A self-assembling selenopeptide targeting vascular cell adhesion molecule-1, a selenopeptide nanomedicine, and its preparation method and application

Abstract

The present invention relates to a self-assembling selenopeptide targeting vascular cell adhesion molecule-1 (VCAM-1), a selenopeptide nanomedicine, and a preparation method and application thereof. The self-assembling selenopeptide targeting vascular cell adhesion molecule-1 includes an oxidation response unit and a vascular cell adhesion molecule-1 targeting unit sequentially connected from the N-terminus to the C-terminus; the oxidation response unit contains a selenocysteine substituted with a fatty chain. The self-assembling selenopeptide has the ability to sensitively respond and eliminate reactive oxygen species, and can target vascular cell adhesion factor-1 overexpressed in plaque tissue, with excellent active targeting. It can be used alone as a drug for treating atherosclerosis or as a carrier to effectively deliver drugs such as statins to plaque tissue, thereby jointly improving the inflammatory microenvironment of plaque tissue. In addition, the self-assembling selenopeptide as a nanocarrier also significantly improves the enrichment of statins in the liver and increases the bioavailability of statins.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a self-assembling selenopeptide targeting vascular cell adhesion molecule-1 (VCAM-1) and a preparation method thereof, a selenopeptide nanomedicine targeting vascular cell adhesion molecule-1 (VCAM-1) and a preparation method thereof, and the use of the self-assembling selenopeptide or selenopeptide nanomedicine in the preparation of a drug for treating atherosclerosis. Background Art

[0002] With socioeconomic development, improved living standards, and an aging population, the incidence of cardiovascular diseases continues to rise. Atherosclerosis is the most common cardiovascular disease and the leading cause of coronary heart disease, cerebral infarction, and peripheral vascular disease. Studies have shown that oxidative stress is a central factor in the development and progression of atherosclerosis. Eliminating reactive oxygen species in atherosclerotic plaques can reduce oxidative stress and inflammation, thereby slowing the progression of atherosclerosis. Currently, treatment for atherosclerosis relies primarily on medication, with surgery and interventional therapies also being considered. However, conventional drug therapies and delivery methods result in very low drug concentrations in plaques, making it difficult to achieve effective direct plaque treatment. Targeted nanoparticle drug delivery systems have garnered significant attention in recent years. By specifically recognizing targeting groups with receptors in lesion tissue, they can enhance drug accumulation in lesion tissue and reduce toxic side effects in healthy tissues. Therefore, the development of plaque-targeting nanomaterials with anti-inflammatory and antioxidant properties is promising for improving the therapeutic efficacy of atherosclerosis.

[0003] Selenium is an essential element for the human body. It is mainly present in the form of selenopeptides as a core component of selenoproteins (such as glutathione peroxidase 4). It can maintain the body's redox balance, eliminate oxidative stress damage to tissues and cells caused by reactive oxygen species, and regulate thyroid metabolism and the immune system. There are reports on the use of inorganic selenium nanoparticles as antioxidants to treat arteriosclerosis. However, inorganic selenium nanomaterials often face challenges in biocompatibility or chemical structure controllability. As a homologous substance of selenoprotein, selenopeptides have good biocompatibility, molecular structure and self-assembly controllability, and are expected to become new anti-inflammatory and antioxidant bio-nanomaterials. However, there are few reports on the application of selenopeptide nanomaterials in the treatment of atherosclerosis. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the present invention aims to provide a self-assembling selenopeptide targeting vascular cell adhesion molecule-1 and a preparation method thereof, a selenopeptide nanomedicine targeting vascular cell adhesion molecule-1 and a preparation method thereof, and the use of the above-mentioned self-assembling selenopeptide or selenopeptide nanomedicine in the preparation of drugs for treating atherosclerosis.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a self-assembling selenopeptide targeting vascular cell adhesion molecule-1, wherein the self-assembling selenopeptide targeting vascular cell adhesion molecule-1 comprises an oxidation response unit and a vascular cell adhesion molecule-1 targeting unit sequentially connected from the N-terminus to the C-terminus;

[0007] The oxidation response unit comprises a fatty chain substituted selenocysteine.

[0008] The present invention innovatively develops a self-assembling selenopeptide, which is amphiphilic, has a low critical micelle concentration, and has good self-assembly ability. It has the ability to sensitively respond to and eliminate reactive oxygen species (hydrogen peroxide, superoxide anion, hydroxyl radicals). Its ability to eliminate hydrogen peroxide is significantly better than vitamin C and ebselen, and its ability to eliminate superoxide anions and hydroxyl radicals is better than ebselen. It can sensitively respond to reactive oxygen concentrations close to the level of inflammatory tissue in the body. At the same time, it can target vascular cell adhesion factor-1 overexpressed in plaque tissue, has excellent active targeting, and can be used alone as a drug for treating atherosclerosis or as a carrier to effectively deliver drugs such as statins to plaque tissue, thereby jointly improving the inflammatory microenvironment of plaque tissue. In addition, the self-assembling selenopeptide as a nanocarrier also significantly improves the enrichment of statins in the liver and increases the bioavailability of statins.

