Therapeutic molecules and nanoparticles for locating and stabilizing atherosclerotic plaques, and their preparation methods.

Abstract

This invention discloses a therapeutic molecule, nanoparticles, and their preparation method for locating and stabilizing atherosclerotic plaques. The therapeutic molecule is synthesized based on cholanonic acid and copper peptide, and the nanoparticles are formed by the self-assembly of the therapeutic molecule. The preparation method involves aldehyde modification of both ends of cholanonic acid and coupling it with copper peptide via a Schiff base reaction to obtain a therapeutic molecule with both diagnostic and therapeutic functions. The copper peptide structure in the therapeutic molecule serves as the hydrophilic segment, and the cholanonic acid structure serves as the hydrophilic segment, allowing it to further self-assemble into nanoparticles in water. These nanoparticles exhibit acid responsiveness and can disintegrate in the slightly acidic environment of atherosclerotic sites. The released copper peptide can stabilize plaques by promoting collagen secretion and inhibiting matrix metalloproteinase expression. Simultaneously, the X-ray imaging characteristics of cholanonic acid can be used to locate and diagnose the distribution of plaques, thus achieving integrated stabilization and localization of atherosclerotic plaques.

Description

Technical Field

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

[0002] Atherosclerosis is the most important cause of coronary artery-related cardiovascular disease. Its main characteristic is the formation and gradual enlargement of plaques on the inner wall of blood vessels, leading to narrowing of the lumen or rupture and subsequent vascular occlusion. In the classification of atherosclerosis, vulnerable plaques are typically characterized by large necrotic cores, thin fibrous caps, and abundant inflammatory cell infiltration, making them fragile. Clinically, rupture of vulnerable plaques is the most common cause of acute coronary syndrome, often resulting in very poor clinical outcomes. At the site of atherosclerotic plaques, in-situ smooth muscle cells secrete various extracellular matrix molecules, such as collagen, elastin, and proteoglycans, which become the main components of the plaque's fibrous cap. However, activated macrophages can simultaneously produce various matrix metalloproteinases, which can degrade interstitial collagen that maintains the strength of the fibrous cap, causing thinning of the fibrous cap, weakening its structural strength, or promoting the degradation of the extracellular matrix at the plaque site, increasing the risk and vulnerability of plaque rupture. Current clinical interventions mainly include lipid-lowering, antiplatelet, and anti-inflammatory therapies, but these methods are not particularly effective in stabilizing atherosclerotic plaques. Therefore, plaque identification and stabilization therapy for vulnerable plaques have become crucial tasks in the diagnosis and treatment of atherosclerosis.

[0003] The rapid development of nanotechnology has provided a new approach for the diagnosis and treatment of diseases in clinical practice. Nanocarriers can load or encapsulate various substances with diagnostic or therapeutic activities. On the one hand, nanocarriers can reach the lesion area under stable loading conditions, thereby protecting the internal environment from the effects 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 reagents for the diagnosis of atherosclerotic diseases in clinical practice. They rely on high-density signals under X-CT or X-ray fluoroscopy to display the morphology of blood vessel cavities, thereby identifying plaques. However, the amount of iodine imaging agents used in traditional techniques such as CTA and angiography is still relatively large, posing a significant burden on renal function and the potential risk of contrast-induced nephropathy. In the field of nanodiagnosis of atherosclerosis, various nanotechnologies and targeted structural designs enable iodine imaging agents to specifically accumulate at plaque sites, allowing direct visualization of plaques via X-ray imaging. This achieves good imaging results while significantly reducing the amount of iodine imaging agent used. While existing nanomedicines for treating atherosclerosis can improve drug utilization and reduce toxicity to some extent, they are still often limited by problems such as uncontrollable drug leakage and the inability to release drugs quickly at the lesion site, which hinders their widespread application.

[0004] Therefore, developing innovative diagnostic and therapeutic nanoparticles for the localization and stabilization of atherosclerotic plaques is of great clinical significance. Summary of the Invention

[0005] To address the aforementioned shortcomings in existing technologies, this invention provides a therapeutic molecule, nanoparticles, and a method for preparing the same for locating and stabilizing atherosclerotic plaques. The nanoparticles composed of this therapeutic molecule are acid-responsive and can disintegrate in the slightly acidic environment of atherosclerotic sites. The released copper peptides can stabilize atherosclerotic plaques by promoting collagen secretion and inhibiting matrix metalloproteinase expression. Simultaneously, by utilizing the X-ray imaging characteristics of choledocholic acid, the distribution of plaques can be located and diagnosed, thereby achieving integrated diagnosis and treatment of atherosclerosis.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A therapeutic molecule for locating and stabilizing atherosclerotic plaques, said therapeutic molecule being synthesized based on cholanonic acid and copper peptide, the chemical structural formula of said therapeutic molecule being:

[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: Cholesaccharide, 4-hydroxymethylbenzaldehyde, dicyclohexylcarbodiimide and 4-dimethylaminopyridine in a molar ratio of 1:2:3:0.2 were dissolved in an organic solvent and stirred to allow the reaction to proceed fully. The resulting solution was dialyzed in deionized water to remove the organic solvent. Then, the solution was freeze-dried to remove the deionized water, yielding aldehyde-modified cholesaccharide.

