Diagnosis and treatment molecule and nanoparticle for positioning and stabilizing atherosclerotic plaque and preparation method of diagnosis and treatment molecule and nanoparticle

Abstract

The invention discloses a diagnosis and treatment molecule for positioning and stabilizing atherosclerotic plaque, a nanoparticle and a preparation method thereof, the diagnosis and treatment molecule is synthesized based on cholic acid and blue copper peptide, and the nanoparticle is formed by self-assembling the diagnosis and treatment molecule. The preparation method comprises the following steps: carrying out hydroformylation modification on two ends of cholic acid, and coupling the cholic acid with blue copper peptide through Schiff base reaction to obtain the diagnosis and treatment molecule with diagnosis and treatment functions at the same time. The blue copper peptide structure in the diagnosis and treatment molecule is used as a hydrophilic segment, and the cholic acid structure is used as a water delivery segment, so that the diagnosis and treatment molecule can be further self-assembled in water to form nanoparticles. The nanoparticles have acid responsiveness and can be disintegrated in a subacid environment at the atherosclerosis, and the released blue copper peptide can stabilize plaques by means of promoting collagen secretion, inhibiting matrix metalloproteinase expression and the like; and meanwhile, the distribution of the plaque can be positioned and diagnosed by utilizing the X-ray imaging characteristics of the cholic acid, so that the stabilization and positioning integration of the atherosclerotic plaque is realized.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a diagnostic and therapeutic molecule, nanoparticles and a preparation method thereof for locating and stabilizing atherosclerotic plaques. Background Art

[0002] Atherosclerosis is the most important cause of coronary artery-related cardiovascular disease. Its main characteristics are the formation of plaques on the inner wall of the blood vessel, which gradually enlarge and narrow the vascular lumen, or rupture and cause blood vessel blockage. In the classification of atherosclerosis, vulnerable plaques usually become fragile due to their large necrotic core, thin fibrous cap, and abundant inflammatory cell infiltration. Clinically, the rupture of vulnerable plaques is the most common cause of acute coronary syndrome and often leads to very poor clinical prognosis. In atherosclerotic plaques, in situ smooth muscle cells secrete a variety of extracellular matrix molecules, such as collagen, elastin, and proteoglycans, which become the main components of the plaque fibrous cap. However, activated macrophages can also produce a variety of matrix metalloproteinases, which can degrade interstitial collagen that maintains the strength of the fibrous cap, causing the thinning of the fibrous cap, weakening of the structural strength, or promoting the degradation of the extracellular matrix at the plaque site, increasing the risk and vulnerability of plaque rupture. Existing clinical intervention measures mainly include lipid-lowering, antiplatelet and anti-inflammatory treatments, which are not clearly targeted at stabilizing atherosclerotic plaques. Therefore, identification of plaques and stabilization of vulnerable plaques have become extremely important key tasks in the diagnosis and treatment of atherosclerosis.

[0003] The booming development of nanotechnology has provided a new method for the diagnosis and treatment of clinical diseases. Nanocarriers can load or encapsulate a variety of substances with diagnostic or therapeutic activity. On the one hand, nanocarriers can reach the lesion area under the premise of stable loading, thereby protecting the internal environment from the influence of harmful drugs. On the other hand, efficient encapsulation and transportation can improve drug utilization, reduce drug degradation, and evade the recognition and clearance of the human immune system. Iodine imaging agents are the most widely used diagnostic agents for atherosclerotic diseases in clinical practice. They rely on high-density signals under X-CT or X-ray fluoroscopy to display the morphology of the vascular cavity and identify plaques. However, the amount of iodine imaging agents used in traditional CTA and angiography is still large, which has a large burden on renal function and the risk of possible contrast-induced nephropathy. In the field of nanodiagnosis of atherosclerosis, various nanotechnologies and targeted structure designs are used to make iodine imaging agents specifically enriched in plaque sites, and plaques can be directly displayed through X-ray imaging, which greatly reduces the amount of iodine imaging agents while achieving good imaging effects. Among the existing nanomedicines used to treat atherosclerosis, although they can improve drug utilization and reduce toxicity to a certain extent, they are still often limited by problems such as uncontrollable drug leakage and the inability to quickly release the drug when it reaches the lesion, making it difficult to be widely used.