[0009] Preferably, the oxidation response unit comprises two fatty chain substituted selenocysteine.

[0010] Preferably, the number of carbon atoms in the fatty chain is an integer of 10-20, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0011] Preferably, the fatty chain is a saturated hydrocarbon chain.

[0012] Preferably, the fatty chain is a straight hydrocarbon chain.

[0013] Preferably, the vascular cell adhesion molecule-1 targeting unit comprises the polypeptide sequence VHPKQHR.

[0014] Preferably, the oxidation response unit comprises a modified polypeptide sequence K(NH2)-K[Sec(C18)]2, wherein Sec(C18) is a C18 alkyl-substituted selenocysteine. The molecular structure of K(NH2)-K[Sec(C18)]2 is as follows:

[0015]

[0016] Preferably, the self-assembling selenopeptide further comprises a spacer amino acid located between the oxidation response unit and the vascular cell adhesion molecule-1 targeting unit.

[0017] Preferably, the spacer amino acids are 1-3 (eg, 1, 2, 3) natural amino acids or unnatural amino acids.

[0018] Preferably, the natural amino acid comprises glycine.

[0019] In a second aspect, the present invention provides a method for preparing the self-assembling selenopeptide targeting vascular cell adhesion molecule-1 according to the first aspect, the preparation method comprising:

[0020] A Boc group-protected fatty chain-substituted selenocysteine is prepared, and then each amino acid raw material is sequentially coupled to the resin by solid-phase synthesis, and eluted to obtain the self-assembling selenopeptide targeting vascular cell adhesion molecule-1.

[0021] Solid-phase peptide synthesis technology is a conventional technique in this field, characterized by mature technology, simple operation, and high yield. It is easy for technicians in related fields to master and can achieve batch production on a scale of grams or above in laboratories or factories.

[0022] The preparation method of the above-mentioned Boc group-protected fatty chain-substituted selenocysteine can be carried out by referring to the method described in patent CN112472815B, which will not be described in detail here.

[0023] Preferably, the solid phase synthesis step comprises:

[0024] The resin is swelled, the Fmoc group of the resin amino acid is removed, and then the amino acid and the oxidation response group are coupled to the resin in sequence.

[0025] Preferably, the swelling of the resin comprises soaking the resin in anhydrous DMF and placing the resin on a shaker.

[0026] Preferably, the step of removing the Fmoc group is further included before coupling the amino acid to the resin.

[0027] Preferably, the step of removing the Boc group is further included before coupling the oxidation responsive group to the resin.

[0028] In a third aspect, the present invention provides a selenopeptide nanomedicine targeting vascular cell adhesion molecule-1, wherein the selenopeptide nanomedicine comprises the self-assembled selenopeptide targeting vascular cell adhesion molecule-1 described in the first aspect and a drug encapsulated therein.

[0029] The targeting unit in the selenopeptide nanomedicine involved in the present invention can target the vascular cell adhesion factor-1 (VCAM-1) overexpressed in arterial plaque tissue, significantly improving the enrichment of the drug in the lesion area; after the drug reaches the lesion site, the highly expressed reactive oxygen species can oxidize the selenocysteine in the oxidation response unit, causing its hydrophobicity to change, resulting in the internal drug deaggregation and release of the drug, thereby achieving the purpose of treating atherosclerosis. Experiments have shown that the selenopeptide nanomedicine targeting vascular cell adhesion molecule-1 can significantly inhibit the development of atherosclerotic plaques in mouse models.

[0030] The seleno-peptide nanomedicine of the present invention can rapidly release the loaded drug in a 100 μM hydrogen peroxide environment; it can reduce the expression of inflammatory factors in pro-inflammatory macrophages, including TNF-α, IL-1β and IL-6; and compared with nanomedicines without VCAM-1 targeting ability, it has a better effect in atherosclerosis model mice (high-fat diet ApoE gene knockout (ApoE)) by intravenous injection. - / - ) male mice) increased the amount of plaque tissue in the aorta by 1 times; after intravenous injection, the drug content in the liver of C57BL / 6 experimental mice increased by 24 times after 24 hours compared with the drug injected alone.

[0031] Preferably, the drug comprises a statin.

[0032] Preferably, the statin includes any one of lovastatin, pravastatin, simvastatin, fluvastatin, atorvastatin, rosuvastatin or pitavastatin, or a combination of at least two thereof.

[0033] Preferably, the selenopeptide nanomedicine further comprises the additive DMG-PEG2000.

[0034] Preferably, the molar ratio of DMG-PEG2000 to the self-assembling selenopeptide targeting vascular cell adhesion molecule-1 is (1-10):(90-99), for example, 1:99, 3:97, 5:95, 6:94, 10:90, etc.

[0035] Preferably, the particle size of the selenopeptide nanomedicine is 50-200 nm, for example, 50 nm, 60 nm, 80 nm, 100 nm, 120 nm, 150 nm, 180 nm, 200 nm, etc.

