CN117298050A

CN117298050A - Echinacoside liposome and LRP1 targeted Echinacoside-loaded liposome as well as preparation methods and applications thereof

Info

Publication number: CN117298050A
Application number: CN202311320134.7A
Authority: CN
Inventors: 王锦玉; 刘德文; 游云; 仝燕; 欧则民
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2023-10-12
Filing date: 2023-10-12
Publication date: 2023-12-29

Abstract

The application is suitable for the technical field of medicines, and provides an echinacoside liposome, an LRP1 targeted echinacoside-loaded liposome, a preparation method and application thereof, and the preparation method of the LRP1 targeted echinacoside-loaded liposome provided by the embodiment of the application can realize enrichment concentration of echinacoside in brain tissues reaching 230.22 +/-33.76 ng/g by constructing the LRP1 targeted liposome, thereby obviously improving neuroprotection effect of ECH on MPTP mice, obviously up-regulating the level of dopamine and metabolites in striatum areas, obviously improving oxidative stress injury in striatum areas of the MPTP mice, and showing good treatment effect on the MPTP mice. In a mine test, ECH@ANG-Lip is used for remarkably improving the exercise behavior of an MPTP mouse, and remarkably improving the total exercise path, the exercise rate and the central area exercise time of the MPTP mouse.

Description

Echinacoside liposome and LRP1 targeted Echinacoside-loaded liposome as well as preparation methods and applications thereof

Technical Field

The application belongs to the technical field of medicines, and particularly relates to an echinacoside liposome, an LRP1 targeted echinacoside liposome and a preparation method and application thereof.

Background

Echinacoside (ECH) is derived from the rhizomes of Echinacea plants. Echinacea is a perennial herb of Compositae, and contains a variety of active ingredients with great medicinal value, including polysaccharides, glycoproteins, alkylamides, etc. Echinacoside is obtained by separating root of Echinacea angustifolia, which is one of main effective components of Echinacea angustifolia, and can be separated from other Chinese herbal medicines such as herba cistanches, radix rehmanniae, radix scrophulariae, herba Orobanches, and herba Hirudo. Studies show that the pharmacological actions of echinacoside mainly comprise antioxidation, anti-inflammatory, anti-tumor, liver protection, nerve protection, wound healing promotion, bone protection, learning and memory improvement, immune regulation and the like.

Additional studies have shown that echinacoside can reduce 6-hydroxydopamine mediated damage to striatal dopamine neurons, which also makes it likely to play a role in the prevention and treatment of Parkinson's Disease (PD). WANG et al demonstrated in a 6-OHDA-induced neurotoxicity model that echinacoside can effect treatment of PD due to destruction of cellular mitochondria and inflammatory response by scavenging ROS products. The adopted environment toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (1-methyl-4-phenyl-1, 2,3, 6-tetrahydropalmidine, MPTP) induced mouse PD model of ZHAO and the like can reduce the ratio of apoptosis and Bax/Bcl-2 after oral echinacoside preparation, obviously improves the phenomenon of unstable gait of mice in 1 week and 2 weeks of administration, proves that echinacoside is a neurotrophic factor capable of being taken orally, and provides a new idea for PD treatment. However, while ECH has good neuroprotection against dopaminergic neurons in vitro, its clinical application is severely limited by an oral absolute bioavailability of 0.83% and extremely low blood brain barrier permeability.

Disclosure of Invention

The embodiment of the application aims to provide a preparation method of echinacoside liposome, which aims to improve the clinical application value of echinacoside.

The embodiment of the application is realized in such a way that the preparation method of the echinacoside liposome comprises the following steps:

dissolving soybean lecithin, cholesterol, phospholipid-polyethylene glycol and echinacoside in an organic solvent for film treatment, and carrying out hydration dissolution film treatment, ultrasonic treatment and filtration treatment to obtain the echinacoside liposome;

wherein the mass ratio of the soybean lecithin to the cholesterol is 1:2-20; the concentration of the soybean lecithin is 10-30 mg.mL ^-1 The method comprises the steps of carrying out a first treatment on the surface of the The mass ratio of the echinacoside to the soybean lecithin is 1:10-40; the mass ratio of the phospholipid-polyethylene glycol to the soybean lecithin is 1:2-5.

Another object of the embodiments of the present application is to provide an echinacoside liposome, which is prepared by the preparation method of the above-mentioned echinacoside liposome.

Another object of an embodiment of the present application is to provide a method for preparing LRP1 (Low-density lipoprotein receptor-associated protein-1, low-density lipoprotein receptor-related protein 1) targeted echinacoside-loaded liposome, comprising:

Dissolving soybean lecithin, cholesterol, phospholipid-polyethylene glycol-maleimide and echinacoside in organic solvent for film treatment, dissolving film by hydration, ultrasonic treatment, and filtering;

adding Angiopep-2 polypeptide into the obtained filtrate, and stirring to obtain LRP1 targeted echinacoside-loaded liposome;

wherein the mass ratio of the soybean lecithin to the cholesterol is 1:2-20; the concentration of the soybean lecithin is 10-30 mg.mL ^-1 The method comprises the steps of carrying out a first treatment on the surface of the The mass ratio of the echinacoside to the soybean lecithin is 1:10-40; the mass ratio of the phospholipid-polyethylene glycol-maleimide to the soybean lecithin is 1:2-5; the reaction mole ratio of the phospholipid-polyethylene glycol-maleimide and the Angiopep-2 polypeptide is 1:0.21-1.65.

Another object of the embodiment of the present application is to provide LRP 1-targeted echinacoside-loaded liposome, where the LRP 1-targeted echinacoside-loaded liposome is prepared by the preparation method of the LRP 1-targeted echinacoside-loaded liposome.

Another object of the embodiment of the present application is to use of the echinacoside liposome or the LRP 1-targeted echinacoside-loaded liposome in preparing a medicament for treating parkinson's disease.

The preparation method of the echinacoside liposome (ECH@Lip) provided by the embodiment of the application is simple in process and short in preparation period, in the process of preparing the ECH@Lip, the hydrated solution is semitransparent with light blue opalescence, the ECH@Lip solution after ultrasound is transparent with light blue opalescence, the Tyndall phenomenon is obvious, and the obtained ECH@Lip is high in encapsulation rate and has the effects of delaying drug metabolism and maintaining drug stability.

According to the preparation method of the LRP1 targeted echinacoside-loaded liposome (ECH@ANG-Lip), provided by the embodiment of the application, the enrichment concentration of the echinacoside in brain tissues can reach 230.22 +/-33.76 ng/g through constructing the LRP1 targeted liposome, the neuroprotection effect of ECH on MPTP mice is remarkably improved, the obtained ECH@ANG-Lip can remarkably up-regulate the level of dopamine and metabolites thereof in striatum areas, the oxidative stress injury of the striatum areas of the MPTP mice is remarkably improved, and good treatment effects are shown on the MPTP mice. In a mine test, ECH@ANG-Lip is used for remarkably improving the exercise behavior of an MPTP mouse, and remarkably improving the total exercise path, the exercise rate and the central area exercise time of the MPTP mouse.

