Preparation method of ginsenoside microvesicles and application of ginsenoside microvesicles and ultrasound in targeted local treatment of atherosclerotic plaques

Abstract

The invention discloses a preparation method of ginsenoside microvesicles and application of the ginsenoside microvesicles in targeted local treatment of atherosclerotic plaques through combination of the ginsenoside microvesicles and ultrasound, the ginsenoside microvesicles Rb3NPs (at) MBs prepared through the preparation method are evenly dispersed and consistent in size, and through combination of the ginsenoside microvesicles and the ultrasound, the effect of treating atherosclerotic plaques is achieved. The method is applied to targeted local treatment of atherosclerotic plaque, the plaque area is reduced by 41.84% relative to a model group, and the combined method effectively reduces atherosclerotic plaque at the aortic arch of a targeted mouse.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a method for preparing ginsenoside microbubbles and an application of ginsenoside microbubbles in combination with ultrasound in targeted local treatment of atherosclerotic plaques. Background Art

[0002] In 2023, the American Journal of Heart Disease reported that the global burden of cardiovascular disease has increased, and with the arrival of an aging society in the future, the incidence of cardiovascular disease will continue to rise. Atherosclerosis (AS) is the pathological basis for the occurrence of cardiovascular disease, causing a series of cardiovascular and cerebrovascular diseases such as coronary heart disease and stroke. Studies have shown that due to the faster blood flow rate and drastic shear force in the aortic arch, the AS situation in the aortic arch is often more serious. For the treatment of AS, for different clinical phenotypes, there are corresponding clinical drugs such as antithrombotic drugs, antilipidemic drugs, and anti-myocardial ischemic drugs. The research and development of target drugs such as PCSK9 inhibitors and inflammatory factor monoclonal antibodies Canakinumab have entered the clinical trial stage, but the most widely used are still statins for lipid-lowering. The disadvantage of statins is that they are systemic medications and the efficiency of local plaque removal is poor. Therefore, it is very important to propose a method for precise local treatment of the aortic arch with severe AS lesions.

[0003] At present, the precise local treatment of AS is mostly achieved with the help of visualization technology, such as MRI, CT, virtual histology intravascular ultrasound VH-IVUS, etc., to release drugs in the plaque area. However, these technologies have high implementation requirements, are difficult to operate, and are expensive.

[0004] The ginsenoside Rb3 used in the present invention is an active substance derived from the traditional Chinese medicine ginseng, which has good biocompatibility and low biotoxicity. Previous studies have reported its application in cardiovascular disease model experiments such as myocardial fibrosis, and it exerts its therapeutic function by downregulating the mechanism of oxidative stress. However, the application of Rb3 is still at the stage of systemic administration, and its targeting is poor. Summary of the invention

[0005] The purpose of the present invention is to provide an application of ginsenoside microbubbles combined with ultrasound in targeted local treatment of atherosclerotic plaques.

[0006] Another object of the present invention is to provide a method for preparing ginsenoside microbubbles, which specifically comprises the following steps: dissolving ginsenoside Rb3, DSPC and DSPE-PEG2000 in chloroform respectively, mixing the three in a thumb bottle at a molar ratio of 1.9 to 9.5:9:1, and then preparing ginsenoside liposomes Rb3NPs-Lipo by a thin film hydration method; filling the liposomes with inert gas through a gas replacement device to form a gas core, thereby constructing ginsenoside microbubbles Rb3NPs@MBs.

[0007] Furthermore, the molar ratio of ginsenoside Rb3, DSPC, and DSPE-PEG2000 is 1.9:9:1 or 3.8:9:1 or 9.5:9:1.

[0008] Furthermore, the particle size of the Rb3NPs-Lipo is 165.7nm±

[0009] 21.52nm~209.6nm±21.52nm.

[0010] Furthermore, the PDI coefficient of the Rb3NPs-Lipo is 0.1479-0.3766.

[0011] Furthermore, the particle size of the Rb3NPs@MBs is 1.70±0.036 μm.

[0012] Furthermore, the concentration of Rb3NPs@MBs is 2.1×10 9 Pieces / mL.

[0013] Compared with the prior art, the present invention has the following advantages and effects:

[0014] 1. Ultrasound, as a diagnostic and treatment technology for traditional vascular imaging and auxiliary plaque diagnosis, combined with drug microbubbles, has many advantages in the precise treatment of AS, such as economy, accuracy, and convenience.

