a complex based on neutrophil nanovesicles that combines anti-inflammatory and anti-angiogenic bidirectional trapping with autoluminescence dynamics, its preparation method, and its application. Background Art

[0002] The cornea is completely transparent and avascular under normal conditions, serving as a refractive organ to transmit light signals to the retina. In certain pathological conditions, such as keratitis, trauma, and alkali burns, invasion of capillaries from the limbal plexus often leads to corneal neovascularization (CorNV), the second leading cause of blindness worldwide, severely reducing patients' quality of life and urgently requiring effective treatments.

[0003] Because the progression of corneal neovascularization (CorNV) is a multi-step process, it mainly involves inflammation-induced cytokine release, vascular endothelial cell activation and proliferation induced by vascular endothelial growth factor (VEGF) production, angiogenesis, and pericyte recruitment. Currently, drugs targeting VEGF are first-line treatments; however, these protein drugs are small in size, easily excreted from the body, require repeated administration, and are prone to drug resistance. Furthermore, given the orderly and complex nature of corneal neovascularization, single-target therapy cannot completely inhibit neovascularization. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a complex (NCCA) based on neutrophil nanovesicles that combines anti-inflammatory and anti-angiogenic bidirectional trapping with autoluminescent dynamics, which achieves anti-inflammatory and anti-angiogenic effects by neutralizing inflammatory factors and VEGF and chemiluminescent photodynamic therapy (PDT), thereby effectively treating ocular-related diseases.

[0005] This invention provides a complex of bidirectional anti-inflammatory and anti-angiogenic trapping combined with autoluminescent dynamics, comprising neutrophil nanovesicles and bi(2,4,5-trichlorosalicylate n-pentyl) oxalate (CPPO) and dihydroporphyrin E6 (Ce6) loaded between phospholipid bilayers of the neutrophil nanovesicles, as well as ranibizumab attached to the outer surface of the neutrophil nanovesicles;

[0006] The ranibizumab is linked to the neutrophil nanovesicles via ketothiols.

[0007] Preferably, one end of the ketithiothiol (TK) is connected to a maleamide group (MAL), and the other end is connected to distearate phosphatidylethanolamine (DSPE).

[0008] The ketithiolide is linked to neutrophil nanovesicles via the phospholipid portion of distearate phosphatidylethanolamine.

[0009] The ketithiolide is linked to ranibizumab via an amide bond formed by the maleamide group and the thiol group on ranibizumab.

[0010] Preferably, the neutrophil nanovesicles have a particle size of 150–200 nm.

[0011] Preferably, each 1×10 10 Each neutrophil nanovesicle contains 15–20 μg of bis(2,4,5-trichlorosalicylate n-pentyl) oxalate, 4–6 μg of dihydroporphyrin E6, and 5–10 μg of ranibizumab.

[0012] This invention provides a method for preparing the aforementioned complex, comprising the following steps:

[0013] Neutrophil nanovesicles, bis(2,4,5-trichlorosalicylate n-pentyl oxalate) and dihydroporphyrin E6 were first mixed and co-incubated, and neutrophil nanovesicles loaded with bis(2,4,5-trichlorosalicylate n-pentyl oxalate) and dihydroporphyrin E6 were collected.

[0014] Ranibizumab was linked to ketothiols to obtain ketothiols linked with ranibizumab.

[0015] The ketothiolate conjugated with ranibizumab and the neutrophil nanovesicles loaded with bis(2,4,5-trichlorosalicylic acid n-pentyl oxalate) and dihydroporphyrin E6 were co-incubated in a second mixture to obtain the complex.

[0016] Preferably, the method for preparing the neutrophil nanovesicles includes sequentially extruding a neutrophil suspension through polycarbonate membranes of 1 μm, 400 nm and 100 nm to collect the nanovesicles.

[0017] The nanovesicles were subjected to density gradient centrifugation with OptiPrep at a volume concentration of 10%–30%, and the 10% and 30% separation layer components were collected to obtain neutrophil nanovesicles.

[0018] Preferably, each 1×10 10 Each neutrophil nanovesicle contains 15–20 μg of bis(2,4,5-trichlorosalicylic acid n-pentyl) oxalate and 4–6 μg of dihydroporphyrin E6.

[0019] The molar ratio of ranibizumab to ketothiolate is 1:(50-100);

[0020] The concentration of ranibizumab is 6–10 mg / mL;

[0021] The molar ratio of the ketothiolate linked with ranibizumab to the neutrophil nanovesicles loaded with bis(2,4,5-trichlorosalicylic acid n-pentyl) oxalate and dihydroporphyrin E6 is (50-100):1.

[0022] This invention provides the use of the complex or the complex prepared by the preparation method in the preparation of anti-inflammatory and / or anti-corneal angiogenesis drugs.

[0023] This invention provides the use of the complex or the complex prepared by the method described above in the preparation of medicaments for the prevention and / or treatment of corneal injury-related diseases.

[0024] The present invention provides an eye drop comprising the complex or the complex prepared by the preparation method and pharmaceutically acceptable excipients.

