Use of a lipid-gold nanoparticle complex in the preparation of a medicament for treating proteinuria

Abstract

The application belongs to the technical field of proteinuria treatment drugs, and relates to application of a lipid gold nanoparticle complex in preparation of a proteinuria treatment drug. The lipid gold nanoparticle complex is composed of a cationic liposome, a gold nanoparticle complex and a targeting peptide. The cationic liposome coats the gold nanoparticle complex, and the targeting peptide is loaded on the surface of the cationic liposome after coating the gold nanoparticle complex. The gold nanoparticle complex is a complex of a gold nanoparticle and a VE-Cadherin overexpression plasmid. The sequence of the VE-Cadherin overexpression plasmid is shown as SEQ ID NO. 1, and the sequence of the targeting peptide is shown as SEQ ID NO. 2. The lipid gold nanoparticle complex provided by the application can repair the glomerular filtration barrier, thereby improving permeability, and further achieving treatment of proteinuria.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of proteinuria treatment drugs, and relates to application of a lipid gold nanoparticle complex in preparation of a proteinuria treatment drug. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing an understanding of the general background of the application and does not necessarily constitute an acknowledgement or a suggestion that the information forms part of the prior art already known to a person of ordinary skill in the art.

[0003] Proteinuria refers to an abnormal increase in protein levels in urine. The protein level in urine of a normal person is not more than 150 mg per day. Proteinuria is a diagnostic marker of kidney diseases, such as diabetes and chronic kidney disease, etc. In addition, proteinuria itself can cause progressive renal dysfunction and is an independent risk factor for the progression of chronic kidney disease. Glomerular proteinuria is the most common subtype of proteinuria and is caused by high permeability of albumin and abnormal filtration due to damage to the glomerular filtration barrier (GFB). The glomerular filtration barrier is composed of three layers of glomerular endothelium, glomerular basement membrane and epithelial cells. Previous studies have mainly focused on the dysfunction caused by damage to epithelial cells.

[0004] According to the research of the inventors, damage to the glomerular endothelium can also cause proteinuria. However, there is currently no drug that can repair the glomerular filtration barrier by repairing the glomerular endothelium to treat proteinuria. SUMMARY

[0005] In order to solve the problems of the prior art, the purpose of the present application is to provide application of a lipid gold nanoparticle complex in preparation of a proteinuria treatment drug, which can repair the glomerular filtration barrier, thereby improving permeability and achieving treatment of proteinuria.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0007] The application of a lipid gold nanoparticle complex in preparation of a proteinuria treatment drug, wherein the lipid gold nanoparticle complex is composed of a cationic liposome, a gold nanoparticle complex and a targeting peptide, the cationic liposome coats the gold nanoparticle complex, and the targeting peptide is loaded on the surface of the cationic liposome after coating the gold nanoparticle complex, the gold nanoparticle complex is a complex of a gold nanoparticle and a VE-Cadherin overexpression plasmid, the sequence of the VE-Cadherin overexpression plasmid is shown as SEQ ID NO. 1, and the sequence of the targeting peptide is shown as SEQ ID NO. 2.

[0008] The glomerular endothelium is composed of a continuous monolayer of cells arranged on the inner surface of the glomerular basement membrane. The connection between glomerular endothelial cells includes tight junction and gap junction, and the main connection mode for regulating the permeability of glomerular endothelial cells is adhesive junction. The adhesive junction is often destroyed under pathological conditions, leading to damage of the glomerular filtration barrier, increased permeability and formation of proteinuria. The VE-Cadherin protein is a key protein of the intercellular adhesive junction. The interaction of VE-Cadherin protein between adjacent endothelial cells forms an adhesive junction, which jointly regulates the material in and out. Therefore, the present application uses a VE-Cadherin overexpression plasmid to up-regulate the expression of VE-Cadherin protein by overexpression, so as to repair the damaged adhesive junction and reduce the vascular permeability. In order to improve the efficiency of the VE-Cadherin overexpression plasmid transported into the cell, the present application uses gold nanoparticles and cationic liposomes to load the VE-Cadherin overexpression plasmid, wherein the cationic liposomes wrap the nucleic acid molecules in the inside of the cationic liposomes by electrostatic adsorption, so as to avoid the decomposition of nuclease and improve the concentration of nucleic acid reaching the target tissue. In addition, the cationic liposomes with positive charge are more easily interacted with the cell membrane with negative charge outside the membrane, and are more easily endocytosed into the cell, and then the VE-Cadherin overexpression plasmid carried by the gold nanoparticles is released from the cationic liposomes, and then overexpression is carried out. At the same time, the addition of the targeting peptide can make the lipid gold nanoparticle complex directional transport to the vascular endothelial cells, and the polypeptide with the function of targeting endothelial vascular cells has many kinds, such as REDV, HHHRHSF, but experiments show that the selection of the targeting peptide also affects the effect of the lipid gold nanoparticle complex on the treatment of proteinuria, and only when HHHRHSF is selected as the targeting peptide, the selection delivery efficiency is higher, so that the damaged adhesive junction of the glomerular endothelial cells can be effectively repaired, and the proteinuria can be treated. When other polypeptides with the function of targeting endothelial vascular cells, such as REDV, are selected as the targeting peptide, the selection delivery efficiency is very low, and it is difficult to repair the damaged adhesive junction of the glomerular endothelial cells, so that it is difficult to treat the proteinuria.