[0015] 2. The present invention successfully designed and developed a ginsenoside microbubble Rb3NPs@MBs combined with ultrasound for targeted local treatment of atherosclerotic plaques. The plaque area was reduced by 41.84% compared with the model group. This combined method effectively reduced the atherosclerotic plaques in the aortic arch of targeted mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the production process of ginsenoside microbubbles (Rb3NPs@MBs) provided by the present invention;

[0017] Figure 2The results of using DLS to detect the three different molar ratios of ginsenoside liposomes (Rb3NPs-Lipo) prepared in Example 1 of the present invention;

[0018] Figure 3 The results of using PSS to detect the ginsenoside microbubbles (Rb3NPs@MBs) prepared in Example 3 of the present invention;

[0019] Figure 4 This is a schematic diagram of the process of treating AS mice with Rb3NPs@MBs+UTMD provided in Example 6 of the present invention;

[0020] Figure 5 This is a comparison chart of the therapeutic effects of Rb3NPs@MBs+UTMD and Rb3NPs provided in Example 6 of the present invention on the AS model group (Model) induced by high fat;

[0021] Figure 6 This is the HE staining and VCAM-1 staining of the aortic valve slice provided in Example 6 of the present invention. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and effect of the present application clearer and more specific, the present application is further described in detail below with reference to the accompanying drawings and examples.

[0023] Example 1

[0024] Preparation of ginsenoside liposomes (Rb3NPs-Lipo):

[0025] (1) Weighing and preparing clear solutions: Use an electronic balance to accurately weigh a certain amount of DSPC (Avanti, USA; CAS No.: 816-94-4) and DSPE-PEG2000 (Avanti, USA; CAS No.: 474922-77-5) powders. Under a fume hood, use chloroform to dissolve them into clear solutions with concentrations of 7.90 mg / mL and 29.92 mg / mL, respectively, for later use. Rb3 (Chengdu Pufeide Biotechnology Co., Ltd.; Catalog No. JOT-10260) was dissolved into 2 mg / mL, 4 mg / mL, and 10 mg / mL clear solutions with methanol:chloroform (1:1) for later use.

[0026] (2) Rotary evaporation: Take a 10 mL thumb bottle and add three lipid solutions (Rb3 500 μL, DSPC 272 μL, DSPE-PEG2000 30 μL) according to the molar ratio of Rb3: DSPC: DSPE-PEG2000 of 1.9:9:1; molar ratio of 3.8:9:1; molar ratio of 9.5:9:1; ultrasonic 5s (temperature 25°C) to make the lipids disperse evenly. Connect the thumb bottle to the rotary evaporator and seal the connection with a sealing film to prevent gas from entering; turn on the vacuum pump of the rotary evaporator to evacuate, adjust the pressure to 200 mbar, adjust the speed to 250-280 rpm, and keep the water bath heating pot at a constant temperature of 55°C; immerse the thumb bottle in the water bath and evaporate for 30-40 minutes. After the chloroform is completely evaporated, a thin and uniform white lipid film will form on the bottom of the thumb bottle, and the instrument will be turned off.

[0027] (3) Hydration: 1 mL of PBS (pH 7.2-7.6, the same below) was added to the thumb bottle and hydrated in an ultrasonic water bath at a temperature of 60-65°C. The lipid membrane began to dissolve and the solution became turbid. The hydration was continued for 5 min. The lipid membrane was completely dissolved and the lipids were redispersed under the action of ultrasound. The solution changed from turbid to clear. Three types of ginsenoside liposomes (Rb3NPs-Lipo) with a drug loading concentration of 1 mg / mL were obtained. The schematic diagram of the preparation process is shown in the figure. Figure 1 shown.

[0028] Example 2

[0029] Characterization of ginsenoside liposomes (Rb3NPs-Lipo):

[0030] The professional cuvette was cleaned with ddH2O and anhydrous ethanol and then dried with nitrogen. 990 μL of PBS solution was added, and 10 μL of the three Rb3NPs-Lipo solutions prepared in Example 1 were added respectively. After being mixed by pipetting, the particle size and polydispersity index (PDI) of Rb3NPs-Lipo were measured using a dynamic light scattering instrument (DLS, Malvern, UK, Lab Blue). The characterization results are shown in Figure 2. Figure 2 As shown in Table 1. The results showed that Rb3NPs-Lipo constructed with different molar ratios were all uniformly dispersed liposome solutions with consistent size.

[0031] The following examples all use a Rb3NPs-Lipo solution with a molar ratio of Rb3:DSPC:DSPE-PEG2000 of 1.9:9:1.

[0032] Table 1 Particle size and PDI of ginsenoside liposomes with three different molar ratios

[0033] Molar Ratio Particle size (nm) PDI 1.9：9:1 165.7±21.52 0.3766 3.8：9：1 209.6±21.52 0.1479 9.5：9：1 199.5±10.09 0.3106

[0034] Example 3

[0035] Preparation of ginsenoside microbubbles (Rb3NPs@MBs):

[0036] (1) Gas replacement: Transfer the Rb3NPs-Lipo solution (molar ratio of 1.9:9:1) into a vial and seal it. Then connect the vial to a vacuum pump. First, evacuate the vial for 5 minutes, and shake the vial left and right while evacuating the vial for 1 minute. After evacuating the vial, fill the vial with perfluoropropane (C3F8) gas, and shake the vial left and right for 1 minute to allow the gas and liquid to fully contact. Repeat this process three times.