[0025] This invention provides a complex combining anti-inflammatory and anti-angiogenic bidirectional trapping with autoluminescent dynamics. Neutrophil nanovesicles (NVs) serve as carriers, with photodynamic therapy (PDT)-related drugs CPPO and Ce6 encapsulated between their phospholipid bilayers. Simultaneously, ranibizumab is modified on its surface with ROS-responsive ketothiols. The complex exerts its anti-inflammatory and angiogenesis-inducing effects through multiple pathways. First, due to the presence of chemokine receptors, the neutrophil nanovesicles can penetrate corneal epithelial cells and spontaneously target inflamed sites, thus rapidly reaching the lesion site of corneal neovascularization. When the complex enters the cornea, the inflammatory factor receptors on the neutrophil nanovesicles actively trap inflammatory factors, thus exerting an anti-inflammatory effect. Second, the ranibizumab linked by the thiol ketone (TK) bond in the complex is released in the highly reactive oxygen species (ROS) microenvironment at the corneal angiogenesis lesion site, where ranibizumab is specifically targeted. The complex captures vascular endothelial growth factor (VEGF) to inhibit the activation and proliferation of vascular endothelial cells. Simultaneously, the lipid solubility of neutrophil vesicles allows ranibizumab in the complex to have good pre-corneal retention capacity, prolonging the pre-corneal retention time and improving drug penetration into the cornea for efficacy. Thirdly, during the angiogenesis and pericyte recruitment phases, the photodynamic therapy (PDT)-related drug CPPO generates chemical energy in a hyperoxic environment, thereby activating the photosensitizer Ce6 to produce singlet oxygen, which is toxic to vascular endothelial cells, affecting their tube-forming ability and thus eliminating newly formed blood vessels. Experiments in this invention show that the nanovesicles can significantly reduce the concentration of inflammatory factors, supporting the complex's effect as an inducer to eliminate inflammatory factors at the lesion site. Furthermore, in this embodiment, the complex was administered to a model mouse with corneal injury. Results showed that compared to the existing corneal neovascularization (CorNV) regimen (ranibizumab), NCCA prolonged the duration of action in the eye; simultaneously, the complex could spontaneously target the neovascularization lesion site. Simultaneously, cell and animal experiments demonstrated that NCCA exhibits stronger anti-angiogenic efficacy compared to existing corneal neovascularization regimens (ranibizumab). Therefore, the complex provided by this invention can achieve multi-target, multi-step treatment of corneal angiogenesis, offering an effective treatment for various diseases caused by corneal damage. Attached Figure Description

[0026] Figure 1 This invention provides a schematic diagram of the synthesis and structure of NCCA.

[0027] Figure 2 The image shows the therapeutic effect of NCCA prepared in Example 1 on inhibiting corneal angiogenesis.

[0028] Figure 3 The image shows the therapeutic effect of NCCA prepared in Example 3 on inhibiting corneal angiogenesis.

[0029] Figure 4The image shows the therapeutic effect of NCCA prepared in Example 4 on inhibiting corneal angiogenesis.

[0030] Figure 5 The images show the morphology of NCCA prepared in Example 4, where the left image is the TEM image of NV and the right image is the TEM image of NCCA.

[0031] Figure 6 The hydrated particle size and zeta potential characterization results of NCCA prepared in Example 4 are shown, where a. particle size distribution of NV and NCCA; b. zeta potential of NV and NCCA;

[0032] Figure 7 The fluorescence colocalization map of NCCA prepared in Example 4;

[0033] Figure 8 UV absorption spectra of Ce6 and NCCA prepared in Example 4;

[0034] Figure 9 for 1 O2 detection reagent ABDA verification Example 4: NCCA prepared under different concentrations of H2O2 produces 1 The result of O2;

[0035] Figure 10 The stability results of NCCA prepared in Example 4 are shown in the figure.

[0036] Figure 11 ROS-responsive release of NCCA prepared in Example 4

[0037] Figure 12 The results of Western blotting were used to verify the inflammatory factor trapping properties of NV; a) is the gel image; b) is the statistical results.

[0038] Figure 13 The results of NCCA retention on the ocular surface include: a. NCCA fluorescence images in the eyes of mouse models; b. Quantitative results of fluorescence signals in the eyes of mouse models at different time points.

[0039] Figure 14 Results of NCCA targeting ability assessment; a. Two-photon fluorescence microscopy image of the mouse model eye 2 days after exposure; b. Two-photon fluorescence microscopy image of the mouse model eye 3 days after exposure;

[0040] Figure 15 The results of NCCA inhibiting HUVEC angiogenesis under high ROS conditions are shown; where a is an image of angiogenesis after different treatments taken with an inverted microscope, b is the quantitative result of node number, c is the quantitative result of grid number, and d is the quantitative result of total branch length.

[0041] Figure 16 This image shows the in vivo therapeutic effect of NCCA on a mouse model of alkali burn-induced corneal neovascularization. Note: Representative photographs of the alkali burn-induced corneal neovascularization model after 0, 3, and 7 days of ranibizumab and NCCA intervention. Detailed Implementation

[0042] This invention provides a complex combining anti-inflammatory and anti-angiogenic bidirectional trapping with autoluminescent dynamics, comprising neutrophil nanovesicles and bi(2,4,5-trichlorosalicylate n-pentyl) oxalate and dihydroporphyrin E6 encapsulated between phospholipid bilayers of the neutrophil nanovesicles, and ranibizumab attached to the outer surface of the neutrophil nanovesicles; the ranibizumab is attached to the neutrophil nanovesicles via ketothiols.

[0043] In this invention, the complex is vesicle-shaped with a particle size of 210–230 nm. The complex uses neutrophil nanovesicles as a carrier. The particle size of the neutrophil nanovesicles is preferably 150–200 nm. The neutrophil nanovesicles possess inflammatory factor receptors. Due to the strong inflammatory response at the site of corneal neovascularization, the complex can spontaneously target the lesion site of corneal neovascularization by utilizing the binding properties of inflammatory factor receptors and inflammatory factors, thereby improving the targeting of the carried drug. Simultaneously, the neutrophil nanovesicles use inflammatory factor receptors to trap inflammatory factors, exerting an anti-inflammatory effect. Furthermore, the neutrophil nanovesicles have good lipid solubility. Using neutrophil nanovesicles as a carrier increases the residence time of the complex at the site of corneal angiogenesis, which is beneficial for improving the efficacy of the carried drug.