[0009] The present application has the following advantages:

[0010] The liposome-gold nanoparticle complex provided by the application has a carrier comprising cationic liposomes and gold nanoparticles, the plasmid concentration is concentrated through the gold nanoparticles, and then the AuNPs are wrapped with the plasmid by using the cationic liposomes, so that the delivery efficiency of the plasmid DNA is improved not only by concentrating the plasmid concentration and protecting the plasmid DNA from degradation, but also by using the cationic liposomes to increase the ability of entering cells and endosome escape. Meanwhile, the cationic liposomes and the gold nanoparticles have good biocompatibility and relatively high delivery efficiency. In addition, the targeting peptide HHHRHSF is selected to improve the targeting property of the liposome-gold nanoparticle complex, and it is found by accident that the targeting peptide can effectively improve the selective delivery efficiency of the liposome-gold nanoparticle complex, so that the damaged adhesion junction of the glomerular endothelial cell can be effectively repaired, and proteinuria can be treated. BRIEF DESCRIPTION OF DRAWINGS

[0011] The drawings constituting a part of the specification of the application are used to provide a further understanding of the application, and the illustrative embodiments of the application and the description thereof are used to explain the application, and do not constitute an improper limitation on the application.

[0012] Figure 1 Fig. 1 is a characterization result of the synthesis process of the liposome-gold nanoparticle complex Ang1-Lipo-AuNPs-VE-Cadherin in the embodiment of the application; (a) the particle size of AuNPs; (b) the particle size of the complex Ang1-Lipo-AuNPs-VE-Cadherin; (c) the change of Zeta potential in the synthesis process of the complex Ang1-Lipo-AuNPs-VE-Cadherin; (d) the encapsulation rate and release rate of the complex Ang1-Lipo-AuNPs-VE-Cadherin on the VE-Cadherin overexpression plasmid; (e) the TEM image of AuNPs; (f) the TEM image of the complex Ang1-Lipo-AuNPs-VE-Cadherin.

[0013] Figure 2 Fig. 2 is a stability result graph of Ang1-Lipo-AuNPs-VE-Cadherin and REDVC-Lipo-AuNPs-VE-Cadherin in the embodiment of the application; (a) the change of the particle size of the complex Ang1-Lipo-AuNPs-VE-Cadherin in an aqueous solution for 7 days; (b) the change of the particle size of the complex REDVC-Lipo-AuNPs-VE-Cadherin in an aqueous solution for 7 days; (c) the change of the particle size of the complex Ang1-Lipo-AuNPs-VE-Cadherin in DMEM containing fetal bovine serum for 3 days; (d) the change of the particle size of the complex REDVC-Lipo-AuNPs-VE-Cadherin in DMEM containing fetal bovine serum for 3 days.

[0014] Figure 3 The following figures illustrate the delivery capabilities of Ang1-Lipo-AuNPs-VE-Cadherin and REDVC-Lipo-AuNPs-VE-Cadherin overexpression plasmids for VE-Cadherin in the embodiments of the present invention: (a) compares the expression levels of VE-Cadherin protein in glomerular endothelial cells by the complexes Ang1-Lipo-AuNPs-VE-Cadherin and REDVC-Lipo-AuNPs-VE-Cadherin; (b) compares the expression levels of VE-Cadherin mRNA in glomerular endothelial cells by the complexes Ang1-Lipo-AuNPs-VE-Cadherin and REDVC-Lipo-AuNPs-VE-Cadherin.

[0015] Figure 4 This describes the cytotoxicity of the complex Ang1-Lipo-AuNPs-VE-Cadherin in the embodiments of the present invention.

[0016] Figure 5 This embodiment of the invention describes the effect of the complex Ang1-Lipo-AuNPs-VE-Cadherin on the expression level of VE-Cadherin protein in human glomerular endothelial cells after TNF-α treatment.

[0017] Figure 6 This invention describes the effect of the complex Ang1-Lipo-AuNPs-VE-Cadherin on the permeability of monolayer glomerular endothelial cells in a Transwell-FITC-Dexran experiment after TNF-α treatment of human glomerular endothelial cells in this embodiment of the invention. (n=9, ***P<0.001).

[0018] Figure 7 In the embodiments of the present invention, (a) is a graph showing the effect of the complex Ang1-Lipo-AuNPs-VE-Cadherin on the expression level of VE-Cadherin protein in glomerular endothelial cells in a proteinuria mouse model constructed by the LPS method; (b) is a schematic diagram showing the effect of treatment with the complex Ang1-Lipo-AuNPs-VE-Cadherin on the urinary protein excretion of proteinuria mice constructed by the LPS method; and (c) is a graph showing the effect of the complex Ang1-Lipo-AuNPs-VE-Cadherin on the albumin excretion in a proteinuria mouse model constructed by the LPS method. Detailed Implementation

[0019] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0020] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0021] Given that there are currently no drugs that treat proteinuria by repairing glomerular endothelium, this invention proposes the application of a lipid-gold nanoparticle complex in the preparation of drugs for treating proteinuria.

