Heart homing peptide synergistic colchicine phospholipid compound as well as preparation method and application thereof

Abstract

The invention provides a heart homing peptide synergistic colchicine phospholipid compound as well as a preparation method and application thereof. The heart homing peptide and colchicine phospholipid complex nanoparticles prepared by the invention have a positive effect on heart tissue repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical preparation technology, specifically relating to a cardiac homing peptide synergistic colchicine phospholipid complex, its preparation method, and its application. Background Technology

[0002] Colchicine, a traditional anti-inflammatory drug, was initially used to treat gout and pericarditis. Colchicine binds to α-tubulin and TUBB dimers, thereby inhibiting microtubule polymerization. This prevents microtubules from assembling properly or maintaining their stability, thus interfering with cell structure and function. It accumulates in inflammatory cells with restricted P-glycoprotein expression, such as neutrophils. Its potential in reducing cardiovascular risk has been demonstrated in several clinical trials. However, colchicine's narrow therapeutic window and significant adverse reactions limit its widespread application. Currently, improving the therapeutic efficacy and reducing adverse reactions of colchicine through novel drug delivery systems is a key research focus. Cardiac homing peptide (CSTSMLKAC) is a peptide molecule screened using phage display technology. It specifically binds to areas of cardiac injury. Chemical modification and structural optimization of the cardiac homing peptide sequence can significantly enhance its targeting and binding ability to areas of cardiac injury. In animal models such as mice and pigs, cardiac homing peptides have been shown to effectively target areas of cardiac injury, and their targeting effect has been verified by fluorescent labeling and radioactive tracing techniques.

[0003] Currently, there is considerable research on colchicine and cardiac homing peptides individually, but fundamental research on their combination, such as drug interaction mechanisms and optimal dosage ratios for combined use, is relatively lacking, resulting in a lack of theoretical support for their combined application. Due to the different chemical structures and physical properties of colchicine and cardiac homing peptides, combining them to formulate suitable drug dosage forms may face technical challenges, including ensuring their stability in formulations and achieving synergistic release. Summary of the Invention

[0004] The purpose of this invention is to provide a cardiac homing peptide synergistic colchicine phospholipid complex, its preparation method and application, wherein the cardiac homing peptide synergistic colchicine phospholipid complex has low biotoxicity and a positive effect on cardiac tissue repair.

[0005] The technical solution adopted in this invention is as follows:

[0006] A method for preparing a cardiac homing peptide-collixine phospholipid complex includes the following steps:

[0007] (1) Colchicine, cholesterol and lecithin are dissolved in a first organic solvent to obtain a first mixed solution. After water bath reaction, the solution is purified and dried to obtain colchicine phospholipid complex.

[0008] The mass ratio of colchicine, cholesterol and lecithin is 0.8-1:1:7-10, the water bath reaction temperature is 20-75℃, and the water bath reaction time is 4-8h.

[0009] (2) Dissolve the colchicine phospholipid complex and the cardiac homing peptide in water, and then react at room temperature for 4-10 h to obtain a second mixed solution of cardiac homing peptide and colchicine phospholipid. After drying, the cardiac homing peptide and colchicine phospholipid complex is obtained.

[0010] The mass ratio of colchicine phospholipid complex to cardiac homing peptide is 18-22:1.

[0011] In some preferred embodiments, the mass-to-volume ratio of colchicine to the first organic solvent is 0.8-1 mg:1 mL.

[0012] In some preferred embodiments, the concentration of cardiac homing peptide in the second mixed solution is 0.45-0.55 mg / mL.

[0013] In some preferred embodiments, the lecithin is egg yolk lecithin and / or soybean lecithin.

[0014] In some preferred embodiments, the first organic solvent is at least one selected from methanol, ethanol, chloroform, diethyl ether, acetone, and tetrahydrofuran.

[0015] In some preferred embodiments, the purification process involves: removing a first organic solvent, then adding a second organic solvent and removing substances insoluble in the second organic solvent, and finally removing the second organic solvent, which is n-hexane and / or petroleum ether.

[0016] In some preferred embodiments, the mass-to-volume ratio of lecithin and the second organic solvent is 0.1-10 mg: 1 mL.

[0017] A cardiac homing peptide-collocytic phospholipid complex was prepared by the above-described method.

[0018] A cardiac homing peptide synergistic colchicine phospholipid complex nanoparticle, comprising the aforementioned cardiac homing peptide synergistic colchicine phospholipid complex, wherein the average particle size of the cardiac homing peptide synergistic colchicine phospholipid complex nanoparticle is 0.15-0.3 μm.

[0019] A method for preparing the above-mentioned cardiac homing peptide synergistic colchicine phospholipid complex nanoparticles includes the following steps: mixing the cardiac homing peptide synergistic colchicine phospholipid complex with deionized water to obtain a second mixed solution, shaking the solution in a water bath at 30-60°C until the cardiac homing peptide synergistic colchicine phospholipid complex dissolves, then ultrasonically dispersing the solution, and finally filtering the solution with a microporous membrane to obtain the cardiac homing peptide synergistic colchicine phospholipid complex nanoparticles, wherein the pore size of the microporous membrane is 0.15-0.3 μm.

[0020] In some preferred embodiments, the concentration of the cardiac homing peptide-collixine phospholipid complex in the second mixed solution is 0.1-100 mg / mL.

