CN113975245B - A kind of preparation method of biomimetic nano drug delivery system based on ginsenoside - Google Patents

Abstract

The invention belongs to the field of nano-drugs, and particularly relates to a preparation method of a bionic nano-drug delivery system based on ginsenoside. The invention takes nano-selenium with the functions of resisting oxidation and reducing blood fat as a drug carrier, delivers drug ginsenoside with the functions of promoting lipid metabolism and resisting inflammation, and simultaneously prepares a bionic nano drug delivery system with low toxicity, good biocompatibility and capability of targeting a focus part by combining the inherent plaque targeting capability of a platelet membrane.

Description

A kind of preparation method of biomimetic nano drug delivery system based on ginsenoside

technical field

The invention belongs to the field of nano-drugs, and particularly relates to a preparation method of a biomimetic nano-drug delivery system based on ginsenosides.

Background technique

Cardiovascular disease is the "number one killer" that threatens national health. The main causes of cardiovascular disease are as follows: hypertension, smoking, abnormal serum lipids, diabetes, obesity, insufficient physical activity, air pollution, etc. These factors eventually lead to heart disease through the common pathological basis, atherosclerosis. The occurrence of vascular disease. At present, the main strategy of drugs for the treatment of atherosclerosis is through lipid regulation and anti-inflammatory, such as statins, which have the effects of reducing lipid levels, inhibiting plaque inflammation, and anti-oxidation; platelet inhibitor drugs for advanced AS aspirin and clopidogrel. Although these drugs have been proved to have a certain improvement effect through clinical practice, these drugs generally have the treatment defects of low bioavailability, poor targeting, and large toxic and side effects. Therefore, there is an urgent need to find a drug with low toxicity, good biocompatibility, and can target the lesion site to solve the above problems.

Ginseng is a widely used traditional Chinese herbal medicine that has been in use for over 2000 years and is widely used in Korea, China and Japan. Ginsenoside is the main extract of ginseng, and it is also the key component of ginseng to exert its medicinal effect. Studies have shown that ginsenoside can improve endothelial dysfunction and vascular inflammation. At the same time, it can reduce the accumulation of lipids in macrophage-derived foam cells and enhance the The stability of AS plaques indicates that ginsenosides have certain potential in the treatment of atherosclerotic diseases, but the poor water solubility and low bioavailability of ginsenosides limit their clinical application. At present, there are only few studies on the application of ginsenoside nanometers to disease treatment. For example, Chinese patent document CN103271891A discloses a ginsenoside nanomicelle and its preparation method, application and pharmaceutical composition. The ginsenoside nanomicelle is used as a fat-soluble compound. It can be used as a co-solvent or as a pharmaceutical carrier, replacing the existing pharmaceutical carriers and co-solvents. Although nano-sized ginsenosides can improve the efficacy and bioavailability, the types and contents of the main active components of total ginsenosides are affected by various factors such as species, harvesting, and extraction processes. The ginsenoside monomer is beneficial to the follow-up molecular mechanism research.

It is necessary to develop nanomedicines that can reduce immune rejection, better biocompatibility and safer.

Based on the existing nanotechnology status, the present invention intends to provide a biomimetic nano-drug delivery system based on ginsenosides. In the drug delivery system of the present invention, ginsenosides and nano-selenium are co-assembled into a nano-core, and the surface is coated with a platelet membrane. The biomimetic nano-drug delivery system has excellent small size and biocompatibility, and can deliver drugs to the atherosclerotic lesions in a targeted manner, improve the drug concentration and therapeutic effect at the plaque site, and reduce the side effects of the drugs. application prospects.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the deficiencies of existing cardiovascular drugs and preparations, and to provide a biomimetic nano-drug delivery system with enhanced therapeutic effect. The system has excellent particle size and biocompatibility, and can enable targeted delivery of drugs to plaques. It can improve the therapeutic effect of atherosclerosis and reduce the toxic and side effects.

In order to achieve the above object, a technical scheme adopted by a biomimetic nano-drug delivery system based on ginsenosides comprises the following steps:

(1) Preparation of selenium nanoparticles;

(2) Preparation of ginsenoside/selenium nanoparticles;

(3) Using platelet membrane to coat ginsenoside/selenium nanoparticles.

Further, the described method for preparing selenium nanometers is: adding a 1% stabilizer Pluronic F-127 aqueous solution to a small beaker, under constant stirring, dropwise add the glutathione aqueous solution, and then drip Add sodium selenite aqueous solution, adjust the pH of the reaction solution to weak alkaline (pH=7.4~8.5) with sodium hydroxide, the reaction solution can be seen from colorless to orange, indicating the formation of selenium nanoparticles;

The preparation method of the ginsenoside/selenium nanoparticles is as follows: adding ginsenoside methanol solution to the selenium nano solution, stirring for 6 hours, until the methanol is completely volatilized. The solution obtained by the reaction was put into a dialysis bag with a molecular weight of 3500 (MWCO: 3500 Da), and the residual organic solvent was removed by dialysis for 24 h. After dialysis, the liquid was placed in an ultrafiltration tube with a MWCO of 10,000 Da, and the nano-solution was concentrated by centrifuging at 3,000 rpm for 20 min to obtain a ginsenoside/selenium nanoparticle solution.