[0022] A typical embodiment of the present invention provides the application of a lipid-gold nanoparticle complex in the preparation of a drug for treating proteinuria. The lipid-gold nanoparticle complex is composed of cationic liposomes, a gold nanoparticle complex, and a targeting peptide. The cationic liposomes coat the gold nanoparticle complex, and the targeting peptide is loaded on the surface of the cationic liposomes after coating the gold nanoparticle complex. The gold nanoparticle complex is a complex of gold nanoparticles and a VE-Cadherin overexpression plasmid. The sequence of the VE-Cadherin overexpression plasmid is shown in SEQ ID NO.1, and the sequence of the targeting peptide is shown in SEQ ID NO.2.

[0023] This invention modifies the surface of cationic liposomes with the targeting peptide HHHRHSF. Through the specific interaction of the HHHRHSF peptide with the Tie-2 receptor on the endothelial cell membrane, the gene vector can specifically target endothelial cells. After the gene vector delivers plasmid DNA into glomerular endothelial cells, plasmid DNA expression and VE-Cadherin expression increase, rebuilding the glomerular filtration barrier, reducing vascular permeability, and thus achieving the purpose of treating proteinuria.

[0024] In some embodiments, the targeting peptide is coupled to DSPE-PEG via the thiol group of cysteine. 2000 -The double bond of maleimide in MAL undergoes an addition reaction to produce DSPE-PEG. 2000 -CHHHRHSF,DSPE-PEG 2000 -CHHHRHSF is inserted into the surface of cationic liposomes. DSPE-PEG 2000 The mass of -CHHHRHSF is 5-20% of the phospholipid content in cationic liposomes.

[0025] In some embodiments, the cationic gold nanoparticles are complexed with the VE-Cadherin overexpression plasmid by electrostatic adsorption. Specifically, the ratio of the cationic gold nanoparticles to the VE-Cadherin overexpression plasmid is (35-40 ng):(6-10 mg).

[0026] In one or more embodiments, the cationic gold nanoparticles are obtained from gold nanoparticles by β-mercaptoethylamine (β-MEA). The molar ratio of the gold nanoparticles to the β-MEA is (0.5-5 nM):(50-65 mM).

[0027] In some embodiments, the cationic liposome is formed from soybean lecithin, cholesterol and 2-dioleoylhydroxypropyl-3-N,N,N-trimethylammonium chloride. The mass ratio of the soybean lecithin, cholesterol and 2-dioleoylhydroxypropyl-3-N,N,N-trimethylammonium chloride is 0.2-0.6:0.05-0.3:0.01-0.09.

[0028] In some embodiments, the method for preparing the lipid gold nanoparticle complex comprises the following steps:

[0029] The cationic gold nanoparticles are mixed with the VE-Cadherin overexpression plasmid in solution to allow the VE-Cadherin overexpression plasmid to be completely complexed with the cationic gold nanoparticles by electrostatic adsorption, thereby obtaining an AuNPs-VE-Cadherin complex;

[0030] DSPE-PEG 2000 -MAL and CHHHRHSF are subjected to addition reaction of thiol and double bond, thereby obtaining DSPE-PEG 2000 -CHHHRHSF.

[0031] The soybean lecithin, cholesterol and 2-dioleoylhydroxypropyl-3-N,N,N-trimethylammonium chloride are prepared into cationic liposomes, and then the AuNPs-VE-Cadherin complex is added, and the mixture is subjected to oscillation and ultrasonic treatment to allow the cationic liposomes to coat the AuNPs-VE-Cadherin complex;

[0032] DSPE-PEG 2000 -CHHHRHSF is added to the solution of the cationic liposome-coated AuNPs-VE-Cadherin complex, and the mixture is mixed to allow the lipophilic DSPE to be completely inserted into the liposome.

[0033] In one or more embodiments, the preparation method of the cationic gold nanoparticles is as follows: sodium dodecyl sulfate is added to a dispersion of gold nanoparticles, uniformly dispersed, then β-mercaptoethylamine is added, stirred for 5-12 hours, filtered, concentrated by centrifugation, purified by dialysis, and the pH is adjusted to 4-5 using HCl.

[0034] In one or more embodiments, the conditions of the addition reaction are as follows: 3-5°C for 8-15 hours.

[0035] In some embodiments, the VE-Cadherin overexpression plasmid is obtained by culturing a strain from which the VE-Cadherin overexpression plasmid is isolated.

[0036] In some embodiments, the drug for treating proteinuria comprises a pharmaceutical carrier. The carrier comprises water, a buffer solution, physiological saline, etc.

[0037] In some embodiments, the drug for treating proteinuria is administered by subcutaneous injection, intravenous injection, etc.

[0038] In order to enable a person skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with specific examples and comparative examples.

[0039] The extraction method of the VE-Cadherin overexpression plasmid is as follows:

[0040] Preparation:

[0041] Wash the conical flask used for shaking culture, and seal the bottle opening with a sealing film with air holes. Prepare LB medium (5 g of tryptone + 5 g of sodium chloride + 2.5 g of yeast extract, add double distilled water (ddH2O) to 500 mL) and sterilize in a pressure cooker. After the high pressure is over, wait for the medium and the conical flask to cool to room temperature, then pour an appropriate amount of medium into the conical flask, then add 1:1000 Kana antibiotic and 2 mL of VE-Cadherin overexpression plasmid strain (DH5α competent cells from TIANGEN) according to the amount of medium in the bottle, and seal the bottle opening with a sealing film again. Place the conical flask in a fixed position on the shaker, and set the shaking speed to 150 rpm / min and the shaking time to 12-16 hours.