[0021] Application of the above-mentioned cardiac homing peptide synergistic colchicine phospholipid complex in cardiac tissue repair.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The cardiac homing peptide synergistic colchicine phospholipid complex nanoparticles prepared in this invention have a positive effect on cardiac tissue repair.

[0024] 2. The cardiac homing peptide synergistic colchicine phospholipid complex nanoparticles prepared in this invention use lecithin as a carrier, are spherical in shape, and have a particle size distribution between 50-300 nm. They exhibit good stability and excellent biocompatibility. The cardiac homing peptide synergistic colchicine phospholipid complex nanoparticles can deliver drug molecules according to specific targeting needs, thereby regulating the distribution of drugs in the body, improving the precision and targeting of treatment, and possessing advantages such as low toxicity and long-lasting effect not found in naked drugs. At the same time, they can avoid the loss of drug activity, which is beneficial for drug storage and transportation.

[0025] 3. The cardiac homing peptide synergistic colchicine phospholipid complex nanoparticles prepared in this invention serve as drug carriers for in vivo delivery. These nanoparticles can deliver drug molecules according to specific targeting needs, thereby regulating drug distribution within the body and improving the precision and targeting of treatment. Furthermore, while small molecule drugs primarily cross cell membranes via simple diffusion, nanoparticles enter cells through endocytosis, thereby improving drug utilization while reducing drug dosage and adverse reactions.

[0026] 4. The cardiac homing peptide synergistic colchicine phospholipid complex nanoparticles prepared by this invention are easily soluble in physiological saline or glucose sodium chloride injection, and are suitable for intravenous injection or intravenous bolus injection. They have the advantages of long circulation, low toxicity and good therapeutic effect. Attached Figure Description

[0027] Figure 1 This is a transmission electron microscope image of the colchicine phospholipid complex nanoparticles of Example 1 of the present invention.

[0028] Figure 2 This is a transmission electron microscope image of the cardiac homing peptide synergistic colchicine phospholipid complex nanoparticles of Example 1 of the present invention.

[0029] Figure 3 This is a transmission electron microscope image of the colchicine phospholipid complex nanoparticles of Example 2 of the present invention;

[0030] Figure 4 This is a transmission electron microscope image of the cardiac homing peptide synergistic colchicine phospholipid complex nanoparticles of Example 2 of the present invention.

[0031] Figure 5 The infrared spectra of colchicine (COL), cholesterol (CHL), soybean lecithin (SPC), the mixture of the first three (MIX), and colchicine phospholipid complex (CP) in Example 2 of the present invention are shown.

[0032] Figure 6 The 1H NMR spectra of COL, CHL, SPC, MIX, and Complex in Example 2 of this invention;

[0033] Figure 7 This is a transmission electron microscope image of the colchicine phospholipid complex nanoparticles of Example 3 of the present invention.

[0034] Figure 8 This is a transmission electron microscope image of the cardiac homing peptide synergistic colchicine phospholipid complex nanoparticles of Example 3 of the present invention.

[0035] Figure 9 This is a transmission electron microscope image of the cardiac homing peptide synergistic colchicine phospholipid complex nanoparticles of Example 4 of the present invention.

[0036] Figure 10 This is a transmission electron microscope image of the cardiac homing peptide synergistic colchicine phospholipid complex nanoparticles of Example 5 of the present invention.

[0037] Figure 11 This is a transmission electron microscope image of the cardiac homing peptide synergistic colchicine phospholipid complex nanoparticles of Example 6 of the present invention.

[0038] Figure 12 The average particle size diagram of the colchicine phospholipid complex (CP) of Example X of the present invention is shown.

[0039] Figure 13 The average particle size diagram of the cardiac homing peptide synergistic colchicine phospholipid complex nanoparticles (CCP) of Embodiment X of the present invention is shown.

[0040] Figure 14The Zeta potential diagram shows the colchicine phospholipid complex (CP) and cardiac homing peptide synergistic colchicine phospholipid complex nanoparticles (CCP) of Embodiment X of the present invention.

[0041] Figure 15 The echocardiograms, left atrial diameter (LAD), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular ejection fraction (LVEF), and left ventricular fraction of shortening (LVFS) of rats in each group after 28 days of intervention were measured.

[0042] Figure 16 H&E staining images of organ sections from rats in each group 14 days after intervention;

[0043] Figure 17 The images show the staining results of the heart tissue of rats in each group 14 days after intervention. A represents the MASSON staining result, and B represents the Sirius red staining result. Detailed Implementation

[0044] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in more detail below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0045] Example 1

[0046] A cardiac homing peptide-collixine phospholipid complex nanoparticle, the preparation method of which includes the following steps:

[0047] (1) Colchicine, cholesterol, and soybean lecithin were dissolved in methanol, a first organic solvent, to obtain a first mixed solution. After a water bath reaction, the first organic solvent was removed by vacuum rotary evaporation. Then, hexane, a second organic solvent, was added and insoluble matter was removed. Finally, the second organic solvent was removed by vacuum rotary evaporation to obtain the colchicine-phospholipid complex. Its transmission electron microscopy image is shown below. Figure 1 As shown.

[0048] In the first mixed solution, the concentration of colchicine was 1 mg / mL, the concentration of soybean lecithin was 10 mg / mL, the water bath reaction temperature was 50℃, the water bath reaction time was 4 h, and the mass-volume ratio of soybean lecithin to the second organic solvent was 2 mg: 1 mL.

