CN113975245A

CN113975245A - Preparation method of bionic nano drug delivery system based on ginsenoside

Info

Publication number: CN113975245A
Application number: CN202111242030.XA
Authority: CN
Inventors: 邵敬伟; 尹梦蝶; 林娟芳
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2021-10-25
Filing date: 2021-10-25
Publication date: 2022-01-28
Anticipated expiration: 2041-10-25
Also published as: CN113975245B

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

Preparation method of bionic 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 bionic nano-drug delivery system based on ginsenoside.

Background

Cardiovascular diseases are the first killers threatening the national health. The main causes of cardiovascular diseases are the following: hypertension, smoking, serum lipid disorders, diabetes, obesity, physical inactivity, atmospheric pollution, etc., which ultimately lead to the development of cardiovascular disease through a common pathological basis, i.e., atherosclerosis. The main strategies of the current drugs for treating atherosclerosis are through lipid regulation and anti-inflammation, such as statins with the effects of reducing lipid level, inhibiting plaque inflammation, and resisting oxidation; used for platelet inhibitor drugs such AS aspirin and clopidogrel in the advanced AS. Although clinical practices prove that the medicines have certain improvement effect, the medicines generally have the treatment defects of low bioavailability, poor targeting property and large toxic and side effect. Therefore, there is an urgent need to find a drug with low toxicity and good biocompatibility, which can target the focal site to solve the above problems.

Ginseng, a widely used traditional Chinese medicinal material, has been used for over 2000 years and is widely used in korea, china and japan. The ginsenoside is a main extract of the ginseng and is also a key component of the ginseng playing the drug effect, and researches show that the ginsenoside can improve endothelial dysfunction and vasculitis, and simultaneously can reduce the accumulation of lipid in macrophage-derived foam cells and enhance the stability of AS plaques, so that the ginsenoside has certain potential in the aspect of treating atherosclerosis, but the ginsenoside has poor water solubility and low bioavailability, thereby limiting the clinical application of the ginsenoside. Currently, the application of ginsenoside nano-micelle to disease treatment has only a few researches, for example, chinese patent document CN103271891A discloses a ginsenoside nano-micelle, a preparation method, an application and a pharmaceutical composition thereof, the ginsenoside nano-micelle is used as a cosolvent of a fat-soluble compound or as a pharmaceutical carrier, and replaces the existing pharmaceutical carriers and cosolvents. Although the realization of the nanocrystallization of the ginsenoside can improve the drug effect and the bioavailability, the types and the contents of the main effective components of the ginsenoside are influenced by various factors such as species, harvesting, extraction process and the like. The ginsenoside monomer is beneficial to the subsequent molecular mechanism research.

It is necessary to develop nano-drugs that can reduce immune rejection, have better biocompatibility and are safer.

Based on the current state of nanometer technology, the invention provides a bionic nanometer drug delivery system based on ginsenoside. The bionic nano drug delivery system has excellent small size and biocompatibility, can deliver the drug to the atherosclerotic focus part in a targeted manner, improves the drug concentration and the treatment effect of the plaque part, reduces the side effect generated by the drug, and has wide application prospect.

Disclosure of Invention

The invention aims to overcome the defects of the existing cardiovascular drugs and preparations and provide a bionic nano drug delivery system for enhancing the treatment effect, the system has excellent particle size and biocompatibility, and can deliver the drugs to plaque parts in a targeted manner, improve the treatment effect of atherosclerosis and reduce toxic and side effects.

In order to realize the aim, the technical scheme adopted by the bionic nano drug delivery system based on the ginsenoside comprises the following steps:

(1) preparing selenium nano-particles;

(2) preparing ginsenoside/selenium nanoparticles;

(3) the ginsenoside/selenium nanoparticles are coated by platelet membrane.

