Preparation method and application of supramolecular double-antibody type nano atherosclerosis diagnosis and treatment agent

Abstract

The invention discloses a preparation method and application of a supramolecular double-antibody type nano atherosclerosis diagnosis and treatment agent, according to the method, a polypeptide sequence PBP: tamra-beta-Ala-YpYYGDAEWVDVS with a fluorescence function, a self-assembly function and a targeting function is innovatively synthesized, and the polypeptide sequence PBP: tamra-beta-Ala-YpYYGDAEWVDVS and VLA-4 protein are co-assembled into a fibrous nano diagnosis and treatment agent PBP-coated VLA-4. According to the nanofiber prepared by the invention, by targeting CD62p and CD106 highly expressed by inflammatory endothelial cells at a plaque part, an atherosclerosis lesion part is accurately positioned, and the position of the plaque is judged by utilizing a fluorescence imaging technology. Besides, the nanofiber inhibits adhesion and recruitment of monocytes in atherosclerosis by closing CD62p and CD106, reduces formation of foam macrophages and release of inflammatory factors, achieves the effect of inhibiting the progress of atherosclerosis, and provides a new thought for early diagnosis and precise treatment of atherosclerosis.

Description

Technical Field

[0001] The present invention belongs to the field of pharmacy, and specifically relates to a preparation method and application of a supramolecular dual-antibody nano-atherosclerosis diagnostic and therapeutic agent. Background Art

[0002] Atherosclerosis is a chronic progressive vascular inflammatory disease. Rupture of plaques can lead to acute cardiovascular disease, making it the main cause of death. Vulnerable plaques have some typical pathological features, mainly including a large lipid necrotic core and a thin fibrous cap, with a large number of active macrophages and inflammatory cells infiltrating. The macrophages accumulated in the plaque site mainly originate from monocytes from the circulating blood. They play an important role in the inflammatory response associated with atherosclerosis and participate in all stages of atherosclerosis. Monocytes that chemotaxis to the plaque site are activated and differentiated into activated macrophages. They participate in the uptake and metabolism of lipoproteins, the secretion of inflammatory factors, the production of enzymes and toxic metabolites, promote the instability of the fibrous cap and the rupture of the plaque, and further recruit monocytes in the circulating blood, forming a vicious cycle. Therefore, blocking the adhesion and aggregation of monocytes to inflammatory endothelial cells becomes a key step in inhibiting the progression of atherosclerosis.

[0003] As common immune cells in the human body, monocytes account for about 1 / 5 of peripheral blood mononuclear cells and play a unique biological role in the body's inflammatory response. Studies have shown that monocytes in circulating blood specifically bind to CD106 and CD62p, which are highly expressed by inflammatory vascular endothelial cells in the plaque site, and differentiate into macrophages through endothelial cells. Macrophages recognize and phagocytize excess oxLDL in the plaque site to form foam cells, thereby expanding the plaque area and increasing the instability of the plaque. In addition, differentiated macrophages will also release inflammatory factors to further recruit monocytes in the blood, forming a vicious cycle. It can be seen that blocking the CD62p and CD106 receptor proteins highly expressed by endothelial cells can effectively inhibit the chemotaxis and adhesion of monocytes to the plaque site, which is expected to become a key step in inhibiting the formation of foam cells.

[0004] At present, drugs and surgery are effective methods for treating atherosclerosis. However, the relatively low accumulation efficiency of drugs in the lesion area, the toxic side effects associated with long-term use of drugs, and complications such as stent thrombosis during surgery have hindered the further development of drugs and surgery in the treatment of atherosclerosis. In recent years, the rise of the concept of "precision medicine" has gradually attracted people's attention to nanomedicines, especially the delivery of diagnostic and therapeutic drugs to inflammatory tissues, which can effectively improve the diagnostic or therapeutic effect and reduce side effects. However, existing nanomedicines mainly rely on single antibodies to inhibit the adhesion of monocytes with a single targeting effect, so that the accumulation of foam macrophages in the plaque site is not effectively resolved, resulting in the persistence of inflammatory response in the plaque site. Therefore, it is a meaningful challenge to develop a nanomedicine that can simultaneously target and bind to multiple monocyte adhesion sites to more effectively block the chemotactic adhesion of monocytes to the plaque site.