[0044] In one embodiment of the present invention, the trapping properties of neutrophil nanovesicles for inflammatory factors were verified using the Western-blotting method. The results showed that, compared with the PBS control, the concentrations of MMP2, IL-6 and TNF-α in the neutrophil nanovesicle group were significantly reduced, which proves the feasibility of using neutrophil nanovesicles as bait for NCCA to eliminate inflammatory cytokines at the lesion site.

[0045] In this invention, the complex comprises a photodynamic therapy (PDT) related drug CPPO and a photosensitizer Ce6. The CPPO generates chemical energy upon contact with an oxidant, which excites the photosensitizer Ce6 to produce singlet oxygen (…). 1 O2), the singlet oxygen reacts with organic molecules in the cell, causing oxidative damage to biological macromolecules such as cell membranes, proteins and DNA, ultimately leading to cell death.

[0046] In one embodiment of the present invention, the complex is generated under different concentrations of H2O2. 1O2 was detected, while no significant changes were observed in the PBS, CPPO, and Ce6 groups. This demonstrates that CPPO in the complex can react with H2O2 to generate chemical energy that activates Ce6 production. 1 O2.

[0047] In this invention, the complex comprises ranibizumab targeting vascular endothelial growth factor (VEGF). The ranibizumab is linked to a carrier via a ROS-responsive ketothiolate. One end of the ketothiolate (TK) is preferably linked to a maleamide group (MAL), and the other end is preferably linked to distearylphosphatidylethanolamine (DSPE), denoted as DSPE-TK-MAL. The ketothiolate is linked to neutrophil nanovesicles via the phospholipid portion of the distearylphosphatidylethanolamine. The ketothiolate is linked to ranibizumab via an amide bond formed by the maleamide group and the thiol group on the ranibizumab. Corneal angiogenesis lesions are located in a highly reactive oxygen species (ROS) microenvironment. After the complex enters the corneal lesion site, ranibizumab is released from the carrier under the action of ROS, targeting and binding VEGF, thereby blocking VEGF-induced vascular endothelial cell activation and proliferation, angiogenesis, and pericyte recruitment.

[0048] In another embodiment of the present invention, the ROS responsiveness of the complex was investigated. Results showed that, compared to the PBS group, the release efficiency of ranibizumab from NCCA under H2O2 stimulation was higher, reaching 92% at 48 hours. This indicates that NCCA possesses the ability to release ranibizumab in a ROS-responsive manner.

[0049] In this invention, each 1×10 10 Each neutrophil nanovesicle contains 15–20 μg of bis(2,4,5-trichlorosalicylic acid n-pentyl) oxalate, 4–6 μg of dihydroporphyrin E6, and 5–10 μg of ranibizumab. 1×10 10 Each neutrophil nanovesicle is composed of 20 mL of a concentration of 4–6 × 10⁻⁶. -6 The suspension of mouse neutrophils (MNHC) was prepared by using cells per mL.

[0050] This invention provides a method for preparing the aforementioned complex, see [link to method]. Figure 1 Specifically, it includes the following steps:

[0051] Neutrophil nanovesicles, bis(2,4,5-trichlorosalicylate n-pentyl oxalate) and dihydroporphyrin E6 were first mixed and co-incubated, and neutrophil nanovesicles loaded with bis(2,4,5-trichlorosalicylate n-pentyl oxalate) and dihydroporphyrin E6 were collected.

[0052] Ranibizumab was linked to ketothiols to obtain ketothiols linked with ranibizumab.

[0053] The ketothiolate conjugated with ranibizumab and the neutrophil nanovesicles loaded with bis(2,4,5-trichlorosalicylic acid n-pentyl oxalate) and dihydroporphyrin E6 were co-incubated in a second mixture to obtain the complex.

[0054] In this invention, neutrophil nanovesicles, bis(2,4,5-trichlorosalicylate n-pentyl oxalate) and dihydroporphyrin E6 are first mixed and co-incubated to collect neutrophil nanovesicles loaded with bis(2,4,5-trichlorosalicylate n-pentyl oxalate) and dihydroporphyrin E6.

[0055] In this invention, the preferred method for preparing the neutrophil nanovesicles includes sequentially extruding a neutrophil suspension through polycarbonate membranes of 1 μm, 400 nm, and 100 nm to collect the nanovesicles; and then performing density gradient centrifugation on the nanovesicles using OptiPrep with a volume concentration of 10%–30% to collect 10% of the separation layer and 30% of the separation layer components to obtain the neutrophil nanovesicles.

[0056] In this invention, the concentration of the neutrophil suspension is preferably 4 × 10⁻⁶. ～6 Cells / mL ~ 7×10 ～6 Cells / mL, which can be 5 × 10 ～6 The density gradient centrifugation is preferably performed using a microliposome extruder. The centrifugal force is preferably 100,000 g for 0.5–1 h. Morphological characterization results show that the neutrophil nanovesicles have a particle size of 150–200 nm, which can be 180 nm.