[0049] (2) The colchicine phospholipid complex and the cardiac homing peptide were dissolved in deionized water and reacted at room temperature for 4 hours to obtain a second mixed solution of cardiac homing peptide and colchicine phospholipid. After drying, the cardiac homing peptide and colchicine phospholipid complex was obtained.

[0050] The mass ratio of colchicine phospholipid complex to cardiac homing peptide is 20:1, and the concentration of cardiac homing peptide in deionized water is 0.5 mg / mL.

[0051] (3) The cardiac homing peptide-collocytic phospholipid complex was mixed with deionized water to obtain a second mixture solution, wherein the concentration of the cardiac homing peptide-collocytic phospholipid complex was 1 mg / mL. The second mixture solution was placed in a shaking water bath at 50°C until completely dissolved, and then ultrasonically dispersed. The mixture was passed through a microporous membrane with a pore size of 0.22 μm to obtain cardiac homing peptide-collocytic phospholipid complex nanoparticles with an average particle size of 213.3 nm and a colchicine loading rate of 8.3%. Its transmission electron microscopy image is shown below. Figure 2 As shown.

[0052] Example 2

[0053] A cardiac homing peptide-collixine phospholipid complex nanoparticle, the preparation method of which includes the following steps:

[0054] (1) Colchicine, cholesterol, and lecithin were dissolved in methanol, a first organic solvent, to obtain a first mixed solution. After a water bath reaction, the first organic solvent was removed by vacuum rotary evaporation. Then, hexane, a second organic solvent, was added and insoluble matter was removed. Finally, the second organic solvent was removed by vacuum rotary evaporation to obtain the colchicine-phospholipid complex (CP). Its transmission electron microscopy image is shown below. Figure 3 As shown.

[0055] In the first mixed solution, the concentration of colchicine was 0.8 mg / mL, the concentration of soybean lecithin was 9 mg / mL, the water bath reaction temperature was 55℃, the water bath reaction time was 4.5 h, and the mass-volume ratio of soybean lecithin to the second organic solvent was 2 mg: 1 mL.

[0056] (2) The colchicine phospholipid complex and the cardiac homing peptide were dissolved in deionized water and reacted at room temperature for 5 hours to obtain a second mixed solution of cardiac homing peptide and colchicine phospholipid. After drying, the cardiac homing peptide and colchicine phospholipid complex was obtained.

[0057] The mass ratio of colchicine phospholipid complex to cardiac homing peptide was 19:1, and the concentration of cardiac homing peptide in deionized water was 0.475 mg / mL.

[0058] (3) The cardiac homing peptide-collocytic phospholipid complex was mixed with deionized water to obtain a second mixture solution, wherein the concentration of the cardiac homing peptide-collocytic phospholipid complex was 1 mg / mL. The second mixture solution was placed in a shaking water bath at 50°C until completely dissolved, and then ultrasonically dispersed. The mixture was filtered through a microporous membrane with a pore size of 0.22 μm to obtain cardiac homing peptide-collocytic phospholipid complex nanoparticles (CCP), with an average particle size of 200.3 nm and a colchicine loading rate of 8.9%. Its transmission electron microscopy image is shown below. Figure 4 As shown.

[0059] Figure 5 Infrared spectra of colchicine (COL), cholesterol (CHL), soybean lecithin (SPC), the mixture of the first three (MIX), and colchicine phospholipid complex (CP) used in this embodiment;

[0060] Figure 6 The X-ray diffraction patterns of colchicine (COL), cholesterol (CHL), soybean lecithin (SPC), the mixture of the first three (MIX), and the colchicine phospholipid complex (CP) in this embodiment are shown.

[0061] Example 3

[0062] A cardiac homing peptide-collixine phospholipid complex nanoparticle, the preparation method of which includes the following steps:

[0063] (1) Colchicine, cholesterol, and lecithin were dissolved in methanol, a first organic solvent, to obtain a first mixed solution. After a water bath reaction, the first organic solvent was removed by vacuum rotary evaporation. Then, hexane, a second organic solvent, was added and insoluble matter was removed. Finally, the second organic solvent was removed by vacuum rotary evaporation to obtain the colchicine-phospholipid complex. Its transmission electron microscopy image is shown below. Figure 7 As shown.

[0064] In the first mixed solution, the concentration of colchicine was 0.7 mg / mL, the concentration of soybean lecithin was 10 mg / mL, the water bath reaction temperature was 60℃, the water bath reaction time was 5 h, and the mass-volume ratio of soybean lecithin to the second organic solvent was 2 mg: 1 mL.

[0065] (2) The colchicine phospholipid complex and the cardiac homing peptide were dissolved in deionized water and then reacted at room temperature for 6 hours to obtain a second mixed solution of cardiac homing peptide and colchicine phospholipid. After drying, the cardiac homing peptide and colchicine phospholipid complex was obtained.

[0066] The mass ratio of colchicine phospholipid complex to cardiac homing peptide was 21:1, and the concentration of cardiac homing peptide in deionized water was 0.525 mg / mL.