Drawings

Fig. 1 is a graph showing the results of characterization of physical and chemical properties of liposomes according to the examples provided herein: (A) Particle size distribution of liposome, (D) Zeta potential distribution, (B, E) TEM morphology, (C) liposome sample, (F) Tyndall effect of liposome sample solution, scale bars is 500 μm;

fig. 2 is a liposome stability and drug release profile provided in the examples herein: (A) Stability of liposomes in serum (n=3), (B, C) liposome storage stability: particle size and encapsulation efficiency (n=3), (D) cumulative drug release profile of drug (n=3);

Fig. 3 is a graph showing the results of a hemolysis test provided in the examples of the present application: (a) incubation for 3h after centrifugation, (B) microscopic image of erythrocyte suspension, (C) hemolysis rate (n=3), scale bar 50 μm;

fig. 4 is a schematic diagram of (a) an in vitro BBB model provided in the examples of the present application, (B) an in vitro trans-BBB model transmittance (n=3), (C) a measurement of resistance values before and after the experiment (n=3), (D) cytotoxicity of liposomes (n=3);

FIG. 5 shows a blank cell without seeding cells and (B, C) a cell with seeding bEnd.3 cells provided in the examples of the present application;

fig. 6 shows the change in resistance (n=3) of the BBB model in vitro provided in the examples of the present application over 7 days;

FIG. 7 is a 20-fold mirror image of BBB cell growth in vitro, scale bar of 50 μm, provided in the examples of the present application

Fig. 8 is an experimental result of autonomous activity count (n=6) provided in the embodiment of the present application;

fig. 9 shows the result of the pole climbing experiment (n=6) provided in the embodiment of the present application

Fig. 10 shows the Nissl staining results (n=3) of the substantia nigra compact provided in the examples of the present application

FIG. 11 shows the results of (A) dense portion TH immunohistochemical staining of the substantia nigra, (B) statistics of TH-positive neuron optical density values (n=3)

Fig. 12 is a fluorescence image provided in an embodiment of the present application: (a) fluorescence in vivo imaging of mice, (B) fluorescence imaging of ex vivo tissues, (C) quantification of fluorescence of ex vivo tissues (n=3, dir@ang-Lip is normal mice);

Fig. 13 is an in vitro brain tissue slice diagram provided in the example of the present application (n=3, dir@ang-Lip is a normal mouse);

fig. 14 is an extracted ion flow diagram provided in an embodiment of the present application: (A) Blank brain tissue sample, (B) ECH control and internal standard sample (C) _ECH ＝10.5ng·mL ^-1 ,C _Verbascoside ＝210ng·mL ^-1 ) (C) blank brain tissue sample+ECH control and internal standard (C) _ECH ＝10.5ng·mL ^-1 ,C _Verbascoside ＝210ng·mL ^-1 ) (D) a brain tissue sample;

fig. 15 is an extracted ion flow diagram provided in an embodiment of the present application: (A) High concentration ECH control (C) _ECH ＝2100ng·mL ^-1 ，C _Verbascoside ＝210ng·mL ^-1 ) (B) a blank brain tissue sample;

FIG. 16 is an ECH standard curve provided in an embodiment of the present application;

fig. 17 is a biodistribution of echinacoside in a mouse provided in the examples herein (n=5);

FIG. 18 is a schematic illustration of an experimental animal treatment protocol provided in an example of the present application;

fig. 19 shows the results of the (a) mine test provided in the examples of the present application: (a) motion profile, (b) total motion path, (c) average motion rate, (d) central zone activity time ratio. (B) neurotransmitter content determination results: (a) dopamine, (b) 3, 4-dihydroxyphenylacetic acid, and (c) homovanillic acid. * p <0.05, # p <0.01, # p <0.001vs the control, # p <0.05, # p <0.01, # p <0.001vs the MPTP-process group, n=6 to 8;

fig. 20 is a graph showing the results of striatal oxidative stress level measurement provided in the examples of the present application: (a) MDA level measurement, (B) GSH-PX level measurement, (C) SOD level measurement, p <0.05, p <0.01, p <0.001vs the control, #p <0.05, #p <0.01, #p <0.001vs the MPTP-process group, n=6 to 8.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application.

The embodiment of the application provides a preparation method of echinacoside liposome, which comprises the following steps: soy lecithin, cholesterol, phospholipid-polyethylene glycol (DSPE-PEG) _2K ) Dissolving Echinacoside in organic solvent, performing film formation treatment, dissolving film by hydration, ultrasonic treating, and filtering to obtain Echinacoside liposome.

Wherein, the organic solvent can be absolute ethyl alcohol, and the organic solvent is used in the earlier stage of the applicationThe selection of the category of (C) is examined by weighing soybean lecithin 120mg, cholesterol 40mg and DSPE-PEG respectively _2K The effect of anhydrous ethanol and mixed solvent (chloroform: methanol=2:1) on the encapsulation efficiency of ech@lip was examined in a 10mL EP tube at 40mg and 6mg of echinacoside, and the results indicate that the above two organic solvents have no significant effect on the encapsulation efficiency of ech@lip, and therefore, anhydrous ethanol is preferable as the organic solvent in view of safety.

In the early stage of the application, absolute ethyl alcohol is used as an organic solvent, and the influence of egg yolk lecithin and soybean lecithin on the encapsulation efficiency of ECH@lip is examined. The results show that the encapsulation efficiency (58.45%) of ech@lip prepared from soybean lecithin is higher than that of egg yolk lecithin (49.49%), and that soybean lecithin has higher stability than egg yolk lecithin, so that soybean lecithin is determined to be preferable as the ech@lip preparation material.