[0057] In this invention, each 1×10 10 Each neutrophil nanovesicle contains 15–20 μg and 4–6 μg of bis(2,4,5-trichlorosalicylic acid n-pentyl) oxalate and dihydroporphyrin E6, respectively; the concentration of the neutrophil nanovesicle is 10 × 10⁻⁶. 10 The concentration is 1 / mL. The temperature for the co-incubation is preferably 36–38°C, and can be 37°C. The time for the first co-incubation is preferably 2.5–3.5 h, and can be 3 h. After co-incubation, the unencapsulated bis(2,4,5-trichlorosalicylic acid n-pentyl) oxalate and dihydroporphyrin E6 are removed, preferably using an ultrafiltration tube. The pore size of the ultrafiltration tube is preferably 100 kDa. The retentate in the ultrafiltration tube is collected for later use.

[0058] This invention links ranibizumab to ketothiols to obtain ketothiols linked with ranibizumab.

[0059] In this invention, the molar ratio of ranibizumab to ketothiocarbaze is preferably 1:(50-100), and can be 1:80. The concentration of ranibizumab is preferably 6-10 mg / mL. The ketothiocarbaze exists in the form of DSPE-TK-MAL. The solvent used to dissolve the DSPE-TK-MAL is DMF. The preferred reaction conditions for the ligation are: incubation at 4°C for 24 h. The ligation is achieved by the condensation reaction between the MAL group on the ketothiocarbaze and the amino group of ranibizumab to form an amide bond, thereby achieving the ligation of ketothiocarbaze and ranibizumab. After ligation, ultrafiltration is preferably used to remove the ketothiocarbaze that has not been ligated with ranibizumab. The pore size of the ultrafiltration membrane is preferably 10 kDa, and a portion of the components inside the filter tube is collected for later use.

[0060] In this invention, the ketothiolate conjugated with ranibizumab and the neutrophil nanovesicles loaded with bis(2,4,5-trichlorosalicylic acid n-pentyl oxalate) and dihydroporphyrin E6 are subjected to a second co-incubation to obtain a complex.

[0061] In this invention, the preferred molar ratio of the ketothioglycol conjugated with ranibizumab and the neutrophil nanovesicles loaded with bis(2,4,5-trichlorosalicylic acid n-pentyl oxalate) and dihydroporphyrin E6 is (50-100):1. The preferred temperature for co-incubation is 36-38°C, and can be 37°C. The preferred incubation time is 0.4-0.6 h, and can be 0.5 h. The second co-incubation involves the ketothioglycol conjugated with ranibizumab being linked to the neutrophil nanovesicles via the phospholipid portion of DSPE.

[0062] The present invention provides an eye drop comprising the complex or the complex prepared by the preparation method and pharmaceutically acceptable excipients.

[0063] This invention provides the use of the complex or the complex prepared by the preparation method in the preparation of anti-inflammatory and / or anti-corneal angiogenesis drugs.

[0064] In this invention, the drug preferably comprises at least one of the following external dosage forms: eye drops, ophthalmic gel, and ophthalmic ointment. The concentration of the complex in the drug is not less than 10 mg / mL, and can be 15 mg / mL, 20 mg / mL, 25 mg / mL, or 30 mg / mL. This invention does not impose any particular limitations on the preparation method and usage method of the drug; any drug preparation method and usage method well known in the art can be used.

[0065] In this invention, the anti-inflammatory effect preferably includes reducing the content of inflammatory factors at the site of ocular lesions. The anti-corneal angiogenesis effect includes inhibiting the formation of corneal vessels and eliminating formed corneal vessels.

[0066] In this embodiment of the invention, in vitro and in vivo experiments have shown that the complex prepared by this invention can effectively reduce the number of corneal vessels in a mouse model of alkali burn-induced corneal neovascularization.

[0067] This invention provides the use of the complex or the complex prepared by the method described above in the preparation of medicaments for the prevention and / or treatment of corneal injury-related diseases.

[0068] In this invention, the corneal injury-related diseases include keratitis, mechanical injury, and chemical corneal injury.

[0069] The following detailed description, in conjunction with embodiments, illustrates a complex based on neutrophil nanovesicles that combines anti-inflammatory and anti-angiogenic bidirectional trapping with autoluminescent dynamics, its preparation method, and its application. However, these descriptions should not be construed as limiting the scope of protection of this invention.

[0070] Example 1

[0071] A method for preparing a complex based on neutrophil nanovesicles that combines anti-inflammatory and anti-angiogenic bidirectional trapping with autoluminescent dynamics.

[0072] The concentration is 4×10 -6 20 mL of mouse neutrophil (MNHC) cell suspension was sequentially extruded through polycarbonate membranes with pore sizes of 1 μm, 400 nm, and 100 nm using a microliposome extruder to obtain nanoscale extracellular vesicles (NVs). The NVs were ultracentrifuged at a gradient density of 10%–30% OptiPrep layers at 100,000 g and 4 °C for 0.5 h, and quantified using a BCA protein assay kit.

[0073] The NV obtained above was mixed with 15 μg of the photodynamic therapy (PDT) related drug bis(2,4,5-trichloro-2-[(pentyloxy)carbonyl]phenyl}oxalate (CPPO) and 4 μg of dihydroporphyrin E6 (Chlorin E6, Ce6) in PBS and incubated with shaking at 37°C for 3 h. Then, residual unencapsulated CPPO and Ce6 were removed using an ultrafiltration tube to obtain the NV encapsulated with CPPO and Ce6, denoted as NCC.

[0074] A ROS-responsive ketothiol (TK) with maleamide (MAL) and distearate phosphatidylethanolamine (DSPE) groups attached to both ends, denoted as DSPE-TK-MAL, was dissolved in DMF. Then, ranibizumab solution (10 mg / ml) was added at a molar ratio of TK to ranibizumab of 50:1. After incubation at 4°C for 24 h, unbound DSPE-TK-MAL was removed using ultrafiltration to obtain ranibizumab-bound DSPE-TK-MAL, denoted as TK-ranibizumab. The purified TK-ranibizumab was then resuspended in PBS.