[0067] (3) The cardiac homing peptide-collocytic phospholipid complex was mixed with deionized water to obtain a second mixture solution, wherein the concentration of the cardiac homing peptide-collocytic phospholipid complex was 1 mg / mL. The second mixture solution was placed in a shaking water bath at 50°C until completely dissolved, and then ultrasonically dispersed. The mixture was passed through a microporous membrane with a pore size of 0.22 μm to obtain cardiac homing peptide-collocytic phospholipid complex nanoparticles with an average particle size of 232 nm and a colchicine loading rate of 7.8%. Its transmission electron microscopy image is shown below. Figure 8 As shown.

[0068] Example 4

[0069] A cardiac homing peptide-collixine phospholipid complex nanoparticle, the preparation method of which includes the following steps:

[0070] (1) Colchicine, cholesterol and lecithin were dissolved in methanol, a first organic solvent, to obtain a first mixed solution. After water bath reaction, the first organic solvent was removed by vacuum rotary evaporation. Then, hexane, a second organic solvent, was added and insoluble matter was removed. Finally, the second organic solvent was removed by vacuum rotary evaporation to obtain colchicine phospholipid complex.

[0071] In the first mixed solution, the concentration of colchicine was 0.75 mg / mL, the concentration of soybean lecithin was 9 mg / mL, the water bath reaction temperature was 65℃, the water bath reaction time was 5.5 h, and the mass-volume ratio of soybean lecithin to the second organic solvent was 2 mg: 1 mL.

[0072] (2) The colchicine phospholipid complex and the cardiac homing peptide were dissolved in deionized water and then reacted at room temperature for 6 hours to obtain a second mixed solution of cardiac homing peptide and colchicine phospholipid. After drying, the cardiac homing peptide and colchicine phospholipid complex was obtained.

[0073] The mass ratio of colchicine phospholipid complex to cardiac homing peptide is 20:1, and the concentration of cardiac homing peptide in deionized water is 0.5 mg / mL.

[0074] (3) The cardiac homing peptide-collocytic phospholipid complex was mixed with deionized water to obtain a second mixture solution, wherein the concentration of the cardiac homing peptide-collocytic phospholipid complex was 1 mg / mL. The second mixture solution was placed in a shaking water bath at 50°C until completely dissolved, and then ultrasonically dispersed. The mixture was passed through a microporous membrane with a pore size of 0.22 μm to obtain cardiac homing peptide-collocytic phospholipid complex nanoparticles with an average particle size of 217.3 nm and a colchicine loading rate of 8.3%. Its transmission electron microscopy image is shown below. Figure 9 As shown.

[0075] Example 5

[0076] A cardiac homing peptide-collixine phospholipid complex nanoparticle, the preparation method of which includes the following steps:

[0077] (1) Colchicine, cholesterol and lecithin were dissolved in methanol, a first organic solvent, to obtain a first mixed solution. After water bath reaction, the first organic solvent was removed by vacuum rotary evaporation. Then, hexane, a second organic solvent, was added and insoluble matter was removed. Finally, the second organic solvent was removed by vacuum rotary evaporation to obtain colchicine phospholipid complex.

[0078] In the first mixed solution, the concentration of colchicine was 0.65 mg / mL, the concentration of soybean lecithin was 9 mg / mL, the water bath reaction temperature was 70℃, the water bath reaction time was 6 h, and the mass-volume ratio of soybean lecithin to the second organic solvent was 2 mg: 1 mL.

[0079] (2) The colchicine phospholipid complex and the cardiac homing peptide were dissolved in deionized water and then reacted at room temperature for 6 hours to obtain a second mixed solution of cardiac homing peptide and colchicine phospholipid. After drying, the cardiac homing peptide and colchicine phospholipid complex was obtained.

[0080] The mass ratio of colchicine phospholipid complex to cardiac homing peptide was 18:1, and the concentration of cardiac homing peptide in deionized water was 0.45 mg / mL.

[0081] (3) The cardiac homing peptide-collocytic phospholipid complex was mixed with deionized water to obtain a second mixture solution, wherein the concentration of the cardiac homing peptide-collocytic phospholipid complex was 1 mg / mL. The second mixture solution was placed in a shaking water bath at 50°C until completely dissolved, and then ultrasonically dispersed. The mixture was passed through a microporous membrane with a pore size of 0.22 μm to obtain cardiac homing peptide-collocytic phospholipid complex nanoparticles with an average particle size of 229.3 nm and a colchicine loading rate of 7.7%. Its transmission electron microscopy image is shown below. Figure 10 As shown.

[0082] Example 6

[0083] A cardiac homing peptide-collixine phospholipid complex nanoparticle, the preparation method of which includes the following steps:

[0084] (1) Colchicine, cholesterol and lecithin were dissolved in methanol, a first organic solvent, to obtain a first mixed solution. After water bath reaction, the first organic solvent was removed by vacuum rotary evaporation. Then, hexane, a second organic solvent, was added and insoluble matter was removed. Finally, the second organic solvent was removed by vacuum rotary evaporation to obtain colchicine phospholipid complex.

[0085] In the first mixed solution, the concentration of colchicine was 0.85 mg / mL, the concentration of soybean lecithin was 9 mg / mL, the water bath reaction temperature was 75℃, the water bath reaction time was 6.5 h, and the mass-volume ratio of soybean lecithin to the second organic solvent was 2 mg: 1 mL.

[0086] (2) The colchicine phospholipid complex and the cardiac homing peptide were dissolved in deionized water and then reacted at room temperature for 6 hours to obtain a second mixed solution of cardiac homing peptide and colchicine phospholipid. After drying, the cardiac homing peptide and colchicine phospholipid complex was obtained.