[0011] Step 2: Dissolve the aldehyde-modified choline and copper peptide obtained in Step 1 in the organic solvent at a molar ratio of (1-10):(2-30), stir to allow them to react fully, dialyze the resulting solution in deionized water to remove the organic solvent, and then freeze-dry to remove the deionized water to obtain the therapeutic molecule.

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

[0013] Nanoparticles made from therapeutic molecules for locating and stabilizing atherosclerotic plaques are formed by self-assembly of therapeutic molecules based on choledocholic acid and copper peptide.

[0014] Furthermore, the therapeutic molecules based on choleretic acid and copper peptide were dissolved in an organic solvent, and the solution was added dropwise to physiological saline at a constant rate while stirring. Finally, the organic solvent was removed by dialysis to obtain nanoparticles.

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

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

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

[0018] (2) The therapeutic nanoparticles prepared by the present invention can be assembled from a single component of therapeutic molecules. The therapeutic component, blue copper peptide, is directly linked to the nanoparticles in the form of chemical bonds, which can effectively avoid problems such as drug leakage when it enters the body.

[0019] (3) The diagnostic and therapeutic nanoparticles prepared in this invention can be passively enriched in the plaque through the pathologically damaged endothelial structure at the site of atherosclerosis, and the plaque can be located and diagnosed by utilizing the X-ray imaging characteristics of iodine imaging agent.

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

[0021] (5) The nanoparticles prepared in this invention can promote collagen production and inhibit matrix metalloproteinase secretion by regulating smooth muscle cells and foam cells at the plaque site through blue copper peptides. In this way, the plaque can be stabilized by remodeling the extracellular matrix at the plaque site, thereby reducing the occurrence of adverse prognosis. Attached Figure Description

[0022] Figure 1 The proton NMR spectrum of the diagnostic molecules.

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

[0024] Figure 3 This is a graph showing the particle size variation of nanoparticles under acidic conditions.

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

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

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

[0028] Figure 7 Figure showing the results of in vivo plaque stabilization therapy using nanoparticles. Detailed Implementation

[0029] The present invention will be described in detail below with reference to the accompanying drawings and preferred embodiments. The purpose and effects of the present invention will become clearer. It should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention.

[0030] Example 1

[0031] Cholescic acid (1.14 g), 4-hydroxymethylbenzaldehyde (0.27 g), dicyclohexylcarbodiimide (0.62 g), and 4-dimethylaminopyridine (24 mg) were dissolved in dimethyl sulfoxide and stirred at 25 °C for 48 h. The resulting solution was dialyzed against deionized water and then freeze-dried to obtain aldehyde-modified cholanscic acid. The obtained aldehyde-modified cholanscic acid (1.37 g, 1 mmol) and copper peptide (0.84 g, 2 mmol) were dissolved in dimethyl sulfoxide and stirred at 25 °C for 48 h. The resulting solution was dialyzed against deionized water and then freeze-dried to obtain the therapeutic molecule. The proton NMR spectrum of this molecule is shown below. Figure 1 .

[0032] Example 2

[0033] Cholescic acid (2.28 g), 4-hydroxymethylbenzaldehyde (0.54 g), dicyclohexylcarbodiimide (1.24 g), and 4-dimethylaminopyridine (48 mg) were dissolved in dimethyl sulfoxide and stirred at 25 °C for 48 h. The resulting solution was dialyzed against deionized water and then freeze-dried to obtain aldehyde-modified cholanscic acid. The obtained aldehyde-modified cholanscic acid (1.37 g, 1 mmol) and copper peptide (0.084 g, 0.2 mmol) were dissolved in dimethyl sulfoxide and stirred at 25 °C for 48 h. The resulting solution was dialyzed against deionized water and then freeze-dried to obtain the therapeutic molecule.

[0034] Example 3

[0035] Cholescic acid (0.38 g), 4-hydroxymethylbenzaldehyde (0.09 g), dicyclohexylcarbodiimide (0.207 g), and 4-dimethylaminopyridine (8 mg) were dissolved in dimethyl sulfoxide and stirred at 25 °C for 48 h. The resulting solution was dialyzed against deionized water and then freeze-dried to obtain aldehyde-modified cholanscic acid. The obtained aldehyde-modified cholanscic acid (0.137 g, 0.1 mmol) and copper peptide (1.26 g, 3 mmol) were dissolved in dimethyl sulfoxide and stirred at 25 °C for 48 h. The resulting solution was dialyzed against deionized water and then freeze-dried to obtain the therapeutic molecule.