[0075] NCC and TK-ranibizumab solution were incubated at 37°C for 0.5 h at a molar ratio of 1:50. Then, the unbound TK-ranibizumab was removed by ultrafiltration to obtain NCC bound to TK-ranibizumab, which was denoted as NCCA drug.

[0076] Example 2

[0077] To verify the ability of NCCA prepared in Example 1 to resist corneal neovascularization in vivo, a mouse model of alkali-induced corneal neovascularization was established by burning mouse corneas with 2.0 mm filter paper moistened with 1M NaOH solution, referring to internationally accepted modeling methods.

[0078] The mouse model of alkali burn was divided into two groups. One group was injected with 10 μl of the drug solvent (PBS), denoted as the model group; the other group was treated with 10 μl of NCCA, denoted as the treatment group. The mice were treated with eye drops three times a day, and the condition of the eyes of the two groups of mice was observed and photographed every other day.

[0079] Figure 2 This shows the corneal angiogenesis in two groups of mice. Figure 2 It was observed that the control group showed extensive red blood vessels on the surface of their eyes, indicating the formation of numerous corneal neovascularizations, thus successfully establishing a corneal neovascularization mouse model. The drug-treated group showed mild red blood vessels on the surface of their eyes, and compared to the control group, the drug-treated group exhibited significantly reduced corneal neovascularization.

[0080] Example 3

[0081] A method for preparing a complex based on neutrophil nanovesicles that combines anti-inflammatory and anti-angiogenic bidirectional trapping with autoluminescent dynamics.

[0082] The concentration is 5×10 -620 mL of mouse neutrophil (MNHC) cell suspension was sequentially extruded through polycarbonate membranes with pore sizes of 1 μm, 400 nm, and 100 nm using a microliposome extruder to obtain nanoscale extracellular vesicles. The NVs were then ultracentrifuged at a gradient density of 15% OptiPrep layer at 100,000 g for 0.8 h at 4 °C, and quantified using a BCA protein assay kit.

[0083] The NV prepared above was mixed with 16 μg CPPO and 5 μg Ce6 in PBS and incubated at 37°C with shaking for 3 h. Then, residual unloaded CPPO and Ce6 were removed using an ultrafiltration tube to obtain NCC. DSPE-TK-MAL was dissolved in DMF, and then ranibizumab solution (10 mg / ml) was added at a molar ratio of 80:1 (TK:ranibizumab). After incubation at 4°C for 24 h, the peptide was linked to ranibizumab via a condensation reaction between the MAL group and the amino group of the linker. Residual DSPE-TK-MAL was removed using an ultrafiltration tube. The purified TK-ranibizumab was resuspended in PBS. NCC and TK-ranibizumab were mixed at a molar ratio of 1:80 and incubated at 37°C for 0.5 h. Then, residual TK-ranibizumab was removed using an ultrafiltration tube to obtain NCCA.

[0084] The prepared NCCA was administered to a mouse model of corneal neovascularization according to the method in Example 2 to verify its efficacy. The results are shown below. Figure 3 The results showed that, compared with the model group, corneal angiogenesis was significantly reduced in the drug-treated mice.

[0085] Example 4

[0086] A method for preparing a complex based on neutrophil nanovesicles that combines anti-inflammatory and anti-angiogenic bidirectional trapping with autoluminescent dynamics.

[0087] The concentration is 7×10 -6 20 mL of mouse neutrophil (MNHC) cell suspension was sequentially extruded through polycarbonate membranes with pore sizes of 1 μm, 400 nm, and 100 nm using a microliposome extruder to obtain nanoscale extracellular vesicles. The NVs were then ultracentrifuged at a gradient density of 20% OptiPrep layer at 100,000 g for 1 h at 4 °C, and quantified using a BCA protein assay kit.

[0088] The NV prepared above was mixed with 18 μg CPPO and 6 μg CE6 in PBS and incubated at 37°C with shaking for 3 h. Then, residual unencapsulated CPPO and Ce6 were removed using an ultrafiltration tube to obtain NCC. DSPE-TK-MAL was dissolved in DMF, and then ranibizumab solution (10 mg / ml) was added at a molar ratio of 100:1 (TK:ranibizumab). After incubation at 4°C for 24 h, the peptide was linked to ranibizumab via a condensation reaction between the MAL group and the amino group of the linker. Residual DSPE-TK-MAL was removed using an ultrafiltration tube. The purified TK-ranibizumab was resuspended in PBS. NCC and TK-ranibizumab were incubated at 37°C for 0.5 h at a molar ratio of 1:100. Then, residual TK-ranibizumab was removed using an ultrafiltration tube to obtain NCCA.

[0089] The prepared NCCA was administered to a mouse model of corneal neovascularization according to the method in Example 2 to verify its efficacy. The results are shown below. Figure 4 The results showed that, compared with the model group, corneal angiogenesis was significantly reduced in the drug-treated mice.

[0090] Example 5

[0091] Characterization of the structure and properties of NCCA prepared in Example 4

[0092] 1. Characterization of NCCA's morphology and other properties

[0093] The prepared NCCA was diluted to 1 mL with PBS, and 5 μL was added to a copper grid on a carbon support film. After drying, the morphology of the NCCA was captured using a 120 kV transmission electron microscope (TEM). Simultaneously, the prepared NV was characterized using the same method.