[0036] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0037] In a fourth aspect, the present invention provides a method for preparing the selenopeptide nanomedicine targeting vascular cell adhesion molecule-1 according to the third aspect, the preparation method comprising:

[0038] An organic solvent containing the drug is mixed with a solution containing the self-assembling selenopeptide for emulsification, the organic solvent is removed, and the supernatant is collected by centrifugation to obtain a solution of the selenopeptide nanomedicine targeting vascular cell adhesion molecule-1.

[0039] Preferably, the emulsification treatment is performed by stirring or shaking.

[0040] Preferably, the removal of the organic solvent is performed by rotary evaporation.

[0041] Preferably, the organic solvent is any volatile organic solvent that can dissolve the drug, such as any one of ethanol, isopropanol, dichloromethane, acetone or cyclohexane, or a combination of at least two thereof.

[0042] In a fifth aspect, the present invention provides the use of the self-assembling selenopeptide targeting vascular cell adhesion molecule-1 described in the first aspect or the selenopeptide nanomedicine targeting vascular cell adhesion molecule-1 described in the third aspect in the preparation of a drug for treating atherosclerosis.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] The present invention innovatively develops a self-assembling selenopeptide, which is amphiphilic, has a low critical micelle concentration, and has good self-assembly ability. It has the ability to sensitively respond to and eliminate reactive oxygen species (hydrogen peroxide, superoxide anion, hydroxyl radicals). Its ability to eliminate hydrogen peroxide is significantly better than vitamin C and ebselen, and its ability to eliminate superoxide anions and hydroxyl radicals is better than ebselen. It can sensitively respond to reactive oxygen concentrations close to the level of inflammatory tissue in the body. At the same time, it can target vascular cell adhesion factor-1 overexpressed in plaque tissue, has excellent active targeting, and can be used alone as a drug for treating atherosclerosis or as a carrier to effectively deliver drugs such as statins to plaque tissue, thereby jointly improving the inflammatory microenvironment of plaque tissue. In addition, the self-assembling selenopeptide as a nanocarrier also significantly improves the enrichment of statins in the liver and increases the bioavailability of statins.

[0045] The seleno-peptide nanomedicine of the present invention can rapidly release the loaded drug in a 100 μM hydrogen peroxide environment; it can reduce the expression of inflammatory factors in pro-inflammatory macrophages, including TNF-α, IL-1β and IL-6; by intravenous injection, compared with the nanomedicine without VCAM-1 targeting ability, it has a better effect in atherosclerosis model mice (high-fat diet (ApoE - / - ) male mice) increased the amount of plaque tissue in the aorta by 1 times; after intravenous injection, the drug content in the liver of C57BL / 6 experimental mice increased by 24 times after 24 hours compared with the drug injected alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a high performance liquid chromatography (HPLC) detection chart of the self-assembling selenopeptide involved in the present invention;

[0047] Figure 2 This is a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) detection diagram of the self-assembling selenopeptide involved in the present invention;

[0048] Figure 3 This is a graph showing the results of the oxidation response property test of the self-assembled selenopeptide involved in the present invention;

[0049] Figure 4 It is a high-resolution quadrupole mass spectrum of the product after the oxidation response of the self-assembled selenopeptide involved in the present invention;

[0050] Figure 5 This is a transmission electron microscope (TEM) observation diagram of the selenopeptide nanomedicine involved in the present invention before and after oxidation response;

[0051] Figure 6 This is a drug release curve diagram of the selenopeptide nanomedicine involved in the present invention;

[0052] Figure 7 This is a graph showing the results of a Western blot experiment on the anti-inflammatory effect of the selenopeptide nanomedicine involved in the present invention;

[0053] Figure 8 This is a fluorescence result diagram showing the aggregation of the selenopeptide nanomedicine involved in the present invention in the diseased blood vessels of atherosclerosis model mice;

[0054] Figure 9 This is a statistical graph of the fluorescence results of the selenium peptide nanomedicine involved in the present invention gathering in the diseased blood vessels of atherosclerosis model mice;

[0055] Figure 10 This is a diagram showing the staining results of plaques in blood vessels of atherosclerosis model rats after treatment with the selenopeptide nanomedicine involved in the present invention;

[0056] Figure 11 This is a statistical graph of plaque staining results in blood vessels of atherosclerosis model rats after treatment with selenopeptide nanomedicines involved in the present invention. DETAILED DESCRIPTION

[0057] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0058] All animal experiments involved in the present invention have been reviewed and approved by the Laboratory Animal Ethics Committee of the National Center for Nanoscience and Technology (approval number NCNST21-2109-0402), and comply with the experimental animal welfare and ethics requirements and relevant legal provisions.

[0059] Example 1

[0060] This embodiment provides a self-assembling selenopeptide as shown in the following formula, and its preparation method is as follows:

[0061]

[0062] (1) Weigh 300 mg of Rink amide AM resin (amino acid loading rate 0.35 mmol / g) into a 10 mL peptide synthesis tube, add 8 mL of anhydrous N,N-dimethylformamide (DMF), and shake on a shaker for 12 hours (shaking speed 2500 rpm) to allow the resin to fully swell. Then remove the DMF using a suction pump.

