Anti-inflammatory and anti-vascular bidirectional trapping combined self-luminous power compound based on neutrophil nano-vesicles as well as preparation method and application of anti-inflammatory and anti-vascular bidirectional trapping combined self-luminous power compound

Abstract

The invention provides an anti-inflammatory and anti-vascular bidirectional trapping combined self-luminous power compound based on neutrophil nano-vesicles as well as a preparation method and application thereof, and belongs to the technical field of biological medicine manufacturing. The compound takes a neutrophil nano-vesicle as a carrier, and is also loaded with bis (2, 4, 5-trichlorosalicylic acid n-amyl ester) oxalate (CPPO) and a photosensitizer Ce6, and meanwhile, the surface of the compound is modified with ranibizumab through ketal mercaptan responded by ROS (reactive oxygen species). The compound has the characteristics of trapping anti-inflammatory factors and spontaneously targeting corneal neovascularization diseased regions, Ranibizumab is responsively released on the basis of ROS to be combined with vascular endothelial growth factors, the anti-angiogenesis function is achieved, meanwhile, chemical energy generated by CPPO under the high-oxygen condition stimulates Ce6 to generate singlet oxygen, and therefore the vascular endothelial cell tubulogenesis capacity is inhibited. The invention provides a new means for treatment of eye disease related diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical manufacturing, and specifically relates to a neutrophil nanovesicle-based anti-inflammatory and anti-angiogenic bidirectional trapping combined with self-luminous power complex, and a preparation method and application thereof. Background Art

[0002] The cornea is completely transparent and avascular in its normal state, and acts 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 vascular plexus often leads to corneal neovascularization (CorNV), which is the second leading cause of blindness worldwide, severely reducing the quality of life of patients, and an effective treatment is urgently needed.

[0003] Since the progression of 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), 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. In addition, given the orderliness and complexity of the corneal neovascularization process, single-target therapy cannot completely inhibit the occurrence of neovascularization. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide an anti-inflammatory and anti-angiogenic bidirectional trapping and self-luminescent dynamic complex (NCCA) based on neutrophil nanovesicles, which achieves anti-inflammatory and anti-angiogenesis by neutralizing inflammatory factors and VEGF and chemically stimulated photodynamic therapy (PDT), thereby effectively treating eye-related diseases.

[0005] The present invention provides an anti-inflammatory and anti-angiogenic bidirectional trapping combined with self-luminous power complex, comprising neutrophil nanovesicles, bis(2,4,5-trichlorosalicylic acid n-pentyl ester) oxalate (CPPO) and chlorin E6 (Ce6) encapsulated between the phospholipid bilayers of the neutrophil nanovesicles, and ranibizumab (Ranibizumab) connected to the outer surface of the neutrophil nanovesicles;

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

[0007] Preferably, one end of the thioketal (TK) is connected to a maleamide group (MAL), and the other end is connected to distearoylphosphatidylethanolamine (DSPE);

[0008] The thioketal is linked to the neutrophil nanovesicles via the phospholipid portion of distearoylphosphatidylethanolamine;

[0009] The thioketal is linked to ranibizumab via an amide bond formed by the maleimide group and the sulfhydryl group on ranibizumab.

[0010] Preferably, the particle size of the neutrophil nanovesicles is 150 to 200 nm.

[0011] Preferably, each 1×10 10 Each neutrophil nanovesicle contained 15-20 μg of bis(2,4,5-trichlorosalicylic acid n-pentyl ester) oxalate, 4-6 μg of dihydrochlorin E6 and 5-10 μg of ranibizumab.

[0012] The present invention provides a method for preparing the composite, comprising the following steps:

[0013] The neutrophil nanovesicles, bis(2,4,5-trichlorosalicylic acid n-pentyl) oxalate and dihydrochlorin E6 are first mixed and co-incubated, and the neutrophil nanovesicles loaded with bis(2,4,5-trichlorosalicylic acid n-pentyl) oxalate and dihydrochlorin E6 are collected;

[0014] connecting ranibizumab to the thioketal to obtain the thioketal connected with ranibizumab;

[0015] The thioketal linked to ranibizumab and the neutrophil nanovesicles loaded with bis(2,4,5-trichlorosalicylic acid n-pentyl ester) oxalate and dihydrochlorin E6 are mixed and co-incubated for a second time to obtain a complex.

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

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

[0018] Preferably, each 1×10 10 Each neutrophil nanovesicle contained 15-20 μg of bis(2,4,5-trichlorosalicylic acid n-pentyl ester) oxalate and 4-6 μg of dihydrochlorin E6;

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

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

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

[0022] The present invention provides application of the complex or the complex prepared by the preparation method in preparing anti-inflammatory and / or anti-corneal angiogenesis drugs.

[0023] The present invention provides application of the complex or the complex prepared by the preparation method in preparing medicines for preventing and / or treating diseases related to corneal damage of the eye.