Wherein the mass ratio of the soybean lecithin to the cholesterol is 1:2-20; in the early stage of the application, absolute ethyl alcohol is used as solvent to fix soybean lecithin and DSPE-PEG _2K The effect of the cholesterol ratio on the ECH@Lip encapsulation efficiency is examined while the echinacoside content is unchanged, and the result shows that the encapsulation efficiency corresponding to the mass ratio of the cholesterol to the lecithin is 56.99%, 55.81%, 59%, 65.24% and 67.4% when the mass ratio of the cholesterol to the lecithin is 1:2, 1:3, 1:5, 1:10 and 1:20 respectively, namely, the ECH@Lip encapsulation efficiency is gradually increased along with the reduction of the cholesterol ratio, and the stability of the liposome is influenced by the fact that the too low ratio of the cholesterol to the soybean lecithin is considered, so the preferable ratio of the cholesterol to the lecithin is determined to be 1:5.

wherein the concentration of the soybean lecithin is 10-30 mg.mL ^-1 The method comprises the steps of carrying out a first treatment on the surface of the In the early stage of the application, absolute ethyl alcohol is used as a solvent to fix the soybean lecithin: cholesterol: DSPE-PEG _2K Echinacoside=60:12:20:3, other conditions are unchanged, the influence of the concentration of soybean lecithin on ECH@lip encapsulation efficiency is examined, and the result shows that the lecithin concentrations are respectively 10 mg.mL ^-1 、20mg·mL ^-1 、30mg·mL ^-1 The corresponding encapsulation rates are 55.32%, 67.86% and 72.65%, namely, with the increase of the concentration of soybean lecithin, the encapsulation rate of ECH@Lip is gradually increased, but the higher concentration of lecithin can cause hemolysis and aggregation of erythrocytes and the particle size of liposome Increasing and aggregate sedimentation, and higher lecithin concentrations reduce the relative drug loading of the liposomes, resulting in increased actual drug loading. Therefore, under the condition of ensuring that the encapsulation efficiency is in a reasonable range, the dosage of the lecithin should be reduced as much as possible, so the concentration of the soybean lecithin is determined to be 20 mg/mL ^-1 。

Wherein the mass ratio of the echinacoside to the soybean lecithin is 1:10-40; in the earlier stage of the application, the concentration of the soybean lecithin is 20 mg.mL ^-1 Other conditions were unchanged and the effect of the ratio of the drug to the lipid on the encapsulation efficiency of ech@lip was examined. The results show that the medicine-fat ratio is 1:10, 1: 20. the corresponding encapsulation efficiency at 1:40 is 64.08%, 73.78% and 80.07%, namely, with the decrease of the drug-to-lipid ratio, the encapsulation efficiency of ECH@lip is gradually increased, and the drug-to-lipid ratio is determined to be 1 according to the requirement of intravenous injection dosage of mice: 40.

wherein the mass ratio of the phospholipid-polyethylene glycol to the soybean lecithin is 1:2-5. The medicine-fat ratio is 1 at the earlier stage: 40, other conditions were unchanged, and surfactant (DSPE-PEG was examined _2K ) Effect of ratio to soybean lecithin on ech@lip encapsulation efficiency. The results show that DSPE-PEG _2K The encapsulation rates corresponding to soybean lecithin are 78.01%, 80.05% and 82.39% when the encapsulation rates are 1:2, 1:3 and 1:5 respectively, namely, the encapsulation rate is highest when the dosage of the surfactant is 1:5, so DSPE-PEG is determined to be selected for use _2K The ratio of the soybean lecithin to the soybean lecithin is 1:5.

In the embodiment of the application, the preparation method of the echinacoside liposome specifically comprises the following steps: dissolving soybean lecithin, cholesterol, phospholipid-polyethylene glycol and Echinacea glycosides in organic solvent, evaporating the organic solvent in water bath, adding physiological saline to hydrate the dissolved film, and performing ultrasonic treatment and filtration treatment to obtain Echinacea glycosides liposome.

Wherein, the water bath temperature (drug carrying temperature) is 40-60 ℃, the proportion of DSPE-PEG 2K and soybean lecithin is 1:5 in the earlier stage of the application, other conditions are unchanged, and the influence of the drug carrying temperature on the ECH@lip encapsulation rate is examined. The results show that the drug carrying temperatures are respectively 77.58%, 79.92% and 79.91% of the encapsulation efficiency corresponding to 40 ℃, 50 and 60 ℃, namely the drug carrying temperature has little influence on the encapsulation efficiency of ECH@Lip, but in the process of thinning by using absolute ethyl alcohol as a solvent, when the temperature is lower than 60 ℃, the film formed at the bottom of the eggplant-shaped bottle is thicker, and the hydration time is more than 20 minutes, so that the drug carrying temperature is determined to be 60 ℃.

The hydration time (drug carrying time) is 10-60min, the drug carrying temperature is 60 ℃ in the early stage of the application, other conditions are unchanged, and the influence of the hydration time on the ECH@lip encapsulation efficiency is examined. The results show that the drug loading time is respectively 76.23%, 75.20% and 74.90% corresponding to the drug loading time of 10min, 30 min and 60min, namely the drug loading time has no obvious influence on the ECH@lip encapsulation rate, so that the drug loading time is determined to be 10min.

Wherein the ultrasonic power is 30-90W; in the early stage of the application, the drug loading time is 10min, other conditions are unchanged, and the influence of ultrasonic power on the ECH@lip encapsulation efficiency is examined. The results are shown in the following table 1, which shows that the ultrasonic power has no obvious effect on the encapsulation efficiency of the echinacoside, when the ultrasonic power is increased to 60W, the particle size is reduced to 116.7+/-1.6 nm, the absolute value of the potential is not changed greatly, and the absolute value of the potential of a nanoparticle system is considered to be more than 30, so that the distribution repulsive force of the nanoparticles is larger, and the system is relatively stable. Nanoparticles with smaller particle sizes may pass through the BBB more easily, and as a result, when the ultrasonic power is increased to above 60W, the particle size of ech@lip is not changed obviously, and the loss of the ultrasonic probe caused by higher ultrasonic power is larger, so that the selection of ultrasonic power to be 60W is determined.

Table 1 investigation of ultrasonic power

Further, the embodiment of the application provides a preparation method of LRP1 targeted echinacoside-loaded liposome, which comprises the following steps: dissolving soybean lecithin, cholesterol, phospholipid-polyethylene glycol-maleimide and echinacoside in a mixed solvent for film treatment, and carrying out hydration dissolution film treatment, ultrasonic treatment and filtration treatment; adding Angiopep-2 polypeptide into the obtained filtrate, and stirring to obtain LRP1 targeted echinacoside-loaded liposome.

Optionally, dissolving soybean lecithin, cholesterol, phospholipid-polyethylene glycol-maleimide and echinacoside in an organic solvent for film treatment, and carrying out hydration dissolution film treatment, ultrasonic treatment and filtration treatment, wherein the method comprises the following steps of: dissolving soybean lecithin, cholesterol, phospholipid-polyethylene glycol-maleimide and echinacoside in organic solvent, evaporating the organic solvent in water bath, adding physiological saline to hydrate and dissolve the film, and performing ultrasonic treatment and filtration treatment; the ultrasonic power is 30-90W; the water bath temperature is 40-60 ℃, and the hydration time is 10-60min.

The dosage requirements of soybean lecithin, cholesterol, phospholipid-polyethylene glycol-maleimide and echinacoside and the parameter limit requirements of ultrasonic power, water bath temperature and hydration time are consistent with the preparation method of the echinacoside liposome.