[0005] Peptides are a kind of life-active substances between amino acids and proteins, which are composed of amino acids that make up organisms in different arrangements. In recent years, peptides have shown strong advantages in the biomedical field due to their inherent properties, such as strong targeting, easy design and functionalization, good biocompatibility and biodegradability. It is worth noting that hydrophobic short peptides can form co-assembled supramolecular nanofibers with enzymes such as alkaline phosphatase or protein bovine serum albumin, and show a series of biomedical applications. Due to the non-covalent interaction between them, supramolecular nanofibers show rapid response to external stimuli and show great application prospects in drug delivery, sensing, tissue engineering, immune response regulation and cell fate control. Recent studies have shown that self-assembling peptides can form co-assembled nanofibers with proteins during the self-assembly process, and the resulting co-assembled nanofibers and hydrogels are relatively efficient in the delivery of proteins / antigens. Moreover, co-assembled peptides based on single antibodies can not only retain the functional activity of single-target peptides, but also increase the stability and half-life of proteins. Compared with single-target peptides, bifunctional peptides can simultaneously play a role in different signal transduction of organisms, and are expected to achieve a 1+1>2 effect. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a supramolecular dual-antibody nano-atherosclerosis diagnostic and therapeutic agent to ensure that under dual targeting and blocking effects, the effect of effectively inhibiting the progression of atherosclerosis is achieved.

[0007] CD62p and CD106 are highly expressed in inflammatory endothelial cells and can serve as adhesion sites for monocytes to recruit monocytes in the circulating blood. Therefore, it is expected that blocking CD62p and CD106 can inhibit the recruitment of monocytes and thus effectively inhibit the progression of atherosclerosis.

[0008] The fibrous nano-diagnostic agent innovatively synthesizes tetramethylrhodamine with fluorescence function, β-Ala with connection function, YpYYG with self-assembly function, and functional polypeptide sequence PBP: tamra-β-Ala-YpYYGDAEWVDVS of CD62p targeting part DAEWVDVS, and assembles it with CD106 receptor affinity protein VLA-4 to form supramolecular dual-antibody nano-atherosclerosis diagnostic agent PBP@VLA-4. By targeting CD62p and CD106 highly expressed by inflammatory endothelial cells in the plaque site, the nano-diagnostic agent PBP@VLA-4 accurately locates the atherosclerotic lesion site and determines the location of the plaque using fluorescence imaging technology. Afterwards, by blocking CD62p and CD106, the nano-diagnostic agent PBP@VLA-4 inhibits the adhesion and recruitment of monocytes in atherosclerosis, reduces the formation of foam macrophages and the release of inflammatory factors, and achieves the effect of inhibiting the progression of atherosclerosis, which is expected to provide new ideas for the early diagnosis and precise treatment of atherosclerosis.

[0009] A method for preparing a supramolecular dual-antibody nano-atherosclerosis diagnostic and therapeutic agent comprises the following steps:

[0010] (1) Dissolve 0.1-2 g of 2-chlorotrityl chloride resin and 0.1-100 mg of the first amino acid at the C-terminus (Fmoc-Ser(tBu)-OH) in 1-5 mL of dichloromethane; after the solid is completely dissolved, add 0.1-1 mL of N,N-diisopropylethylamine to the solution and react on a shaker at 20-40°C for 1-4 hours. Then, add 0.2-0.8 mL of N,N-diisopropylethylamine, 0.2-0.8 mL of methanol and 1-8 mL of dichloromethane to the solution and continue the reaction for 10-40 minutes to block the unreacted groups on the resin; after the reaction is completed, wash with 5-50 mL of N,N-dimethylformamide and drain to prepare Fmoc-Ser(tBu) amino acid resin;

[0011] (2) removing the Fmoc protecting group of the amino acid resin prepared in step (1) with a mixed solution of 1 to 10 mL of piperidine and N,N-dimethylformamide (DMF), and washing the amino acid from which the protecting group has been removed with 5 to 50 mL of DMF and draining the mixture until the Fmoc protecting group has been removed successfully, thereby preparing a deaminated H2N-Ser(tBu)-resin;

[0012] (3) dissolving 0.1-2 g of the obtained H2N-Ser(tBu)-resin with the Fmoc protecting group removed, 0.1-100 mg of the second amino acid at the C-terminus (Fmoc-Val-OH), 0.01-100 mg of 1-hydroxybenzotriazole and 0.01-1 g of N,N'-diisopropylcarbodiimide in 1-10 mL of DMF, and reacting in a shaker at 20-40° C. for 0.5-2 h; after the reaction is completed, washing the resin with DMF and draining it to obtain Fmoc-Val-Ser(tBu)-resin;

[0013] (4) removing the Fmoc protection from the Fmoc-Val-Ser(tBu)-resin prepared in step (3) using a mixed solution of piperidine and N,N-dimethylformamide used in step (2) to obtain H2N-Val-Ser(tBu)-resin;

[0014] (5) replacing the "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) with "H2N-Val-Ser(tBu)-resin and Fmoc-Asp(otBu)-OH", respectively, and the other steps remain the same as steps (3) and (4), to obtain H2N-Asp(otBu)-Val-Ser(tBu)-resin;

[0015] Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) are replaced by "H2N-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Val-OH", respectively, and the other steps are kept consistent with steps (3) and (4), to obtain H2N-Val-Asp(otBu)-Val-Ser(tBu)-resin;