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

[0025] The present invention provides a complex of anti-inflammatory and anti-angiogenic bidirectional trapping combined with self-luminous power, using neutrophil nanovesicles (NVs) as carriers, encapsulating photodynamic therapy (PDT) related drugs CPPO and Ce6 between its phospholipid bilayers, and at the same time, its surface is modified with ranibizumab through ROS-responsive ketalthiols. The complex exerts its anti-inflammatory and neovascularization efficacy through multiple pathways. First, due to the presence of chemokine receptors, the neutrophil nanovesicles can penetrate corneal epithelial cells and spontaneously target the inflammatory site, thereby quickly reaching the lesion site of corneal neovascularization; when the complex enters the cornea, the inflammatory factor receptors on the neutrophil vesicles actively trap inflammatory factors to play an anti-inflammatory role; second, the ranibizumab connected by the thiol ketone (TK) bond in the complex is released in the high reactive oxygen species (ROS) microenvironment of the corneal angiogenesis lesion site, and the ranibizumab special The heterosexual capture of vascular endothelial growth factor inhibits the activation and proliferation of vascular endothelial cells, and at the same time, the lipid solubility of neutrophil vesicles is used to make the ranibizumab in the complex have good corneal retention ability, prolong the corneal retention time, and improve the drug penetration into the cornea to exert its efficacy; third, in the stage of angiogenesis and pericyte recruitment, the photodynamic therapy (PDT)-related drug CPPO produces chemical energy in a peroxic environment, thereby activating the photosensitizer Ce6 to produce singlet oxygen, which is toxic to vascular endothelial cells and affects its ability to form tubes, thereby eliminating the formed new blood vessels. The experiment of the present invention shows that the nanovesicle can significantly reduce the concentration of inflammatory factors, supporting the effect of the complex as an induction to eliminate inflammatory factors in the lesion site. In addition, in the embodiment of the present invention, the complex is also administered to a model mouse with corneal damage. The results show that compared with the existing scheme (ranibizumab) of corneal neovascularization (CorNV), NCCA prolongs the action time in the eye; at the same time, the complex can spontaneously target the neovascular lesion site. At the same time, cell experiments and animal experiments show that compared with the existing solution for corneal neovascularization (ranibizumab), NCCA shows stronger efficacy in anti-angiogenesis ability. It can be seen that the complex provided by the present invention can achieve the treatment of corneal angiogenesis in multiple targets and steps, providing an effective treatment for various diseases caused by corneal damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The synthesis and structural diagram of NCCA provided by the present invention;

[0027] Figure 2 This is a diagram showing the therapeutic effect of NCCA prepared in Example 1 in inhibiting corneal angiogenesis;

[0028] Figure 3 This is a diagram showing the therapeutic effect of NCCA prepared in Example 3 in inhibiting corneal angiogenesis;

[0029] Figure 4This is a diagram showing the therapeutic effect of NCCA prepared in Example 4 in inhibiting corneal angiogenesis;

[0030] Figure 5 The morphology characterization diagram of NCCA prepared in Example 4, wherein the left figure is a TEM image of NV, and the right figure is a TEM image of NCCA;

[0031] Figure 6 The hydration particle size and Zeta potential characterization results of NCCA prepared in Example 4, including a. particle size distribution of NV and NCCA; b. Zeta potential of NV and NCCA;

[0032] Figure 7 This is the NCCA fluorescence co-localization image prepared in Example 4;

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

[0034] Fig. 9 for 1 O2 detection reagent ABDA verifies the generation of NCCA prepared in Example 4 under different concentrations of H2O2 1 Results for O2;

[0035] Fig.10 This is a graph showing the stability results of NCCA prepared in Example 4;

[0036] Fig.11 ROS responsive release of NCCA prepared in Example 4

[0037] Fig.12 The results of Western-blotting to verify the inflammatory factor trapping characteristics of NV; a is the detection gel image; b is the statistical result image;

[0038] Fig.13 The results of the evaluation of NCCA ocular surface retention include: a. NCCA fluorescence images of the mouse model eye; b. Quantitative results of fluorescence signals of the mouse model eye at different time points;

[0039] Fig.14 Results of NCCA targeting ability assessment; a. 2D two-photon fluorescence microscopy fluorescence image of mouse model eye; b. 3D two-photon fluorescence microscopy fluorescence image of mouse model eye;

[0040] Fig.15 The results of NCCA inhibiting HUVEC angiogenesis in a high ROS environment; a is a photo of angiogenesis taken with an inverted microscope after different treatments, b is the quantitative result of the number of nodes, c is the quantitative result of the number of grids, and d is the quantitative result of the total branch length;

[0041] Fig.16 The in vivo therapeutic effect of NCCA on the alkali burn-induced corneal neovascularization mouse model; Note: Representative photos of the alkali burn-induced corneal neovascularization model after 0, 3, and 7 days of intervention with ranibizumab and NCCA. DETAILED DESCRIPTION

[0042] The present invention provides an anti-inflammatory and anti-angiogenic bidirectional trapping combined with self-luminous power complex, comprising neutrophil nanovesicles, bis(2,4,5-trichlorosalicylic acid n-pentyl ester) oxalate and dihydrochlorin E6 encapsulated between the phospholipid bilayers of the neutrophil nanovesicles, and ranibizumab connected to the outer surface of the neutrophil nanovesicles; the ranibizumab is connected to the neutrophil nanovesicles via thioketal.