[0087] The mass ratio of colchicine phospholipid complex to cardiac homing peptide was 22:1, and the concentration of cardiac homing peptide in deionized water was 0.55 mg / mL.

[0088] (3) The cardiac homing peptide-collocytic phospholipid complex was mixed with deionized water to obtain a second mixture solution, wherein the concentration of the cardiac homing peptide-collocytic phospholipid complex was 1 mg / mL. The second mixture solution was placed in a shaking water bath at 50°C until completely dissolved, and then ultrasonically dispersed. The mixture was passed through a microporous membrane with a pore size of 0.22 μm to obtain cardiac homing peptide-collocytic phospholipid complex nanoparticles with an average particle size of 231.2 nm and a colchicine loading rate of 6.3%. Its transmission electron microscopy image is shown below. Figure 11 As shown.

[0089] Example 7: Basic property testing of cardiac homing peptide synergistic colchicine phospholipid complex nanoparticles

[0090] The particle size of the colchicine phospholipid complex (CP) and cardiac homing peptide synergistic colchicine phospholipid complex nanoparticles (CCP) prepared in Example 3 was tested, such as... Figure 12 and Figure 13 As shown, the average particle size of CCP is 232±8.07 nm, and the polydispersity index (PDI) is 0.299±0.051; the average particle size of CP is 183.5±2.159 nm, and the PDI is 0.196±0.014. It can be seen that the particle size of CCP is significantly larger than that of CP, indicating that the cardiac homing peptide and colchicine phospholipid complex were successfully combined in CCP.

[0091] In addition, such as Figure 14 As shown, the Zeta potential of CCP is -14.95±0.25mV, and the Zeta potential of CP is -10.71±1.52mV, indicating that CCP has higher stability.

[0092] Example 8: Construction and In vivo validation of a myocardial infarction (MI) model in SD rats.

[0093] (1) Model building method

[0094] Ninety healthy male SD rats (weighing 200±10g) were purchased from the Animal Experiment Center of Xinjiang Medical University. The rats were randomly divided into six groups according to the experimental design: sham-operated group (Sham group), MI group, LP group, COL group, CP group, and CCP group. Each group received a tail vein injection of liposome nanoparticles at a dose of 200μg / (kg·time). All animals were housed under constant temperature and humidity conditions with stable diurnal rhythms, and were given standard feed on an ad libitum basis. After a 7-day acclimatization period, the experimental phase began, as detailed below:

[0095] ① Fast for 12 hours before surgery, but drink water freely.

[0096] ② Intramuscular injection of anesthetic (Symptomycin and Sodium) was administered. After the rats were fully anesthetized, they were fixed in a supine position on a constant-temperature operating table.

[0097] ③ Perform endotracheal intubation using a pen-shaped intravenous catheter. Place the scalpel handle near the trachea and observe the aerosol to confirm successful intubation. Connect the animal to a ventilator, set the tidal volume to 10 mL / kg, respiratory rate to 50 breaths / min, and inspiratory:expiratory ratio to 1:2.

[0098] ④ Remove chest hair, disinfect with iodine-soaked cotton balls, and cut the skin between the 3rd and 4th ribs on the left side.

[0099] ⑤ After bluntly separating the superficial and deep pectoral muscles, use hemostatic forceps to separate the muscles below the third rib. Then, lift the rib with your left hand and cut the rib with your right hand.

[0100] ⑥ Use hemostatic forceps to clamp the cut ribs and widen them, insert an eyelid opener, and at the same time peel off the pericardium;

[0101] ⑦ Pull the thymus gland to fully expose the heart;

[0102] ⑧ Except for the Sham group, all other groups used 6-0 medical atraumatic sutures, which were passed under the coronary artery and a latex tube of about 5 mm was inserted. The sutures were tightened to compress and close the coronary artery, inducing acute myocardial ischemia, which was manifested by the whitening of the myocardium. After the chest cavity was closed, electrocardiogram monitoring was performed on the day after the myocardial infarction.

[0103] ⑨ Close the chest cavity, expel the air from the chest cavity, and suture the muscles and skin layer by layer.

[0104] ⑩ The LP, COL, CP, and CCP groups were administered blank liposomes (cholesterol and lecithin), colchicine, colchicine-phospholipid complex, and cardiac homing peptide-collchicine-phospholipid complex via tail vein injection at a dose of 200 μg / (kg·time), respectively; the Sham and MI groups were injected with an equal volume of physiological saline. Before injection, the rat tail was disinfected with alcohol to expose the tail vein. The rat's tail was held and straightened with the left hand, and a 1 mL syringe was held in the right hand, inserted at an angle of less than 30°, locating the vein at the end of the tail for injection. Smooth and unobstructed injection indicated successful needle entry into the blood vessel.

[0105] ⑪ Twelve-lead electrocardiograms were performed on rats preoperatively (excluding individuals with abnormal electrocardiograms), 30 minutes postoperatively, 24 hours postoperatively, and before tissue collection. After anesthesia, rats were fixed to a rat board, and needle electrodes were inserted to the corresponding sites on the chest wall and limbs to record electrocardiogram waveforms. Dorsiflexion of the ST segment and the presence of at least five pathological Q waves in leads I, II, aVL, and V1-V6 were used as criteria for successful MI modeling.