Still further, the preparation method of the platelet membrane-coated ginsenoside/selenium nanoparticle solution is as follows: the ginsenoside/selenium nanoparticle solution and the platelet membrane are mixed with equal volumes of ultrasound for 5 minutes and then stirred overnight to obtain the biomimetic nano-drug delivery. system.

The platelet membranes used were derived from the blood of healthy rats, which were purified by centrifugation and repeatedly freeze-thawed.

Compared with the prior art, the beneficial effects of the present invention are:

(1) Selenium nanoparticles are prepared by a simple redox method, which not only has excellent particle size and specific surface area, but also has a good anti-oxidative therapeutic effect.

(2) Nanoparticles prepared by co-assembly of ginsenosides and selenium nanoparticles greatly improved the bioavailability of ginsenosides in clinical applications, and the composite nanoparticles could more effectively inhibit cell adhesion.

(3) The encapsulation of platelet membrane can not only reduce the phagocytosis of macrophages, but also can bring the synthesized nanoparticles to the atherosclerotic lesions, and realize the efficient accumulation of nanoparticles in the plaque. The present invention exhibits low toxicity, improved carrier biocompatibility, and aggregation at plaque sites, and can be used for drug delivery at atherosclerotic lesions.

Description of drawings

Fig. 1 is the particle size potential distribution diagram of platelet membrane-encapsulated nano-groups;

Figure 2 is an atomic force image of platelet membrane-encapsulated nanogroups;

Figure 3 is the cell adhesion of platelet membrane-encapsulated nanogroups;

Figure 4 shows the proportion of plaques in ApoE-/- mice atherosclerotic by platelet membrane-encapsulated nanogroups.

Detailed ways

The technical solutions of the present invention will be further described according to the specific embodiments, but it should not be understood that the scope of the above-mentioned subject matter of the present invention is limited to the following examples. Without departing from the above-mentioned technical idea of the present invention, various substitutions and changes can be made according to the common technical knowledge and conventional means in the field, which should be included in the scope of the present invention.

Example 1

Preparation of a biomimetic nano-drug delivery system based on ginsenosides:

A: Preparation of ginsenoside/selenium nanoparticles (Se/Rb1 NPs): 3 mL of 1% F-127 stock solution was added to a 10 mL small beaker, and 100 μL of glutathione was added dropwise with constant stirring (40 mM) aqueous solution, followed by dropwise addition of 100 μL of sodium selenite (10 mM) aqueous solution, the reaction solution was adjusted to weakly alkaline (pH=7.4) with sodium hydroxide, and the reaction solution changed from colorless to orange to the naked eye, Indicating the formation of nano-selenium (Se NPs), 500 μL of ginsenoside Rb1 methanol solution (10 mM) was added, and stirring was continued for 6 h until the methanol evaporated completely. The solution obtained by the reaction was put into a dialysis bag with a molecular weight of 3500 (MWCO: 3500 Da), and the residual organic solvent was removed by dialysis for 24 h. After the dialysis, the liquid was placed in an ultrafiltration tube with a MWCO of 10,000 Da, centrifuged at 3,000 rpm for 20 min to concentrate the nano-solution to obtain Se/Rb1 NPs.

B: Preparation of platelet membrane (PM): Take the blood of normal rats, put it at room temperature for 10 minutes, centrifuge at 100 g for 20 minutes at 25°C, and the upper pale yellow serum is the required platelets; Centrifuge at ℃ for 20 min to remove the sediment at the bottom of the EP tube; add PBS (pH 7.4) containing 1 mM EDTA to the removed supernatant, wash with gentle pipetting, centrifuge at 800 g for 20 min at 25 ℃, remove the supernatant. Resuspend the platelet pellet in PBS containing 1 mM EDTA, add protease inhibitors, freeze in a -80 °C refrigerator, and then thaw at room temperature, after repeated freezing and thawing 4-5 times, 4000 g, 4 Centrifuge at ℃ for 3 min to get the pellet, wash 2-3 times with PBS containing protease inhibitor, and resuspend in ddH ₂ O to obtain platelet membrane (PM).

C: Preparation of biomimetic ginsenoside/selenium nanoparticles: Mix an equal volume of platelet membrane with the ginsenoside/selenium nanosolution obtained in step A, sonicate for 5 min, and continue stirring overnight to obtain PM@Se/Rb1 NPs.