[0043] In the present invention, the complex is in the form of a vesicle, and the particle size is 210 to 230 nm. The complex is based on neutrophil nanovesicles as carriers. The particle size of the neutrophil nanovesicles is preferably 150 to 200 nm. Inflammatory factor receptors exist on the neutrophil nanovesicles. Due to the strong inflammatory response at the site of corneal neovascularization, the binding characteristics of inflammatory factor receptors and inflammatory factors are utilized to enable the complex to spontaneously target the lesion site of corneal neovascularization, thereby improving the targeting of the carried drug; at the same time, the neutrophil nanovesicles use inflammatory factor receptors to trap inflammatory factors, thereby playing an anti-inflammatory role. In addition, the neutrophil nanovesicles have good lipid solubility, and the complex uses neutrophil nanovesicles as carriers, which increases the residence time of the complex at the site of corneal angiogenesis, which is beneficial to improving the efficacy of the carried drug.

[0044] In one embodiment of the present invention, the Western-blotting method was used to verify the trapping properties of neutrophil nanovesicles for inflammatory factors. 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 proved the feasibility of using neutrophil nanovesicles as bait for NCCA to eliminate inflammatory cytokines in lesions.

[0045] In the present invention, the complex includes CPPO, a drug related to photodynamic therapy (PDT), and Ce6, a photosensitizer. When the CPPO contacts an oxidant, chemical energy is generated, and the chemical energy excites Ce6 to generate singlet oxygen ( 1 O2), the singlet oxygen reacts with organic molecules in cells, causing oxidative damage to biological macromolecules such as cell membranes, proteins and DNA, and ultimately leading to cell death.

[0046] In one embodiment of the present invention, the complex produces 1O2, while there was no significant change in PBS, CPPO, and Ce6 groups. This proves that CPPO in the composite can react with H2O2 to generate chemical energy to activate Ce6 to produce 1 O2.

[0047] In the present invention, the complex includes ranibizumab targeting vascular endothelial growth factor (VEGF). The ranibizumab is connected to a carrier via a thioketal responsive to ROS. One end of the thioketal (TK) is preferably connected to a maleimide group (MAL), and the other end is preferably connected to distearoylphosphatidylethanolamine (DSPE), which is recorded as DSPE-TK-MAL. The thioketal is connected to neutrophil nanovesicles via the phospholipid part of distearoylphosphatidylethanolamine. The thioketal is connected to ranibizumab via an amide bond formed by a maleimide group and the sulfhydryl group on the ranibizumab. The corneal vascularization lesion site belongs to a highly reactive oxygen (ROS) microenvironment. After the complex enters the corneal lesion site, ranibizumab is released from the carrier under the action of ROS, and targeted binding to VEGF is blocked, thereby blocking VEGF-induced vascular endothelial cell activation and proliferation, blood vessel formation and pericyte recruitment, etc.

[0048] In another embodiment of the present invention, the ROS responsiveness of the complex was studied, and the results showed that compared with the PBS group, the release efficiency of ranibizumab in NCCA under H2O2 stimulation was higher, and the release rate could reach 92% at 48h. This indicates that NCCA has the ability to release ranibizumab in a ROS-responsive manner.

[0049] In the present invention, each 1×10 10 Each neutrophil nanovesicle contains 15-20 μg of bis(2,4,5-trichlorosalicylic acid n-pentyl ester) oxalate, 4-6 μg of chlorin E6 and 5-10 μg of ranibizumab. 10 Neutrophil nanovesicles are composed of 20 mL of 4-6×10 -6 A mouse neutrophil (MNHC) cell suspension with a concentration of 100 cells / mL was prepared.

[0050] The present invention provides a method for preparing the composite. Figure 1 , specifically including the following steps:

[0051] The neutrophil nanovesicles, bis(2,4,5-trichlorosalicylic acid n-pentyl) oxalate and dihydrochlorin E6 are first mixed and co-incubated, and the neutrophil nanovesicles loaded with bis(2,4,5-trichlorosalicylic acid n-pentyl) oxalate and dihydrochlorin E6 are collected;

[0052] connecting ranibizumab to the thioketal to obtain the thioketal connected with ranibizumab;

[0053] The thioketal linked to ranibizumab and the neutrophil nanovesicles loaded with bis(2,4,5-trichlorosalicylic acid n-pentyl ester) oxalate and dihydrochlorin E6 are mixed and co-incubated for a second time to obtain a complex.

[0054] The present invention performs a first mixed co-incubation on neutrophil nanovesicles, bis(2,4,5-trichlorosalicylic acid n-pentyl ester) oxalate and dihydrochlorin E6, and collects neutrophil nanovesicles loaded with bis(2,4,5-trichlorosalicylic acid n-pentyl ester) oxalate and dihydrochlorin E6.

[0055] In the present invention, the preparation method of the neutrophil nanovesicles preferably includes continuously extruding the neutrophil suspension through 1 μm, 400 nm and 100 nm polycarbonate membranes in sequence to collect the nanovesicles; subjecting the nanovesicles to density gradient centrifugation using OptiPrep with a volume concentration of 10% to 30%, collecting 10% separation layer and 30% separation layer components to obtain neutrophil nanovesicles.