[0106] (2) Echocardiography to detect the effect of CCP on cardiac function in MI rats

[0107] After successful MI modeling, the LP group, COL group, CP group and CCP group were given tail vein injection of the therapeutic drug at a dose of 200 μg / (kg·time) daily; the Sham group and MI group were injected with an equal volume of normal saline. The following tests were performed after 28 days of intervention.

[0108] All experiments used a GEVIVID7 transthoracic Doppler ultrasound imaging system and an i13-L scanning probe for echocardiographic examination. The examinations were performed by an experienced sonographer unaware of the experimental groupings. After anesthesia, the fur on the anterior chest region of the rats was removed. The rats were placed in a left lateral decubitus position on a small animal temperature-controlled heating pad. Cardiac structure and function were assessed, and the left atrial diameter (LAD), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS) were measured. The average of three cardiac cycles for each group was used as the final result.

[0109] The results are as follows Figure 15 As shown, the cardiac function test results after 28 days of intervention in each group showed that the LVEF in the Sham group was [(74.05±3.623)%], which was significantly higher than that in the MI group [(46.25±6.169)%] (P<0.001). After treatment with CCP, CP, and COL, the LVEFs were [(69.88±4.634)%], [(65.31±1.955)%], and [(65.02±2.21)%], respectively, all of which were statistically significant compared with the MI group (P<0.001). The LVFS in the MI group was [(17.8±3.928)%], which was significantly lower than that in the Sham group [(39.49±3.635)%], P<0.001. After CCP and CP intervention, LVFS was increased by [(33.56±2.353)%] and [(30.32±1.015)%] respectively compared with the MI group (P<0.001). In conclusion, CCP has a significant ameliorative effect on cardiac function in MI rats.

[0110] (3) Pathological tissue staining examination

[0111] Each group of SD rats was injected with liposome nanoparticles via the tail vein according to the aforementioned method and dosage. After 14 consecutive days of administration, the rats were sacrificed, and their organs (heart, liver, spleen, lung, and kidney) were collected for pathological staining and analysis of the staining results.

[0112] ① Slicing method

[0113] After the in vivo experiments were completed, the rat hearts were quickly removed and perfused through the aortic sinus ostium with heparinized sodium saline until the hearts turned white. They were then repeatedly rinsed with PBS solution to thoroughly remove any residual blood. The auricle tissue was preserved and placed in a 10 mL centrifuge tube containing 4% cell fixative. The centrifuge tube was placed on a shaker and fixed overnight at room temperature with uniform shaking for subsequent pathological staining analysis.

[0114] Preparation of paraffin tissue sections: After fixation, the tissue samples were removed and subjected to gradient dehydration (50%, 70%, 80%, 90%, 95%, 100%), clearing with a 1:1 mixture of xylene and alcohol, paraffin infiltration, embedding, sectioning, and mounting. Ten sections were cut consecutively from each paraffin block, with each section thickness controlled at 5 μm.

[0115] ②H&E dyeing

[0116] Paraffin sections were dried overnight in a 60°C oven, and H&E staining was performed the following day. The specific steps are as follows:

[0117] (I) Dewaxing and hydration of sections: The sections were immersed in xylene I (15 min) and xylene II (10 min) in sequence, and then in anhydrous ethanol, 95% ethanol, 90% ethanol, 80% ethanol and 70% ethanol in sequence (10 s each time), and finally rinsed with distilled water (1 min).

[0118] (II) Hematoxylin staining: The sections were immersed in hematoxylin staining solution for 5 minutes, and then rinsed with tap water for 2 minutes;

[0119] (III) Differentiation and Blue Reversal: After rinsing with tap water for 2 minutes, immerse in 1% hydrochloric acid alcohol for 10 seconds for differentiation treatment, and then rinse with tap water for 2 minutes again for blue reversal treatment.

[0120] (IV) Eosin staining: The sections were immersed in eosin staining solution for 1 min, and then rinsed with tap water for 30 s;

[0121] (V) Dehydration, clearing and mounting: The sections were sequentially dehydrated in 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol and anhydrous ethanol (10s each time). Then they were sequentially immersed in xylene II and xylene (15s) and allowed to air dry naturally before being mounted with neutral resin.

[0122] (VI) Microscopic observation and image acquisition.

[0123] After each group underwent tail vein injection intervention for 14 days, the H&E staining results were as follows: Figure 16 As shown.

[0124] First, examine the cardiac tissue structure of the rats in each group: The cardiac tissue structure of the rats in the Sham group was intact, with myocardial fibers arranged tightly and orderly, and running in a regular pattern, without any fiber breakage; the myocardial cell nuclei were located in the center of the cell, oval in shape, and stained evenly and clearly. There was no obvious dilation or edema in the interstitium, and no pathological changes such as inflammatory cell infiltration, myocardial necrosis, or fibrosis.

[0125] In both the MI and LP groups, some areas showed disordered arrangement of myocardial fibers, with fiber breakage and dissolution; some myocardial cell nuclei exhibited condensation, darkened staining, or disappearance, indicating nuclear dissolution. Extensive neutrophil infiltration was observed, accompanied by varying degrees of myocardial necrosis.

[0126] Compared with the MI group, the myocardial tissue pathological changes in the CP group and CCP group were significantly reduced. The myocardial fibers were arranged more regularly than those in the MI group, the necrotic area was significantly reduced, and the degree of fibrosis was significantly reduced. The myocardial cell nuclei were clear and intact, the inflammatory cell infiltration was less, the inflammatory response was milder, the myocardial interstitial structure tended to be normal, and the fibrotic lesions were significantly relieved.