[0094] Figure 5 TEM images reveal the exosome structure of NV, namely a core-shell nanostructure composed of a bilayer membrane and a protein cavity. Figure 5 (Middle left image). Figure 5 The right-middle image shows the TEM image from NCCA. The results show that ultrafiltration and incubation did not change the overall structure of the NV, maintaining its integrity.

[0095] 2. Particle size and potential characterization of NCCA

[0096] NCCA was diluted 100-fold with deionized water and placed in a sample cell. The hydrated particle size and zeta potential were measured using a Malvern nanoparticle size potentiostat. NV particle size and potential were also measured simultaneously.

[0097] See results Figure 6The hydrated particle size and zeta potential of NCCA were measured using a nanoparticle size analyzer. Loading with CPPO, Ce6, and ranibizumab reduced the potential.

[0098] 3. Detection of fluorescence co-localization of Ce6, ranibizumab, and NV

[0099] The synthesis was verified by recording the co-localization of Ce6 (autofluorescence), Cy3-ranibizumab, and DIR-NV (DIR is a fluorescently labeled molecule) in NCCA using confocal microscopy.

[0100] like Figure 7 As shown, Ce6, Cy3-ranibizumab, and DIR-NV exhibit significant co-localization under confocal microscopy, with a Pearson coefficient of 84%, verifying the successful loading of Ce6 and ranibizumab onto NV.

[0101] 4. Ce6 package included in NCCA verification experiments

[0102] The UV absorption spectra of Ce6 and NCCA were measured using an ELISA reader. Results are shown below. Figure 8 The characteristic absorption peak of Ce6 at 400 nm can be observed in the ultraviolet absorption spectra of both, which proves that Ce6 has been successfully loaded onto NCCA.

[0103] 5. Functions of CPPO in NCCA

[0104] use 1 O2 detection reagent ABDA verifies NCCA production under different concentrations of H2O2. 1 O2. ABDA can react with the system. 1 The O2 reaction causes a decrease in absorbance at 378 nm, which is used to detect the presence of O2 in the system. 1 O2 is generated and is concentration-dependent on H2O2.

[0105] from Figure 9 It can be seen that the A / A0 ratio in the NCCA group decreased with increasing H2O2 concentration, while there was no significant change in the PBS, CPPO, and Ce6 groups. This proves that CPPO in NCCA can react with H2O2 to generate chemical energy that activates Ce6 production. 1 O2 and exhibits a concentration dependence of H2O2.

[0106] 6. NCCA Stability Testing

[0107] The hydrated particle size of NCCA diluted 100 times in PBS or FBS solution was measured on days 0, 1, 2, and 3 using a nanoparticle size analyzer to determine its stability.

[0108] like Figure 10As shown, the particle size of the NCCA solution did not change significantly in PBS and FBS within 3 days of storage, demonstrating its good stability, which is beneficial for its storage and maintenance of its activity.

[0109] 7. Study on in vitro ROS-responsive release of NCCA

[0110] 100kDa dialysis tubes containing NCCA were placed in PBS and 10mM H2O2, respectively, and stirred continuously on a magnetic stirrer for 48 hours. 1mL of the suspension was collected at different time points. Ranibizumab release was detected at different time points within 48 hours using a ranibizumab ELISA assay kit.

[0111]

[0112] Where E is the release efficiency of ranibizumab; V1 is the sampling volume (1 mL); V0 is the total release volume (10 mL). Ci is the concentration of ranibizumab in the release solution at the i-th sampling (pg / mL); mRan is the total mass of ranibizumab released from the nanoparticles (pg); n is the number of samplings; and Cn is the concentration of ranibizumab in the release solution at the n-th sampling.

[0113] See results Figure 11 Compared with the PBS group, the release efficiency of ranibizumab in NCCA was higher under H2O2 stimulation, reaching 92% at 48h, demonstrating that NCCA has the ability to release ranibizumab in response to ROS.

[0114] 8. Validation of NV's ability to capture inflammatory cytokines

[0115] Mouse neutrophils were multiplied to 80% in culture medium, and total protein was extracted. All operations were performed on ice. After washing three times with PBS, the cells were aspirated and dried. Cell lysis buffer (RIPA lysis buffer with 1 / 100 PMSF and phosphatase inhibitor added) was added, and the cells were lysed on ice for 10 min. The lysate was then transferred to EP tubes and lysed for another 30 min, with shaking every 10 min. After centrifugation at 4°C and 12000 rpm for 30 min, the supernatant was obtained as the target protein. The protein concentration was determined using BCA assay. 5× Loading buffer was added to the remaining protein sample and mixed. The sample was then heated in a metal bath (95°C, 10 min) to denature the protein. After cooling, the protein sample was aliquoted and stored at -80°C for later use. Freshly extruded NV was ultracentrifuged, and the precipitate was resuspended in 100 μL PBS. The NV protein concentration was determined using BCA assay. 1.5 mm thick gel plates were carefully washed with long and short glass plates and dried before being assembled on electrophoresis clamps. Add water to check for tight assembly to prevent leakage, discard the water, and blot dry with filter paper. Prepare the lower gel mixture by mixing 4 mL of the lower gel solution, 4 mL of the lower gel buffer, and 80 μL of the coagulant. Pour the mixture into a glass plate, press with anhydrous ethanol, and allow to solidify at room temperature for 20 minutes. Discard the anhydrous ethanol and blot dry with filter paper. Next, mix 1 mL of the upper gel solution, 1 mL of the upper gel buffer, and 20 μL of the coagulant, add to the glass plate, insert the comb, and allow to stand for 20 minutes until the upper gel solidifies. Remove the comb and prepare for electrophoresis. Assemble the electrophoresis apparatus and pour in electrophoresis buffer until the liquid level is level with the top of the electrophoresis plate. Add the pre-stained protein marker and protein sample sequentially, and accurately connect the positive and negative electrodes. First, adjust the electrophoresis apparatus voltage to 80V for electrophoresis. Once the pre-stained protein markers have separated into clear protein bands, change the voltage to 120V and continue electrophoresis until the protein bands reach the bottom of the electrophoresis tank. After electrophoresis, cut the desired gel for staining. Place the gel in an appropriately sized container, add approximately 20 mL of Coomassie Brilliant Blue staining solution, and stain for 30 minutes. Discard the staining solution, add Coomassie Brilliant Blue destaining solution, and shake on a shaker for destaining. Every 10 minutes or so, carefully discard the destaining solution and add fresh destaining solution until a low-background Coomassie Brilliant Blue stained target band is obtained. Use a chemiluminescence imaging system to capture images of the Coomassie Brilliant Blue stained gel.