[0056] In the present invention, the concentration of the neutrophil suspension is preferably 4×10 ～6 / mL~7×10 ～6 / mL, which can be 5×10 ～6 / mL. The extrusion is preferably completed in a microliposome extruder. The centrifugal force of the density gradient centrifugation is preferably 100000g, and the time is 0.5-1h. The morphological characterization results show that the particle size of the neutrophil nanovesicles is 150-200nm, and can be 180nm.

[0057] In the present invention, every 1×10 10 Each neutrophil nanovesicle contains 15-20 μg and 4-6 μg of bis(2,4,5-trichlorosalicylic acid n-pentyl ester) oxalate and dihydrochlorin E6, respectively; the concentration of the neutrophil nanovesicle is 10×10 10 / ml. The temperature of the mixed incubation is preferably 36-38°C, and can be 37°C. The time of the first mixed incubation is preferably 2.5-3.5h, and can be 3h. After the mixed incubation, it also includes removing the unencapsulated bis(2,4,5-trichlorosalicylic acid n-pentyl ester) oxalate and dihydrochlorin E6, preferably using an ultrafiltration tube. The pore size of the filter membrane of the ultrafiltration tube is preferably 100kDa. Collect the retentate in the ultrafiltration tube for later use.

[0058] The present invention connects ranibizumab to thioketal to obtain the thioketal connected with ranibizumab.

[0059] In the present invention, the molar ratio of ranibizumab to thioketal is preferably 1:(50-100), which may be 1:80. The concentration of ranibizumab is preferably 6-10 mg / mL. The thioketal is in the form of DSPE-TK-MAL. The solvent for dissolving DSPE-TK-MAL is DMF. The reaction conditions for the connection are preferably: incubation at 4°C for 24 hours. The connection is a condensation reaction between the MAL group on the thioketal and the amino group of ranibizumab to form an amide bond, thereby achieving the connection between the thioketal and ranibizumab. After connection, it is preferred to use an ultrafiltration method to remove the thioketal that is not connected to ranibizumab. The pore size of the filter membrane of the ultrafiltration tube used in the ultrafiltration method is preferably 10 kDa, and some components in the filter tube are collected for standby use.

[0060] In the present invention, the thioketal connected with ranibizumab and the neutrophil nanovesicles loaded with bis(2,4,5-trichlorosalicylic acid n-pentyl ester) oxalate and dihydrochlorin E6 are subjected to a second mixing and co-incubation to obtain a complex.

[0061] In the present invention, the mixing molar ratio of the thioketal connected with ranibizumab and the neutrophil nanovesicles loaded with bis(2,4,5-trichlorosalicylic acid n-pentyl ester) oxalate and dihydrochlorin E6 is preferably (50-100): 1. The temperature of the mixed co-incubation is preferably 36-38°C, and can be 37°C. The time of the mixed co-incubation is preferably 0.4-0.6h, and can be 0.5h. The second mixed co-incubation is that the thioketal connected with ranibizumab is connected to the neutrophil nanovesicle through the phospholipid part of DSPE.

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

[0063] The present invention provides application of the complex or the complex prepared by the preparation method in preparing anti-inflammatory and / or anti-corneal angiogenesis drugs.

[0064] In the present invention, the drug preferably includes at least one of the following external dosage forms: eye drops, eye gels and eye ointments. In the drug, the concentration of the complex is not less than 10 mg / mL, and can be 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL. The present invention has no special restrictions on the preparation method and use method of the drug, and the preparation method and use method of the drug known in the art can be used.

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

[0066] In the examples of the present invention, in vitro and in vivo experiments show that the composite prepared by the present invention can effectively reduce the number of corneal blood vessels in the corneal neovascularization mouse model induced by alkali burns.

[0067] The present invention provides application of the complex or the complex prepared by the preparation method in preparing medicines for preventing and / or treating diseases related to corneal damage of the eye.

[0068] In the present invention, the diseases related to corneal damage include keratitis, mechanical damage and chemical corneal damage.

[0069] The following is a detailed description of the anti-inflammatory and anti-vascular bidirectional trapping and self-luminous power complex based on neutrophil nanovesicles provided by the present invention, as well as its preparation method and application, in conjunction with the examples. However, they should not be construed as limiting the scope of protection of the present invention.

[0070] Example 1

[0071] A method for preparing a complex of anti-inflammatory and anti-angiogenic bidirectional trapping combined with self-luminous power based on neutrophil nanovesicles

[0072] The concentration was 4×10 -6 20 mL of mouse neutrophil (MNHC) cell suspension with a pore size of 1 μm, 400 nm and 100 nm was extruded continuously by a microliposome extruder to obtain nano-scale extracellular vesicles (NV). NV was ultracentrifuged at a gradient density consisting of 10% to 30% OptiPrep layer at 100,000 g and 4°C for 0.5 h, and quantified using a BCA protein analysis kit.

[0073] The NV obtained above was mixed with 15 μg of photodynamic therapy (PDT)-related drug Bis{3,4,6-trichloro-2-[(pentyloxy)carbonyl]phenyl}oxalate (CPPO) and 4 μg of Chlorin E6 (Ce6) in PBS and incubated with shaking at 37°C for 3 h. Then, the residual unencapsulated CPPO and Ce6 were removed by ultrafiltration tube to obtain NV encapsulated with CPPO and Ce6, which was recorded as NCC.