[0127] Furthermore, H&E staining results showed that the hepatocytes in the CCP group were regularly arranged, with uniform cytoplasm and neatly positioned nuclei within the liver lobules, showing no obvious cell damage or inflammation. The white and red pulp of the spleen were clearly demarcated, with the white pulp exhibiting distinct lymphoid follicle structures and tightly and regularly arranged cells. The red pulp showed clear sinusoids without significant abnormalities. The alveoli were structurally normal, with fine alveolar walls and clear air spaces, showing no inflammatory infiltration or edema, and the gas exchange areas remained intact. The renal tubules and glomeruli were structurally clear, with regularly arranged tubules, normal tubular epithelial cell morphology, no significant glomerular swelling or damage, and no inflammatory or fibrotic changes in the kidneys. In conclusion, H&E staining of the heart, liver, spleen, lungs, and kidneys in the CCP group all showed the tissue morphology of normal rats after the MI model, without causing additional tissue damage, demonstrating good biocompatibility.

[0128] ③Masson staining

[0129] Paraffin sections were dried in a 60°C oven overnight, and stained the following day using the Masson trichrome staining kit. The specific steps are as follows:

[0130] (I) Dewaxing and hydration of sections: the method is the same as H&E staining;

[0131] (II) Masson complex staining: Add Masson complex staining solution (solution A) to cover the tissue, incubate for 5 min, and then wash with distilled water;

[0132] (III) Phosphomolybdic acid treatment: Add phosphomolybdic acid solution (solution B) dropwise to cover the tissue, incubate for 5 min, pour off and shake dry.

[0133] (IV) Aniline blue staining: Add aniline blue solution (solution C) to cover the tissue, incubate for 5 min, and then rinse with distilled water.

[0134] (V) Acetic acid differentiation: Add acetic acid differentiation solution (solution D) to cover the tissue, incubate for 30-60 seconds, and then pour it off;

[0135] (VI) Dehydration, clearing and mounting: The method is the same as H&E staining;

[0136] (VII) Microscopic observation and image acquisition;

[0137] (VIII) Calculation of fibrosis ratio: The collagen volume fraction (CVF) was calculated using ImageJ (version 1.8.0, National Institutes of Health) software. CVF = collagen area / total area × 100%.

[0138] After 14 days of tail vein injection intervention in each group, the MASSON staining results were as follows: Figure 17 As shown in A:

[0139] The myocardial fibers in the Sham group were neatly arranged, evenly stained, and intact, with almost no collagen fiber deposition. It was normal myocardial tissue that was not damaged or fibrotic.

[0140] In the MI group, the blue area within the infarct region is extensive and dense, with abundant collagen deposition and fibrosis; the myocardial tissue is almost completely replaced by connective tissue. Collagen fiber deposition extends from the periphery to the core of the infarct edge. The distribution of connective tissue is uneven, with some areas showing excessive fibrosis. Myocardial fibers are significantly reduced or even completely absent, indicating cardiomyocyte necrosis. The remaining myocardial fibers are disordered and partially broken, indicating significant myocardial tissue damage. There are significant gaps or loose tissue within the infarct area, representing interstitial spaces after the removal of necrotic tissue. The boundary between the fibrotic area and the viable myocardium is clear, but the transition zone shows a gradual increase in collagen deposition.

[0141] Compared to the MI group, both the CP and CCP groups reduced myocardial fibrosis and promoted myocardial tissue repair to varying degrees, with the CCP group showing the most significant therapeutic effect. The blue-stained area in the infarct core was significantly reduced, indicating decreased collagen deposition and a lower degree of fibrosis, suggesting that the CCP group's treatment intervention inhibited myocardial fibrosis. Collagen fibers were more sparsely distributed and stained lighter, inhibiting connective tissue proliferation and improving the fiber remodeling process. More myocardial fibers were preserved, arranged more tightly and regularly, with good continuity in some areas. In conclusion, the CCP group promoted myocardial repair, reduced interfibrillary collagen deposition, and resulted in a tighter tissue structure, demonstrating the best therapeutic effect.

[0142] ④Sirius Red staining

[0143] Paraffin sections were dried in a 60℃ oven overnight, and then stained with Sirius red the following day. The specific steps are as follows:

[0144] (I) Dewaxing and hydration of sections: the method is the same as H&E staining;

[0145] (II) Iron hematoxylin staining: Prepare iron hematoxylin staining solution, add staining solution for 10 min, and wash with distilled water for 20 s;

[0146] (III) Soak in tap water for 10 minutes, then rinse with distilled water 3 times, 10 seconds each time;

[0147] (IV) Sirius red staining: Add Sirius red staining solution and stain for 20 min;

[0148] (V) Rinse with running water: Remove residual staining solution from the surface of the slides;

[0149] (VI) Dehydration, clearing and mounting: The method is the same as H&E staining;

[0150] (VII) Microscope observation and image acquisition;

[0151] (VIII) Calculation of fibrosis ratio: Same as Masson staining above.