[0042] Experimental steps:

[0043] After the end of the shaking, the conical flask is removed from the shaker. The shaken bacterial solution is transferred in batches to 50 mL enzyme-free centrifuge tubes and centrifuged at 4000 rpm / min for 10 min at room temperature. The supernatant is discarded and the centrifugation is repeated until the bacterial solution in the conical flask is completely separated. 10 mL of Solution I / Rnase A is added to the centrifuge tube from which the supernatant has been discarded, and vortexed or pipetted to mix evenly. At this time, the solution becomes turbid. Then 10 mL of Solution II solution is added to the solution, and the solution is inverted 8-10 times to make it completely clear. This operation should be completed within 2-3 min.

[0044] 5 mL of pre-cooled N3 buffer is added to the solution, and the solution is inverted to mix thoroughly. At this time, a large amount of white flocculent precipitate appears in the centrifuge tube. The plug of the filter is opened, and the lysate from the previous step is quickly transferred to the filter, and the lysate is gently transferred to a new 50 mL enzyme-free centrifuge tube with the plug. Add 0.1 times the volume of the above centrifuge tube solution of ERT Solution, and gently invert the solution to make the lysate turbid. Then the centrifuge tube is incubated on ice for 10 min, and the solution is inverted every 2-3 min during the incubation. At this time, the solution becomes clear. After the end of the incubation, the centrifuge tube is incubated in a 42°C water bath for 5 min, and the lysate becomes turbid again. After the water bath, the centrifuge tube is placed in the centrifuge, and centrifuged at 4000 rpm / min at room temperature for 5 min, at which time a blue precipitate appears at the bottom. The upper aqueous phase is carefully aspirated into a new 50 mL enzyme-free centrifuge tube, and the centrifugation is repeated several times until no blue precipitate appears at the bottom. Add 0.5 times the volume of anhydrous ethanol, and gently invert 6-7 times, which is completed within 1-2 min at room temperature.

[0045] The centrifuge tube containing the HiBind DNA Maxi Column is opened, and 3 mL of GPS buffer is added to the Column, which is placed at room temperature for 4 min, and then centrifuged at 4000 rpm / min at room temperature for 3 min. The filtrate is discarded, and the centrifuge tube is ready for use. 20 mL of the solution after the addition of anhydrous ethanol in the previous step is poured into the Column, which is centrifuged at 4000 rpm / min at room temperature for 3 min, and the filtrate is discarded. The above operation is repeated until the solution is completely separated. 10 mL of HBC buffer diluted with isopropanol in advance is added to the Column, which is centrifuged at 4000 rpm / min at room temperature for 3 min, and the filtrate is discarded. 15 mL of DNA wash buffer diluted with anhydrous ethanol in advance is added to the Column, which is centrifuged at 4000 rpm / min at room temperature for 3 min, and the filtrate is discarded. 10 mL of DNA wash buffer diluted with anhydrous ethanol in advance is added to the Column, which is centrifuged at 4000 rpm / min at room temperature for 3 min, and the filtrate is discarded. The Column is centrifuged at 4000 rpm / min for 10 min to prevent ethanol residue.

[0046] The Column was transferred to a new 50 mL enzyme-free centrifuge tube. 500 μL of Endo-Free Elution buffer was evenly added to the column membrane of the Column, and it was placed at room temperature for 5 min. The centrifuge tube was centrifuged at 4000 rpm / min for 5 min, and the filtrate after centrifugation was sucked out and added to the column membrane of the Column for secondary centrifugation. The liquid centrifuged out was collected, which was the VE-Cadherin overexpression plasmid (referred to as DNA plasmid) and stored at -80°C. The sequence of the VE-Cadherin overexpression plasmid is as follows:

[0047]

[0048] Example 1

[0049] The preparation steps of the lipid gold nanoparticle complex Ang1-Lipo-AuNPs-VE-Cadherin are as follows:

[0050] 1) Preparation of AuNPs

[0051] The glass instruments used are immersed in aqua regia for 2-3 days, then washed with distilled water three times, and dried for use. Add 30 μL of 25.6% gold tetrachloride solution and 60 mL of distilled water to a 100 mL two-necked flask, and heat to boiling. Then quickly add 8 mL of 1% trisodium citrate solution, continue to heat, at which time the solution changes from light yellow to wine red, and stop heating after boiling for 20 min. Gradually cool to room temperature to obtain 13±2 nm AuNPs. Store at 4°C in the dark.

[0052] 2) Preparation of cationic AuNPs

[0053] Add 0.3 g of SDS to the obtained AuNPs solution and stir for 2 h. Then add 0.45 g of β-MEA and continue to stir for 10 h. Filter the solution three times with a 0.22 μm filter membrane to remove the precipitated SDS. Concentrate by centrifugation at 16800 rpm, and place the concentrated solution in a dialysis bag with a cut-off of 3500 Da for purification by dialysis. Adjust the pH to 4 with HCl to obtain a cationic AuNPs solution, and store at 4°C in the dark.