[0036] Example 4

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

[0038] Experimental Example 1: Acid-responsiveness study 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 embodiment exhibit good stability under physiological pH conditions (7.4), while they can rapidly disintegrate under acidic conditions (5.5), resulting in an increase in particle size. Furthermore, by simulating a slightly acidic environment (pH 6.8) at the plaque site, it was found that the nanoparticles can also disintegrate, with an increase in particle size, and their response rate is considerable, enabling them to rapidly disintegrate after accumulation in plaques and play a diagnostic and therapeutic role.

[0040] Example 2: The 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 lipopolysaccharide-activated smooth muscle for 48 hours, and the collagen production level was measured using an ELISA kit.

[0042] Depend on Figure 4 It can be seen that, compared with the blank control, lipopolysaccharide-activated smooth muscle (positive control) exhibited a pathological reaction, with a significant reduction in collagen synthesis. However, compared with the positive control, the nanoparticles prepared in this invention can promote the rate of collagen synthesis in smooth muscle cells.

[0043] Example 3: The 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. At the same time, a Transwell invasion model was constructed. The solution and macrophages activated by lipopolysaccharide were added to the invasion chamber. After culturing for 24 hours, the cells that invaded the chamber were stained with crystal violet to determine the degradation level of the cells on the matrix gel.

[0045] Depend on Figure 5It was found that, compared with the blank control group, macrophages activated by lipopolysaccharide (positive control) secreted a large amount of matrix metalloproteinases and invaded through the chambers, as evidenced by an increase in crystal violet-stained cells. In contrast, macrophages treated with nanoparticles showed less invasion, suggesting that nanoparticles can effectively reduce the secretion of matrix metalloproteinases by activated macrophages.

[0046] Example 4: In vivo diagnostic capability of nanoparticles for atherosclerosis

[0047] A mouse model of atherosclerosis was established, and nine weeks after model establishment, mice were treated with a 1 mg / ml nanoparticle solution via tail vein injection. The mouse aorta was dissected at different time points after administration, and the distribution of nanoparticle signals in the aorta was observed by X-ray. Figure 6 As shown.

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

[0049] Example 5: The in vivo stabilizing therapeutic effect of nanoparticles on atherosclerosis

[0050] A mouse model of atherosclerosis was established, and nine weeks after model establishment, mice were treated with a 1 mg / ml nanoparticle solution via tail vein injection. Six weeks after administration, the mouse hearts were dissected, and tissue sections of the aortic root were prepared and stained with Masson's stain. Figure 7 As shown.

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

[0052] It will be understood by those skilled in the art that the above descriptions are merely preferred examples of the invention and are not intended to limit the invention. Although the invention has been described in detail with reference to the foregoing examples, those skilled in the art can still modify the technical solutions described in the foregoing examples or make equivalent substitutions for some of the technical features. All modifications and equivalent substitutions made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A diagnostic molecule for locating and stabilizing atherosclerotic plaques, characterized in that, The therapeutic molecule is synthesized based on choleretic acid and copper peptide, and its chemical structural formula is as follows: 。 2. A method for preparing a therapeutic molecule for locating and stabilizing atherosclerotic plaques as described in claim 1, characterized in that, The method includes the following steps: Step 1: Cholesaccharide, 4-hydroxymethylbenzaldehyde, dicyclohexylcarbodiimide and 4-dimethylaminopyridine in a molar ratio of 1:2:3:0.2 were dissolved in an organic solvent and stirred to allow the reaction to proceed fully. The resulting solution was dialyzed in deionized water to remove the organic solvent. Then, the solution was freeze-dried to remove the deionized water, yielding aldehyde-modified cholesaccharide. Step 2: Dissolve the aldehyde-modified choline and copper peptide obtained in Step 1 in the organic solvent at a molar ratio of (1~10):(2~30), stir to allow them to react fully, dialyze the resulting solution in deionized water to remove the organic solvent, and then freeze-dry to remove the deionized water to obtain the therapeutic molecule.

3. The method for preparing the 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 therapeutic molecule of claim 1 for positioning and stabilizing atherosclerotic plaques, characterized in that, It is formed by the self-assembly of therapeutic molecules based on choleretic acid and copper peptide.

5. Nanoparticles made from the therapeutic molecules according to claim 4 for locating and stabilizing atherosclerotic plaques, characterized in that, Therapeutic molecules based on choleretic acid and copper peptides were dissolved in an organic solvent, and the solution was added dropwise to physiological saline at a constant rate while stirring. Finally, the organic solvent was removed by dialysis to obtain nanoparticles.

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