Liposomes that actively target atherosclerotic plaques and their application in diagnosis

Abstract

The present invention belongs to the field of medical technology, and discloses a liposome that actively targets atherosclerotic plaques and its application in diagnosis. The carrier material forming the liposome includes a targeting peptide R8 modified with methoxy polyethylene glycol phospholipid (R8-DSPE-PEG), polysulfide propylene-polyethylene glycol (PPS-PEG) and cholesterol (CH), and the sequence of the targeting peptide R8 is Asp-Ser-Ser-Ser-Gln-Cys-His-Arg. The liposome of the present invention can increase the enrichment concentration of fluorescent dyes or drugs in the local lesion, has the advantages of low toxicity and high efficiency, and the obtained targeted liposome has a good effect on the diagnosis of atherosclerosis.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a liposome that actively targets atherosclerotic plaques and an application thereof in diagnosis. Background Art

[0002] Atherosclerosis refers to a disease characterized by thickening, hardening and reduced elasticity of the arterial wall, and the formation of atherosclerotic plaques in the arterial intima. It is the main cause of coronary heart disease, cerebral infarction and peripheral vascular disease. Regarding the pathogenesis of the disease, most scholars in recent years support the "endothelial injury response theory", believing that the various major risk factors of the disease ultimately damage the arterial intima, and the formation of atherosclerotic lesions is the result of the inflammatory-fibroproliferative response of the artery to the intima damage.

[0003] Damage to the arterial intima can be a functional disorder or anatomic injury. In the case of long-term hyperlipidemia, the increased oxidatively modified low-density lipoprotein (ox LDL) and cholesterol in the lipoproteins cause functional damage to the arterial intima, causing changes in the surface characteristics of endothelial cells and leukocytes (monocytes and lymphocytes), and increased expression of adhesion factors. The number of monocytes adhering to endothelial cells increases, and they move from between endothelial cells to the subintima to become macrophages, which engulf oxLDL through scavenger receptors and transform into foam cells, forming the earliest lipid streaks of atherosclerotic lesions. Macrophages can oxidize LDL, form peroxides and superoxide ions, and synthesize and secrete at least 6 cytokines. Under the action of these cytokines, fatty streaks are promoted to evolve into fibrofatty lesions and then develop into fibrous plaques.

[0004] When hemodynamic changes occur, such as increased blood pressure, turbulence caused by local vascular stenosis, and changes in shear stress, the continuity between endothelial cells in the arterial intima is interrupted, and the endothelial cells retract, exposing the subintimal tissue. At this time, platelet-activating factor (PAF) activates platelets in the blood, causing them to adhere and aggregate on the intima to form mural thrombi. Platelets can release many cytokines, which enter the arterial wall and promote the proliferation of smooth muscle cells in atherosclerotic lesions.

[0005] In order to achieve dynamic monitoring of plaque formation, sensitive release of ROS, high penetration of plaques, targeted enrichment of plaques, etc., it is necessary to prepare liposomes that can actively target atherosclerotic plaques. Summary of the invention

[0006] The present invention provides a liposome that actively targets atherosclerotic plaques and its application in diagnosis. The present invention adopts the following technical solutions:

[0007] On the one hand, the present invention relates to a liposome that actively targets atherosclerotic plaques. The carrier material forming the liposome includes a targeting peptide R8 modified with methoxy polyethylene glycol phospholipid (R8-DSPE-PEG), polysulfide propylene-polyethylene glycol (PPS-PEG) and cholesterol (CH). The sequence of the targeting peptide R8 is Asp-Ser-Ser-Ser-Gln-Cys-His-Arg.

[0008] In a preferred embodiment of the present invention, the average particle size of the liposome is less than 500 nm; preferably less than 200 nm.

[0009] In a preferred embodiment of the present invention, the mass ratio of the targeting peptide R8 modified methoxy polyethylene glycol phospholipid (R8-DSPE-PEG), polypropylene sulfide-polyethylene glycol (PPS-PEG) and cholesterol (CH) is (3-5): (1-1.5): (1-1.5).

[0010] In a preferred embodiment of the present invention, the liposome encapsulates a fluorescent dye.

[0011] In a preferred embodiment of the present invention, the fluorescent dye is Cy5.5 dye.

[0012] Another aspect of the present invention also relates to a method for preparing the above-mentioned liposomes, comprising the following steps: dissolving a carrier material with 40-60 times the mass of chloroform, and then adding a fluorescent dye accounting for 0.5-2 wt% of the carrier material; removing the organic solvent by rotary evaporation to form a lipid film on the container wall; adding deionized water 40-60 times the mass of the carrier material in a constant temperature water bath at 40-50°C to fully hydrate the lipid film to form a crude liposome suspension; and using an extruder to extrude through 200nm and 100nm polycarbonate membranes respectively.