[0016] Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) are replaced by "H2N-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Trp(Boc)-OH", respectively, and the other steps are kept consistent with steps (3) and (4), to obtain H2N-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin;

[0017] Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) are replaced by "H2N-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Glu(otBu)-OH", respectively, and the other steps are kept consistent with steps (3) and (4), to obtain H2N-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin;

[0018] Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) are replaced with "H2N-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Ala-OH", and the other steps are kept consistent with steps (3) and (4) to obtain H2N-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin;

[0019] Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) are replaced by "H2N-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Asp(otBu)-OH", respectively, and the other steps are kept consistent with steps (3) and (4), to obtain H2N-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin;

[0020] Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) are replaced by "H2N-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Gly-OH", respectively, and the other steps are kept consistent with steps (3) and (4), to obtain H2N-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin;

[0021] Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) are replaced with "H2N-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Tyr(tBu)-OH", and the other steps are kept consistent with steps (3) and (4) to obtain H2N-Tyr(tBu)-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin;

[0022] Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) were replaced with "H2N-Tyr(tBu)-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Tyr(HPO3BZL)-OH", and the other steps were kept consistent with steps (3) and (4) to obtain H2N-Tyr(HPO3BZL)-Tyr(tBu)-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin;

[0023] Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) were replaced with "H2N-Tyr(HPO3BZL)-Tyr(tBu)-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Tyr(tBu)-OH", and the other steps were kept consistent with steps (3) and (4) to obtain H2N-Tyr(tBu)-Tyr(HPO3BZL)-Tyr(tBu)-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin;

[0024] Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) were replaced by "H2N-Tyr(tBu)-Tyr(HPO3BZL)-Tyr(tBu)-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-β-Ala-OH", and the other steps were kept consistent with steps (3) and (4) to obtain amino acid coupling resin: β-Ala-Tyr(tBu)-Tyr(HPO3BZL)-Tyr(tBu)-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin;

[0025] (6) 0.1-1 g of the amino acid coupling resin obtained in step (5), 0.1-100 mg of tetramethylrhodamine (tamra) and 0.1-100 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate are added to a mixture of N,N-dimethylformamide and N,N-diisopropylethylamine in sequence, and the mixture is reacted in a shaker at 25-40° C. in the dark for 1-4 hours; after the condensation reaction is complete, the mixture is washed with 5-50 mL of DMF and dried to obtain a fluorescent resin peptide: tamra-β-Ala-Y(HPO3BZL)Y(tBu)Y(tBu)GD(otBu)AE(otBu)W(Boc)VD(otBu)VS(tBu);

[0026] (7) Dissolve 0.5-1.5 g of the fluorescent resin peptide tamra-β-Ala-Y(HPO3BZL)Y(tBu)Y(tBu)GD(otBu)AE(otBu)W(Boc)VD(otBu)VS(tBu) in a cleavage solution consisting of trifluoroacetic acid, water and triisopropylsilane, and react with stirring at 20-40° C. in the dark for 1-4 h; after complete cleavage, wash with 5-50 mL of ice ether, and dry under reduced pressure at 20-40° C. to obtain a crude polypeptide PBP; then, purify the crude polypeptide by Shimadzu semi-preparative method to obtain a fluorescent polypeptide PBP: tamra-β-Ala YpYYGDAEWVDVS;

[0027] (8) After dissolving 0.1-100 mg of the fluorescent polypeptide PBP prepared in step (7) into 0.1-100 mL of phosphate buffer (PBS) solution, 0.01-10 mg of the recombinant protein (VLA-4) and 1-100 U of alkaline phosphatase are added to the PBS solution in sequence, and stirred at 25-50° C. for 3-6 hours to obtain the supramolecular dual-antibody nanodiagnostic agent PBP@VLA-4.

[0028] Furthermore, in the step (1), the volume ratio of N,N-diisopropylethylamine, methanol and dichloromethane is 1:1:1 to 1:5:5.

[0029] Furthermore, in the step (6), the mass ratio of tetramethylrhodamine to benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate is 1:0.5 to 1:1.5.

[0030] Furthermore, in step (7), the volume ratio of the resin peptide to the lysate is 1:8 to 1:12.

[0031] Furthermore, the dephosphorylation rate of the polypeptide PBP prepared in step (7) is 97.6%, and the affinity K for CD62p protein is D The value was 29.6 μM.

[0032] Furthermore, the particle size of the polypeptide PBP prepared in step (7) is 8 to 16 nm.

[0033] Furthermore, in step (8), the mass ratio of polypeptide PBP to VLA-4 is 5:1 to 20:1, and the concentration of alkaline phosphatase is 5 to 15 U / mL.

[0034] Furthermore, the PBP@VLA-4 prepared in step (8) is in the form of nanofibers with a size of 22 to 40 nm.