[0116] Western blotting analysis showed that chemokine receptors (CCR2, CXCR2, and CXCR4) and inflammatory cytokine receptors (IL-1R, IL-6R, and TNF-αR) were detectable in both MNHC and NV, indicating that NV carries receptors that trap inflammatory cytokines. The expression of these inflammatory cytokine receptors endows NCCA with anti-inflammatory properties by specifically capturing inflammatory cytokines. Figure 12 (a)

[0117] To verify this, changes in cytokine concentrations after incubation with NV in vitro were examined. As expected, the concentrations of MMP2, IL-6, and TNF-α were significantly reduced in the NV treatment group. Figure 12 (b) This strongly supports the feasibility of using NCCA as a decoy to eliminate inflammatory cytokines at the lesion site.

[0118] Example 6

[0119] Compared to existing corneal neovascularization (CorNV) regimens (ranibizumab), NCCA prolongs the duration of action in the eye.

[0120] The corneal pharmacodynamics of NCCA was evaluated in an alkali burn-induced corneal neovascularization (CorNV) mouse model. To assess the ability of NCCA to prolong ocular surface retention time, ranibizumab was fluorescently labeled. After establishing the alkali burn-induced corneal neovascularization mouse model, mice were randomly divided into ranibizumab treatment group and NCCA treatment group. After instilling NCCA into the eyes at a concentration of 10 mg / mL, fluorescence imaging of the eyes was performed using a small animal imaging system.

[0121] The results are as follows Figure 13 As shown, the fluorescence intensity of ranibizumab alone decreased rapidly, dropping by more than 50% within 30 minutes, indicating a short corneal retention time. Conversely, the fluorescence signal of the NCCA group remained above 80% within 40 minutes, and the fluorescence signal of the NCCA group was consistently stronger than that of the ranibizumab group. This demonstrates that the lipid-soluble NCCA has better anterior corneal retention capacity. These results suggest that NV is a promising drug carrier that can prolong anterior corneal retention time, allowing for more time for drug penetration into the cornea.

[0122] Example 7

[0123] Compared to CorNV's existing regimen (ranibizumab), NCCA achieves spontaneous targeting to the lesion site.

[0124] Two-photon fluorescence microscopy experiment, 2D ( Figure 14 a) and 3D Figure 14 (b) Reconstructed fluorescence images show that the fluorescence signal of NCCA is much stronger than that of Rani, further confirming that the pharmacodynamics of NCCA in the cornea is superior to that of free ranibizumab. Furthermore, the fluorescence signal of NCCA is highly colocalized with that of neovascularization, indicating that NCCA has a good ability to migrate and target to the lesion site.

[0125] Example 8

[0126] To investigate the in vitro anti-endothelial cell tube-forming ability of NCCA, an in vitro tube-forming experiment was conducted. The specific steps of the HUVEC tube-forming experiment are as follows:

[0127] (1) After the base adhesive is taken out of the -20℃ refrigerator, it is placed in the 4℃ refrigerator overnight to melt.

[0128] (2) Before performing the experiment, pre-cool the pipette tips, centrifuge tubes and 96-well plates in a refrigerator. Disinfect the remaining experimental supplies with alcohol and place them in a clean bench for at least 30 minutes of UV irradiation.

[0129] (3) After the matrix gel has completely melted, dispense and plate it using pre-cooled pipette tips and centrifuge tubes. Use 50L of matrix gel per well of a 96-well plate, avoiding air bubbles, and operate on ice throughout the process. After plating, place the plate in a 37°C incubator for 1 hour to allow the matrix gel to solidify.

[0130] (4) After the matrix gel solidifies, HUVECs are plated, with 20,000-30,000 cells per well. Different treatment drugs are added to the wells.

[0131] The experimental groups were as follows:

[0132] Control group: ECM medium containing 1% FEB;

[0133] H2O2 group: ECM medium containing 0.1 μM H2O2 + 1% FBS;

[0134] Ranibizumab group: ECM medium containing 0.1 μM H2O2 + ranibizumab + 1% FBS;

[0135] NV-Rabbitzumab group: ECM medium containing 0.1 μM H2O2 + NV-Rabbitzumab + 1% FBS;

[0136] NCCA group: ECM medium containing 0.1 μM H2O2 + NCCA (prepared in Example 4) + 1% FBS.