Further, the method for preparing the selenium nano-meter comprises the following steps: adding 1% stabilizer Pluronic F-127 aqueous solution into a small beaker, dropwise adding glutathione aqueous solution under the condition of continuously stirring, then dropwise adding sodium selenite aqueous solution, adjusting the pH of the reaction solution to be alkalescent (pH = 7.4-8.5) by using sodium hydroxide, and changing the reaction solution from colorless to orange by naked eyes to indicate the generation of selenium nano;

the preparation method of the ginsenoside/selenium nanoparticles comprises the following steps: adding ginsenoside methanol solution into selenium nanometer solution, stirring for 6 hr until methanol is completely volatilized. The solution obtained by the reaction was filled into a dialysis bag having a molecular weight of 3500 (MWCO: 3500 Da) and dialyzed for 24 hours to remove the residual organic solvent. After dialysis, the liquid is filled into an ultrafiltration tube with MWCO of 10000 Da, and the nano solution is concentrated by a centrifuge at 3000 rpm for 20 min, thus obtaining the ginsenoside/selenium nano particle solution.

Still further, the preparation method of the platelet membrane coated ginsenoside/selenium nanoparticles comprises the following steps: and mixing the ginsenoside/selenium nanoparticle solution and the platelet membrane in equal volume, performing ultrasonic treatment for 5 minutes, and stirring overnight to obtain the bionic nano drug delivery system.

The platelet membrane is derived from blood of healthy rat, and is prepared by centrifuging, purifying and repeatedly freezing and thawing.

Compared with the prior art, the invention has the beneficial effects that:

(1) the selenium nanometer is prepared by a simple oxidation-reduction method, has excellent particle size and specific surface area, and has good antioxidant treatment effect.

(2) The nano-particles prepared by co-assembling ginsenoside and selenium nano-particles greatly improve the bioavailability of ginsenoside in clinical application, and simultaneously, the composite nano-particles can more effectively inhibit cell adhesion.

(3) The platelet membrane is wrapped, so that phagocytosis of macrophages can be reduced, the synthesized nanoparticles can be brought to the atherosclerotic lesion site, and the nanoparticles can be efficiently accumulated at the plaque site. The invention exhibits low toxicity, improved carrier biocompatibility and aggregation at plaque sites, and can be used for drug delivery at atherosclerotic lesion sites.

Drawings

FIG. 1 is a graph of the particle size potential of a platelet membrane-encapsulated nano-population;

FIG. 2 is an atomic force image of a platelet membrane-wrapped nano-group;

FIG. 3 is cell adhesion of platelet membrane-wrapped nanoarrays;

FIG. 4 is the plaque proportion of platelet membrane-encapsulated subgroups on ApoE-/-mouse atherosclerosis.

Detailed Description

The technical solutions of the present invention are further described according to specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples. Various alterations and modifications can be made without departing from the technical idea of the invention, and all changes and modifications made by the ordinary skill in the art can be made without departing from the scope of the invention.

Example 1

A bionic nanometer drug delivery system based on ginsenoside is prepared by the following steps:

a: preparation of ginsenoside/selenium nanoparticles (Se/Rb 1 NPs): adding 3 mL of 1% F-127 mother liquor into a 10 mL small beaker, dropwise adding 100 μ L of glutathione (40 mM) aqueous solution under the condition of continuous stirring, then dropwise adding 100 μ L of sodium selenite (10 mM) aqueous solution, adjusting the reaction solution to be alkalescent (pH = 7.4) by using sodium hydroxide, changing the reaction solution from colorless to orange by naked eyes to indicate the generation of nano selenium (Se NPs), then adding 500 μ L of ginsenoside Rb1 methanol solution (10 mM), and continuing stirring for 6 h until the methanol is completely volatilized. The solution obtained by the reaction was filled into a dialysis bag having a molecular weight of 3500 (MWCO: 3500 Da) and dialyzed for 24 hours to remove the residual organic solvent. After the dialysis is finished, the liquid is filled into an ultrafiltration tube with MWCO of 10000 Da, and the nano solution is concentrated by a centrifuge at 3000 rpm for 20 min to obtain Se/Rb1 NPs.

B: preparation of Platelet Membrane (PM): collecting blood of normal rat, and standing at room temperatureCentrifuging at 25 deg.C for 20 min at 100 g after 10 min to obtain yellowish upper serum as the desired platelet; then centrifuging for 20 min at 25 ℃ by 100 g again, and removing the sediment at the bottom of the EP tube; adding PBS (pH 7.4) containing 1 mM EDTA into the supernatant, gently blowing, beating and washing, centrifuging at 25 deg.C for 20 min, and removing the supernatant; suspending the platelet precipitate in PBS containing 1 mM EDTA, adding protease inhibitor, freezing at-80 deg.C, thawing at room temperature, repeatedly freezing and thawing for 4-5 times, centrifuging at 4000 g and 4 deg.C for 3 min to obtain precipitate, washing with PBS containing protease inhibitor for 2-3 times, and suspending in ddH₂And O, obtaining the Platelet Membrane (PM).