[0152] After 14 days of tail vein injection intervention in each group, the Sirius red staining results were as follows: Figure 17 As shown in B:

[0153] In the Sham group, collagen fibers in the heart tissue were evenly distributed, mainly in the basement membrane and myocardial interstitium. Collagen fiber content was low, with no significant accumulation. Sirius red staining intensity was weak. The staining was mainly light red or orange-red, indicating a relatively low collagen fiber content. The heart tissue structure was relatively intact, with regular arrangement of myocardial fibers, no obvious fibrosis or damage, and no significant changes in the cardiac matrix.

[0154] The heart tissue of the MI group showed a large amount of collagen fiber deposition, especially in the MI area, where collagen fiber accumulation was obvious, and the heart tissue underwent a significant fibrotic reaction after being damaged.

[0155] Collagen fiber deposition in the cardiac tissue of the COL group was significantly reduced compared to the MI group, but significant collagen accumulation still existed. It was concentrated in the interstitial portion of the heart, indicating that COL treatment promoted collagen deposition.

[0156] In the LP group, collagen fiber deposition was significantly increased in the cardiac tissue, and collagen deposition was significantly enhanced in the infarcted area, with obvious staining reaction.

[0157] Collagen deposition increased in the CP group compared to the Sham group, but was less than that in the MI and LP groups. Collagen was mainly distributed in the interstitium, with relatively localized deposition. No obvious damage or extensive fibrosis was observed, and the overall structure remained relatively intact. Collagen deposition was relatively localized and did not show excessive fibrosis.

[0158] Collagen deposition was significantly increased in the CCP group, showing a marked increase compared to the Sham and CP groups. Collagen fiber deposition was widely distributed, indicating that CCP treatment exacerbated collagen accumulation. In the CCP group, collagen fibers were more evenly distributed, mainly in the basement membrane and myocardial interstitium, with significantly lower deposition levels than in the MI group. The tissue structure was relatively intact, without obvious fibrosis or scarring from collagen deposition. The degree of tissue fibrosis was low, and the myocardial fibers were arranged relatively regularly.

[0159] In conclusion, the study shows that CCP intervention has a positive effect on cardiac tissue repair and does not cause significant fibrotic reactions.

[0160] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a cardiac homing peptide synergistic colchicine phospholipid complex, characterized in that, Includes the following steps: (1) Colchicine, cholesterol and lecithin are dissolved in a first organic solvent to obtain a first mixed solution. After water bath reaction, the solution is purified and dried to obtain colchicine phospholipid complex. The mass ratio of colchicine, cholesterol, and lecithin is 0.8-1:1:7-10, the water bath reaction temperature is 20-75℃, and the water bath reaction time is 4-8 hours. (2) Dissolve the colchicine phospholipid complex and the cardiac homing peptide in water, and then react at room temperature for 4-10 h to obtain a second mixed solution of cardiac homing peptide and colchicine phospholipid. After drying, the cardiac homing peptide and colchicine phospholipid complex is obtained. The mass ratio of the colchicine phospholipid complex to the cardiac homing peptide is 18-22:

1.

2. The preparation method of the cardiac homing peptide synergistic colchicine phospholipid complex as described in claim 1, characterized in that, The concentration of colchicine in the first mixed solution is 0.8-1 mg / mL; And / or, the concentration of the cardiac homing peptide in the second mixed solution is 0.39-0.55 mg / mL.

3. The preparation method of the cardiac homing peptide synergistic colchicine phospholipid complex as described in claim 1, characterized in that, The lecithin is egg yolk lecithin and / or soybean lecithin; The first organic solvent is at least one of methanol, ethanol, chloroform, diethyl ether, acetone and tetrahydrofuran.

4. The method for preparing the cardiac homing peptide synergistic colchicine phospholipid complex as described in claim 1, characterized in that, The purification process is as follows: removing the first organic solvent, then adding a second organic solvent and removing substances insoluble in the second organic solvent, and finally removing the second organic solvent, wherein the second organic solvent is n-hexane and / or petroleum ether.

5. The preparation method of the cardiac homing peptide synergistic colchicine phospholipid complex as described in claim 4, characterized in that, The mass-to-volume ratio of the lecithin to the second organic solvent is 0.1-10 mg: 1 mL.

6. A cardiac homing peptide synergistic colchicine phospholipid complex, characterized in that, It is prepared by any one of claims 1-5.

7. A cardiac homing peptide synergistic colchicine phospholipid complex nanoparticle, characterized in that, It includes the cardiac homing peptide synergistic colchicine phospholipid complex as described in claim 6, wherein the average particle size of the cardiac homing peptide synergistic colchicine phospholipid complex nanoparticles is 0.15-0.3 μm.

8. A method for preparing cardiac homing peptide synergistic colchicine phospholipid complex nanoparticles as described in claim 7, characterized in that, The method includes the following steps: mixing the cardiac homing peptide synergistic colchicine phospholipid complex of claim 6 with deionized water to obtain a second mixed solution, shaking the solution in a water bath at 30-60°C until the cardiac homing peptide synergistic colchicine phospholipid complex dissolves, then ultrasonically dispersing the solution, and finally filtering the solution with a microporous membrane to obtain the cardiac homing peptide synergistic colchicine phospholipid complex nanoparticles, wherein the microporous membrane has a pore size of 50-300 nm.

9. The preparation method according to claim 8, characterized in that, The concentration of the cardiac homing peptide synergistic colchicine phospholipid complex in the second mixed solution is 0.1-100 mg / mL.

10. The application of the cardiac homing peptide synergistic colchicine phospholipid complex according to claim 6 in cardiac tissue repair.

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