[0013] Another aspect of the present invention relates to the use of the above liposome in preparing a diagnostic reagent that actively targets atherosclerotic plaques.

[0014] The beneficial effects of the present invention are: the liposome of the present invention can increase the enrichment concentration of fluorescent dyes or drugs in the local lesion, has the advantages of low toxicity and high efficiency, and the obtained targeted liposome has a good effect on the diagnosis of atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Comparison of fluorescence values ​​of cell lysate in targeted validation experiments;

[0016] Figure 2 : Fluorescence microscope photos of live cell detection in the targeted verification experiment of the experimental group;

[0017] Figure 3: Fluorescence microscope photos of live cell detection in the control group targeted validation experiment. DETAILED DESCRIPTION

[0018] Unless otherwise specified, all reagents involved in the embodiments of the present invention are commercially available products and can be purchased through commercial channels.

[0019] Example: 1.1 Targeting peptide modified with methoxy polyethylene glycol phospholipid (DSPE-PEG):

[0020] DSPE-PEG2000-Mal (10 mg) was dissolved in chloroform (4 mL) and triethylamine (2 μL) was added. Cys-R8 (wherein the sequence of R8 is Asp-Ser-Ser-Ser-Gln-Cys-His-Arg, and the molar ratio of DSPE-PEG2000-Mal / Cys-R8 = 1 / 1.5) was dissolved in methanol (2 mL). Under nitrogen protection, the methanol solution of Cys-R8 was added to the chloroform solution of DSPE-PEG2000-Mal and gently stirred at 25°C in the dark for 24 hours. The residue was redissolved in chloroform and the solution was dialyzed in water to purify the product. The purified solution was lyophilized to collect DSPE-PEG2000-R8.

[0021] 1.2 ROS-responsive poly(propylene sulfide)-poly(ethylene glycol) (PPS-PEG):

[0022] 0.448 mL of 1, 8-diazabicyclo[5.4.0]undec-7-ene was dissolved in 15 mL of tetrahydrofuran and transferred to a dry 50 mL round-bottom flask. Under nitrogen protection, the reaction system was reacted at 0°C for 30 min. Similarly, 10 mL of tetrahydrofuran and 0.070 mL of 1-butanethiol were also degassed for 30 min and added dropwise to the above flask to react and dissolve for 30 min. Then, 4.68 mL of PPS was added to the above mixture and placed at 0°C for 2h. 0.37 g of iodoacetic acid was added to terminate the reaction and stirred at room temperature overnight. After 24 h, the precipitated salt was filtered to remove it, and it was further purified by three precipitations in cooled methanol and vacuum dried to obtain a colorless viscous polymer PPS-COOH. Weigh 4g NH2-PEG2000 into a 50mL round-bottom flask, add 15mL dichloromethane to completely dissolve it, then add 3g PPS-COOH, 185mg 1-(3-dimethylaminopropyl) and 49mg 4-dimethylaminopyridine respectively and react for 24h under stirring at room temperature. Transfer the above mixture into a dialysis bag (MWCO=5000Da), dialyze with deionized water for 2 days and then freeze-dry to obtain the product PPS-PEG.

[0023] 1.3 Preparation of liposomes:

[0024] Preparation of targeted liposomes: Targeting peptide R8 modified methoxy polyethylene glycol phospholipids (R8-DSPE-PEG), polysulfide propylene-polyethylene glycol (PPS-PEG) and cholesterol (CH) (mass ratio 3:1:1) were used as carrier materials, dissolved in 50 times the mass of chloroform, and then 1wt% of Cy5.5 dye was added to the carrier material. The organic solvent was removed by slow rotary evaporation (55℃ water bath, 90r / min, 30min) to form a lipid film on the container wall. Deionized water 50 times the mass of the carrier material was added, and the lipid film was fully hydrated in a constant temperature water bath at 45℃ to form a crude liposome suspension. The extruder was used to extrude through 200nm and 100nm polycarbonate membranes respectively.

[0025] Comparative Example: As a comparative example, DSPE-PEG of the same mass was used instead of R8-DSPE-PEG, and liposomes were prepared according to the same steps.

[0026] 1.4 Targeted validation experiment:

[0027] Since atherosclerotic plaques are rich in macrophages and highly express CD44 receptors, THP1 macrophages are usually selected and activated with lipopolysaccharide, incubated with fluorescently labeled R8 targeted liposomes or non-targeted comparative liposomes, and then live cell microscopic observation and fluorescence value detection are performed. By comparing the results of liposomes with R8 target heads and non-targeted liposomes, the targeting of HA targeted liposomes to macrophages activated by highly expressing CD44 receptors is evaluated.