[0004] Therefore, the development of innovative therapeutic nanoparticles for localizing and stabilizing atherosclerotic plaques is of great clinical significance. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a diagnostic and therapeutic molecule, nanoparticles and a preparation method thereof for locating and stabilizing atherosclerotic plaques. The nanoparticles composed of the diagnostic and therapeutic molecules are acid-responsive and can disintegrate in the slightly acidic environment of atherosclerosis. The released blue copper peptide can stabilize atherosclerotic plaques by promoting collagen secretion, inhibiting the expression of matrix metalloproteinases, etc. At the same time, the X-ray imaging characteristics of cholic acid can be used to locate and diagnose the distribution of plaques, thereby realizing the integrated diagnosis and treatment of atherosclerosis.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A diagnostic and therapeutic molecule for locating and stabilizing atherosclerotic plaques, wherein the diagnostic and therapeutic molecule is synthesized based on cholinesterase and blue copper peptide, and the chemical structure of the diagnostic and therapeutic molecule is:

[0008]

[0009] A method for preparing a diagnostic and therapeutic molecule for locating and stabilizing atherosclerotic plaques, the method comprising the following steps:

[0010] Step 1: dissolving cholinesterase, 4-hydroxymethylbenzaldehyde, dicyclohexylcarbodiimide and 4-dimethylaminopyridine in a molar ratio of 1:2:3:0.2 in an organic solvent, stirring to fully react, dialyzing the resulting solution in deionized water to remove the organic solvent; then freeze-drying, removing the deionized water, and obtaining aldehyde-modified cholinesterase;

[0011] Step 2: dissolving the aldehyde-modified cholinesterase and blue copper peptide obtained in step 1 in the organic solvent at a molar ratio of (1-10): (2-30), stirring to fully react, dialyzing the resulting solution in deionized water to remove the organic solvent; then freeze-drying, removing the deionized water, and obtaining the diagnostic molecule.

[0012] Furthermore, the organic solvent is dimethyl sulfoxide.

[0013] A nanoparticle made of therapeutic molecules for locating and stabilizing atherosclerotic plaques, which is formed by self-assembly of therapeutic molecules based on cholinesterase inhibitors and blue copper peptides.

[0014] Furthermore, the diagnostic and therapeutic molecules based on cholinesterase and blue copper peptide are dissolved in an organic solvent, and the solution is added dropwise into normal saline at a constant speed and stirred, and finally the organic solvent is removed by dialyzation to obtain nanoparticles.

[0015] Application of nanoparticles in the localization diagnosis and stabilization treatment of atherosclerotic plaques.

[0016] The beneficial effects of the present invention are as follows:

[0017] (1) The present invention prepares diagnostic and therapeutic molecules based on cholinesterase and blue copper peptide through simple esterification reaction and Schiff base reaction. The reaction conditions are mild and easy to prepare in large quantities.

[0018] (2) The diagnostic and therapeutic nanoparticles prepared by the present invention can be assembled from a single component, namely the diagnostic and therapeutic molecule. The therapeutic component, the blue copper peptide, is directly connected to the nanoparticles in the form of a chemical bond, which can effectively avoid problems such as drug leakage when entering the body.

[0019] (3) The diagnostic and therapeutic nanoparticles prepared by the present invention can be passively enriched in plaques through the pathologically damaged endothelial structure at the site of atherosclerosis, and the X-ray imaging properties of the iodine imaging agent can be used to locate and diagnose the plaques.

[0020] (4) The nanoparticles prepared by the present invention are acid-sensitive and can disintegrate in the slightly acidic environment of the plaque, quickly and efficiently releasing the blue copper peptide for treatment, thus solving the problem that the nanocarrier cannot release the drug in time when it reaches the lesion.

[0021] (5) The nanoparticles prepared by the present invention can regulate smooth muscle cells and foam cells at the plaque site through blue copper peptide, promote collagen production, and inhibit the secretion of matrix metalloproteinases, thereby innovatively achieving plaque stabilization by remodeling the extracellular matrix at the plaque site and reducing the occurrence of adverse prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the hydrogen nuclear magnetic resonance spectrum of the diagnostic molecule.

[0023] Figure 2 This is the particle size distribution diagram of diagnostic nanoparticles.

[0024] Figure 3 The graph shows the change in particle size of nanoparticles under acidic conditions.

[0025] Figure 4 This figure shows the results of nanoparticles promoting collagen synthesis in cells in vitro.

[0026] Figure 5 This figure shows the results of the nanoparticles' inhibition of extracellular matrix degradation in vitro.

[0027] Figure 6This is a graph showing the results of nanoparticle localization to plaques in vivo.