[0054] 3) Preparation of AuNPs-VE-Cadherin complex (AV)

[0055] Add 8 mg of VE-Cadherin overexpression plasmid to the cationic AuNPs solution and stir overnight to obtain an AuNPs-VE-Cadherin solution.

[0056] 4) Preparation of Lipo-AuNPs-VE-Cadherin complex (LAV)

[0057] Soybean lecithin 0.2 g, cholesterol 0.05 g and 2-dioleoylhydroxypropyl-3-N,N,N-trimethylammonium chloride (DOTAP) 0.025 g were taken respectively into a 500 mL tomato-shaped bottle, 200 mL methanol solution was added, and the three were dissolved by heating to 80°C and stirring to mix. After cooling to room temperature, the methanol was removed on a rotary evaporator, and a uniform layer of liposomes was formed on the bottle wall. The AV complex solution was added to the tomato-shaped bottle, and hydrated for 2 h by shaking. Then the large liposomes were broken by an ultrasonic disruptor, with a power of 200 W, a working time of 5 s, an intermittent time of 5 s, and a total time of 10 min. Subsequently, centrifugal purification was performed. 12000 rpm, 10 min. The cationic liposome-encapsulated AuNPs and VE-Cadherin plasmid (LAV) were obtained by filtering three times with a 0.22 μm filter membrane.

[0058] 5) Preparation of Ang1-Lipo-AuNPs-VE-Cadherin complex (Ang1-LAV)

[0059] First, DSPE-PEG 2000 -MAL and CHHHRHSF (as shown in SEQ ID NO. 2) were dissolved in water at a molar ratio of 1.5:1, and reacted overnight at 4°C to obtain DSPE-PEG 2000 -CHHHRHSF.

[0060] The DSPE-PEG 2000 -CHHHRHSF was modified to the surface of the liposome by "post-insertion method". The DSPE-PEG 2000 -CHHHRHSF solution was added to the liposome gold nanoparticle (LAV) solution, and the lipophilic DSPE was inserted into the liposome by gentle shaking for 6 h, to obtain the Ang1-Lipo-AuNPs-VE-Cadherin complex (Ang1-LAV).

[0061] Example 2

[0062] The preparation steps of the liposome gold nanoparticle complex Ang1-Lipo-AuNPs-VE-Cadherin are as follows:

[0063] 1) Preparation of AuNPs

[0064] Glassware used was immersed in aqua regia for 2-3 days, then washed with distilled water for 3 times, and dried for use. 10 μL of 25.6% gold chloride solution and 40 mL of distilled water were added into a 100 mL flask, and heated to boiling. Then 3 mL of 1% trisodium citrate solution was quickly added, and the solution was heated. The solution changed from light yellow to wine red. The heating was stopped after 15 minutes of boiling, and the solution was gradually cooled to room temperature to obtain AuNPs with a size of 13±2 nm. The solution was stored at 4°C in the dark.

[0065] 2) Preparation of cationic AuNPs

[0066] 0.5 g of SDS was added into the obtained AuNPs solution, and stirred for 3 hours. Then 0.3 g of β-MEA was added, and stirred for 4 hours. The solution was filtered with a 0.22 μm filter membrane for 3 times to remove the precipitated SDS. The solution was concentrated by centrifugation at 12000 rpm, and the concentrated solution was put into a dialysis bag with a cut-off of 4000 Da for dialysis purification. The pH value was adjusted to 5 with HCl to obtain a cationic AuNPs solution, which was stored at 4°C in the dark.

[0067] 3) Preparation of AuNPs-VE-Cadherin complex (AV)

[0068] 5 mg of VE-Cadherin overexpression plasmid was added into the cationic AuNPs solution, and stirred overnight to obtain an AV solution.

[0069] 4) Preparation of Lipo-AuNPs-VE-Cadherin complex (LAV)

[0070] 0.2 g of soybean lecithin, 0.05 g of cholesterol and 0.025 g of 2-dioleoylhydroxypropyl-3-N,N,N-trimethylammonium chloride (DOTAP) were respectively put into a 500 mL flask, 200 mL of methanol solution was added, and the three were dissolved by heating to 80°C and stirring. After the solution was cooled to room temperature, the methanol was removed on a rotary evaporator, and a uniform layer of liposomes was formed on the wall of the flask. The AV complex solution was added into the flask, and hydrated by shaking for 2 hours. Then the large liposomes were broken by an ultrasonic disrupter at a power of 200 W for 5 s, and the total working time was 10 min. Then the solution was purified by centrifugation at 12000 rpm for 10 min. The solution was filtered with a 0.22 μm filter membrane for 3 times to obtain cationic liposome-encapsulated AuNPs and VE-Cadherin plasmid (LAV).

[0071] 5) Preparation of Ang1-Lipo-AuNPs-VE-Cadherin complex (Ang1-LAV)

[0072] Firstly, DSPE-PEG 2000MAL and CHHHRHSF were dissolved in water at a molar ratio of 1.3:1, and reacted overnight at 4℃ to obtain DSPE-PEG 2000 CHHHRHSF.

[0073] DSPE-PEG 2000 CHHHRHSF was modified to the surface of the liposome. DSPE-PEG 2000 The CHHHRHSF solution was added to the liposome gold nanoparticle (LAV) solution, and gently shaken for 6h to allow the lipophilic DSPE to fully insert into the liposome, thereby obtaining an Ang1-LAV complex.