[0074] The ROS-responsive thioketal (TK) with maleimide group (MAL) and distearoylphosphatidylethanolamine (DSPE) connected at both ends was recorded as DSPE-TK-MAL, dissolved in DMF, and 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 hours, DSPE-TK-MAL that was not bound to ranibizumab was removed by ultrafiltration tube to obtain DSPE-TK-MAL bound to ranibizumab, recorded as TK-ranibizumab. The purified TK-ranibizumab was suspended in PBS.

[0075] The NCC and TK-ranibizumab solution were incubated at a molar ratio of 1:50 at 37° C. for 0.5 h. Then, the TK-ranibizumab not bound to the NCC was removed by an ultrafiltration tube to obtain the NCC bound to TK-ranibizumab, which was recorded as NCCA drug.

[0076] Example 2

[0077] In order to verify the anti-corneal neovascularization ability of NCCA prepared in Example 1 in vivo, an alkali burn-induced corneal neovascularization mouse model was established by alkali-burning the mouse cornea with 2.0 mm filter paper soaked in 1 M NaOH solution, referring to the internationally common modeling method.

[0078] The mouse model after alkali burn was divided into two groups, one group was injected with 10μl of drug solvent (PBS), recorded as the model group; the other group was given 10μl NCCA drug treatment, recorded as administration. Eye drops were given three times a day, and the eye conditions of the two groups of mice were observed and photographed every other day.

[0079] Figure 2 Figure 2 shows the corneal angiogenesis of the two groups of mice. Figure 2 It can be seen that the control group had a large area of ​​red blood on the surface of the eyeball, indicating that a large number of corneal neovascularization was formed on the mouse cornea, and the corneal neovascularization mouse model was successfully established. The drug-treated group had slight red blood on the surface of the eyeball of the model mouse, and compared with the control group, the corneal neovascularization of the drug-treated group was significantly reduced.

[0080] Example 3

[0081] A method for preparing a complex of anti-inflammatory and anti-angiogenic bidirectional trapping combined with self-luminous power based on neutrophil nanovesicles

[0082] The concentration was 5×10 -620 mL of mouse neutrophil (MNHC) cell suspension with a pore size of 1 μm, 400 nm and 100 nm was extruded continuously by a microliposome extruder to obtain nano-scale extracellular vesicles. Then, NV was ultracentrifuged with a gradient density consisting of 15% OptiPrep layer, 100,000 g, 4°C for 0.8 h, and quantified using a BCA protein analysis kit.

[0083] The NV prepared above was mixed with 16 μg CPPO and 5 μg Ce6 in PBS and incubated with shaking at 37°C for 3 h. Then, the residual unencapsulated CPPO and Ce6 were removed by ultrafiltration tube to obtain NCC. DSPE-TK-MAL was dissolved in DMF, and then a 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 through a condensation reaction between the MAL group of the linker and the amino group of ranibizumab. The residual DSPE-TK-MAL was removed by ultrafiltration tube. The purified TK-ranibizumab was suspended in PBS. NCC and TK-ranibizumab were mixed and incubated at 37°C for 0.5 h at a molar ratio of 1:80. Then, the residual TK-ranibizumab was removed by ultrafiltration tube to obtain NCCA.

[0084] The prepared NCCA was used to test the effect of administration in the corneal neovascularization mouse model according to the method of Example 2. The results are shown in Figure 3 The results showed that compared with the model group, the corneal angiogenesis in the mice in the drug-treated group was significantly reduced.

[0085] Example 4

[0086] A method for preparing a complex of anti-inflammatory and anti-angiogenic bidirectional trapping combined with self-luminous power based on neutrophil nanovesicles

[0087] The concentration was 7×10 -6 20 mL of mouse neutrophil (MNHC) cell suspension with a pore size of 1 μm, 400 nm and 100 nm was extruded continuously by a microliposome extruder to obtain nano-scale extracellular vesicles. Then, NV was ultracentrifuged with a gradient density consisting of 20% OptiPrep layer, 100,000 g, 4°C for 1 hour, and quantified using a BCA protein analysis kit.

[0088] The prepared NV was mixed with 18 μg CPPO and 6 μg CE6 in PBS and incubated with shaking at 37°C for 3 h. Then, the residual unencapsulated CPPO and Ce6 were removed by ultrafiltration tube to obtain NCC. DSPE-TK-MAL was dissolved in DMF, and then a 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 through a condensation reaction between the MAL group of the linker and the amino group of ranibizumab. The residual DSPE-TK-MAL was removed by ultrafiltration tube. The purified TK-ranibizumab was suspended in PBS. NCC was incubated with TK-ranibizumab at a molar ratio of 1:100 at 37°C for 0.5 h. Then, the residual TK-ranibizumab was removed by ultrafiltration tube to obtain NCCA.

[0089] The prepared NCCA was used to test the effect of administration in the corneal neovascularization mouse model according to the method of Example 2. The results are shown in Figure 4 The results showed that compared with the model group, the corneal angiogenesis in the mice in the drug-treated group was significantly reduced.

[0090] Example 5

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

[0092] 1. Characterization of NCCA morphology and other characteristics

[0093] Take the prepared NCCA, dilute it to 1 mL with PBS, drop 5 μL onto the carbon support film copper grid, and collect the morphology of NCCA using a 120 kV transmission electron microscope (TEM) after drying. At the same time, the prepared NV is characterized by the same method.