C: preparing bionic ginsenoside/selenium nanoparticles: and D, uniformly mixing the platelet membrane with the same volume with the ginsenoside/selenium nano solution obtained in the step A, performing ultrasonic treatment for 5 min, and continuously stirring overnight to obtain PM @ Se/Rb1 NPs.

Example 2

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

the particle size distribution and potential size of PM, Se/Rb1 NPs and PM @ Se/Rb1 NPs are respectively measured by a Malvern particle sizer. Observing the final form of PM @ Se/Rb1 NPs by adopting an atomic force microscope, dripping the nano solution into the center of a mica sheet, drying the mica sheet by using nitrogen, and then using ddH₂And (4) performing O-drip washing for multiple times, and finally performing shooting by using an atomic force microscope.

As a result, as shown in FIG. 1, the particle size of untreated platelets per Se was larger than 3000 nm, the particle size of Se/Rb1 NPs was 51.5. + -. 1.1 nm, and the particle size of PM @ Se/Rb1 NPs after cell membrane coating was increased to 58.7. + -. 1.4 nm, indicating that the thickness of PM coated on the surface of Se/Rb1 NPs was about 7 nm. Meanwhile, the data of the electric potential show that the electric potential of Se/Rb1 NPs is reduced from-4.6 +/-0.4 mV to-16.4 +/-1.1 mV after being wrapped by PM. The atomic force microscope in FIG. 2 shows that the PM @ Se/Rb1 NPs are distributed uniformly, are oblate spheres as a whole, have a diameter of about 60 nm, and are consistent with the particle size measured by a particle size analyzer.

Example 3

Cell adhesion of a biomimetic nano drug delivery system based on ginsenosides:

study of drug adhesion to HUVEC and matrigel: 200. mu.L of Matrigel diluted one-fold with unformulated DMEM medium was spread in a 24-well plate and placed in an incubator to be completely coagulated. The unsolidified Matrigel was then discarded and blocked with 1% BSA for 1 h. The blocking solution was discarded and washed 3 times with PBS. After HUVEC digestion after 4 h stimulation with TNF- α at a concentration of 10 ng/mL, the cells were incubated for 0.5 h in a dark incubator with rhodamine 123 reagent which allowed staining of whole cells. The HUVEC cell resuspension containing the different drugs was pipetted into the well plate and incubated for an additional 45 min with a blank medium control. After washing the nonadherent 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 were randomly selected for each well.

As shown in A and C in FIG. 3, Rb1 has the weakest inhibition effect on the adhesion between HUVEC and Matrigel, and the inhibition rate is only 10%, while the inhibition effect of Se NPs is 35%, and the inhibition effect of PM @ Se/Rb1 NPs is the strongest and reaches 43%.

Investigation of drug adhesion to HUVEC and U937 cells: after digestion of 70-80% HUVEC, 2X 10⁵cells/mL, plated in 24-well plates at 0.5 mL per well, and incubated overnight. Stimulating for 4 h by TNF-alpha with the concentration of 10 ng/mL, after incubating suspension cell U937 cells for 30 min by a culture medium containing rhodamine 123 serving as a fluorescent dye, centrifuging for 5 min at 1500 rpm, keeping precipitates, re-suspending the U937 cells by a culture medium containing Rb1, Se NPs and PM @ Se/Rb1 NPs, taking a blank culture medium as a control group, adding the control group into HUVEC washed by PBS, after interacting for 45 min, adding 4% paraformaldehyde to fix the cells, and taking a picture of adhesion between the U937 cells and the HUVEC cells under a microscope. The average adhesion rate was calculated by taking 20 fields per well, outputting the number of adhered cells by Image J software, and the final adhesion rate was obtained by the following calculation formula: relative adhesion rate (%) = (number of adherent cells in sample group/number of adherent cells in control group) × 100.