Wherein the reaction molar ratio of the phospholipid-polyethylene glycol-maleimide to the Angiopep-2 polypeptide is 3:2.

The organic solvent is a mixed solvent of ethanol and methanol or a solvent of chloroform and methanol. Preferably, it may be composed of chloroform and methanol in a volume ratio of 2:1, due to DSPE-PEG _2K MAL cannot be dissolved in absolute ethanol, so a mixed solvent is used instead.

Examples of certain embodiments of the present application are given below, which are not intended to limit the scope of the present application.

Example 1: preparation of Echinacoside Liposome (ECH@Lip)

Respectively weighing soybean lecithin 120mg, cholesterol 40mg and DSPE-PEG _2K 40mg of echinacoside 6mg is added into an EP tube with 10mL of absolute ethyl alcohol, the solution is completely dissolved by ultrasonic treatment for 5min, the solution is distilled in a water bath with 60 ℃ for 30min, the absolute ethyl alcohol is volatilized, 6mL of physiological saline (preheated in the water bath with 60 ℃) is added for hydration for 10min to dissolve a film, the ultrasonic treatment is carried out for 6min, the ultrasonic treatment condition is 62.5W, the ultrasonic treatment is carried out for 5s, the ultrasonic treatment is carried out for 3s, and the solution with ECH@Lip is obtained by filtering through a filter membrane with 0.22 mu m for 5 times.

Example 2: preparation of LRP1 targeted echinacoside-loaded liposome (ECH@ANG-Lip)

Respectively weighing soybean lecithin 60mg, cholesterol 12mg and DSPE-PEG _2K MAL12 mg, echinacoside 6mg in a 10mL EP tube, adding mixed solvent (chloroform: methanol=2:1) 6mL, water-bathing at 50deg.C,volatilizing the organic solvent at low pressure, adding 6mL of physiological saline to hydrate the film to obtain semitransparent milky white solution, performing ultrasonic treatment for 8min (ultrasonic treatment is performed for 5s and stopping for 3 s) at 100W to obtain transparent liposome solution (ECH@Mal Lip) with light blue opalescence, and filtering the solution with a 0.22 μm filter membrane for 5 times. 3mL of filtrate was taken and 5mg of ANG polypeptide (molar ratio of reaction DSPE-PEG) _2K -MAL: ang=3:2), magnetically stirring for 12h to obtain an ech@ang-Lip solution.

The physicochemical properties of ECH@Lip and ECH@ANG-Lip obtained above were characterized (particle size, measurement of Zeta potential, measurement of encapsulation efficiency and drug loading, detection of appearance) and the results are shown in FIG. 1, wherein A represents the particle size distribution of the liposome, D represents the Zeta potential distribution, B, E represents the TEM appearance map, C represents the liposome sample, F represents the tyndall effect of the liposome sample solution, and scale bars are 500. Mu.m.

As shown in fig. 1 (C, F), the ech@lip and the ech@ang-Lip are transparent solutions with light blue opalescence, and a significant tyndall effect is generated when the laser penetrates through the solutions.

As shown in FIG. 1 (A, D) and Table 2, the liposome was modified by ANG to slightly increase the particle size, the PDI was not greatly changed, and the potential was changed from-22.7.+ -. 0.59 to 1.0.+ -. 0.17. After ANG was modified on the surface of the liposome, the liposome surface was positively charged.

TABLE 2 particle size, PDI and Zeta potential of liposomesn＝3)

Wherein, the encapsulation rates of ECH@lip and ECH@ANG-Lip are 55.20 +/-0.12% and 52.83+/-0.31%, and the drug loading rates are 4.77+/-0.14% and 4.25+/-0.12% respectively.

As shown in fig. 1 (B, E), the liposome is in a sphere-like shape, has uniform size distribution, and has small shape and particle size change after ANG modification.

In vitro drug release and liposome stability examination are carried out on the ECH@lip and ECH@ANG-Lip, and the results are shown in the liposome stability and drug release curves in FIG. 2: (A) Stability of liposomes in serum (n=3), (B, C) liposome storage stability: particle size and encapsulation efficiency (n=3), (D) cumulative drug release profile (n=3).

The results show that the free ECH released most rapidly in physiological saline as shown in FIG. 2 (D), the drug release rate of 0.5h reaches 39.08%, and the drug release rate of 4h reaches 83.56%. After 4 hours, the cumulative drug release rate of ECH was reduced due to poor stability of ECH in solution and partial degradation. The cumulative drug release behaviors of the liposome group ECH@lip and ECH@ANG-Lip are basically consistent, the 0.5h drug release rate is 10.93% and 15.02% respectively, and the 4h cumulative drug release rate reaches the highest values, namely 68.99% and 69.86% respectively.

As shown in FIG. 2 (A), the ECH@Lip and ECH@ANG-Lip showed no significant change in particle size within 48h in fetal calf serum, but the particle size of the liposome increased significantly at 72h, indicating that aggregation of the liposome may occur between 48h and 72h, and the liposome stability was reduced.

As shown in fig. 2 (B, C), the ech@lip and ech@ang-Lip have no obvious change in particle size and encapsulation rate within 5 days under the storage condition of 4 ℃; the particle size is obviously increased on the 7 th day, and the encapsulation efficiency is obviously reduced. Interestingly, the size of the ECH@ANG-Lip appears to be reduced to be basically consistent with the ECH@lip and then increased to be basically consistent with the ECH@lip between the 3 rd day and the 7 th day; while the encapsulation efficiency increases and then decreases.

Further, the examination results of the lyoprotectant are shown in table 3, the effect of sucrose as the lyoprotectant is obviously better than mannitol, and the 3% sucrose has better protection effect on the particle size of liposome and the PDI thereof.

TABLE 3 investigation of lyoprotectants @n＝3)

Further, hemolysis tests were performed on the ECH@Lip and ECH@ANG-Lip obtained above. As shown in FIG. 3 (A), the ECH@Lip and ECH@ANG-Lip groups are 0.4-2.0 mg.multidot.mL ^-1 Within the concentration range, no hemolysis was observed after incubation for 3h sampling centrifugation, and the hemolysis rates of ECH@Lip and ECH@ANG-Lip were both lower than 2% (FIG. 3 (C)). After centrifugation, the sample was shaken well, and no erythrocyte aggregation was observed under a microscope (FIG. 3 (B)). The hemolysis test examination result shows that the intravenous injection administration of ECH@Lip and ECH@ANG-Lip does not cause hemolysis and erythrocyte coagulation reaction.

In addition, in the embodiment, the cell uptake time is determined to be 60min through a cell uptake experiment, and when the modification ratio of the ANG on the surface of the liposome is found to be 6% by combining with the flow cytometry, the uptake of the bEnd.3 cell into the liposome reaches the highest, so that the modification ratio of the ANG is determined to be 6%.