[0035] To achieve the purpose of the invention, the present invention also provides the use of the supramolecular dual-antibody nano-atherosclerosis diagnostic and therapeutic agent prepared by the above-mentioned preparation method in accurately identifying early atherosclerotic plaques. The supramolecular dual-antibody nano-atherosclerosis diagnostic and therapeutic agent PBP@VLA-4 simultaneously targets CD62p and CD106 to block multiple binding sites of monocytes, thereby inhibiting the chemotaxis and adhesion of monocytes in the plaque site, and then performing specific fluorescence imaging and precise treatment of the plaque.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] (1) The present invention is based on non-covalent interaction, using the polypeptide sequence PBP as a carrier, and co-assembling with the CD106 receptor affinity protein to form a nanoprobe, thereby avoiding the loss of antibody function caused by covalent modification.

[0038] (2) The present invention blocks the dual targets of CD62p and CD106 at the lesion site, so that the assembled nanopeptide can not only retain the functional activity of the single-target polypeptide, but also increase the stability and half-life of the protein.

[0039] (3) Compared with single-target peptides, bifunctional peptides can simultaneously target different signal transduction pathways in the body, achieving the effect of 1+1>2.

[0040] (4) The preparation method of the present invention is stable and reliable with good repeatability. The prepared PBP has good biocompatibility, safety and strong targeting. It can not only target inflammatory endothelial cells in the plaque site to block CD62p, but also synergize with VLA-4 protein through the peptide targeting sequence to achieve the effect of simultaneously targeting and binding CD62p and CD106, blocking multiple binding sites of monocytes, and achieving the inhibition of monocyte chemotaxis and adhesion in the plaque site. At the same time, specific fluorescence imaging of the plaque is performed, which is expected to provide new ideas for the clinical early diagnosis and precise treatment of atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of this specification or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this specification. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0042] Figure 1 is the chemical structural formula of the PBP polypeptide of the present invention;

[0043] Figure 2 is the mass spectrum of the PBP polypeptide of the present invention;

[0044] Figure 3 is the chromatogram of the PBP polypeptide of the present invention;

[0045] Figure 4 is the affinity K of the PBP polypeptide of the present invention to CD62p protein D The SPR test results of the value;

[0046] Figure 5 This is a result diagram of the dephosphorylation rate of the PBP polypeptide of the present invention;

[0047] Fig. 6A The circular dichroism spectra of the PBP polypeptide of the present invention before and after assembly;

[0048] Figure 6B The circular dichroism secondary structure analysis diagram of the PBP polypeptide of the present invention before and after assembly;

[0049] Fig. 7A This is a transmission electron microscopy image of the PBP polypeptide nanomaterial of the present invention before self-assembly;

[0050] Figure 7B This is a transmission electron microscopy image of the PBP polypeptide nanomaterial after self-assembly;

[0051] Figure 7C is a transmission electron micrograph of the VLA-4 protein of the present invention;

[0052] Fig.7D This is a transmission electron microscopy image of the co-assembled PBP@VLA-4 of the present invention;

[0053] Figure 8 Figure 2 is a particle size diagram of PBP and PBP@VLA-4 of the present invention;

[0054] Fig. 9 This is the gel electrophoresis result of the protective effect of PBP@VLA-4 co-assembly on protein stability of the present invention;

[0055] Fig. 10A is the affinity K of the PBP of the present invention for targeting inflammatory MAEC cells after assembly D Value streaming test results;

[0056] Fig. 10B is the targeting affinity K of PBP@VLA-4 co-assembled to inflammatory MAEC cells D Value streaming test results;

[0057] Fig.11A The results of the inhibitory effects of PBP and PBP@VLA-4 on monocyte migration at different concentrations are shown in the figure;

[0058] Fig. 11B The results of the inhibitory effects of PBP and PBP@VLA-4 on monocyte adhesion at different concentrations are shown in the figure;

[0059] Fig.12 The results of fluorescence imaging of mouse As plaques by PBP and PBP@VLA-4;

[0060] Fig.13 This is the result of gross Oil Red O staining after PBP and PBP@VLA-4 treated As plaques in mice. DETAILED DESCRIPTION

[0061] In order to enable those skilled in the art to better understand the technical solutions in this specification, the technical solutions in the embodiments of this specification will be clearly and completely described below in conjunction with the drawings in the embodiments of this specification. Obviously, the described embodiments are only part of the embodiments of this specification, not all of the embodiments. Based on the embodiments in this specification, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this specification.