[0094] Figure 5 TEM images show the exosome structure of NV, which is a core-shell nanostructure consisting of a double-layer membrane and a protein cavity ( Figure 5 center left). Figure 5 The middle right picture is a TEM image of NCCA. The results show that ultrafiltration and incubation did not change the overall structure of NV and maintained the integrity of NV.

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

[0096] NCCA was diluted 100 times with deionized water and placed in a sample pool. The hydrated particle size and Zeta potential were measured using a Malvern nanoparticle size potentiometer.

[0097] Results Figure 6The hydrated particle size and Zeta potential of NCCA were measured by a nanoparticle size analyzer, and the potential decreased after loading CPPO, Ce6 and ranibizumab.

[0098] 3. Fluorescence co-localization detection of Ce6, ranibizumab and NV

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

[0100] like Figure 7 As shown, Ce6, Cy3-ranibizumab and DIR-NV had obvious colocalization under confocal microscopy, with a Pearson coefficient of 84%, verifying the successful loading of Ce6 and ranibizumab on NV.

[0101] 4. Ce6 package in NCCA verification experiment

[0102] The UV absorption spectra of Ce6 and NCCA were measured by an enzyme marker. Figure 8 The characteristic absorption peak of Ce6 at 400nm can be observed in the UV absorption spectra of both, which proves that Ce6 is successfully loaded onto NCCA.

[0103] 5. Function of CPPO in NCCA

[0104] use 1 O2 detection reagent ABDA verifies the production of NCCA under different concentrations of H2O2 1 O2. ABDA can react with 1 O2 reaction causes the absorbance at 378nm to decrease, which is used to detect whether there is any 1 The production of O2 is dependent on the concentration of H2O2.

[0105] from Fig. 9 It can be seen that the A / A0 of the NCCA group decreased with the increase of H2O2, while there was no significant change in the PBS, CPPO, and Ce6 groups, which proved that CPPO in NCCA can react with H2O2 to generate chemical energy to activate Ce6 to produce 1 O2 and has a concentration dependence on H2O2.

[0106] 6. Stability test of NCCA

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

[0108] like Fig.10As shown, the particle size of the NCCA solution in PBS and FBS did not change significantly after 3 days, which proved that it had good stability, which was beneficial for its storage and maintaining its activity.

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

[0110] The 100 kd dialysis tubes carrying NCCA were placed in PBS and 10 mM H2O2, respectively, and placed on a magnetic stirrer for continuous stirring for 48 h. 1 mL was taken out from the suspension at different time points. The release of ranibizumab at different time points within 48 h was detected using the ranibizumab Elisa detection kit.

[0111]

[0112] Wherein, E is the release efficiency of ranibizumab; V1 is the sampling volume (1 mL); V0 is the total volume of the release solution (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; Cn is the concentration of ranibizumab in the release solution at the n-th sampling.

[0113] Results Fig.11 Compared with the PBS group, the release efficiency of ranibizumab in NCCA under H2O2 stimulation was higher, and the release rate could reach 92% at 48 h, proving that NCCA has the ability to release ranibizumab in a ROS-responsive manner.

[0114] 8. Verification of NV’s ability to capture inflammatory cytokines

[0115] After the mouse neutrophils proliferated to 80% in the culture medium, the total protein was extracted. All operations were performed on ice. After washing with PBS by centrifugation for 3 times, the cells were dried and lysed on ice for 10 minutes by adding cell lysis buffer (1 / 100 PMSF and phosphatase inhibitors were added to the RIPA lysis buffer), and then transferred to an EP tube for further lysis for 30 minutes, with oscillation and mixing every 10 minutes. After centrifugation at 4°C and 12000rpm for 30 minutes, the supernatant obtained was the target protein. The protein concentration was determined using BCA detection solution. Add 5× Loading buffer to the remaining protein sample, mix well, and heat on a metal bath (95°C, 10 minutes) to denature the protein. After the protein sample cools, it is divided into portions and stored in a -80°C refrigerator for later use. After ultracentrifugation of the extruded fresh NV, the precipitate is resuspended in 100 μL PBS and the NV protein concentration is determined using BCA detection solution. Carefully wash the long and short glass plates for 1.5 mm gel thickness and dry them, and assemble them on the electrophoresis clamp. Add water to check whether the assembly is tight to prevent liquid leakage, discard the water and dry with filter paper. Absorb the lower layer glue solution (4mL), lower layer glue buffer (4mL) and coagulant (80μL) and mix them well to prepare the lower layer glue mixture, inject it into the glass plate, press the glue with anhydrous ethanol, solidify at room temperature for 20min, pour out the anhydrous ethanol, and dry with filter paper. Then, absorb the upper layer glue solution (1mL), upper layer glue buffer (1mL) and coagulant (20μL) and mix them evenly, add them to the glass plate, insert the tooth comb, let it stand for 20min, remove the tooth comb, and prepare for sample electrophoresis. Assemble the electrophoresis device and inject the electrophoresis buffer until the liquid level is level with the upper end of the electrophoresis plate. Add the pre-stained protein marker and protein sample in sequence, and accurately connect the positive and negative electrodes of the electrophoresis. First, adjust the electrophoresis instrument voltage to 80V for electrophoresis. After the pre-stained protein Marker is separated into clear protein bands, change the voltage to 120V and continue electrophoresis until the protein bands run to the bottom of the electrophoresis tank. After the electrophoresis is completed, cut the required gel for dyeing. Place the gel in a container of appropriate size, add about 20mL Coomassie Brilliant Blue staining solution, dye for 30min, discard the staining solution, add Coomassie Brilliant Blue destaining solution, and shake on a shaking table for decolorization. Every about 10min, carefully pour out the destaining solution, and then add new destaining solution until the target band of Coomassie Brilliant Blue staining with low background is obtained. Use a chemiluminescence imaging system to take pictures of the gel stained with Coomassie Brilliant Blue.