Example 3: research of LRP1 targeted echinacoside-loaded liposome crossing in-vitro BBB model

The experimental method comprises the following steps:

1. Cell resuscitation

(1) The culture medium, the complete culture medium, is put in an ultra clean bench and rewarmed to room temperature.

(2) A jar of warm water at 37 ℃ was prepared.

(3) Taking out the freezing tube filled with bEnd.3 cells from a liquid nitrogen tank or dry ice, and rapidly rotating the freezing tube in a clockwise single direction in warm water at 37 ℃ by holding the freezing tube, wherein the thawing time is controlled to be 1-2 min.

(4) Wiping the surface of the cryopreservation tube with alcohol cotton ball, transferring into an ultra-clean bench, opening the cryopreservation tube after passing through an alcohol lamp outer flame, transferring the cell suspension in the tube into a centrifuge tube filled with 9mL of culture medium, and transferring to a centrifuge tube at 1000 rpm.min ^-1 Centrifuging for 5min.

(5) After centrifugation, the supernatant was removed, 5mL of complete medium was added to resuspend the cells, and the cell suspension was transferred entirely to a petri dish, supplemented with 2mL of complete medium, and homogenized by cross shaking. Time of labeling, cell name, and operator name.

(6) Observing the state and the quantity of the cells under a microscope, and placing the cells into an incubator for culturing.

(7) After 24h, the cell growth state was observed and the new complete medium was replaced.

(8) When the cell density is up to about 80%, the cells are passaged and transferred to a 3 rd generation mirror to observe that the growth state of the cells is good and stable, and then the cells start to be used for experiments.

2. Cell passage

(1) When bEnd.3 cells grew to about 80% of the bottom of the dish, passaging was performed.

(2) The old media in the dishes was first removed and the remaining media was rinsed by adding 1mL of sterile PBS.

(3) Trypsin was added to thoroughly infiltrate the bottom of the dish and digested for 30s.

(4) And (3) in the digestion process, when the cell morphology is rounded, the cell gaps are enlarged and part of cells float in trypsin digestion liquid under an inverted microscope, adding 2mL of complete culture medium to stop digestion, and blowing and beating uniformly mixed cells with wall removed.

(5) The cell suspension was transferred to a 5mL EP tube, centrifuged, the supernatant discarded, and 2mL of complete medium was added to resuspend the cells.

(6) Taking 0.5mL of cell suspension in a culture dish, supplementing the complete culture medium to 7mL, shaking uniformly in a cross manner, and placing the culture dish in an incubator for continuous culture, wherein the passage ratio is 1:4.

3. Cell cryopreservation

(1) Preparing a frozen stock solution, wherein the frozen stock solution comprises 55% of culture medium, 40% of FBS and 5% of DMSO.

(2) Cell counting was performed on a cell counting plate from 10. Mu.L of the centrifuged cell suspension.

(3) According to 5x10 ⁶ The concentration of the cell freezing solution is not more than 2/3 of that of the tube, otherwise the tube can crack.

(4) Gradient cooling: standing at 4deg.C for 10min, standing at-20deg.C for 30min, standing at-80deg.C overnight, and transferring to liquid nitrogen tank.

4. Cytotoxicity of cells

1) Preparation of drug-containing liposome

Taking ECH-containing 0.5 mg.mL ^-1 Ech@lip of (a), ultrafiltration and centrifugation to remove free materialThe materials and the solution are diluted gradually to 100, 10, 1 and 0.1 mug.mL by complete culture medium ^-1 The preparation of ECH@Lip, ECH@ANG-Lip series concentration is the same as ECH@lip.

2) Cell Activity assay

bEnd.3 is set to 1X10, respectively ⁴ The density of each well was inoculated in 96-well plates, 100. Mu.L of each well was inoculated, and incubated in a constant temperature incubator for 24 hours. After cell attachment, 100 μl of ech@lip solution of the above concentration was added to 96-well plates, 6 duplicate wells were placed in parallel, and negative control (bend. 3+ complete) and reference (complete only) groups were placed in a constant temperature incubator for incubation for 24h. mu.L of CCK-8 reagent was added to each well, incubated in an incubator for 2 hours, and absorbance at 450nm was measured with a microplate reader. Cell activity was calculated as follows.

Note that: a is that _S Represents the absorbance of the experimental well, A _b Absorbance representing reference group, A _c The absorbance of the control group was represented.

5. Construction of in vitro BBB model

1) Gelatin coated Transwell chamber

150 mu L of gelatin with mass fraction of 2% is taken in a Transwell 12-orifice plate (0.4 mu m,12 mm) cell, the gelatin solution is ensured to be fully paved in the cell, the cell is kept stand for 2 hours in an incubator, the gelatin in the cell is sucked out, and 1mL of PBS is added for cleaning once, thus the gel can be used.

2) Cell seed plate

Taking the bEnd.3 in logarithmic growth phase at 1X10 ⁵ Individual/cm ² (1.13×10=11.3 ten thousand/well) was inoculated into the upper chamber of a gelatin-coated Transwell 12-well plate, the volume of the cell suspension was 500 μl, the lower chamber was 1000 μl of complete medium, and incubated in a constant temperature incubator. New media was changed every two days and the cell growth was observed under an inverted microscope. After observing a substantial fusion of cells, the model was subjected to a 4h leakage test, a test of TEER resistance and an fli permeability test to determine if the BBB model construction was successful.

3) 4h leakage experiment

1mL of complete medium was added to the upper chamber of the Transwell, and 1.5mL of complete medium was added to the lower chamber to form a liquid level difference of >0.5cm, and it was observed whether a liquid level difference of >0.5cm could be maintained after 4 hours.

4) Determination of TEER resistance

Cell TEER values were measured once a day after cell inoculation into a Transwell 12-well plate. Standing a Transwell 12 pore plate in an ultra clean bench for 15min to restore cells to room temperature, soaking two ends of an electrode in 75% alcohol for 15min, airing for 15s, flushing the electrode with PBS for 3 times, connecting a probe, turning on a power supply, adjusting the mode to be Ohms, immersing the electrode in a culture medium, immersing the short end in the inner side of the cell, immersing the long end in the bottom of the lower chamber, and ensuring that the electrode is stable and forms 90 degrees with the bottom of the culture plate. The resistance value was recorded, and the resistance value per unit area (measured value x effective film area) was calculated.

5) In vitro observation of BBB model cell growth

Microscopic photographing is carried out 1 to 7 days after cell inoculation, and the growth condition of cells is observed.