[0137] The preparation method of NV-ranibizumab is as follows: DSPE-TK-MAL was dissolved in DMF, and then ranibizumab solution (10 mg / ml) was added at a molar ratio of 100:1 (TK:ranibizumab). After incubation at 4°C for 24 h, the peptide was linked to ranibizumab via a condensation reaction between the MAL group and the amino group of ranibizumab. Residual DSPE-TK-MAL was removed using an ultrafiltration tube. The purified TK-ranibizumab was resuspended in PBS. Neutrophil nanovesicles (NV) and TK-ranibizumab were incubated at 37°C for 0.5 h at a molar ratio of 1:100. Then, residual TK-ranibizumab was removed using an ultrafiltration tube to obtain NV-ranibizumab.

[0138] (5) Take photos at appropriate times and use ImageJ to statistically analyze the pipe situation.

[0139] The results are as follows Figure 15As shown, in the human umbilical vein endothelial cell (HUVEC) tuberculosis assay, NCCA exhibited a significant inhibitory effect on tuberculosis compared to the ranibizumab group. In the presence of H2O2, CPPO reacts with H2O2 to generate chemical energy, activating the singlet oxygen produced by the photosensitizer Ce6, which has a toxic effect on HUVECs, affecting their tuberculosis ability and thus inhibiting angiogenesis. Therefore, in vitro cell experiments have verified that NCCA has a stronger anti-angiogenic ability than existing methods.

[0140] Example 9

[0141] NCCA in vivo efficacy validation experiment

[0142] Cy7-labeled ranibizumab was prepared by incubating Cy7 fluorescent dye solution and ranibizumab solution at a molar ratio of 1:1 overnight at 4°C, followed by centrifugation and washing three times. The final fluorescent labeling material NCCA for Cy7-labeled ranibizumab was prepared using the same method. After successful model establishment, mice anesthetized by alkali burns were randomly divided into groups, and 10 μL of Cy7-labeled ranibizumab and Cy7-labeled NCCA were locally instilled onto the ocular surface, respectively. Fluorescence imaging of the head region was performed using an IVISLumina imaging system (PerkinElmer) equipped with filters (excitation / emission, 750 / 773 nm). Imaging was performed at different time points after administration (every 5 min) up to 40 min after administration. The initial fluorescence intensity was set to 100%, and the fluorescence intensity at each subsequent time point was normalized accordingly.

[0143] See results Figure 16 Compared with the normal control group, corneal angiogenesis gradually increased in the alkali burn group over time. Compared with the alkali burn group, corneal angiogenesis was slightly reduced in the ranibizumab group, while the NCC group showed a slower rate of corneal angiogenesis and a significantly reduced number of corneal vessels compared to the alkali burn group. In vivo animal experiments demonstrate that the NCCA provided by this invention is more effective than ranibizumab in inhibiting angiogenesis.

[0144] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The use of a complex of anti-inflammatory and anti-angiogenic bidirectional trapping combined with autoluminescent dynamics in the preparation of a medicament for the prevention and / or treatment of ocular corneal injury-related diseases, said complex comprising neutrophil nanovesicles and bi(2,4,5-trichlorosalicylate n-pentyl) oxalate and dihydroporphyrin E6 encapsulated between phospholipid bilayers of said neutrophil nanovesicles, and ranibizumab attached to the outer surface of said neutrophil nanovesicles; The ranibizumab is linked to the neutrophil nanovesicles via DSPE-TK-MAL. MAL is maleimide group; DSPE is distearate phosphatidylethanolamine; TK is ketthioglycolate. The corneal injury-related diseases include keratitis and / or corneal neovascularization.

2. The application according to claim 1, characterized in that, The ketithiolide is linked to neutrophil nanovesicles via the phospholipid portion of distearate phosphatidylethanolamine. The ketithiolide is linked to ranibizumab via an amide bond formed by the maleimide group and the thiol group on ranibizumab.

3. The application according to claim 1, characterized in that, The neutrophil nanovesicles have a particle size of 150~200nm.

4. The application according to claim 1, characterized in that, Each 1×10 10 Each neutrophil nanovesicle contains 15–20 µg of bis(2,4,5-trichlorosalicylate n-pentyl) oxalate, 4–6 µg of dihydroporphyrin E6, and 5–10 µg of ranibizumab.

5. The application according to any one of claims 1 to 4, characterized in that, The method for preparing the complex includes the following steps: Neutrophil nanovesicles, bis(2,4,5-trichlorosalicylate n-pentyl oxalate) and dihydroporphyrin E6 were first mixed and co-incubated, and neutrophil nanovesicles loaded with bis(2,4,5-trichlorosalicylate n-pentyl oxalate) and dihydroporphyrin E6 were collected. Ranibizumab was linked to DSPE-TK-MAL to obtain DSPE-TK-MAL with ranibizumab linked to it. The DSPE-TK-MAL conjugated with ranibizumab and the neutrophil nanovesicles loaded with bis(2,4,5-trichlorosalicylic acid n-pentyl oxalate) and dihydroporphyrin E6 were subjected to a second co-incubation to obtain the complex.

6. The application according to claim 5, characterized in that, The method for preparing neutrophil nanovesicles includes sequentially extruding a neutrophil suspension through polycarbonate membranes of 1 μm, 400 nm and 100 nm to collect nanovesicles. The nanovesicles were subjected to density gradient centrifugation with OptiPrep at a volume concentration of 10% to 30%, and 10% and 30% of the separated layer components were collected to obtain neutrophil nanovesicles.

7. The application according to claim 5, characterized in that, The molar ratio of ranibizumab to ketothiolate is 1:(50~100). The concentration of ranibizumab is 6~10 mg / mL; The molar ratio of the DSPE-TK-MAL conjugated with ranibizumab to the neutrophil nanovesicles loaded with bis(2,4,5-trichlorosalicylic acid n-pentyl oxalate) and dihydroporphyrin E6 is (50~100):1.

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