[0116] Western blotting analysis showed that chemokine receptors (CCR2, CXCR2, and CXCR4) and inflammatory factor receptors (IL-1R, IL-6R, and TNF-αR) were detected in both MNHC and NV, indicating that NV carries receptors that trap inflammatory factors, and the expression of inflammatory factor receptors confers anti-inflammatory properties to NCCA by specifically capturing inflammatory cytokines ( Fig.12 (a)

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

[0118] Example 6

[0119] NCCA has a prolonged duration of action in the eye compared to the current regimen (ranibizumab) for corneal neovascularization (CorNV)

[0120] The corneal pharmacodynamics of NCCA was evaluated in the alkali burn CorNV mouse model. To evaluate the ability of NCCA to prolong the ocular surface retention time, the alkali burn-induced corneal neovascularization mouse model was established with fluorescently labeled ranibizumab, and the mice were randomly divided into a ranibizumab-treated group and a NCCA-treated group. After eye drops at a concentration of 10 mg / mL, fluorescence imaging of the eyes was performed using a small animal imager.

[0121] The results are as follows Fig.13 As shown in the figure, the fluorescence intensity of ranibizumab alone decreased rapidly, and decreased by more than 50% within 30 minutes, indicating that ranibizumab alone has a short corneal retention time. In contrast, the fluorescence signal of the NCCA group remained above 80% within 40 minutes, and the fluorescence signal of the NCCA group was always stronger than that of the ranibizumab group. This proves that the fat-soluble NCCA has better anterior corneal retention ability. These results show that NV is a promising drug carrier that can prolong the anterior corneal retention time, which is beneficial for drugs to have more time to penetrate into the cornea.

[0122] Example 7

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

[0124] Two-photon fluorescence microscopy experiments, 2D ( Fig.14 a) and 3D( Fig.14 b) The reconstructed fluorescence image shows that the fluorescence signal of NCCA is much stronger than that of Rani, which once again confirms that the pharmacodynamics of NCCA in the cornea is superior to that of free ranibizumab. In addition, the fluorescence signal of NCCA is highly co-localized with the fluorescence signal of new blood vessels, indicating that NCCA has a good ability to migrate and target to the lesion site.

[0125] Example 8

[0126] In order to study the in vitro anti-endothelial cell tube formation ability of NCCA, an in vitro tube formation experiment was performed. The specific steps of the HUVEC tube formation experiment are as follows:

[0127] (1) Take out the matrix gel from -20℃ and place it in a 4℃ refrigerator to melt overnight.

[0128] (2) Before conducting the experiment, place the pipette tip, centrifuge tube, and 96-well plate in a refrigerator to pre-cool. Spray the remaining experimental supplies with alcohol for disinfection, place them in a clean bench, and irradiate them with ultraviolet light for more than 30 minutes.

[0129] (3) After the matrix gel is completely melted, use pre-cooled pipette tips and centrifuge tubes to dispense and plate the matrix gel. 50 L of matrix gel per well of a 96-well plate is sufficient to avoid bubbles. The whole process should be performed on ice. 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 to 30,000 cells per well. Different treatment drugs are added to the wells.

[0131] The experimental groups are:

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

[0136] NCCA group: ECM culture medium containing 0.1 μM H 2 O 2 + NCCA (prepared in Example 4) + 1% FBS.

[0137] The preparation method of NV-ranibizumab is as follows: DSPE-TK-MAL is dissolved in DMF, and then a ranibizumab solution (10 mg / ml) is added at a molar ratio of 100:1 (TK: ranibizumab). After incubation at 4°C for 24 hours, the polypeptide is connected to ranibizumab through a condensation reaction between the MAL group of the linker and the amino group of ranibizumab. The residual DSPE-TK-MAL is removed by an ultrafiltration tube. The purified TK-ranibizumab is suspended in PBS. The neutrophil nanovesicles NV and TK-ranibizumab are incubated at 37°C for 0.5 hours at a molar ratio of 1:100. Then, the residual TK-ranibizumab is removed by an ultrafiltration tube to obtain NV-ranibizumab. )

[0138] (5) Take photos and record them at appropriate time points, and use ImageJ to count the tube formation status.

[0139] The results are as follows Fig.15As shown in the human umbilical vein endothelial cell (HUVEC) tube formation experiment, NCCA showed a significant inhibitory effect on tube formation compared with the ranibizumab group. In the presence of H2O2, CPPO and H2O2 produce chemical energy, which activates the singlet oxygen produced by the photosensitizer Ce6, which can produce toxic effects on HUVEC, affecting the tube formation ability of HUVEC, thereby inhibiting angiogenesis. It can be seen that the in vitro cell experiment verified that NCCA has a stronger anti-angiogenesis ability than the existing scheme.