6) FLU permeability experiment

Sodium fluorescein (Sodium Fluorescein, FLU) has a molecular weight 376Da, excitation light 428nm, and emission light 536nm. Preparing the total culture medium without phenol red into 1, 3, 5, 7 and 10 mug.mL respectively ^-1 The fluorescence intensity of the sodium fluorescein solution is measured by a fluorescence enzyme-labeled instrument, and a standard curve is drawn. 500. Mu.L of 100. Mu.g.mL was added to the upper chamber of a Transwell 12-well plate ^-1 (266 mu M) of sodium fluorescein solution, adding 1mL of complete culture medium into a lower chamber, placing into a constant temperature incubator for incubation, respectively taking 100 mu L of culture solution from a receptor pool for 15, 30 and 60 minutes, measuring fluorescence intensity by using a fluorescence enzyme-labeled instrument, and calculating the permeabilities of sodium fluorescein of a model group and a cell-free control group.

dQ/dt is the rate at which FLU passes the BBB, C ₀ For the initial concentration of FLU in the upper chamber, A is the area of the Transwell upper chamber filter.

The permeability coefficient of FLU passing through the BBB model and the permeability coefficient of FLU passing through the cell-free control group are calculated according to the formula respectively, and the permeability coefficient of FLU in vitro permeation bEnd.3 single-layer BBB model can be calculated according to the following formula.

Wherein PS is _t Permeability coefficient x surface area, PS for the flux across the BBB _f Permeation coefficient x surface area for FLU through cell-free control. The membrane area of the Transwell 12-well plate cell was 1.13cm ² 。

6. Investigation of the transmittance of liposomes across the in vitro BBB model

Through the evaluation, a qualified Transwell chamber is selected for the investigation of the liposome transmittance. After ECH@lip and ECH@ANG-Lip are subjected to ultrafiltration and centrifugation, the mixture is diluted to corresponding concentrations by using a complete culture medium, 500 mu L of the mixture is added into an upper chamber of a Transwell 12-well plate, and 1mL of the culture medium is added into a lower chamber. The experiment is divided into 3 groups, namely ECH@lip, ECH@ANG-Lip and ANG+ECH@ANG-Lip, wherein the treatment process of the ANG+ECH@ANG-Lip group is as follows: 200. Mu.L ANG (10. Mu.g.mL) was added ^-1 ) The solution presaturates cells in the Transwell cells, and after incubation for 1h in a constant temperature incubator, 500. Mu.L of ECH@ANG-Lip is added into the upper chamber of the Transwell 6-well plate. 200. Mu.L of liquid was removed from the chamber at 0.5, 1.0, 2.0, 4.0, 6.0h, respectively, and 200. Mu.L of complete medium was supplemented, and the collected samples were immediately quantitatively analyzed by UPLC. The transmittance calculation formula:

C _t for the concentration at the time of sampling of the lower chamber, C ₀ For the initial concentration of the upper chamber, V _acceptor For the volume of the lower chamber solution, V _donor Is the upper chamber solution volume.

Experimental results:

1. cytotoxicity of cells

1) Cell Activity assay

In fig. 4, (a) schematic diagram of in vitro BBB model, (B) in vitro trans-BBB model transmittance (n=3), (C) measurement of resistance values before and after experiments (n=3), (D) cytotoxicity of liposomes (n=3). As shown in FIG. 4 (D), when the drug concentration was 1 to 300. Mu.M, both liposomes were not significantly cytotoxic.

2. Construction of in vitro BBB model

1) 4h leakage experiment

As a result, as shown in FIG. 5, (A) in FIG. 5, a blank cell without cells was inoculated, and (B, C) in FIG. 5, a cell with bEnd.3 cells was inoculated. After 4 hours, the liquid level of the cell A rises, the liquid level difference between the inner cell and the outer cell is not generated, and the liquid level difference of the cell B, C can still be maintained to be more than 0.5cm, which shows that the cell inoculated with bEnd.3 cells has obvious barrier property and the cells growing on the wall are tightly connected.

2) Determination of TEER resistance

As a result, as shown in FIG. 6, the TEER value reached 205.38. Omega. Cm 3 days after inoculation of bEnd.3 cells ^-2 The TEER values remained relatively stable after 3 days, indicating dense growth of the bend.3 cell layer and barrier function of the BBB model in vitro.

3) In vitro observation of BBB model cell growth

As a result, as shown in FIG. 7, there was a clear gap between bEnd.3 cells on the first day after cell seeding, and the cell arrangement exhibited a typical "paving stone" shape or shuttle shape. The next day after cell seeding, the cells were closely packed and no significant gaps were observed between cells. On the third day after cell seeding, no obvious cell contours were observed and the cells had grown to a confluent state. The fourth day after cell inoculation, the cells showed packed growth. Five days after cell seeding, cell stacking growth was more evident.

4) FLU permeability experiment

The permeability coefficients of the in vitro BBB model are 5.93+/-1.39x10 respectively after 15 min, 30 min and 60min are calculated ^-6 、4.31±2.42x10 ^-6 、6.92±3.92x10 ^-6 。

3. Investigation of the transmittance of liposomes across the in vitro BBB model

As shown in FIG. 4 (B), the transmittance of ECH@ANG-Lip at each time point is obviously higher than that of the ECH@lip group, which indicates that the transfer efficiency of ECH@ANG-Lip across the BBB in vitro is higher. However, when cells in BBB cells were pre-saturated with ANG in vitro, the transport of ECH@ANG-Lip was significantly inhibited, indicating that uptake of ECH@ANG-Lip by bEnd.3 cells might be predominantly by LRP1 receptor mediated transcytosis.

After the in vitro BBB model investigation of the liposome is finished, the resistance value of the Transwell cell TEER is measured again, and the result is shown in fig. 4 (C), wherein the resistance value of the in vitro BBB model is not changed obviously after the in vitro BBB model investigation of the liposome is finished, which indicates that the barrier property of the BBB model is not changed obviously after the in vitro BBB model of the liposome is finished.

Example 4: LRP1 targeted echinacoside-loaded liposome in vivo targeting evaluation

The experimental method comprises the following steps:

1. modeling and evaluation of MPTP mice

50 SPF-class C57BL/6J male mice, 8 weeks old, 20-25 g in mass, provided by Beijing vitamin Toril Hua laboratory animal technology Co., ltd., animal quality eligibility certificate: 110011221107842558, license number: SCXK (Beijing) 2021-0006. The raising environment is SPF-level animal raising room with temperature of 20-25 deg.c, humidity of 40-70% and 12 hr light and shade circulation, and the ventilation times are maintained for 15 times/hr.

MPTP is prepared by physiological saline, the model group is continuously injected with 30mg/kg/d MPTP by abdominal cavity for 7 days, the injection volume is 0.25mL according to 25g, and the normal group is injected with the same volume of physiological saline by abdominal cavity. Animal handling methods were approved by the animal ethics committee of the chinese traditional medicine institute, academy of chinese medicine (2021B 130).