[0074] Example 3

[0075] The preparation steps of the liposome gold nanoparticle complex Ang1-Lipo-AuNPs-VE-Cadherin are as follows:

[0076] 1) Preparation of AuNPs

[0077] The glassware used was immersed in aqua regia for 2-3 days, then washed with distilled water three times, and dried for use. 50μL of 25.6% gold tetrachloride solution and 80mL of distilled water were added to a 200mL two-neck flask, and heated to boiling. Then 10mL of 1% trisodium citrate solution was quickly added, and heating was continued. At this time, the solution changed from light yellow to wine red, and boiling was stopped after 30min, and gradually cooled to room temperature, thereby obtaining 13±2nm AuNPs. Store at 4℃ in the dark.

[0078] 2) Preparation of cationic AuNPs

[0079] 0.5g SDS was added to the obtained AuNPs solution, and stirred for 1h. Then 0.7g of β-MEA was added and stirred for 10h. The solution was filtered three times with a 0.22μm filter membrane to remove the precipitated SDS. Concentrate by centrifugation at 18000rpm, and place the concentrate in a dialysis bag with a cut-off of 3000Da for purification by dialysis. Adjust the pH to 4 with HCl, thereby obtaining a cationic AuNPs solution, which was stored at 4℃ in the dark.

[0080] 3) Preparation of AuNPs-VE-Cadherin complex (AV)

[0081] 10mg of VE-Cadherin overexpression plasmid was added to the cationic AuNPs solution, and stirred overnight, thereby obtaining an AV solution.

[0082] 4) Preparation of Lipo-AuNPs-VE-Cadherin complex (LAV)

[0083] Soybean lecithin 0.2 g, cholesterol 0.05 g and 2-dioleoylhydroxypropyl-3-N,N,N-trimethylammonium chloride (DOTAP) 0.025 g were taken respectively into a 500 mL tomato-shaped bottle, 200 mL methanol solution was added, and the three were dissolved by heating to 80°C and stirring to mix. After cooling to room temperature, the methanol was removed on a rotary evaporator, and a uniform layer of liposomes was formed on the bottle wall. The AV complex solution was added to the tomato-shaped bottle, and hydrated for 2 h by shaking. Then the large liposomes were broken by an ultrasonic disrupter, with a power of 200 W, a working time of 5 s, an intermittent time of 5 s, and a total time of 10 min. Subsequently, centrifugal purification was performed. 12000 rpm, 10 min. The cationic liposome-encapsulated AuNPs and VE-Cadherin plasmid (LAV) were obtained by filtering through a 0.22 μm filter membrane three times.

[0084] 5) Preparation of Ang1-Lipo-AuNPs-VE-Cadherin complex (Ang1-LAV)

[0085] First, DSPE-PEG 2000 -MAL and CHHHRHSF were dissolved in water at a molar ratio of 1.7:1, and reacted overnight at 4°C to obtain DSPE-PEG 2000 -CHHHRHSF.

[0086] The DSPE-PEG 2000 -CHHHRHSF was modified to the surface of the liposomes by "post-insertion method". The DSPE-PEG 2000 -CHHHRHSF solution was added to the liposome gold nanoparticle (LAV) solution, and gently shaken for 6 h to allow the lipophilic DSPE to fully insert into the liposomes, thereby obtaining the Ang1-LAV complex.

[0087] The synthesis process of the REDVC-Lipo-AuNPs-VE-Cadherin complex is the same as that of the Ang1-Lipo-AuNPs-VE-Cadherin complex, except that the targeting peptide CHHHRHSF is replaced by REDVC (as shown in SEQ ID NO. 3).

[0088] Experimental Example

[0089] After synthesizing the lipid gold nanoparticle complexes Ang1-Lipo-AuNPs-VE-Cadherin and REDVC-Lipo-AuNPs-VE-Cadherin of different target peptides in Example 1, the present application first uses human renal glomerular endothelial cells (HRGEC) for in vitro experiments to compare the delivery abilities of the complexes Ang1-Lipo-AuNPs-VE-Cadherin and REDVC-Lipo-AuNPs-VE-Cadherin to overexpress plasmid VE-Cadherin, and Western blot and qRT-PCR experiments find that the delivery efficiency of Ang1-Lipo-AuNPs-VE-Cadherin is higher. Then in vivo and in vitro experiments are carried out to prove the effects of the complex Ang1-Lipo-AuNPs-VE-Cadherin on repairing vascular endothelial cell junction and relieving proteinuria.

[0090] Figure 1 a and Figure 1 e indicates that the particle size of the AuNPs prepared in Example 1 is 13±2nm, Figure 1 b and Figure 1 f indicates that the particle size of Ang1-Lipo-AuNPs-VE-Cadherin prepared in Example 1 is 112±2nm. Figure 1 c is the change process of the Zeta potential during the preparation of Ang1-Lipo-AuNPs-VE-Cadherin in Example 1, indicating that the surface of the prepared Ang1-Lipo-AuNPs-VE-Cadherin is positive. Figure 1 d indicates that the encapsulation efficiency of Ang1-Lipo-AuNPs-VE-Cadherin is 98.61±0.15%, and the release rate is 58.98±14.72%.