[0028] Figure 7 Figure 3. Results of in vivo plaque stabilization therapy with nanoparticles. DETAILED DESCRIPTION

[0029] The present invention will be described in detail below based on the accompanying drawings and preferred embodiments, and the purpose and effects of the present invention will become more clear. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] Example 1

[0031] Dissolve choline acid (1.14 g), 4-hydroxymethylbenzaldehyde (0.27 g), dicyclohexylcarbodiimide (0.62 g) and 4-dimethylaminopyridine (24 mg) in dimethyl sulfoxide, stir at 25°C for 48 h, dialyze the resulting solution in deionized water, and then freeze-dry to obtain aldehyde-modified choline acid. Dissolve the obtained aldehyde-modified choline acid (1.37 g, 1 mmol) and blue copper peptide (0.84 g, 2 mmol) in dimethyl sulfoxide, stir at 25°C for 48 h, dialyze the resulting solution in deionized water, and then freeze-dry to obtain a diagnostic molecule. The hydrogen nuclear magnetic resonance spectrum of this molecule is shown in Figure 1 .

[0032] Example 2

[0033] Dissolve choline acid (2.28 g), 4-hydroxymethylbenzaldehyde (0.54 g), dicyclohexylcarbodiimide (1.24 g) and 4-dimethylaminopyridine (48 mg) in dimethyl sulfoxide, stir at 25°C for 48 h, dialyze the resulting solution in deionized water, and then freeze-dry to obtain aldehyde-modified choline acid. Dissolve the obtained aldehyde-modified choline acid (1.37 g, 1 mmol) and blue copper peptide (0.084 g, 0.2 mmol) in dimethyl sulfoxide, stir at 25°C for 48 h, dialyze the resulting solution in deionized water, and then freeze-dry to obtain the diagnostic and therapeutic molecule.

[0034] Example 3

[0035] Dissolve choline acid (0.38 g), 4-hydroxymethylbenzaldehyde (0.09 g), dicyclohexylcarbodiimide (0.207 g) and 4-dimethylaminopyridine (8 mg) in dimethyl sulfoxide, stir at 25°C for 48 h, dialyze the resulting solution in deionized water, and then freeze-dry to obtain aldehyde-modified choline acid. Dissolve the obtained aldehyde-modified choline acid (0.137 g, 0.1 mmol) and blue copper peptide (1.26 g, 3 mmol) in dimethyl sulfoxide, stir at 25°C for 48 h, dialyze the resulting solution in deionized water, and then freeze-dry to obtain the diagnostic and therapeutic molecule.

[0036] Example 4

[0037] The diagnostic and therapeutic molecule prepared in Example 1 was dissolved in 1 ml of dimethyl sulfoxide, and the solution was added dropwise into 3 ml of normal saline with stirring, and finally the organic solvent was removed by dialysis to obtain nanoparticles. The particle size (d.nm) of the nanoparticles was 122.4±8.6, and the particle size distribution of the nanoparticles was shown in FIG. Figure 2 .from Figure 2 It can be seen that the particle size distribution of the obtained nanoparticles is relatively concentrated, indicating that their particle size is relatively uniform and can have good monodispersity in the body, making their circulation period and enrichment characteristics relatively uniform, stable and controllable.

[0038] Experimental Example 1 Study on the Acid Responsiveness of Nanoparticles

[0039] The nanoparticles prepared in Example 4 were placed at 37°C and the particle size changes of the nanoparticles in solutions with different pH values ​​were measured using a dynamic light scattering instrument. The results are as follows: Figure 3 .Depend on Figure 3 It can be seen that the nanoparticles prepared in this example show good stability under physiological pH conditions (7.4), and can be rapidly disintegrated under acidic conditions (5.5), which is manifested by an increase in particle size. Further, by simulating the slightly acidic environment (pH 6.8) at the plaque, it was found that the nanoparticles can also disintegrate, increase in particle size, and have a considerable response rate, which can achieve rapid disintegration after enrichment in the plaque, and play a diagnostic and therapeutic role.

[0040] Experimental Example 2 Ability of Nanoparticles to Promote Collagen Synthesis in In Vitro Cells

[0041] The nanoparticles prepared in Example 4 were prepared into a solution with a concentration of 1 mg / ml. The solution was co-cultured with smooth muscle activated by lipopolysaccharide for 48 hours, and the level of collagen production was determined using an ELISA kit.

[0042] Depend on Figure 4 It can be seen that compared with the blank control, the smooth muscle activated by lipopolysaccharide (positive control) has a pathological reaction and its collagen synthesis is significantly reduced. Compared with the positive control, the nanoparticles prepared in the present invention can promote the synthesis rate of collagen in smooth muscle cells.