Example 2

Characterization of a ginsenoside-based biomimetic nano-drug delivery system:

The particle size distribution and potential of PM, Se/Rb1 NPs and PM@Se/Rb1 NPs were measured by Malvern particle size analyzer. The morphology of the final PM@Se/Rb1 NPs was observed by atomic force microscope. The nano-solution was dropped on the center of the mica sheet, the mica sheet was blown dry with nitrogen, washed with ddH ₂ O for several times, and finally photographed by atomic force microscope.

The results are shown in Fig. 1. The particle size of the untreated platelet itself is greater than 3000 nm, and the particle size of the Se/Rb1 NPs is 51.5 ± 1.1 nm. 1.4 nm, indicating that the thickness of PM wrapping on the surface of Se/Rb1 NPs is about 7 nm. Meanwhile, the potential data showed that the potential of Se/Rb1NPs decreased from -4.6 ± 0.4 mV to -16.4 ± 1.1 mV after being encapsulated by PM. The atomic force microscope in Fig. 2 shows that the PM@Se/Rb1 NPs have a relatively uniform distribution, and the overall shape is oblate spherical with a diameter of about 60 nm, which is consistent with the particle size measured by the particle size analyzer.

Example 3

Cell adhesion of a ginsenoside-based biomimetic nano-drug delivery system:

A: Investigate the adhesion of drugs to HUVEC and Matrigel: Spread 200 μL of Matrigel diluted with unprepared DMEM medium in a 24-well plate, and put it in an incubator to completely solidify. Then, the non-solidified Matrigel was discarded and blocked with 1% BSA for 1 h. Discard blocking solution and wash 3 times with PBS. After digestion of HUVECs stimulated with TNF-α at a concentration of 10 ng/mL for 4 h, they were incubated with Rhodamine 123, a reagent that can stain whole cells, for 0.5 h in a dark incubator. The HUVEC cell resuspension containing different drugs was sucked into the well plate and incubated for 45 min, and the blank medium was used as the control. After washing the non-adherent cells with PBS, 4% paraformaldehyde was added to fix the cells, and the adhesion between HUVEC and Matrigel was photographed with a fluorescence microscope, and 20 fields of view were randomly selected for each well.

As shown in A and C in Fig. 3, Rb1 has the weakest inhibitory effect on the adhesion between HUVEC and Matrigel, with an inhibition rate of only 10%, while that of Se NPs is 35%, and that of PM@Se/Rb1 NPs The strongest, reaching 43%.

B: Investigate the adhesion of the drug to HUVEC and U937 cells: take the HUVEC grown to 70-80% after digestion, 2×10 ⁵ cells/mL, spread into 24-well plate, 0.5 mL per well, and culture overnight. The cells were stimulated with TNF-α at a concentration of 10 ng/mL for 4 h, and U937 cells were suspended in culture medium containing the fluorescent dye Rhodamine 123 for 30 min, centrifuged at 1500 rpm for 5 min, and the pellet was retained. The U937 cells were resuspended in the medium of PM@Se/Rb1 NPs. The blank medium was used as the control group, and was added to the HUVECs washed with PBS. After interaction for 45 min, 4% paraformaldehyde was added to fix the cells, and U937 was photographed under a microscope. Photo of adhesion between HUVEC and HUVEC cells. 20 fields of view per well were taken to calculate the average adhesion rate, the number of adhered cells was output by Image J software, and the final adhesion rate was calculated by the following formula: relative adhesion rate (%) = (sample group adhesion The number of cells/the number of adherent cells in the control group) × 100.

As shown in B and D in Fig. 3, the inhibition rates of Rb1, Se NPs and PM@Se/Rb1 NPs on the adhesion between HUVEC and U937 were 35%, 70% and 85%, respectively, and PM@Se/Rb1 NPs inhibited The strongest adhesion was between HUVEC and U937.

Example 4

A ginsenoside-based biomimetic nano-drug delivery system affects the percentage of atherosclerotic plaque formation area in ApoE-/- mice in the whole arterial area:

Modeling: The animals used were ApoE knockout C57 mice, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. and kept in a common animal room with natural circadian rhythm (12:12), room temperature 25 ± 1 ℃, give sufficient food and water, and change the bedding every 3 days.

Grouping and administration method: 30 male ApoE-/- mice were fed with high-fat diet, and the composition of high-fat diet was: basic material 83.3%, 10% fat, 5% sucrose, 1% cholesterol, 0.5% sodium cholate , 0.2% propylthiouracil. After two months of high-fat diet, 30 ApoE-/- mice were equally divided into the following groups: normal diet (ND), high-fat diet (HFD), Rb1 alone group, Se NPs group, Se/Rb1 NPs group, PM@Se/Rb1 NPs group. The ND group was used as the negative control group (ND control), and the HFD group was used as the positive control group (HFD control). The drug concentration of Rb1 in the Rb1 single administration group, the Se/Rb1 NPs group and the PM@Se/Rb1 NPs group was 5 mg/kg, administered by tail vein injection, once every 3 days, and the treatment cycle was one month. The scheme is shown in A in Figure 4.