[0091] In order to prove the stability of the complexes Ang1-Lipo-AuNPs-VE-Cadherin and REDVC-Lipo-AuNPs-VE-Cadherin, the present application respectively explores the stability of the complexes in aqueous solution for seven days and in DMEM culture medium containing fetal bovine serum for three days. As Figure 2 indicated, the experimental results show that the complexes Ang1-Lipo-AuNPs-VE-Cadherin and REDVC-Lipo-AuNPs-VE-Cadherin have good stability.

[0092] Subsequently, in order to compare the delivery abilities of the different targeting peptides Ang1-Lipo-AuNPs-VE-Cadherin and REDVC-Lipo-AuNPs-VE-Cadherin complexes to the overexpression plasmid VE-Cadherin, the effects of adding 0.001 μg / μL of the Ang1-Lipo-AuNPs-VE-Cadherin and REDVC-Lipo-AuNPs-VE-Cadherin complexes on the expression of VE-Cadherin in the cells were studied by Western blot experiments and qRT-PCR experiments. As shown in Figs. 2a and 2b, compared with the control group, the expression of VE-Cadherin protein and mRNA in the cells increased after adding the Ang1-Lipo-AuNPs-VE-Cadherin and REDVC-Lipo-AuNPs-VE-Cadherin complexes. However, the expression of VE-Cadherin protein and mRNA in the cells added with the Ang1-Lipo-AuNPs-VE-Cadherin complex increased more obviously, indicating that the Ang1-Lipo-AuNPs-VE-Cadherin complex successfully delivered the overexpression plasmid VE-Cadherin to the cells and expressed well. Figure 3 a and 3b, compared with the control group, the expression of VE-Cadherin protein and mRNA in the cells increased after adding the Ang1-Lipo-AuNPs-VE-Cadherin and REDVC-Lipo-AuNPs-VE-Cadherin complexes. However, the expression of VE-Cadherin protein and mRNA in the cells added with the Ang1-Lipo-AuNPs-VE-Cadherin complex increased more obviously, indicating that the Ang1-Lipo-AuNPs-VE-Cadherin complex successfully delivered the overexpression plasmid VE-Cadherin to the cells and expressed well.

[0093] Next, human renal glomerular endothelial cells were selected for cell experiments. First, the cytotoxicity of the Ang1-Lipo-AuNPs-VE-Cadherin complex was explored by the present application. The endothelial cells were cultured and 0-0.01 μg / μL of the Ang1-Lipo-AuNPs-VE-Cadherin complex was added, and the cell activity was observed after 24 h. The experimental results showed that the Ang1-Lipo-AuNPs-VE-Cadherin complex had no obvious cytotoxicity, as shown in Fig. 3. Figure 4

[0094] Subsequently, the effects of adding 0.001 μg / μL of the Ang1-Lipo-AuNPs-VE-Cadherin complex on the expression of VE-Cadherin in the cells were studied by Western blot experiments. As shown in Fig. 4, compared with the control group, the expression of VE-Cadherin protein in the cells increased after adding the Ang1-Lipo-AuNPs-VE-Cadherin complex. Figure 5 ​As shown in a, after the addition of Ang1-Lipo-AuNPs-VE-Cadherin complex, the expression of VE-Cadherin protein in the cells increased significantly, indicating that the Ang1-Lipo-AuNPs-VE-Cadherin complex successfully delivered VE-Cadherin into the cells and expressed. The present application also used a transmembrane cell barrier experiment (Transwell-FITC-Dexran permeation method) to explore the effect of Ang1-Lipo-AuNPs-VE-Cadherin complex on cell permeability. As shown in b, compared with the control group only added with TNF-α, the fluorescence intensity in the lower chamber of the experimental group added with TNF-α and Ang1-Lipo-AuNPs-VE-Cadherin complex decreased significantly, indicating that the vascular barrier permeability after the treatment of Ang1-Lipo-AuNPs-VE-Cadherin complex decreased significantly. Figure 6

[0095] After the completion of the in vitro experiment, the present application also carried out an in vivo experiment. The present application used a lipopolysaccharide injection method (LPS) to construct a proteinuria mouse model.

[0096] Specific operation of constructing a proteinuria mouse by LPS method: Before the experiment, the mice need to be fasted for 12 h. The mouse proteinuria model is prepared by intraperitoneal injection of 5 mg / kg of LPS suspension.

[0097] The proteinuria model mice were randomly divided into two groups, the experimental group was injected with Ang1-Lipo-AuNPs-VE-Cadherin complex with a concentration of 0.1 mg / kg, and the control group was injected with PBS, and the expression of VE-Cadherin protein in the glomerular endothelial cells and the occurrence of proteinuria in the mice were observed after 24 h. As shown in a, the expression of VE-Cadherin protein in the glomerular endothelial cells of the mice treated with Ang1-Lipo-AuNPs-VE-Cadherin complex increased significantly, indicating that Ang1-Lipo-AuNPs-VE-Cadherin complex can reach the glomerular site through blood circulation, successfully deliver the VE-Cadherin overexpression plasmid into the glomerular endothelial cells and express, and increase the expression level of the protein. As shown in b, the excretion amount of urine protein of the proteinuria model mice treated with Ang1-Lipo-AuNPs-VE-Cadherin complex decreased significantly after 24 h, and the content of albumin in the urine also decreased effectively, indicating that Ang1-Lipo-AuNPs-VE-Cadherin can repair the glomerular filtration barrier by increasing the expression level of VE-Cadherin protein, and achieve the purpose of treating proteinuria. Figure 7 Figure 7 Figure 7 c, the excretion amount of urine protein of the proteinuria model mice treated with Ang1-Lipo-AuNPs-VE-Cadherin complex decreased significantly after 24 h, and the content of albumin in the urine also decreased effectively, indicating that Ang1-Lipo-AuNPs-VE-Cadherin can repair the glomerular filtration barrier by increasing the expression level of VE-Cadherin protein, and achieve the purpose of treating proteinuria.​​​