[0063] (2) Add 8 mL of Fmoc deprotecting agent (DMF solution containing 2% 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and 5% piperazine at a mass volume ratio) to the peptide synthesis tube and react on a shaker at 25°C for 10 minutes to remove the Fmoc group of the amino acid on the resin. Then, wash the resin with dichloromethane and DMF alternately three times.

[0064] (3) Place a small amount of resin in a 1.5 mL plastic centrifuge tube, add 50 μL of ninhydrin test solution (formula: 0.5 g ninhydrin, 0.1 g vitamin C, 20 g phenol, 20 mL ethanol), and boil for 1 minute. If the resin turns dark blue or dark purple, Fmoc deprotection is successful and the next step can be carried out; if the color does not change, repeat the deprotection step. Then wash the resin three times with dichloromethane and DMF alternately.

[0065] (4) Weigh 0.28 g of Fmoc-Lys(Dde)-OH, 0.20 g of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), and 0.07 g of 1-hydroxybenzotriazole (HOBT) in a 15 mL plastic centrifuge tube, add 8 mL of coupling agent (DMF solution containing 5% by volume of N-methylmorpholine), and shake on a shaker for 10 minutes. Then add the mixed solution to the above-mentioned peptide synthesis tube and react at room temperature on a shaker for 1 hour. Then use a suction pump to remove the reaction solution in the peptide synthesis tube, wash the resin with dichloromethane and DMF alternately 3 times, and take a small amount of resin in a 1.5 mL plastic centrifuge tube, add ninhydrin test solution and boil for 1 minute. If the resin color does not change, it means that the amino acid coupling is successful; if it turns blue or purple, it means that the amino acid coupling is incomplete and the coupling step of the amino acid needs to be repeated.

[0066] (5) Repeat the above amino acid coupling steps to continue coupling Fmoc-Gly-OH, Fmoc-Arg(pbf)OH, Fmoc-His(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-ProOH, Fmoc-His(Trt)-OH, and Boc-Val-OH to the resin one by one.

[0067] (6) Add 8 mL of DMF solution containing 2% hydrazine to the peptide synthesis tube and shake on a shaker at 25°C for 10 min to remove the Dde protection of the C-terminal Lys(Dde) amino acid. Wash the resin three times with dichloromethane and DMF alternately.

[0068] (7) Following the above amino acid coupling steps, Fmoc-Lys(Fmoc)-OH and Boc-L-Sec(C18)-OH were coupled one by one to the C-terminal lysine side chain.

[0069] (8) After the reaction is completed, the resin is washed alternately with dichloromethane and DMF 3 times, and washed with methanol 3 times, and the resin is taken out and placed in a glass flask. Under ice-water bath conditions, 5 mL of cleavage solution (formula is 4.75 mL trifluoroacetic acid, 125 μL water, 125 μL triisopropylsilane) is added, and the reaction is stirred for 2 hours to cleave the peptide from the resin. Then filter with filter paper, collect the filtrate, and blow the filtrate with nitrogen to one tenth of the original volume. The concentrated filtrate is precipitated in cold ether to obtain a white precipitate, which is the crude selenopeptide.

[0070] (9) The crude selenopeptide was purified by semi-preparative liquid chromatography using a mobile phase consisting of pure water and acetonitrile containing 1% trifluoroacetic acid. The collected mobile phase was rotary evaporated to remove the acetonitrile and freeze-dried to obtain the pure selenopeptide.

[0071] By high performance liquid chromatography (HPLC) detection, such as Figure 1 As shown in FIG, the purity of the prepared selenopeptide is greater than 95%. Detection by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) showed that the purity of the prepared selenopeptide was greater than 95%. Figure 2 As shown, the molecular weight and isotope peak shape of the obtained selenopeptide are completely consistent with the simulation results of ChemDraw software, which further proves the successful preparation of the selenopeptide shown in the above formula.

[0072] (10) A similar method was used to prepare a control peptide with a purity greater than 95% (amino acid sequence: VHPGQHGGK(NH2)-K(C18)2), which was characterized by amino acid mutations in the VCAM-1 targeting fragment of the selenopeptide to reduce the targeting ability, and the peptide did not contain selenopeptides.

[0073] Example 2

[0074] This embodiment provides a selenopeptide nanomedicine, the preparation method of which is as follows:

[0075] (1) In a 4 mL glass bottle, 6.8 mg of the selenopeptide prepared in Example 1 was weighed and dissolved in 200 μL of ethanol.

[0076] (2) In a 20 mL glass bottle, weigh 3.2 mg of PEG2000-DMG and dissolve it in 10 mL of saline. Then, add 1 mL of this solution to the selenopeptide ethanol solution and mix thoroughly using a vortex mixer (3000 rpm).

[0077] (3) In a 10 mL round-bottom flask, weigh 8.3 mg of simvastatin, add 0.5 mL of dichloromethane, and mix to fully dissolve. Then, add the mixed solution of selenopeptide and PEG2000-DMG to the simvastatin solution and mix thoroughly using a vortex mixer (3000 rpm, 5 minutes) until emulsified.