[0062] Example

[0063] (1) Preparation of Fmoc-Ser(tBu) amino acid resin: Weigh 1 g of 2-chlorotrityl chloride resin and 0.5 mg of the first amino acid at the C-terminus (Fmoc-Ser(tBu)-OH) and dissolve them in 5 mL of dichloromethane. After the solid is completely dissolved, add 1 mL of N,N-diisopropylethylamine to the solution and react on a shaker at 37°C for 2 h. Then, add 0.5 mL of N,N-diisopropylethylamine, 0.5 mL of methanol and 2 mL of dichloromethane to the solution and continue the reaction for 20 min to block the unreacted groups on the resin. After the reaction is completed, wash with 50 mL of N,N-dimethylformamide and drain to prepare Fmoc-Ser(tBu) amino acid resin;

[0064] (2) Preparation of amino acid coupling resin sequence: The Fmoc protecting group of the amino acid resin prepared in step (1) was removed with a mixed solution of 5 mL of piperidine and N,N-dimethylformamide (DMF), and the amino acid from which the protecting group was removed was washed and dried with 50 mL of DMF until the Fmoc protecting group was successfully removed, thereby preparing a deaminated H2N-Ser(tBu)-resin;

[0065] (3) 0.5 g of the obtained H2N-Ser(tBu)-resin with the Fmoc protecting group removed, 10 mg of the second amino acid at the C-terminus (Fmoc-Val-OH), 10 mg of 1-hydroxybenzotriazole and 0.5 g of N,N'-diisopropylcarbodiimide were dissolved in 10 mL of DMF and reacted in a shaker at 30° C. for 1 h; after the reaction was completed, the resin was washed with DMF and dried to obtain Fmoc-Val-Ser(tBu)-resin;

[0066] (4) removing the Fmoc protection from the Fmoc-Val-Ser(tBu)-resin prepared in step (3) using a mixed solution of piperidine and N,N-dimethylformamide used in step (2) to obtain H2N-Val-Ser(tBu)-resin;

[0067] (5) replacing the "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) with "H2N-Val-Ser(tBu)-resin and Fmoc-Asp(otBu)-OH", respectively, and the other steps remain the same as steps (3) and (4), to obtain H2N-Asp(otBu)-Val-Ser(tBu)-resin;

[0068] Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) are replaced by "H2N-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Val-OH", respectively, and the other steps are kept consistent with steps (3) and (4), to obtain H2N-Val-Asp(otBu)-Val-Ser(tBu)-resin;

[0069] Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) are replaced by "H2N-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Trp(Boc)-OH", respectively, and the other steps are kept consistent with steps (3) and (4), to obtain H2N-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin;

[0070] Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) are replaced by "H2N-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Glu(otBu)-OH", respectively, and the other steps are kept consistent with steps (3) and (4), to obtain H2N-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin;

[0071] Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) are replaced with "H2N-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Ala-OH", and the other steps are kept consistent with steps (3) and (4) to obtain H2N-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin;

[0072] Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) are replaced by "H2N-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Asp(otBu)-OH", respectively, and the other steps are kept consistent with steps (3) and (4), to obtain H2N-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin;

[0073] Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) are replaced by "H2N-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Gly-OH", respectively, and the other steps are kept consistent with steps (3) and (4), to obtain H2N-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin;

[0074] Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) are replaced with "H2N-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Tyr(tBu)-OH", and the other steps are kept consistent with steps (3) and (4) to obtain H2N-Tyr(tBu)-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin;

[0075] Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) were replaced with "H2N-Tyr(tBu)-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Tyr(HPO3BZL)-OH", and the other steps were kept consistent with steps (3) and (4) to obtain H2N-Tyr(HPO3BZL)-Tyr(tBu)-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin;

[0076] Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) were replaced with "H2N-Tyr(HPO3BZL)-Tyr(tBu)-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Tyr(tBu)-OH", and the other steps were kept consistent with steps (3) and (4) to obtain H2N-Tyr(tBu)-Tyr(HPO3BZL)-Tyr(tBu)-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin;

[0077] Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) were replaced by "H2N-Tyr(tBu)-Tyr(HPO3BZL)-Tyr(tBu)-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-β-Ala-OH", respectively. The other steps were kept consistent with steps (3) and (4), and the amino acid coupling tree was obtained: β-Ala-Tyr(tBu)-Tyr(HPO3BZL)-Tyr(tBu)-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin;

[0078] (6) Preparation of fluorescent resin peptide: 1 g of the amino acid coupling resin obtained in step (5), 10 mg of tetramethylrhodamine (tamra) and 1 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate were added to a mixture of N,N-dimethylformamide and N,N-diisopropylethylamine in sequence, and reacted in a shaker at 30°C in the dark for 2 hours; after the condensation reaction was complete, the mixture was washed with 50 mL of DMF and dried to obtain a fluorescent resin peptide: tamra-β-Ala-Y(HPO3BZL)Y(tBu)Y(tBu)GD(otBu)AE(otBu)W(Boc)VD(otBu)VS(tBu);