[0098] The above descriptions are only the preferred embodiments of the present application, not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall fall within the scope of the present application.

Claims

1. The application of a lipid-gold nanoparticle complex in the preparation of a drug for treating glomerular proteinuria, characterized in that, The lipid-gold nanoparticle complex is composed of cationic liposomes, a gold nanoparticle complex, and a targeting peptide. The cationic liposomes coat the gold nanoparticle complex, and the targeting peptide is loaded on the surface of the cationic liposomes after coating with the gold nanoparticle complex. The gold nanoparticle complex is a complex of gold nanoparticles and a VE-Cadherin overexpression plasmid. The sequence of the VE-Cadherin overexpression plasmid is shown in SEQ ID NO.1, and the sequence of the targeting peptide is shown in SEQ ID NO.

2.

2. The use of the lipid-gold nanoparticle complex as described in claim 1 in the preparation of a drug for treating glomerular proteinuria, characterized in that, The targeting peptide is linked to DSPE-PEG via the thiol group of cysteine. 2000 -The double bond of maleimide in MAL undergoes an addition reaction to produce DSPE-PEG. 2000 -CHHHRHSF, DSPE-PEG 2000 -CHHHRHSF is inserted into the surface of cationic liposomes.

3. The use of the lipid-gold nanoparticle complex as described in claim 1 in the preparation of a drug for treating glomerular proteinuria, characterized in that, Cationic gold nanoparticles are combined with VE-Cadherin overexpression plasmids via electrostatic adsorption.

4. The use of the lipid-gold nanoparticle complex as described in claim 3 in the preparation of a drug for treating glomerular proteinuria, characterized in that, The cationic gold nanoparticles were obtained from gold nanoparticles via β-mercaptoethylamine.

5. The use of the lipid-gold nanoparticle complex as described in claim 1 in the preparation of a drug for treating glomerular proteinuria, characterized in that, The cationic liposomes are formed from soybean lecithin, cholesterol, and 2-dioleoylhydroxypropyl-3-N,N,N-trimethylammonium chloride.

6. The use of the lipid-gold nanoparticle complex as described in claim 1 in the preparation of a drug for treating glomerular proteinuria, characterized in that, The preparation method of the lipid-gold nanoparticle complex includes the following steps: Cationic gold nanoparticles and VE-Cadherin overexpression plasmid were mixed evenly in solution, so that the VE-Cadherin overexpression plasmid was completely complexed with the cationic gold nanoparticles by electrostatic adsorption, thus obtaining AuNP-VE-Cadherin complex. DSPE-PEG 2000 -MAL and CHHHRHSF undergo an addition reaction of thiol to double bond to obtain DSPE-PEG. 2000 -CHHHRHSF; Soybean lecithin, cholesterol, and 2-dioleoylhydroxypropyl-3-N,N,N-trimethylammonium chloride were used to prepare cationic liposomes. Then, AuNPs-VE-Cadherin complex was added, and the mixture was shaken and sonicated to encapsulate the AuNPs-VE-Cadherin complex in the cationic liposomes. DSPE-PEG 2000 -CHHHRHSF is added to a solution of cationic liposomes coated with AuNPs-VE-Cadherin complex, and mixed to allow the lipophilic DSPE to be completely inserted into the liposomes, thus obtaining the final product.

7. The use of the lipid-gold nanoparticle complex as described in claim 6 in the preparation of a drug for treating glomerular proteinuria, characterized in that, The preparation method of cationic gold nanoparticles is as follows: sodium dodecyl sulfate is added to the dispersion of gold nanoparticles and dispersed evenly. Then β-mercaptoethylamine is added and the mixture is stirred for 5-12 h. The mixture is then filtered, centrifuged and concentrated, and purified by dialysis. The pH is adjusted to 4-5 using HCl. Alternatively, the conditions for the addition reaction are: 3~5℃ for 8~15 h.

8. The use of the lipid-gold nanoparticle complex as described in claim 1 in the preparation of a drug for treating glomerular proteinuria, characterized in that, The VE-Cadherin overexpression plasmid was obtained by culturing and isolating the bacterial strain that contained the VE-Cadherin overexpression plasmid.

9. The use of the lipid-gold nanoparticle complex as described in claim 1 in the preparation of a drug for treating glomerular proteinuria, characterized in that, Drugs for treating glomerular proteinuria include drug carriers.

10. The use of the lipid-gold nanoparticle complex as described in claim 1 in the preparation of a drug for treating glomerular proteinuria, characterized in that, The medications for treating glomerular proteinuria are administered via subcutaneous or intravenous injection.

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