[0078] (4) Using a rotary evaporator, distill under reduced pressure at 37°C to remove dichloromethane and ethanol from the mixed emulsion. The resulting solution is then centrifuged (3000 rpm, 10 minutes), and the upper layer is the seleno-peptide nano-drug solution.

[0079] Using dynamic light scattering and Zeta potential analysis, the hydrated particle size of the selenopeptide nanosolution after dilution 20 times was measured to be 75±9 nm, the polydispersity index (PDI) was 0.48±0.09, and the Zeta potential was 6.35±0.73 mV (n=3).

[0080] Test Example 1

[0081] This test example detects the oxidation response properties of the self-assembled selenopeptide prepared in Example 1 and the selenopeptide nanomedicine prepared in Example 2, including the following steps:

[0082] (1) Prepare 100 μM selenopeptide and 200 μM hydrogen peroxide aqueous solution, mix equal volumes, and place in a 37°C reaction.

[0083] At 1, 2, 4, and 6 hours, 50 μL of the reaction solution was taken out and the peak area of the selenopeptide was tested by HPLC. The peak area was compared with the peak area of 50 μM selenopeptide to calculate the proportion of oxidized selenopeptide. Figure 3 As shown, 50 μM selenopeptide can be rapidly oxidized under the condition of low concentration of 100 μM hydrogen peroxide, with the oxidation ratio reaching 87% after 4 hours and 94% after 6 hours (n=3).

[0084] Using a high-resolution quadrupole mass spectrometer, such as Figure 4 As shown, the components of the 6-hour reaction solution include a polypeptide with two 18-alkyl chains removed, a selenopeptide containing only one 18-alkyl chain, and 18-alkyl selenous acid and other substances.

[0085] (2) Take the seleno-peptide nanomedicine and dilute it to a concentration of 60 μM. Then mix it with an equal volume of 200 μM hydrogen peroxide solution and place it at 37°C for reaction. Figure 5 As shown, after 4 hours, the nanoparticles were observed to disintegrate using transmission electron microscopy (TEM).

[0086] Take seleno-peptide nanomedicine and dilute it so that the concentration of seleno-peptide is 60 μM. Then mix it with equal volumes of 200 μM hydrogen peroxide aqueous solution, take 1 mL of the mixed solution and put it into a dialysis bag with a molecular cutoff of 500 Da. Then take a 20 mL glass bottle and put it into a 37°C water bath. Wrap the glass bottle with tin foil to avoid light, and add 5 mL of normal saline containing 100 μM hydrogen peroxide, and then put the dialysis bag into the glass bottle. At 1, 2, 3, 4, 6, 8, and 12 hours, take out 100 μL of liquid outside the dialysis bag to test the content of simvastatin, and add 100 μL of normal saline containing 100 μM hydrogen peroxide. Using HPLC testing, compare with the standard curve of simvastatin, calculate the content of simvastatin in the removed liquid, and further calculate the release ratio of simvastatin over time (n=3). As Figure 6 As shown, the seleno-peptide nanomedicine can rapidly release the loaded simvastatin in a 100 μM hydrogen peroxide environment, with the release amount reaching 84% in 4 hours.

[0087] Test Example 2

[0088] This test example detects the antioxidant capacity of the self-assembled selenopeptide prepared in Example 1, comprising the following steps:

[0089] (1) A 200 μM phosphate buffered saline solution (PBS, pH = 7.2) of selenopeptide, vitamin C, and ebselen was prepared and mixed with an equal volume of a 400 μM hydrogen peroxide solution. The mixture was allowed to react at 25°C in the dark for 12 hours. The absorbance of each solution at 415 nm was measured using a microplate reader according to the operating procedures of the hydrogen peroxide content detection kit (Solarbio, Cat. No. BC3595). The residual hydrogen peroxide content and clearance rate in the sample were calculated (n = 3).

[0090] (2) Prepare 100 μM PBS (pH = 7.2) solutions of selenopeptide, vitamin C, and ebselen. Follow the operating procedures of the superoxide anion scavenging ability test kit (Solar Bio, Cat. No. BC1415) and measure the absorbance of each solution at 530 nm using a microplate reader. Calculate the superoxide anion scavenging rate of the sample (n = 3).

[0091] (3) Prepare 100 μM PBS (pH = 7.2) solutions of selenopeptide, vitamin C, and ebselen. Follow the operating procedures of the hydroxyl radical scavenging ability test kit (Solar Bio, catalog number BC1325) and test the absorbance of each solution at 536 nm using a microplate reader to calculate the scavenging rate of the sample for hydroxyl radicals (n = 3).

[0092] Experimental results showed that under conditions of equal molar concentration, the selenopeptide had an elimination rate of 93.7% for hydrogen peroxide, which was 2.8 times higher than vitamin C (32.5%) and 4.2 times higher than ebselen (22.0%). The selenopeptide had an elimination rate of 25.4% for superoxide anions, which was slightly lower than vitamin C (31.4%) and significantly higher than ebselen (11.9%). The selenopeptide had an elimination rate of 5.5% for hydroxyl radicals, which was slightly lower than vitamin C (7.7%) and slightly higher than ebselen (4.8%). This indicates that the self-assembling selenopeptide of the present invention has an excellent ability to eliminate the main types of reactive oxygen species in the body.