As shown in B and D in FIG. 3, Rb1, Se NPs and PM @ Se/Rb1 NPs inhibited the adhesion between HUVEC and U937 by 35%, 70% and 85%, respectively, and PM @ Se/Rb1 NPs inhibited the adhesion between HUVEC and U937 most strongly.

Example 4

A bionic nano drug delivery system based on ginsenoside has the following percentage of the area of formed ApoE-/-mouse atherosclerotic plaque to the whole artery:

modeling: the animals used were C57 mice with ApoE gene knockout, purchased from Beijing Wintonlihua laboratory animal technology Co., Ltd, raised in a common animal house, given sufficient food and water at a temperature of 25. + -. 1 ℃ in natural circadian rhythm (12: 12), and bedding was changed every 3 days.

Grouping and administration modes: 30 male ApoE-/-mice were fed a high fat diet consisting of: 83.3 percent of base material, 10 percent of fat, 5 percent of cane sugar, 1 percent of cholesterol, 0.5 percent of sodium cholate and 0.2 percent of propylthiouracil. After two months of high fat diet feeding, 30 ApoE-/-mice were equally divided into the following groups: normal feeding group (Normal diet, ND), High-fat feeding group (High-fat diet, HFD), Rb1 single-administration group, Se NPs group, Se/Rb1 NPs group, PM @ Se/Rb1 NPs group. The ND group was used as a negative control group (ND control), and the HFD group was used as a positive control group (HFD control). The drug Rb1 in the Rb1 single administration group, the Se/Rb1 NPs group and the PM @ Se/Rb1 NPs group has the administration concentration of 5 mg/kg, is administered by tail vein injection once in 3 days, and has the treatment period of one month, and the specific embodiment is shown as A in figure 4.

Example 5

Aorta gross oil red O staining: separating out a large aorta, placing the large aorta in 4% paraformaldehyde, removing tissues at the periphery of the large aorta as much as possible, longitudinally cutting a blood vessel, preparing a ready-prepared oil red O staining solution, placing the large aorta in 60% isopropanol for differentiation for 10 min, transferring the large aorta into an oil red O dye for staining for 2 h, differentiating the large aorta with 70% ethanol for multiple times, removing the redundant dye, and observing the dyeing condition of the oil red O with a body type microscope and taking a picture. The area of the plaque stained for oil red O on the aorta was quantified using Image J software.

As shown in B in FIG. 4, the mean aortic plaque area of mice treated with the 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 a more pronounced oil red O staining area than the normal feeding group. The free Rb1 and Se NPs group has no obvious difference compared with the HFD control group, and no obvious treatment effect is seen. In contrast, the Se/Rb1 NPs and PM @ Se/Rb1 NPs groups observed different degree of reduction of oil red O staining area in aorta, wherein the oil red O staining area of the PM @ Se/Rb1 NPs group is the least, indicating that it has better effect of reducing aortic plaque generation.

In conclusion, in the above embodiments, the selenium nano-particles are used as a carrier and co-assembled with ginsenoside Rb1 to form ginsenoside/selenium nano-particles, and a novel bionic nano-carrier system targeting atherosclerotic plaques is constructed by utilizing a native platelet membrane, wherein the inherent natural targeting effect of the platelet membrane can improve the drug concentration at the focal site, and meanwhile, the particle size of the bionic ginsenoside/selenium nano-system is extremely small, so that the bionic ginsenoside/selenium nano-carrier system can well penetrate a biological membrane and a blood vessel wall, and the circulation time in blood is prolonged, thereby exerting a better treatment effect. Based on the reasons, the nano system prepared in the embodiment has low toxicity, high targeting property, strong penetrating power and better biocompatibility, can enrich the drug concentration of the atherosclerotic plaque part, reduce the drug dosage and reduce the toxic and side effects, thereby providing a new effective way for preventing and treating the atherosclerosis aiming at the focus part.

The above embodiments are preferred implementations of the present invention, and the present invention can be implemented in other ways without departing from the spirit of the present invention.

Claims

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 nanoparticle framework inner core and a platelet membrane coated on the surface of the inner core.

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 platelet membrane is derived from blood of healthy rat, and is prepared by centrifuging, purifying and repeatedly freezing and thawing.

3. 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 method specifically comprises the following steps:

A. preparing 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.

4. The preparation method of the biomimetic nano drug delivery system based on ginsenoside according to claim 3, 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.