[0079] (7) Preparation of PBP: Dissolve 1 g of the fluorescent resin peptide tamra-β-Ala-Y(HPO3BZL)Y(tBu)Y(tBu)GD(otBu)AE(otBu)W(Boc)VD(otBu)VS(tBu) prepared in step (6) in 5 mL of a cleavage solution consisting of trifluoroacetic acid, water and triisopropylsilane, and react with stirring at 25°C in the dark for 2 h; after complete cleavage, wash with 50 mL of ice ether and dry under reduced pressure at 37°C to obtain a crude polypeptide PBP; then, purify the crude polypeptide using the Shimadzu semi-preparative method to obtain the fluorescent polypeptide PBP: tamra-β-Ala YpYYGDAEWVDVS. Figure 1 As shown in the chemical structure of the polypeptide PBP, the phosphorylation site is located on the second tyrosine. Figure 2 and Figure 3 ), the prepared polypeptide has the same molecular weight as the current one and the purity is >95%. The affinity of PBP to CD62p protein was detected by surface plasmon resonance technology. D The relevant fluorescence intensity increases with the increase of material concentration, and K D The value is 29.6μM ( Figure 4 ), indicating that PBP has a high affinity for CD62p. Under the action of alkaline phosphatase, the dephosphorylation rate of polypeptide PBP gradually increased with time and reached the highest value (97.6%, Figure 5 ). The good dephosphorylation effect of peptide PBP is the basis for its self-assembly. It can be seen that peptide PBP is expected to self-assemble efficiently to form spherical nanoprobes. In addition, by comparing the changes in the secondary structure of PBP before and after self-assembly through circular dichroism, it can be seen that the α-helix composition of PBP after self-assembly is significantly increased, the proportion of random coils is reduced, and the secondary structure is more ordered ( Fig. 6A and Figure 6B ), further verifying the good self-assembly performance of PBP.

[0080] (8) Preparation of nano-diagnostic agent PBP@VLA-4: After dissolving 0.5 mg of the fluorescent peptide PBP prepared in step (7) into 0.5 mL of phosphate buffered saline (PBS) solution, 0.05 mg of recombinant protein (VLA-4) and 5 U of alkaline phosphatase were added to the PBS solution in sequence, and stirred at 37°C for 4 hours to obtain a fibrous supramolecular dual-antibody nano-diagnostic agent PBP@VLA-4. After comparing the morphology of PBP before and after self-assembly, it was found that PBP was in a loose and disordered state under the microscope ( Fig. 7A ), and can self-assemble into short worm-like nanomaterials under the action of alkaline phosphatase ( Figure 7B ), indicating that the dephosphorylated PBP has excellent self-assembly ability. By comparing the morphology of VLA-4 protein and PBP@VLA-4, it was found that VLA-4 protein was monodispersed spherical before assembly ( Figure 7C ), while the morphology of the PBP@VLA-4 assembled with the peptide PBP is similar to that of the PBP alone, both of which are nanofiber-like ( Fig.7D ). After measurement and comparison, it was found that the size of the nanofibers assembled by PBP was 45.7nm×9.4nm., while the size of the nanofibers of PBP@VLA-4 was 59.5nm×18.2nm, which was larger and more aggregated ( Figure 8 ), which may be related to the large molecular weight of VLA-4 protein. After 14 days at 37°C, the protein bands in PBP@VLA-4PBS remained basically unchanged, while the protein bands simply dissolved in PBS became significantly lighter ( Fig. 9 ), indicating that the pure protein degraded slowly in PBS, while PBP@VLA-4 showed higher stability.

[0081] Next, flow cytometry was used to compare the targeting and binding abilities of PBP and PBP@VLA-4 to inflammatory endothelial cells before and after co-assembly. Fig. 10A and 10B As shown in Figure 2, the fluorescence intensity of PBP@VLA-4 increases with the increase of material concentration. D The value (3.95±0.8μM) was much lower than the K D The value (18.67±2.62μM) indicates that the nanofibers formed after PBP@VLA-4 co-assembly show better targeting and binding ability to inflammatory endothelial cells. By comparing the inhibitory effects of PBP and PBP@VLA-4 on monocyte migration and adhesion, it was found that PBP had almost no significant inhibitory effect on monocyte migration and adhesion. However, the inhibitory effect of PBP@VLA-4 nanofibers on monocyte migration and adhesion increased with the increase of its concentration, and reached the best effect at a concentration of 10μM ( Fig.11A and 11B), indicating that PBP@VLA-4 can significantly inhibit the migration and adhesion of monocytes to inflammatory endothelial cells, providing a theoretical basis for subsequent animal experiments. - / - After investigating the ability of the prepared PBP@VLA-4 nanofiber probe to target fluorescent imaging of atherosclerosis in mice, it was found that the dual-antibody PBP@VLA-4 nanoprobe could significantly enhance the fluorescence signal intensity of atherosclerosis ( Fig.12 ), further proving that the dual-antibody PBP@VLA-4 nanoprobe is expected to effectively target atherosclerotic plaques and perform fluorescence imaging on them. - / - The mice were divided into groups for treatment and the aorta was stained with Oil Red O. Fig.13 ), compared with the monoclonal antibody nanomaterial PBP, the dual-antibody nanodiagnostic agent PBP@VLA-4 can better inhibit the progression of atherosclerotic plaques in model mice.