[0093] Test Example 3

[0094] This test case examined the anti-inflammatory effect of selenopeptide nanomedicines, including the following steps:

[0095] Mouse mononuclear macrophage leukemia cells (RAW 264.7) were used as the cell experimental model.

[0096] The cell culture conditions included: the culture medium was DMEM containing 10% fetal bovine serum and 1% double-antibody, and the cell culture incubator was set at a temperature of 37°C and a CO2 content of 5%. Specifically, RAW 264.7 cells were cultured at a density of 1×10 5Cells were seeded in 6-well plates at 400 nmol / well and cultured for 12 hours. The culture medium was then replaced with a medium containing 100 ng / mL lipopolysaccharide (LPS) and cultured for 24 hours to induce an inflammatory response in the cells. The old culture medium was then removed, the cells were washed once with PBS, and replaced with a new culture medium containing 10 μM simvastatin, 6.75 μM selenopeptide and its nanomedicine, respectively, and cultured for another 24 hours. In addition, cells without LPS stimulation and cells with LPS stimulation but no drug intervention were cultured as a control group under the same conditions. The drug-intervention cells and the control group cells were washed three times with cold PBS, and the well plates were placed on ice.

[0097] Lysis buffer containing phenylmethylsulfonyl fluoride (PMSF) (prepared as 1 mL of lysis buffer plus 10 μL of PMSF) was added to the well plate containing cells, 400 μL per well, and lysed on ice for 30 minutes. After lysis, the lysate was collected and centrifuged (12,000 rpm, 4°C, 5 minutes). The supernatant was collected and the total protein concentration was determined using a BCA protein concentration assay kit (Solarbio, Cat. No. PC0020). A volume of the solution containing 50 μg of protein was taken and brought to a total volume of 20 μL with loading buffer. The cells were then incubated at 100°C for 5 minutes to denature the protein.

[0098] Add the electrophoresis buffer to the electrophoresis tank. Using a pipette, carefully pipette 10 μL of the protein solution into the sample well of the gradient gel and begin electrophoresis (voltage 80 V, time 90 minutes). After electrophoresis, transfer to the membrane began. First, soak the polyvinylidene fluoride (PVDF) membrane in methanol for 10 minutes to activate it. Then, place the membrane in a transfer sandwich (with a sponge gasket, filter paper, gel, membrane, filter paper, and sponge gasket positioned from the negative pole to the positive pole, respectively). Transfer using a rapid transfer buffer and perform electrophoresis at 400 V for 30 minutes. After successful transfer, place the membrane in 5% skim milk powder and gently shake for 1 hour to block the membrane. The PVDF membrane was then incubated with primary antibodies against IL-1β, IL-6, and TNF-α, respectively, overnight at 4°C. After incubation, remove the primary antibody solution and wash three times with TBST buffer (100 mL of 10× TBS buffer, 1 mL of Tween 20, and 900 mL of purified water), gently shaking for 10 minutes each wash. The membrane was then incubated with secondary antibody for 1 hour, followed by three washes with TBST buffer, gently shaking for 10 minutes each time. ECL luminescent solutions A and B were then mixed in equal amounts and added dropwise to the membrane. After incubation in the dark for 5 minutes, images were taken using a chemiluminescence imaging system (ChemiScope 6000, CLINX).

[0099] like Figure 7As shown in the results, after LPS stimulation, the expression levels of inflammatory factors IL-1β, IL-6, and TNF-α in RAW 264.7 cells increased. However, after intervention with selenopeptides or selenopeptide nanomedicines, the levels of these three inflammatory factors decreased to varying degrees. This indicates that selenopeptide nanomedicines can effectively reduce the secretion of inflammatory factors by inflammatory macrophages and reduce the inflammatory response.

[0100] Test Example 4

[0101] This test case examined the accumulation of seleno-peptide nanoparticles in the liver of experimental mice, and included the following steps:

[0102] A seleno-peptide nanodrug was prepared at a 1.5 mg / mL simvastatin concentration, and 200 μL of this seleno-peptide nanodrug was injected into the tail vein of C57BL / 6 mice (n=3). A control group received 200 μL of a 1.5 mg / mL simvastatin solution (containing 10% DMSO to increase simvastatin solubility). Twenty-four hours later, the mice were asphyxiated in a CO2 atmosphere, and the livers were removed by autopsy. A portion of the liver was excised, weighed, and recorded. After thorough grinding, acetonitrile was added and mixed using a vortex mixer (3000 rpm). After centrifugation (3000 rpm, 10 minutes), the supernatant was collected and filtered through a 200 μm organic filter membrane. Quantitative analysis was performed using LC-MS (precursor ion m / z 441.2, major fragment ion m / z 325.2).