[0043] Experimental Example 3 Inhibitory effect of nanoparticles on extracellular matrix degradation in vitro

[0044] The nanoparticles prepared in Example 4 were prepared into a solution with a concentration of 1 mg / ml, and a Transwell invasion model was constructed. The solution and macrophages activated by lipopolysaccharide were added to the invasion chamber together. After culturing for 24 hours, the cells that invaded the chamber were stained with crystal violet to determine the level of matrix gel degradation by the cells.

[0045] Depend on Figure 5It can be seen that compared with the blank control group, macrophages activated by lipopolysaccharide (positive control) secreted a large amount of matrix metalloproteinases and invaded through the chamber, which was manifested by an increase in crystal violet-stained cells. Compared with the positive control group, macrophages treated with nanoparticles invaded less, suggesting that nanoparticles can effectively reduce the secretion of matrix metalloproteinases by activated macrophages.

[0046] Experimental Example 4 In vivo diagnostic ability of nanoparticles for atherosclerosis

[0047] A mouse atherosclerosis model was constructed, and 9 weeks after the model was constructed, the mice were treated with a 1 mg / ml nanoparticle solution via tail vein injection. The mouse aorta was isolated after different time periods of administration, and the distribution of nanoparticle signals in the aorta was observed by X-ray, such as Figure 6 shown.

[0048] Depend on Figure 6 It can be seen that nanoparticles can be effectively enriched at the plaques in the aorta and can be tracked, located and diagnosed through X-ray imaging.

[0049] Experimental Example 5: In vivo stabilization therapeutic ability of nanoparticles on atherosclerosis

[0050] A mouse atherosclerosis model was established, and 9 weeks after the model was established, the mice were treated with a 1 mg / ml nanoparticle solution by tail vein injection. 6 weeks after administration, the mouse heart was isolated, and the aortic root was sliced ​​and Masson stained. Figure 7 shown.

[0051] Depend on Figure 7 It can be seen that the atherosclerotic plaques at the aortic root of mice treated with nanoparticles have richer collagen components (blue) and denser tissue, indicating that the plaque stability is improved and the risk of poor prognosis is reduced.

[0052] Those skilled in the art can understand that the above are only preferred examples of the invention and are not intended to limit the invention. Although the invention is described in detail with reference to the above examples, those skilled in the art can still modify the technical solutions recorded in the above examples or replace some of the technical features therein with equivalents. Any modification, equivalent replacement, etc. made within the spirit and principle of the invention shall be included in the protection scope of the invention.

Claims

1. A diagnostic and therapeutic molecule for locating and stabilizing atherosclerotic plaques, characterized in that: The diagnostic and therapeutic molecule is synthesized based on cholinesterase and blue copper peptide, and the chemical structural formula of the diagnostic and therapeutic molecule is:

2. A method for preparing a diagnostic and therapeutic molecule for locating and stabilizing atherosclerotic plaques as claimed in claim 1, characterized in that: The method comprises the following steps: Step 1: dissolving cholinesterase, 4-hydroxymethylbenzaldehyde, dicyclohexylcarbodiimide and 4-dimethylaminopyridine in a molar ratio of 1:2:3:0.2 in an organic solvent, stirring to fully react, dialyzing the resulting solution in deionized water to remove the organic solvent; then freeze-drying, removing the deionized water, and obtaining aldehyde-modified cholinesterase; Step 2: dissolving the aldehyde-modified cholinesterase and blue copper peptide obtained in step 1 in the organic solvent at a molar ratio of (1-10): (2-30), stirring to fully react, dialyzing the resulting solution in deionized water to remove the organic solvent; then freeze-drying, removing the deionized water, and obtaining the diagnostic molecule.

3. The method for preparing a diagnostic and therapeutic molecule for locating and stabilizing atherosclerotic plaques according to claim 2, characterized in that: The organic solvent is dimethyl sulfoxide.

4. A nanoparticle made from the diagnostic and therapeutic molecule of claim 1 for locating and stabilizing atherosclerotic plaques, characterized in that: It is formed by self-assembly of diagnostic and therapeutic molecules based on cholinesterase and blue copper peptide.

5. The nanoparticles for locating and stabilizing atherosclerotic plaques made from the diagnostic and therapeutic molecules according to claim 4, characterized in that: The diagnostic and therapeutic molecules based on cholinesterase and blue copper peptide are dissolved in an organic solvent, and the solution is added dropwise into normal saline at a constant speed with stirring, and finally the organic solvent is removed by dialyzation to obtain nanoparticles.

6. Use of the nanoparticles as claimed in claim 4 in the localization diagnosis and stabilization treatment of atherosclerotic plaques.

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