[0037] (2) Shaking: The aerated vial was placed in a dental shaker (Navadha Enterprises, Italy, ADC18) and shaken for 45 seconds to obtain a Rb3NPs@MBs solution. The schematic diagram is shown in FIG. Figure 1 shown.

[0038] Example 4

[0039] Characterization of ginsenoside microbubbles (Rb3NPs@MBs):

[0040] Use a micropipette to take 10 μL of the Rb3NPs@MBs solution obtained in Example 3 and add it to the sample port of the particle counter (Partical Sizing System, PSS, AccuSizer 780AD). Measure the microbubble concentration and particle size. After the measurement, wash the counting bottle with ddH2O. Repeat the measurement three times for each sample and take the average value. The characterization method of Rb3NPs@MBs is the same as above. The characterization results are shown in Figure 3 The results show that the particle size of Rb3NPs@MBs is 1.70±0.036μm and the concentration is 2.1×10 9 The Rb3NPs@MBs were uniformly dispersed and of the same size, indicating that good Rb3NPs@MBs were successfully prepared.

[0041] Example 5

[0042] Preparation of AS model mice:

[0043] ApoE- / - mice and C57BL / 6J mice aged 6 weeks were purchased (Guangdong Yaokang) and raised in the animal room of Peking University / Hong Kong University of Science and Technology Medical Center in Shenzhen. The light cycle was 12h light / 12h dark, the temperature was (21±0.5)℃, and the humidity was (55±2)%. After receiving the mice, they were first fed with ordinary feed for one week and had free access to water. After one week, ApoE- / - mice were fed with high-fat feed (1.25% cholesterol) (Research DIETS, USA), and C57BL / 6J mice were fed with ordinary feed and had a normal diet, and were given free water. The feeding was continued for 12 weeks, and the body weight, blood lipids and aortic plaque formation of the mice were tested after 12 weeks.

[0044] The body weight of AS model mice ApoE- / - mice (hereinafter referred to as model group or Model group) was significantly increased compared with C57BL / 6J mice (hereinafter referred to as control group or Ctrl group) fed with the same period, and the blood lipid level and aortic plaques were significantly increased and increased. The relevant parameters are shown in Table 2.

[0045] Table 2 Particle size and PDI of ginsenoside liposomes with three different molar ratios

[0046]

[0047] Example 6

[0048] Application of ginsenosides and ginsenoside microbubbles in targeted therapy:

[0049] This example is divided into 4 groups: a control group (control), a model group (model), a ginsenoside group (Rb3NPs) and a ginsenoside microbubble combined with ultrasound group (Rb3NPs@MBs+UTMD).

[0050] Group 1: Control group (control):

[0051] C57BL / 6J mice were fed with ordinary diet for 12 weeks, and there were no plaques in the blood vessels. After the 14th week, the mice in the control group were killed, the aorta was completely dissected, and the arterial lipid components were stained to observe the plaque area (staining method and calculation method: the mouse aorta was taken, and the part of the aortic arch was cut after Oil Red O staining, and the proportion of the red plaque area to the total area was calculated using Image J, the same below), and the aortic valve was taken for sectioning and VCAM-1 histochemical staining to observe the expression of VCAM-1 factor (staining method and calculation method: a section of the aortic arch connected to the heart was cut off under a stereo microscope (Olympus, Japan, SZX2-ILLTQ) for tissue immunohistochemical detection. After the picture was exported, the area of ​​the positive area was counted using Image J software. The same below).

[0052] The second group of models:

[0053] After feeding ApoE- / - mice with a high-fat diet for 12 weeks, obvious plaques were seen in the blood vessels of the model mice. After the 14th week, the model group mice were killed, the aorta was completely dissected, the arterial lipid components were stained to observe the plaque area, and the aortic valve was taken for sectioning and VCAM-1 histochemical staining to observe the expression of VCAM-1 factor.

[0054] Group 3 Ginsenoside Group (Rb3NPs):

[0055] 1. Injection of ginsenosides: ApoE- / - mice were fed a high-fat diet for 12 weeks, and obvious plaques were seen in the blood vessels of the model mice. On the first day after the 12th week, 0.1 mL of Rb3 NPs (Rb3 was dissolved in saline to a clear solution with a concentration of 1 mg / mL) was injected into the AS model mice through the tail vein.