[0103] Experimental results showed that the simvastatin content per unit weight in the liver of mice injected intravenously with seleno-peptide nanomedicine was 24 times higher than that of mice injected with simvastatin alone. In clinical pharmacology, statins are hydroxymethylglutaryl-CoA reductase inhibitors that inhibit the synthesis of endogenous cholesterol, achieving the effect of lowering blood lipids. Simvastatin's lipid-lowering effect is primarily exerted in the liver, and after oral administration, the lactone ring in the molecular structure of simvastatin must be opened in the liver to be converted into the active substance. Oral simvastatin also has low bioavailability due to first-pass metabolism, while intravenous injection poses the problem of rapid renal metabolism. Because nanoparticles with a particle size of approximately 100 nm have the characteristic of large-scale accumulation in the liver, seleno-peptide nanomedicines can increase the accumulation of simvastatin in the liver, improving its bioavailability to exert its lipid-lowering function.

[0104] Test Example 5

[0105] This test case examined the plaque targeting effect of selenopeptide nanomedicine in atherosclerosis model mice, including the following steps:

[0106] First, a fluorescently labeled selenopeptide nanodrug was prepared. Specifically, the selenopeptide was mixed with Cy5.5-NHS at a molar ratio of 10:1 and allowed to react in the dark at room temperature for 2 hours for fluorescent labeling. Then, using the fluorescently labeled selenopeptide, a selenopeptide nanodrug was prepared according to the protocol provided in Example 2. As a control, a fluorescently labeled control peptide, VHPGQHGGK(NH2)-K(C18)2, was used to prepare a control nanodrug.

[0107] ApoE was fed with a high-fat diet - / - Mice were 12 weeks old and an atherosclerosis model was established. Fluorescently labeled seleno-peptide nano-drugs and control peptide nano-drugs (n=3) were injected through the tail vein. Six hours later, the mice were anesthetized with isoflurane and an anesthesia machine vaporizer and fixed on the dissection table. Under continuous inhalation of isoflurane anesthesia, the chest cavity of the mouse was cut open and the heart was fully exposed. A small incision was made in the right atrium, the syringe needle was inserted into the left ventricle, and cold PBS was injected for systemic perfusion. When the fluid outflowing from the right atrium turned clear and transparent and the liver turned from red to white, the aorta was removed and the fat tissue of the vascular adventitia was removed using fine tweezers under a microscope. The fluorescence intensity of the aorta was tested using a small animal fluorescence imager (IVIS), where the excitation wavelength was 680nm and the emission wavelength was 700nm.

[0108] The experimental results are as follows Figure 8 and Figure 9 As shown, compared with the control peptide nanomedicine, the aorta of the atherosclerosis model mice injected with the selenopeptide nanomedicine of the present invention has a stronger fluorescence signal, which is 2.2 times that of the control group, indicating that the selenopeptide nanomedicine of the present invention can be more effectively enriched in the diseased blood vessels.

[0109] Test Example 6

[0110] This test case examined the effectiveness of seleno-peptide nanomedicine in slowing the development of vascular plaques in atherosclerosis model mice, including the following steps:

[0111] ApoE was fed with a high-fat diet - / -Nine male mice were 12 weeks old and used to establish an atherosclerosis model. The mice were then switched from a high-fat diet to a regular chow diet and randomly divided into three intervention groups: saline, simvastatin, and seleno-peptide nanomedicine (n=3). Each group received two injections of the corresponding drug twice weekly, at a dose of 15 mg of simvastatin per kg body weight. Mice in the saline group received 200 μL of saline per injection for a total of eight weeks. Following treatment, the mice were anesthetized with isoflurane and an anesthesia vaporizer and fixed on a dissecting table. Under continuous isoflurane inhalation anesthesia, the thoracic cavity was opened to fully expose the heart. 1-2 mL of blood was collected with a syringe for routine blood count and biochemical analysis. Next, a small incision was made in the right atrium, and the needle of the syringe was inserted into the left ventricle. Systemic perfusion with cold PBS was performed. After the right atrial outflow became clear and the liver turned from red to white, the aorta (excluding the aortic arch) was removed and fixed in 4% formaldehyde for at least 12 hours.

[0112] Prepare Oil Red O staining solution: Weigh 0.5g of Oil Red O and add 100ml of isopropanol. Heat in a 90°C water bath for 1 hour, then filter. Mix the filtrate with purified water in a ratio of 3:2 and let it stand for 10 minutes.

[0113] The aorta was removed from the fixative, and any residual fixative was washed off with PBS. Adventitial adipose tissue was removed using fine forceps under a microscope. Oil Red O staining solution was then added and stained at 37°C for 30 minutes. After washing with 75% ethanol and then purified water, the aorta was photographed and plaque area was quantitatively analyzed using Image-Pro Plus software.

[0114] The experimental results are as follows Figure 10 and Figure 11 As shown, after intervention with selenopeptide nanomedicine, the plaque area in the aorta (excluding the aortic arch) of the model mice was significantly reduced, while intravenous simvastatin had no significant effect. In addition, blood routine and blood biochemical index tests showed that the blood routine indicators (including red blood cells, white blood cells, platelets, hemoglobin, etc.), liver function (including alanine aminotransferase, aspartate aminotransferase, etc.), and renal function (including blood creatinine, urea, etc.) of the model mice treated with selenopeptide nanomedicine were all within the normal range, indicating that selenopeptide nanomedicine has no obvious toxicity to the hematopoietic system, liver, or kidney.

