Nanoparticles for quantitatively labeling foam cells in vulnerable plaques of atherosclerosis and preparation method and application thereof

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a nanoparticle for quantitatively labeling foam cells in vulnerable plaques of atherosclerosis, wherein the nanoparticle comprises a platelet membrane, a fluoride and a delivery carrier; the fluoride is used as the core, the delivery carrier is used as the middle layer, and the platelet membrane is used as the outer shell. The delivery carrier is mixed with the fluoride, and after emulsification and centrifugation, particles formed after the delivery carrier is coated with the fluoride are obtained; the particles formed after the delivery carrier is coated with the fluoride are mixed with the platelet membrane to obtain the nanoparticle PFCE@PLGA@PM. The PFCE@PLGA@PM can be effectively aggregated into the atherosclerotic plaque, and taken up by the foam cells in the plaque, so as to achieve quantitative labeling of the foam cells in the plaque.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a nanoparticle for quantitatively marking foam cells in vulnerable plaques of atherosclerosis, and a preparation method and application thereof. Background Art

[0002] Rupture of vulnerable atherosclerotic plaques is a common cause of heart attacks and strokes. Identification of vulnerable plaques is crucial for the treatment of atherosclerosis. Plaque vulnerability is associated with the deposition and apoptosis of foam cells within the plaque. Therefore, quantification of foam cells within plaques is helpful for determining plaque vulnerability. However, due to interference from imaging technology and background, current molecular imaging methods cannot accurately quantify foam cells within plaques.

[0003] In order to accurately quantify foam cells in plaques, introducing new elements without background interference may be a promising method. Fluorine has attracted attention due to its good biosafety and almost no background interference, and has been used to track the dynamics of immune cell infiltration and provide quantitative analysis. Therefore, fluorine is expected to become a promising element for quantitative assessment of atherosclerotic plaques. In addition, an efficient and specific targeted delivery method is needed to deliver fluorine to foam cells. Platelet membrane biomimetic nanoparticles are a highly specific and efficient targeting system for atherosclerotic plaques. Fluorine labeling of foam cells in plaques can be achieved by delivering fluorine to plaque foam cells through platelet membrane biomimetic nanoparticles. Based on this, quantitative analysis of foam cells in plaques can be achieved, which is conducive to the judgment of plaque vulnerability. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a nanoparticle for quantitatively labeling foam cells in vulnerable plaques of atherosclerosis and a preparation method and application thereof in view of the deficiencies in the prior art.

[0005] In order to solve the above technical problems, the present invention discloses a nanoparticle that specifically targets foam cells in atherosclerotic plaques to achieve quantitative labeling of foam cells. The technical solution of the present invention is as follows:

[0006] The present invention provides a nanoparticle for quantitatively labeling foam cells in vulnerable plaques of atherosclerosis, wherein the nanoparticle comprises a platelet membrane, fluoride and a delivery carrier, wherein the fluoride is used as the inner core, the delivery carrier is used as the middle layer, and the platelet membrane is used as the outer shell. The delivery carrier loaded with fluoride is wrapped with the platelet membrane.

[0007] Wherein, the fluorinated compound is a perfluoroether, preferably any one of perfluoro-15-crown-5-ether, perfluoropolyether and perfluorocyclic ether, more preferably perfluoro-15-crown-5-ether (PFCE).

[0008] Wherein, the delivery carrier is any one of polyglycolide lactide, mesoporous silica or liposome; the particle size of the particles formed after the delivery carrier is coated with fluoride is 50-100 nm. Preferably, the delivery carrier is polyglycolide lactide (PLGA).

[0009] Preferably, the platelet membrane (PM) is derived from clinically obtained platelet-rich plasma. The preparation method of the platelet membrane is as follows: centrifuge the platelet-rich plasma (850×g, 4°C, 10min), remove the supernatant; resuspend the precipitate in physiological saline, repeat centrifugation (850×g, 4°C, 10min) and wash three times; after the last wash, resuspend in physiological saline containing protease inhibitors. The washed platelets are frozen at -80°C and thawed at room temperature, and the freeze-thaw is repeated 3-4 times; centrifuge again (4000×g, 4°C, 5min), remove the supernatant; resuspend the precipitate in physiological saline containing protease inhibitors, repeat centrifugation (4000×g, 4°C, 5min) and wash three times to obtain a platelet membrane precipitate, which is dispersed in physiological saline containing protease inhibitors.

[0010] In a second aspect, the present invention provides a method for preparing the nanoparticles according to the first aspect, comprising the following steps:

[0011] (1) mixing the delivery carrier with fluoride, emulsifying and centrifuging to obtain PFCE@PLGA particles formed by the delivery carrier coating the fluoride;

[0012] (2) The PFCE@PLGA particles formed after the delivery carrier is coated with fluoride are mixed with platelet membranes to obtain the nanoparticles PFCE@PLGA@PM.