[0028] After recovery, THP1 macrophages were resuspended at a density of 5 × 10 5 / ml, inoculated in a T75 culture flask. 10ml of THP1 cells growing in suspension were transferred to a 15ml centrifuge tube, centrifuged at 1000rpm for 5 minutes, resuspended in 1ml of culture medium and counted, and prepared into 1×10 5 / 200μl concentration of cell suspension. 5 PMA was added to 200 μl of cell suspension to a final concentration of 100 ng / ml. After mixing, the cell suspension was inoculated in a 96-well plate at 200 μl / well and the stimulation was continued for 72 hours. After 72 hours of stimulation, the original culture medium was discarded and fresh culture medium containing lipopolysaccharide (LPS) was added (LPS solution was diluted 1000 times in fresh culture medium to a final concentration of 2.5 μg / mL). LPS treatment was performed for 2 hours.

[0029] The experiment set up two groups, adding fluorescently labeled R8 targeted liposomes or non-targeted control liposomes respectively. Take 1 ml of liposomes and dilute them 10 times by volume in fresh culture medium. Discard the culture medium containing LPS in the 96-well plate and add the diluted fluorescent labeled test sample. Each group has three replicates and incubate at 37°C for 30 minutes.

[0030] Wash the plate once with PBS, dilute 2 μl of Hoechst33342 stock solution in 10 ml of fresh culture medium to prepare a diluted Hoechst33342 solution, incubate the diluted Hoechst33342 solution with cells for 10 min to stain the nuclei, and take pictures with a live cell imaging analysis system. Discard the staining solution from the well plate after taking pictures, add 100 μl of RIPA cell lysis solution, and use a fluorescence plate reader to detect the fluorescence intensity in the well plate. The experimental data obtained were calculated using Graph Pad Prism 8 software for mean and standard deviation and statistical analysis of t test, with p < 0.05 being statistically significant.

[0031] The targeting validation compared the internalization efficiency of R8-targeted liposomes or non-targeted control liposomes in THP1 cells after LPS stimulation. The experimental results showed (see Figure 1 ), compared with the non-targeted liposome group, the fluorescence value of R8 targeted liposome increased significantly, which was 3.8 times that of the non-targeted liposome group.

[0032] Fluorescence microscopy also showed (see Figure 2 , Figure 3 ), the red fluorescence in the cells of the R8-targeted liposome group was significantly higher than that in the non-targeted control group. This indicates that the endocytosis efficiency of the R8-targeted liposomes by THP1 cells after LPS stimulation is significantly higher than that of the non-targeted liposomes. It can be concluded that liposomes containing the R8 target head can be specifically taken up by activated macrophages that highly express the CD44 receptor.

[0033] The preferred embodiments of the present invention are described above, but they are not intended to limit the present invention. Those skilled in the art may make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present invention.

Claims

1. A liposome that actively targets atherosclerotic plaques, characterized in that: The carrier material forming the liposome includes targeting peptide R8 modified methoxy polyethylene glycol phospholipid, polypropylene sulfide-polyethylene glycol and cholesterol, the sequence of the targeting peptide R8 is Asp-Ser-Ser-Ser-Gln-Cys-His-Arg, and the mass ratio of the targeting peptide R8 modified methoxy polyethylene glycol phospholipid, polypropylene sulfide-polyethylene glycol and cholesterol is (3-5): (1-1.5): (1-1.5).

2. The liposome actively targeting atherosclerotic plaque according to claim 1, characterized in that: The average particle size of the liposome is less than 500 nm.

3. The liposome actively targeting atherosclerotic plaque according to claim 1, characterized in that: The average particle size of the liposome is less than 200 nm.

4. The liposome actively targeting atherosclerotic plaque according to any one of claims 1 to 3, characterized in that: The liposomes encapsulate a fluorescent dye.

5. The liposome actively targeting atherosclerotic plaque according to claim 4, characterized in that: The fluorescent dye is Cy5.5 dye.

6. The method for preparing the liposome actively targeting atherosclerotic plaques according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: dissolving a carrier material with chloroform 40-60 times the mass, and then adding a fluorescent dye accounting for 0.5-2wt% of the carrier material; removing the organic solvent by rotary evaporation to form a lipid film on the container wall; adding deionized water 40-60 times the mass of the carrier material in a constant temperature water bath at 40-50°C to fully hydrate the lipid film to form a crude liposome suspension; and extruding through a 200nm and 100nm polycarbonate membrane using an extruder.

7. Use of the liposome according to any one of claims 1 to 5 in the preparation of a diagnostic agent that actively targets atherosclerotic plaques.

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