[0115] In summary, the self-assembling selenopeptides and selenopeptide nanomedicines of the present invention have antioxidant and anti-inflammatory properties. After intravenous injection, they can target atherosclerotic plaques in the blood vessels of atherosclerotic model mice, slowing the development and progression of atherosclerotic plaques. Furthermore, the self-assembling selenopeptides and selenopeptide nanomedicines have good biosafety and do not exhibit significant blood, liver, or kidney toxicity in experimental mice.

[0116] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the self-assembling selenopeptide targeting vascular cell adhesion molecule-1, selenopeptide nanomedicine, and its preparation method and application, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacement of various raw materials of the present invention, addition of auxiliary ingredients, selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

[0117] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0118] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A self-assembling selenopeptide targeting vascular cell adhesion molecule-1, characterized in that: The self-assembling selenopeptide targeting vascular cell adhesion molecule-1 comprises an oxidation response unit and a vascular cell adhesion molecule-1 targeting unit sequentially connected from the N-terminus to the C-terminus, and a spacer amino acid located between the oxidation response unit and the vascular cell adhesion molecule-1 targeting unit; The oxidation response unit comprises a modified polypeptide sequence K(NH2)-K[Sec(C18)]2, and its molecular structure is shown below: The vascular cell adhesion molecule-1 targeting unit includes the polypeptide sequence VHPKQHR.

2. The self-assembling selenopeptide targeting vascular cell adhesion molecule-1 according to claim 1, characterized in that The spacer amino acids are 1-3 natural amino acids or unnatural amino acids.

3. The self-assembling selenopeptide targeting vascular cell adhesion molecule-1 according to claim 2, characterized in that The natural amino acids include glycine.

4. The method for preparing a self-assembling selenopeptide targeting vascular cell adhesion molecule-1 according to any one of claims 1 to 3, characterized in that: The preparation method comprises: A Boc group-protected fatty chain-substituted selenocysteine is prepared, and then each amino acid raw material is sequentially coupled to the resin by solid-phase synthesis, and eluted to obtain the self-assembling selenopeptide targeting vascular cell adhesion molecule-1.

5. A selenopeptide nanomedicine targeting vascular cell adhesion molecule-1, characterized in that: The selenopeptide nanomedicine comprises the self-assembled selenopeptide targeting vascular cell adhesion molecule-1 according to any one of claims 1 to 3 and a drug encapsulated therein.

6. The selenopeptide nanomedicine targeting vascular cell adhesion molecule-1 according to claim 5, characterized in that: The drugs include statins.

7. The selenopeptide nanomedicine targeting vascular cell adhesion molecule-1 according to claim 6, characterized in that: The statins include any one of lovastatin, pravastatin, simvastatin, fluvastatin, atorvastatin, rosuvastatin or pitavastatin, or a combination of at least two of them.

8. The selenopeptide nanomedicine targeting vascular cell adhesion molecule-1 according to claim 5, characterized in that: The selenopeptide nanomedicine further comprises the additive DMG-PEG2000.

9. The selenopeptide nanomedicine targeting vascular cell adhesion molecule-1 according to claim 8, characterized in that: The molar ratio of the DMG-PEG2000 to the self-assembling selenopeptide targeting vascular cell adhesion molecule-1 is (1-10): (90-99).

10. The selenopeptide nanomedicine targeting vascular cell adhesion molecule-1 according to claim 5, characterized in that: The particle size of the selenopeptide nanomedicine is 50-200 nm.

11. The method for preparing a selenopeptide nanomedicine targeting vascular cell adhesion molecule-1 according to any one of claims 5 to 10, characterized in that: The preparation method comprises: An organic solvent containing the drug is mixed with a solution containing the self-assembling selenopeptide for emulsification, the organic solvent is removed, and the supernatant is collected by centrifugation to obtain a solution of the selenopeptide nanomedicine targeting vascular cell adhesion molecule-1.

12. The method for preparing the selenopeptide nanomedicine targeting vascular cell adhesion molecule-1 according to claim 11, characterized in that: The emulsification process is carried out by stirring or shaking.

13. The method for preparing the selenopeptide nanomedicine targeting vascular cell adhesion molecule-1 according to claim 11, characterized in that: The removal of the organic solvent is performed by rotary evaporation.

14. The method for preparing the selenopeptide nanomedicine targeting vascular cell adhesion molecule-1 according to claim 11, characterized in that: The organic solvent includes any one of ethanol, isopropanol, dichloromethane, acetone or cyclohexane, or a combination of at least two thereof.

15. Use of the self-assembling selenopeptide targeting vascular cell adhesion molecule-1 according to any one of claims 1 to 3 or the selenopeptide nanomedicine targeting vascular cell adhesion molecule-1 according to any one of claims 5 to 10 in the preparation of a drug for treating atherosclerosis.

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