[0013] Wherein, in step (1), the delivery carrier is mixed with the fluoride in the form of a delivery carrier solution, and the solvent of the delivery carrier solution is any one of dichloromethane, DMSO or tetrahydrofuran, preferably dichloromethane. More preferably, the concentration of the delivery carrier solution is 50 mg / mL.

[0014] The dosage of the delivery carrier is calculated by adding 500-600 mg of the delivery carrier to each mL of fluoride; preferably, 500 mg of the delivery carrier is added to each mL of fluoride.

[0015] Wherein, in step (1), the emulsification is performed more than twice, each emulsification lasts for 1 to 2 minutes, and the emulsification conditions are: 30 to 50W, ultrasound for 5 to 6 seconds, and an interval of 2 to 3 seconds. Preferably, the emulsification is performed twice, and the conditions for the first emulsification are emulsification for 1 minute, 40W, ultrasound for 5 seconds, and an interval of 3 seconds, and the conditions for the second emulsification are emulsification for 2 minutes, 40W, ultrasound for 5 seconds, and an interval of 3 seconds. More preferably, the emulsion obtained by the first emulsification is mixed with a polyvinyl alcohol solution and then emulsified for the second time, and the polyvinyl alcohol solution is a polyvinyl alcohol aqueous solution with a polyvinyl alcohol concentration of 20 g / L.

[0016] Wherein, in step (2), the mass ratio of the PFCE@PLGA particles formed after the delivery carrier is coated with fluoride and the platelet membrane is 10:1 to 10:1.5. Preferably, the mass ratio is 10:1. More preferably, the PFCE@PLGA and the platelet membrane are mixed in the form of a PFCE@PLGA aqueous solution and a platelet membrane solution, the concentration of the PFCE@PLGA aqueous solution is 10 mg / mL, the concentration of the platelet membrane solution is 1 mg / mL, and the mixing volume ratio of the PFCE@PLGA aqueous solution and the platelet membrane solution is 1:1.

[0017] In a third aspect, the present invention provides use of the nanoparticles described in the first aspect in the preparation of a reagent for diagnosing the vulnerability of atherosclerotic plaques.

[0018] Wherein, the diagnosis of atherosclerotic plaque vulnerability is performed by quantitatively labeling foam cells in atherosclerotic plaques.

[0019] Beneficial effects:

[0020] Compared with the molecular imaging method in the prior art which cannot accurately quantify foam cells in plaques, the beneficial effects of the present invention are as follows:

[0021] (1) In order to achieve quantitative analysis of foam cells in atherosclerotic plaques, the present invention provides a nanoparticle, wherein the nanoparticle comprises a platelet membrane (PM), a fluoride, and a delivery vehicle; the fluoride is used as a core, the delivery vehicle is used as an intermediate layer, and the platelet membrane is used as an outer shell. The platelet membrane is used to wrap the nanoparticle formed by the delivery vehicle loaded with fluoride, thereby achieving quantitative analysis of foam cells in atherosclerotic plaques.

[0022] (2) The present invention provides a platelet membrane-wrapped poly(l-glycolide-lactide) (PLGA) nanoparticle (PFCE@PLGA@PM) loaded with perfluoro-15-crown-5-ether (PFCE), wherein the fluorine element has good biosafety and no background interference, and the platelet membrane-wrapped nanoparticle is a highly specific and efficient targeting system for atherosclerotic plaques, and the PFCE@PLGA@PM of the present invention can effectively aggregate into atherosclerotic plaques; further, the PFCE@PLGA@PM is taken up by foam cells in the plaques, and quantitative labeling of foam cells in the plaques is achieved, and the PFCE@PLGA@PM is targeted to the foam cells in the plaques, and quantitative analysis of the foam cells is achieved by measuring the content of the fluorine element. The PFCE@PLGA@PM of the present invention has a good application prospect in the preparation of reagents for diagnosing the vulnerability of atherosclerotic plaques. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0024] Figure 1 The figure shows the components of PFCE@PLGA@PM nanoparticles, where PM is platelet membrane, PLGA is poly(lactide-glycolide), and PFCE is perfluoro-15-crown-5-ether.

[0025] Figure 2 Confocal laser scanning microscopy images of activated human umbilical vein endothelial cells (HUVECs) and activated mouse macrophages RAW264.7 after co-culture with PFCE@PLGA@PM or PFCE@PLGA, respectively.

[0026] Figure 3 The relationship between HUVECs and foam cells in different mixing ratios and fluorine content. Figure 3 A is the relationship between the non-activated HUVECs and foam cells at different mixing ratios and the fluoride content. Figure 3 B is a graph showing the relationship between activated HUVECs and foam cells at different mixing ratios and fluorine content.