1.1 autonomous Activity count

The frequency of horizontal movement or vertical movement of the mice is detected by using a mouse autonomous activity recorder, and the ability of the mice to autonomously move is evaluated. The experiment was performed once a day starting on the fourth day after the end of dosing, for up to day 7. The mice are put into the device for adaptation for 5min before the experiment is started formally, and the counting time of each autonomous activity is 5min.

1.2 pole climbing experiments

The bradykinesia of model mice was assessed using a pole-climbing experiment. After the administration is finished, pole climbing training is carried out once a day for 3 days. Starting on the fourth day after the end of dosing, experiments were performed once a day, and the time required for the mice to climb from the top of the rod to the bottom of the rod was recorded for up to day 7.

1.3 Experimental animal Material

After the modeling administration and the behavioral evaluation are finished, the 3 normal mice and the 3 model mice are respectively obtained. Firstly, 0.3% pentobarbital sodium 0.1-0.2 mL/10g is given to a mouse, the chest of the mouse is cut off, the heart is exposed, the right auricle is cut off, heart perfusion is carried out on the mouse, a perfusion needle is pricked into the left ventricle from the apex of the heart, and 10mL of physiological saline and 10mL of 10% formalin are sequentially injected. The whole brain of the mouse is taken out and immersed in 10% formalin solution for fixation at normal temperature for 24 hours.

1.4Nissl staining

1.4.1 Paraffin section preparation

After brain tissue fixation, gradient ethanol dehydration, xylene transparency, paraffin embedding and slicing were performed, respectively.

1.4.2Nissl staining

(1) Paraffin sections and dewaxing.

(2) Dyeing: toluidine blue dye liquor is dyed for 10min.

(3) Differentiation: the 95% alcohol differentiates brain tissues until the Nib body turns dark blue, and the background is light blue or colorless; mast cells appear purple-red and the background appears bluish or colorless.

(4) Sealing piece: drying by an electric hair drier or an oven, and sealing the neutral resin by xylene for a plurality of minutes.

1.5 immunohistochemical staining

(1) Paraffin sections and dewaxing.

(2) Antigen retrieval: heating citric acid antigen retrieval buffer solution (pH 6.0) to boil in a microwave oven, and placing the tissue slice into the retrieval solution for antigen retrieval and high fire retrieval for 10min. After natural cooling, the sections were washed 3 times with shaking in PBS (pH 7.4) on a decolorizing shaker for 5min each.

(3) Blocking endogenous peroxidases: the sections were incubated in 3% hydrogen peroxide solution for 30min (at room temperature protected from light). After incubation, the sections were then placed in PBS buffer (pH

7.4 In the above step), the washing is carried out for 3 times each for 5min.

(4) BSA blocking: the filter paper was blotted to remove excess PBS, circled around the tissue with a histochemical pen, 3% BSA was added dropwise to cover the tissue evenly, and the tissue was blocked for 30min at room temperature.

(5) An antibody: removing excessive sealing liquid, dripping primary antibody, and placing slices in a wet box at 4deg.C

Incubate overnight.

(6) And (2) secondary antibody: the sections were placed in PBS (pH 7.4) and washed 3 times with 5min each. The filter paper is used for sucking out redundant PBS, secondary antibody covering tissues corresponding to the primary antibody is dripped, and the tissue is incubated for 50min at room temperature.

(7) DAB color development: the sections were placed in PBS (pH 7.4) and washed 3 times with 5min each. The filter paper absorbs the redundant PBS, the freshly prepared DAB color development liquid is dripped, the color development time is controlled under a microscope, and the positive color is brown yellow.

(8) Counterstaining the nuclei: the Harris hematoxylin is counterstained for 3min, and after washing, the Harris hematoxylin is differentiated by alcohol containing 1% hydrochloric acid for a few seconds, washed, and returned to blue by ammonia water, and washed again.

(9) And (3) removing the water sealing piece: and (3) carrying out gradient dehydration on 75%, 85%, absolute ethyl alcohol I and absolute ethyl alcohol II for 5min respectively, dehydrating and transparentizing in dimethylbenzene I, and sealing with neutral resin.

2. In vivo fluorescence imaging in mice

2.1 preparation of DiR-loaded liposomes

Preparation of DiR@Lip: respectively weighing soybean lecithin 60mg, cholesterol 12mg and DSPE-PEG by adopting a film dispersion method _2K 12 mg、DiR 0.3mg(1mg·mL ^-1 Adding a mixed solvent (chloroform: methanol=2:1) into a 10mL EP tube, carrying out water bath at 50 ℃, vacuumizing, volatilizing the organic solvent, adding 6mL of physiological saline hydration film, carrying out ultrasonic treatment for 8min (ultrasonic treatment is carried out for 5s and 3s is stopped) at 100W to obtain a transparent pale blue liposome solution, and filtering the solution through a 0.22 mu m filter membrane for 5 times to obtain the DiR@Lip solution.

Preparation of DiR@ANG-Lip: respectively weighing soybean lecithin 60mg, cholesterol 12mg, DSPE-PEG2K-MAL12 mg, diR0.3mg (1 mg.mL) ^-1 DiR solution of (C) in a 10mL EP tube, film and hydration process are the same as for DiR@Lip. Liposome after ultrasoundTransferring the solution to a penicillin bottle, adding 10mg of ANG, magnetically stirring for 12h, and filtering the solution with a 0.22 μm filter membrane for 5 times to obtain the DiR@ANG-Lip solution.

2.2 in vivo fluorescence imaging of mice

Model mice were randomly divided into 2 groups of 6 mice each, each injected with 50. Mu.g/mL DiR@Lip and DiR@ANG-Lip at 0.4mL/25g tail vein. In order to examine whether the blood brain barrier characteristics of mice are obviously changed after MPTP molding, a group of normal mice are set for comparison, and the DiR@ANG-Lip is injected in equal doses. Mice were anesthetized (isoflurane anesthetized) at 2, 5, 7, 12, 24 hours after injection, respectively, and in vivo fluorescence imaging images were acquired from the mice via a living animal imaging system. The excitation wavelength was 720nm, the emission wavelength was 830nm, and the exposure time was 20 s/map. To observe the distribution of liposomes in the main organs (brain, heart, liver, spleen, lung and kidney) of mice, three mice were dissected at the time point of highest fluorescence intensity for each group, and the main organs were collected for fluorescence imaging.

3. Establishment of quantitative analysis method of echinacoside in brain tissue

3.1 preparation of control solutions and quality control samples

Precisely weighing ECH reference substance in a volumetric flask, adding methanol solution containing internal standard of acteoside, and diluting with methanol solution containing internal standard of acteoside to obtain reference substance solution with serial concentration. Taking low, medium and high 3 concentration reference substance solutions, drying with nitrogen, adding a certain amount of blank brain tissue sample solution for re-dissolving, and mixing uniformly to obtain the quality control sample.