[0082] For those skilled in the art, the present invention is not limited to the details of the exemplary embodiments described above. The embodiments are exemplary and non-restrictive, and the scope of protection of the present invention is defined by the appended claims rather than the above description, and therefore, any reference numerals in the claims should not be regarded as limiting the claims involved.

[0083] The above description is only an example of one or more embodiments of the present specification and is not intended to limit one or more embodiments of the present specification. For those skilled in the art, one or more embodiments of the present specification may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present specification shall be included in the scope of the claims.

Claims

1. A method for preparing a supramolecular dual-antibody nano-atherosclerosis diagnostic and therapeutic agent, characterized in that: The method comprises the following steps: (1) 0.1-2 g of 2-chlorotrityl chloride resin and 0.1-100 mg of the first amino acid at the C-terminus (Fmoc-Ser(tBu)-OH) are dissolved in 1-5 mL of dichloromethane; after the solid is completely dissolved, 0.1-1 mL of N,N-diisopropylethylamine is added to the solution, and the mixture is reacted on a shaker at 20-40° C. for 1-4 h; then, 0.2-0.8 mL of N,N-diisopropylethylamine, 0.2-0.8 mL of methanol and 1-8 mL of dichloromethane are added to the solution, and the reaction is continued for 10-40 min to block the unreacted groups on the resin; after the reaction is completed, the mixture is washed with 5-50 mL of N,N-dimethylformamide and dried to prepare Fmoc-Ser(tBu) amino acid resin; (2) removing the Fmoc protecting group of the amino acid resin prepared in step (1) with a mixed solution of 1 to 10 mL of piperidine and N,N-dimethylformamide (DMF), and washing the amino acid from which the protecting group has been removed with 5 to 50 mL of DMF and draining the mixture until the Fmoc protecting group has been removed successfully, thereby preparing a deaminated H2N-Ser(tBu)-resin; (3) dissolving 0.1-2 g of the obtained H2N-Ser(tBu)-resin with the Fmoc protecting group removed, 0.1-100 mg of the second amino acid at the C-terminus (Fmoc-Val-OH), 0.01-100 mg of 1-hydroxybenzotriazole and 0.01-1 g of N,N'-diisopropylcarbodiimide in 1-10 mL of DMF, and reacting in a shaker at 20-40° C. for 0.5-2 h; after the reaction is completed, washing the resin with DMF and draining it to obtain Fmoc-Val-Ser(tBu)-resin; (4) removing the Fmoc protection from the Fmoc-Val-Ser(tBu)-resin prepared in step (3) using a mixed solution of piperidine and N,N-dimethylformamide used in step (2) to obtain H2N-Val-Ser(tBu)-resin; (5) replacing the "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) with "H2N-Val-Ser(tBu)-resin and Fmoc-Asp(otBu)-OH", respectively, and the other steps remain the same as steps (3) and (4), to obtain H2N-Asp(otBu)-Val-Ser(tBu)-resin; Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) are replaced by "H2N-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Val-OH", respectively, and the other steps are kept consistent with steps (3) and (4), to obtain H2N-Val-Asp(otBu)-Val-Ser(tBu)-resin; Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) are replaced by "H2N-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Trp(Boc)-OH", respectively, and the other steps are kept consistent with steps (3) and (4), to obtain H2N-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin; Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) are replaced by "H2N-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Glu(otBu)-OH", respectively, and the other steps are kept consistent with steps (3) and (4), to obtain H2N-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin; Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) are replaced with "H2N-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Ala-OH", and the other steps are kept consistent with steps (3) and (4) to obtain H2N-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin; Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) were replaced by "H2N-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Asp(otBu)-OH", respectively, and the other steps were kept consistent with steps (3) and (4), to obtain H2N-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin; Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) were replaced by "H2N-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Gly-OH", respectively, and the other steps were kept consistent with steps (3) and (4) to obtain H2N-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin; Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) were replaced with "H2N-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Tyr(tBu)-OH", and the other steps were kept consistent with steps (3) and (4) to obtain H2N-Tyr(tBu)-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin; Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) were replaced with "H2N-Tyr(tBu)-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Tyr(HPO3BZL)-OH", and the other steps were kept consistent with steps (3) and (4) to obtain H2N-Tyr(HPO3BZL)-Tyr(tBu)-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin; Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) were replaced with "H2N-Tyr(HPO3BZL)-Tyr(tBu)-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-Tyr(tBu)-OH", and the other steps were kept consistent with steps (3) and (4) to obtain H2N-Tyr(tBu)-Tyr(HPO3BZL)-Tyr(tBu)-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin; Then, "H2N-Ser(tBu)-resin and the second amino acid (Fmoc-Val-OH)" in step (3) were replaced by "H2N-Tyr(tBu)-Tyr(HPO3BZL)-Tyr(tBu)-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin and Fmoc-β-Ala-OH", and the other steps were kept consistent with steps (3) and (4) to obtain the amino acid coupling tree: β-Ala-Tyr(tBu)-Tyr(HPO3BZL)-Tyr(tBu)-Gly-Asp(otBu)-Ala-Glu(otBu)-Trp(Boc)-Val-Asp(otBu)-Val-Ser(tBu)-resin; (6) 0.1-1 g of the amino acid coupling resin obtained in step (5), 0.1-100 mg of tetramethylrhodamine (tamra) and 0.1-100 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate are added to a mixture of N,N-dimethylformamide and N,N-diisopropylethylamine in sequence, and the mixture is reacted in a shaker at 25-40° C. in the dark for 1-4 hours; after the condensation reaction is complete, the mixture is washed with 5-50 mL of DMF and dried to obtain a fluorescent resin peptide: tamra-β-Ala-Y(HPO3BZL)Y(tBu)Y(tBu)GD(otBu)AE(otBu)W(Boc)VD(otBu)VS(tBu); (7) Dissolve 0.5-1.5 g of the fluorescent resin peptide tamra-β-Ala-Y(HPO3BZL)Y(tBu)Y(tBu)GD(otBu)AE(otBu)W(Boc)VD(otBu)VS(tBu) in a cleavage solution consisting of trifluoroacetic acid, water and triisopropylsilane, and react with stirring at 20-40° C. in the dark for 1-4 h; after complete cleavage, wash with 5-50 mL of ice ether, and dry under reduced pressure at 20-40° C. to obtain a crude polypeptide PBP; then, purify the crude polypeptide by Shimadzu semi-preparative method to obtain a fluorescent polypeptide PBP: tamra-β-Ala YpYYGDAEWVDVS; (8) After dissolving 0.1-100 mg of the fluorescent polypeptide PBP prepared in step (7) into 0.1-100 mL of phosphate buffer (PBS) solution, 0.01-10 mg of the recombinant protein (VLA-4) and 1-100 U of alkaline phosphatase are added to the PBS solution in sequence, and stirred at 25-50°C for 3-6 hours to obtain a fibrous supramolecular dual-antibody nanodiagnostic agent PBP@VLA-4.