Example 5

Oil red O staining of the aorta: isolate the aorta, put the aorta in 4% paraformaldehyde, remove the tissue around the aorta as much as possible, cut the blood vessel longitudinally, and prepare the oil red O staining solution, the aorta was roughly placed in 60% isopropanol for 10 min for differentiation, transferred to Oil Red O dye for 2 h, differentiated with 70% ethanol for several times, and the excess dye was removed to see the aorta. It can be roughly turned into white, and finally observe the oil red O staining with a stereo microscope and take pictures. The area of the plaques stained with Oil Red O on the aorta was quantified using Image J software.

As shown in B in Figure 4, the average plaque area of the aorta of mice treated with ND control group, HFD control group, Rb1, Se NPs, Se/Rb1 NPs, PM@Se/Rb1 NPs group was: 6.5%, 38.8% , 28.1%, 32.6%, 24.9%, 12.7%. The HFD control group showed more obvious Oil Red O staining area than the normal feeding group. There was no significant difference between the free Rb1 and Se NPs group compared with the HFD control group, and there was no obvious treatment effect. In contrast, the Se/Rb1 NPs and PM@Se/Rb1 NPs groups observed different degrees of reduction in the Oil Red O staining area in the aorta, and the PM@Se/Rb1NPs group had the least Oil Red O staining area, indicating that It has a better effect on reducing the formation of aortic plaque.

To sum up, in the above examples, selenium nanoparticles were used as carriers to co-assemble ginsenoside/selenium nanoparticles with ginsenoside Rb1, and a novel biomimetic nanocarrier targeting atherosclerotic plaques was constructed using the native platelet membrane. system, in which the inherent natural targeting effect of platelet membrane can increase the drug concentration at the lesion site, and at the same time, the particle size of the biomimetic ginsenoside/selenium nanosystem is extremely small, which can well penetrate the biological membrane and blood vessel wall, and prolong the time in the blood. cycle time, so as to play a better therapeutic effect. Based on the above reasons, the nanosystems prepared in the above examples have low toxicity, high targeting, strong penetration and good biocompatibility, and can enrich the drug concentration at the site of atherosclerotic plaques. Reducing the dosage and reducing the toxic and side effects provides a new and effective way for the prevention and treatment of atherosclerosis at the lesion site.

The above-mentioned embodiment is a preferred implementation scheme of the present invention. In addition, the present invention can also be implemented in other ways, and any obvious replacements are within the protection scope of the present invention without departing from the concept of the technical solution.

Claims (2)

1. A preparation method of a bionic nano drug delivery system based on ginsenoside is characterized in that: the bionic nano drug delivery system consists of a ginsenoside/selenium nano particle skeleton inner core and a platelet membrane coated on the surface of the ginsenoside/selenium nano particle skeleton inner core;

the platelet membrane is derived from blood of healthy rat, and is prepared by centrifugal purification and repeated freeze thawing;

the preparation method of the bionic nano drug delivery system based on the ginsenoside specifically comprises the following steps:

A. preparing the ginsenoside/selenium nanoparticles: dropwise adding a glutathione aqueous solution into an aqueous solution of a stabilizer Plannik F-127, then dropwise adding a sodium selenite aqueous solution, adjusting the pH of a reaction solution to be alkalescent by using sodium hydroxide, changing the reaction solution from colorless to orange to show the generation of selenium nano, then adding a ginsenoside methanol solution, continuously stirring for 6 hours until the methanol is completely volatilized, filling the solution obtained by the reaction into a dialysis bag with an MWCO of 3500 Da, dialyzing for 24 hours to remove residual organic solvent, after the dialysis is finished, filling the solution into an ultrafiltration tube with the MWCO of 10000 Da, and concentrating the nano solution by a centrifuge at 3000 rpm for 20 minutes to obtain a ginsenoside/selenium nano particle solution;

B. preparing bionic ginsenoside/selenium nanoparticles: uniformly mixing the platelet membrane with the ginsenoside/selenium nanoparticle solution with the same volume, performing ultrasonic treatment for 5 min, and continuously stirring overnight to obtain the final platelet membrane-coated ginsenoside/selenium nanoparticle, namely the bionic nano drug delivery system.

2. The preparation method of the biomimetic nano drug delivery system based on ginsenoside according to claim 1, wherein the preparation method comprises the following steps: the average grain diameter of the ginsenoside/selenium nanoparticles coated by the platelet membrane is 50-100 nm.

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