[0027] Figure 4 This is the relationship diagram between oxLDL, fluoride content and modeling time. Figure 4 A is the relationship between oxLDL content and modeling time. Figure 4 B is the relationship between fluorine content and modeling time. Figure 4 C is the relationship between oxLDL content and fluoride content. DETAILED DESCRIPTION

[0028] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0029] Example 1 Preparation of platelet membrane (PM)

[0030] Centrifuge human platelet-rich plasma (O-type blood) (850×g, 4°C, 10min), remove the supernatant; resuspend the precipitate in normal saline, repeat centrifugation (850×g, 4°C, 10min) and wash three times; after the last wash, resuspend in normal saline containing protease inhibitors. The washed platelets are frozen at -80°C and thawed at room temperature, and freeze-thawed 3-4 times; centrifuge again (4000×g, 4°C, 5min), remove the supernatant; resuspend the precipitate in normal saline containing protease inhibitors, repeat centrifugation (4000×g, 4°C, 5min) and wash three times to obtain platelet membrane precipitate, which is dispersed in normal saline containing protease inhibitors; use the BCA kit to dilute the platelet membrane solution to 1mg / mL and store it in a -80°C freezer.

[0031] Example 2 Preparation of PFCE@PLGA@PM Nanoparticles

[0032] (1) Preparation of PFCE@PLGA particles formed after the delivery carrier is coated with fluoride: 100 mg PLGA was dissolved in 2 mL of dichloromethane, and 0.2 mL PFCE was added. The mixture was ultrasonically emulsified for 1 min under ice bath conditions, with ultrasonic parameters of 40 W, ultrasonic for 5 s, and intervals of 3 s. The emulsion was then added to 10 mL of 2% polyvinyl alcohol solution (2 g polyvinyl alcohol was dissolved in 100 mL of water), and ultrasonically emulsified again for 2 min under ice bath conditions, with ultrasonic parameters of 40 W, ultrasonic for 5 s, and intervals of 3 s. The emulsion after the second emulsification was stirred at room temperature for 4 hours to evaporate the dichloromethane. The mixture was then centrifuged at 2000 × g for 5 min to remove larger particles, and then centrifuged at 8000 × g for 20 min to collect the precipitate to obtain PFCE@PLGA. The particle size of the final prepared particles ranged from 50 to 100 nm.

[0033] (2) The PFCE@PLGA precipitate collected in step (1) was dissolved in 10 mL of pure water (final concentration was 10 mg / mL), and mixed with the platelet membrane solution prepared in advance at a concentration of 1 mg / mL at a volume ratio of 1:1, and subjected to water bath sonication for 2 min to obtain PFCE@PLGA@PM, the structural schematic of which is shown in FIG. Figure 1 shown.

[0034] Example 3

[0035] Human umbilical vein endothelial cells (HUVECs) and mouse macrophages RAW264.7 were inoculated in glass-bottomed plates filled with culture medium (DMEM+10% FBS+1% P / S) and incubated for 12 hours. After cell attachment, HUVECs were cultured with 50ng / mL tumor necrosis factor α (TNF-α) for 24 hours to simulate damaged endothelial cells. RAW264.7 cells were first treated with 100ng / mL lipopolysaccharide (LPS) for 24 hours, and then treated with 50ng / mL oxidized modified low-density lipoprotein (oxLDL) for 48 hours to simulate foam cells. The activated cells were co-cultured with PFCE@PLGA@PM or PFCE@PLGA (final concentration of 0.05mg / mL) prepared in Example 2 labeled with a cell membrane green fluorescent probe (DiO) for 4 hours, and then the supernatant was aspirated, fixed with 4% paraformaldehyde and labeled with the dye DAPI. Fluorescent images of the cells were taken by confocal laser scanning microscopy (CLSM, Leica SP5) as shown in the figure. Figure 2 As shown. Figure 2 It can be seen that PFCE@PLGA@PM is more easily incorporated into cells compared to PFCE@PLGA without platelet membrane. In addition, activated RAW264.7 cells take up more PFCE@PLGA@PM compared to activated HUVECs.

[0036] Example 4

[0037] The PFCE@PLGA@PM prepared in Example 2 was used to conduct an in vitro targeting foam cell experiment, as follows:

[0038] First, HUVECs and RAW264.7 were inoculated in different partitions of a 2-partition culture dish. Among them, the inoculation ratio of RAW264.7 was 0, 20%, 40%, 60% and 80% of the total inoculation amount, respectively. After the cells adhered to the wall, HUVECs were cultured with 50ng / mL TNF-α for 24 hours to simulate damaged endothelial cells. RAW264.7 cells were first treated with 100ng / mL LPS for 24 hours and then with 50ng / mL oxLDL for 48 hours to simulate foam cells. Using unstimulated HUVECs cells as a control, the above steps were repeated. Subsequently, the culture medium containing PFCE@PLGA@PM (DMEM+10%FBS+1%P / S) was added to each partition, and added to exceed the partition separator, so that the culture medium in each partition was connected and different cells were in the same liquid environment. After another 4-hour culture, the cells were washed, digested and collected with PBS (2.0 mM KH2PO4, 137 mM NaCl, 10.0 mM Na2HPO4, 2.7 mM KCl), and then 19F NMR quantitative analysis, the results are as follows Figure 3 As shown, to test the specific targeting effect on foam cells in vitro, non-activated HUVEC and activated RAW264.7 (foam cells) were mixed and cultured with PFCE@PLGA@PM. Figure 3 As shown in A, in the presence of unactivated HUVECs, the amount of fluoride and the ratio of foam cells showed a good linear relationship (r 2 =0.9745). Figure 3 As shown in B, in the presence of activated HUVECs, the amount of fluoride and the ratio of foam cells also showed a good linear relationship (r 2 =0.9025). These results suggest that PFCE@PLGA@PM has the potential to quantify the number of foam cells in plaques via fluorine signals.

[0039] Example 5

[0040] ApoE- / - mice (male, 6-8 weeks old, about 25g) were fed a high-fat diet for 6 weeks, and then underwent right carotid artery constriction surgery. Six mice were randomly selected every other week and injected with PFCE@PLGA@PM (1 mg / mL, 200 μL / mouse) through the tail vein. 48 hours after administration, the mice were killed by dislocation of the neck, and the right common carotid artery was collected. The same amount of carotid artery tissue was ground, and the oxLDL content in the tissue homogenate was detected by oxLDL kit. 19 The PFCE content in tissue homogenate was determined by F NMR. Figure 4 As shown, Figure 4 As shown in A, with the formation and development of vulnerable carotid plaques, the amount of oxLDL in the plaques gradually increased over time, and the linear relationship between oxLDL content and time was poor (r 2 =0.8365). Figure 4 As shown in B, the fluorine content also increases with time, but it is not a linear increase (r 2 =0.8175). However, if Figure 4 As shown in C, when analyzing the relationship between oxLDL and fluoride content, it was found that the two were linearly correlated (r 2 =0.9672).

[0041] In summary, PFCE@PLGA@PM can be effectively taken up by foam cells in plaques, and the fluorine element without background interference can be used as a marker for foam cells, and the content of foam cells in plaques can be quantified by the fluorine content.

[0042] The present invention provides a method and method for quantitatively labeling foam cells in vulnerable plaques of atherosclerosis and its preparation method and application. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A nanoparticle for quantitatively labeling foam cells in vulnerable plaques of atherosclerosis, characterized in that: The nanoparticles include platelet membrane, fluoride and a delivery carrier; the fluoride is used as the core, the delivery carrier is used as the middle layer, and the platelet membrane is used as the shell; the amount of the delivery carrier is calculated by adding 500-600 mg of the delivery carrier to each mL of fluoride; The mass ratio of the particles formed after the delivery carrier is coated with fluoride and mixed with the platelet membrane is 10:1~10:1.5; The fluoride is perfluoro-15-crown-5-ether; and the delivery carrier is polyglycolide-lactide.

2. The nanoparticle according to claim 1, characterized in that The particles formed after the delivery carrier is coated with fluoride have a particle size of 50~100nm.

3. The method for preparing nanoparticles according to any one of claims 1 to 2, characterized in that: The steps include: (1) Mixing the delivery vehicle and fluoride, emulsifying and centrifuging to obtain particles formed by the delivery vehicle coating the fluoride; wherein the amount of the delivery vehicle is calculated by adding 500-600 mg of the delivery vehicle per mL of fluoride; (2) mixing the particles formed after the delivery carrier is coated with fluoride with the platelet membrane, wherein the mass ratio of the particles formed after the delivery carrier is coated with fluoride to the platelet membrane is 10:1 to 10:1.5; The fluoride is perfluoro-15-crown-5-ether; and the delivery carrier is polyglycolide-lactide.

4. The preparation method according to claim 3, characterized in that: In step (1), the delivery carrier is mixed with the fluoride in the form of a delivery carrier solution, and the solvent of the delivery carrier solution is any one of dichloromethane, DMSO or tetrahydrofuran.

5. The preparation method according to claim 3, characterized in that: In step (1), the emulsification is performed more than twice, each time for 1 to 2 minutes, and the emulsification conditions are: 30 to 50W, ultrasound for 5 to 6 seconds, and an interval of 2 to 3 seconds.

6. Use of the nanoparticles according to any one of claims 1 to 2 in the preparation of a reagent for diagnosing the vulnerability of atherosclerotic plaques; wherein: The diagnosis of atherosclerotic plaque vulnerability is performed by using the nanoparticles to quantitatively label foam cells in atherosclerotic vulnerable plaques.

Images