[0056] 2. Post-treatment: After 14 weeks, the mice in the model group were killed, the aorta was completely dissected, the arterial lipid components were stained to observe the plaque area, and the aortic valve was taken for sectioning and VCAM-1 histochemical staining to observe the expression of VCAM-1 factor.

[0057] Group 4: Ginsenoside microbubble combined with ultrasound group (Rb3NPs@MBs+UTMD):

[0058] 1. Injection of ginsenoside microbubbles: ApoE- / - mice were fed a high-fat diet for 12 weeks, and obvious plaques were seen in the blood vessels of the model mice. On the first day after the 12th week, 0.1 mL of Rb3NPs@MBs (concentration of 1 mg / mL) was injected into the AS model mice through the tail vein.

[0059] 2. Burst: After microbubble injection, the mouse aortic arch was immediately found by ultrasound, and targeted microbubble burst was immediately performed in the directional area (using the burst function of Vevo2100 small animal ultrasound machine, and selecting burst for burst when the microbubble entered the aortic arch through contrast mode), bursting once every 5-10 seconds for one minute. The treatment cycle was 2 weeks, and the treatment frequency was once every 3 days, for a total of 5 treatments.

[0060] 3. Post-blasting treatment: The model mice were killed after the treatment, the aorta was completely dissected, the arterial lipid components were stained to observe the plaque area, and the aortic valve was taken for sectioning and VCAM-1 histochemical staining to observe the expression of VCAM-1 factor.

[0061] Schematic diagram of the treatment process of AS mice with Rb3NPs@MBs+UTMD group Figure 4 shown.

[0062] The results of the 4 groups are as follows Figure 5As shown, the plaque area of ​​the model group in group 2 was 41.66%, the plaque area of ​​the Rb3NPs group in group 3 was 36.50%, and the plaque area of ​​the Rb3NPs@MBs+UTMD group in group 4 was 24.23%. Figure 5 The results showed that the plaques in the aortic arch of the fourth group Rb3NPs@MBs+UTMD group were significantly reduced, which was 41.84% less than that of the model group, while the plaques in the aortic arch of the third group Rb3NPs group without targeted treatment were not significantly reduced, which was only 12.4% less than that of the model group.

[0063] The aortic valve sections in the above four experimental groups were further stained with HE and VCAM-1. Figure 6 As shown, Figure 6 The results showed that the lipid infiltration of aortic valve plaques in the Rb3NPs@MBs+UTMD targeted treatment group was significantly reduced compared with the Rb3NPs group, and the expression of VCAM-1 was decreased.

[0064] The above results demonstrate that the combined application of ginsenoside microbubbles and ultrasound effectively reduces atherosclerotic plaques in the aortic arch of targeted mice.

Claims

1. An application of ginsenoside microbubbles combined with ultrasound in the targeted local treatment of atherosclerotic plaques.

2. The method for preparing ginsenoside microbubbles according to claim 1, characterized in that: Ginsenoside Rb3, DSPC and DSPE-PEG2000 were dissolved in chloroform respectively, and the three were mixed in a thumb bottle at a molar ratio of 1.9-9.5:9:

1. Then, ginsenoside liposomes Rb3NPs-Lipo were prepared by thin film hydration method. Inert gas was filled into the liposomes through a gas replacement device to form a gas core to construct ginsenoside microbubbles Rb3NPs@MBs.

3. The method for preparing ginsenoside microbubbles according to claim 2, characterized in that: The molar ratio of the ginsenoside Rb3, DSPC and DSPE-PEG2000 is 1.9:9:

1.

4. The method for preparing ginsenoside microbubbles according to claim 2, characterized in that: The molar ratio of the ginsenoside Rb3, DSPC and DSPE-PEG2000 is 3.8:9:

1.

5. The method for preparing ginsenoside microbubbles according to claim 2, characterized in that: The molar ratio of the ginsenoside Rb3, DSPC and DSPE-PEG2000 is 9.5:9:

1.

6. The method for preparing ginsenoside microbubbles according to claim 2, characterized in that: The inert gas is perfluoropropane.

7. The method for preparing ginsenoside microbubbles according to claim 2, characterized in that: The particle size of the Rb3NPs-Lipo is 165.7nm±21.52nm~209.6nm±21.52nm.

8. The method for preparing ginsenoside microbubbles according to claim 2, characterized in that: The PDI coefficient of the Rb3NPs-Lipo is 0.1479-0.3766.

9. The method for preparing ginsenoside microbubbles according to claim 2, characterized in that: The particle size of the Rb3NPs@MBs is 1.70±0.036μm.

10. The method for preparing ginsenoside microbubbles according to claim 2, characterized in that: The concentration of Rb3NPs@MBs is 2.1×10 9 Pieces / mL.

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