[0140] Example 9

[0141] NCCA in vivo efficacy validation experiment

[0142] The Cy7 fluorescent dye solution and the ranibizumab solution were incubated in a 4°C refrigerator at a molar ratio of 1:1 overnight, and the Cy7-labeled ranibizumab was prepared by centrifugation and washing three times. The fluorescent labeled final material NCCA of Cy7-labeled ranibizumab was prepared according to the above method. After the model was successfully established, the alkali-burned mice were anesthetized and randomly divided into groups, and 10 μL Cy7-labeled ranibizumab and Cy7-labeled NCCA were topically instilled on the surface of the eyeball. Fluorescence imaging of the head area was performed using an IVISLumina imaging system (PerkinElmer) equipped with a filter (excitation / emission, 750 / 773 nm). Imaging was performed at different time points (every 5 minutes) after administration to 40 minutes after administration. The initial fluorescence intensity was set to 100%, and the fluorescence intensity at each time point was then normalized accordingly.

[0143] Results Fig.16 . Compared with the normal control group, the alkali burn group showed a gradual increase in corneal angiogenesis over time. Compared with the alkali burn group, the corneal angiogenesis of mice in the ranibizumab group was slightly reduced, while the rate of corneal angiogenesis in the NCC group was reduced compared with the alkali burn group, and the number of corneal angiogenesis was also significantly reduced. In vivo animal experiments showed that the NCCA provided by the present invention has more advantages than ranibizumab treatment in anti-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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A complex of anti-inflammatory and anti-angiogenic dual-direction trapping combined with self-luminous power, characterized in that: It comprises neutrophil nanovesicles, bis(2,4,5-trichlorosalicylic acid n-pentyl ester) oxalate and chlorin E6 encapsulated between the phospholipid bilayers of the neutrophil nanovesicles, and ranibizumab connected to the outer surface of the neutrophil nanovesicles; The ranibizumab is linked to the neutrophil nanovesicles via thioketal.

2. The composite according to claim 1, characterized in that One end of the thioketal is connected to a maleamide group, and the other end is connected to distearoyl phosphatidylethanolamine; The thioketal is linked to the neutrophil nanovesicles via the phospholipid portion of distearoylphosphatidylethanolamine; The thioketal is linked to ranibizumab via an amide bond formed by the maleimide group and the sulfhydryl group on ranibizumab.

3. The composite according to claim 1, characterized in that The particle size of the neutrophil nanovesicle is 150-200 nm.

4. The composite according to claim 1, characterized in that 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 dihydrochlorin E6, and 5-10 μg of ranibizumab.

5. A method for preparing the composite according to any one of claims 1 to 4, characterized in that: The following steps are involved: The neutrophil nanovesicles, bis(2,4,5-trichlorosalicylic acid n-pentyl) oxalate and dihydrochlorin E6 are first mixed and co-incubated, and the neutrophil nanovesicles loaded with bis(2,4,5-trichlorosalicylic acid n-pentyl) oxalate and dihydrochlorin E6 are collected; connecting ranibizumab to the thioketal to obtain the thioketal connected with ranibizumab; The thioketal linked to ranibizumab and the neutrophil nanovesicles loaded with bis(2,4,5-trichlorosalicylic acid n-pentyl ester) oxalate and dihydrochlorin E6 are mixed and co-incubated for a second time to obtain a complex.

6. The preparation method according to claim 5, characterized in that: The method for preparing the neutrophil nanovesicles comprises sequentially extruding a neutrophil suspension with 1 μm, 400 nm and 100 nm polycarbonate membranes to collect the nanovesicles; The nanovesicles are subjected to density gradient centrifugation using OptiPrep with a volume concentration of 10% to 30%, and the 10% separation layer and 30% separation layer components are collected to obtain neutrophil nanovesicles.

7. The preparation method according to claim 5, characterized in that: Each 1×10 10 Each neutrophil nanovesicle contained 15-20 μg of bis(2,4,5-trichlorosalicylic acid n-pentyl) oxalate and 4-6 μg of chlorin E6; The molar ratio of ranibizumab to thioketal is 1:(50-100); The concentration of ranibizumab is 6 to 10 mg / mL; The molar ratio of the thioketal connected with ranibizumab to the neutrophil nanovesicles loaded with bis(2,4,5-trichlorosalicylic acid n-pentyl ester) oxalate and dihydrochlorin E6 is (50-100):

1.

8. Use of the complex according to any one of claims 1 to 4 or the complex prepared by the preparation method according to any one of claims 5 to 7 in the preparation of anti-inflammatory and / or anti-corneal angiogenesis drugs.

9. Use of the complex according to any one of claims 1 to 4 or the complex prepared by the preparation method according to any one of claims 5 to 7 in the preparation of a drug for preventing and / or treating diseases related to corneal damage.

10. An eye drop, characterized in that: The invention comprises the complex described in any one of claims 1 to 4 or the complex prepared by the preparation method described in any one of claims 5 to 7 and pharmaceutically acceptable excipients.

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