2. The method for preparing a supramolecular dual-antibody nano-atherosclerosis diagnostic and therapeutic agent as claimed in claim 1, characterized in that: In the step (1), the volume ratio of N,N-diisopropylethylamine, methanol and dichloromethane is 1:1:1 to 1:5:

5.

3. The method for preparing a supramolecular dual-antibody nano-atherosclerosis diagnostic and therapeutic agent as claimed in claim 1, characterized in that: In the step (6), the mass ratio of tetramethylrhodamine to benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate is 1:0.5 to 1:1.

5.

4. The method for preparing a supramolecular dual-antibody nano-atherosclerosis diagnostic and therapeutic agent as claimed in claim 1, characterized in that: In the step (7), the volume ratio of the resin peptide to the lysate is 1:8 to 1:

12.

5. The method for preparing a supramolecular dual-antibody nano-atherosclerosis diagnostic and therapeutic agent as claimed in claim 1, characterized in that: The dephosphorylation rate of the polypeptide PBP prepared in step (7) is 97.6%, and the affinity K for CD62p protein is D The value was 29.6 μM.

6. The method for preparing a supramolecular dual-antibody nano-atherosclerosis diagnostic and therapeutic agent as claimed in claim 1, characterized in that: The particle size of the polypeptide PBP prepared in step (7) is 8 to 16 nm.

7. The method for preparing a supramolecular dual-antibody nano-atherosclerosis diagnostic and therapeutic agent as claimed in claim 1, characterized in that: In the step (8), the mass ratio of polypeptide PBP to VLA-4 is 5:1 to 20:1, and the concentration of alkaline phosphatase is 5 to 15 U / mL.

8. The method for preparing a supramolecular dual-antibody nano-atherosclerosis diagnostic and therapeutic agent as claimed in claim 1, characterized in that: The PBP@VLA-4 prepared in step (8) is in the form of nanofibers with a size of 22 to 40 nm.

9. Application of the supramolecular dual-antibody nano-atherosclerosis diagnostic and therapeutic agent prepared by the preparation method according to any one of claims 1 to 8 in accurately identifying early atherosclerotic plaques, wherein the supramolecular dual-antibody nano-atherosclerosis diagnostic and therapeutic agent PBP@VLA-4 simultaneously targets CD62p and CD106 to block multiple binding sites of monocytes, thereby inhibiting the chemotaxis and adhesion of monocytes in the plaque site, thereby performing specific fluorescence imaging and precise treatment of the plaque.

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