3.2 chromatographic conditions

Ultra-high performance liquid chromatograph, ACQUTY UPLC BEH C ₁₈ Chromatographic column (2.1x50 mm,1.7 μm); column temperature: 40 ℃; a (0.1% formic acid water), B (acetonitrile); flow rate: 0.3mL min ^-1 The method comprises the steps of carrying out a first treatment on the surface of the Gradient elution: 0-6 min, 10-30% B;6 to 7.1min,30 to 90 percent of B;7.1 to 8.1min,90 percent of B;8.1 to 8.2min,90 to 10 percent of B; 8.2-10 min,10% B. Sample injection amount: 2. Mu.L.

3.3 Mass Spectrometry conditions

Triple quadrupole tandem mass spectrometer 6500, electrospray ion source, parameters were as follows: air curtain gas (CUR) 40Arb, collision gas (CAD) 6Pa, spray voltage (IS) -4500V, auxiliary gas heating Temperature (TEM) 550 ℃, auxiliary gas 1 (GS 1) 55Arb, auxiliary gas 2 (GS 2) 55Arb, inlet voltage (EP) -10V. The negative ion mode and the MRM mode are used for detection, and the ion pair information is shown in Table 4.

Table 4 ion pair information

3.4 sample processing method

Brain tissue samples were taken, according to weight (g): volume (mL) =1: 4, adding 50% methanol, adding 20 μl antioxidant (2% ascorbic acid and 0.1% disodium edentate aqueous solution), operating the tissue homogenizer at 60Hz frequency for 3min (10 steel balls of 3mm are added), adding 1600 μl homogenate into 1600 μl methanol containing internal standard, and swirling for 120s. Centrifuging at 10000rpm and 4 ℃ for 10min, transferring supernatant, blow-drying with nitrogen, adding 100 mu L of methanol for re-dissolution, blow-mixing, centrifuging again, collecting supernatant, and performing UPLC-MS/MS sample injection analysis.

3.5 Selectivity

(A) Taking 6 blank brain tissue samples with different sources, and performing sample injection analysis (blank brain tissue) after treatment according to a sample pretreatment method; (B) Taking working solution of echinacoside and internal standard (acteoside) at the lower limit of the ration, and carrying out sample injection analysis (reference substance and internal standard); (C) Taking 6 blank brain tissue samples with different sources, adding working solution at the lower limit of the standard curve quantification to obtain a dosing brain tissue sample containing echinacoside and an internal standard, and performing sample injection analysis (dosing brain tissue sample) after treatment according to a sample pretreatment method; (D) Brain tissue samples were taken and analyzed by sample injection after treatment according to the sample pretreatment method (brain tissue samples).

3.6 residual Rate

Sample introduction 2100 ng/mL ^-1 After the high concentration control, a blank sample was taken to estimate the residue.

3.7 Standard Curve and quantitative Limit

Preparation of the blank biological sample solutions at concentrations of 10.5, 105, 262.5, 525, 1050, 2100ng mL, respectively ^-1 Echinacoside and internal standard workAnd (3) carrying out liquid sample injection analysis to obtain a standard curve of the echinacoside and the internal standard, wherein the quantitative limit is more than or equal to 10. Using weighting (1/X ² ) The least squares method uses ECH peak area (Y) to perform curve fitting regression on the sample mass concentration (X).

3.8 precision and accuracy

(A) 5 parts of quality control samples at the lower limit of quantification, low concentration quality control samples at the concentration which is not higher than 3 times of the lower limit of quantification, medium concentration quality control samples near the middle part of the standard curve range and high concentration quality control samples at the position about 75% of the upper limit of the standard curve range are prepared in parallel respectively, sample injection analysis is carried out, each sample measurement is completed within 1 batch, and the daily precision is calculated. (B) And preparing 5 parts of each component quantitative lower limit and each of low, medium and high concentration quality control sample on continuously different dates, carrying out 3 batches of measurement, carrying out a standard curve on each batch, and calculating the daytime precision. The ratio of the obtained mass concentration to the actual mass concentration is the accuracy (relative recovery rate).

3.9 dilution reliability

Taking 5 blank brain tissue samples subjected to sample pretreatment, adding ECH reference substance solution to the blank brain tissue samples until the ECH reference substance solution is higher than the quantitative upper limit concentration, diluting the blank brain tissue samples by 10 times, and calculating the dilution accuracy and precision.

3.10 matric effect and extraction recovery

Taking 6 blank brain tissue samples with different sources and subjected to sample pretreatment, respectively adding a quality control sample with low concentration and high concentration (the concentration of the lower limit of quantification is 3 times or less and the concentration of the high concentration quality control sample is close to the position of the upper limit of quantification is 75 percent) and an internal standard, carrying out sample injection analysis, and calculating a matrix effect by using the ratio of the peak area B to the peak area A of the working solution with corresponding concentration of each untreated component, namely B/A. Taking blank brain tissue, respectively adding a low-concentration quality control sample and a high-concentration quality control sample and an internal standard, carrying out sample injection analysis after treatment according to a sample pretreatment method, and calculating the extraction recovery rate by the ratio of the peak area C to the peak area A of the working solution with the corresponding concentration of each untreated component, namely C/A. And meanwhile, the matrix effect and the recovery rate of the internal standard are required to be calculated.

3.11 stability

Taking a blank brain tissue sample, adding echinacoside and an internal standard working solution, uniformly mixing by vortex for 30s, preparing into low-concentration and high-concentration (the concentration of the lower limit of quantification is within 3 times and the concentration of the lower limit of quantification is close to the upper limit of quantification) echinacoside and an internal standard drug-containing brain tissue sample, and carrying out parallel 5 parts, wherein the samples are respectively processed according to different methods: placing (A) at room temperature for 8h, (B) placing in a sample tray (15 ℃) for 24h, then carrying out sample injection analysis, and calculating the echinacoside content according to the standard curve of the current day, freezing at the ultralow temperature of-80 ℃ and thawing at room temperature for 3 times, and (D) freezing at the ultralow temperature of-80 ℃ for 1 month. Under each investigation condition, 5 parts of low-concentration and high-concentration drug-containing brain tissues are parallel, sample injection analysis is carried out after the treatment according to a sample pretreatment method, and the content of echinacoside is calculated according to a daily following standard curve.

4. Biological distribution of echinacoside in mice

To further verify whether liposomes can deliver ECH across the BBB into the brain, the mouse brain tissue extracts were assayed for content using UPLC-MS/MS. MPTP model mice are randomly divided into 3 groups, 5 mice in each group are respectively injected with ECH, ECH@Lip and ECH@ANG-Lip with equal doses, and the injection dose is 24mg/kg. After 1h of administration, the heart is anesthetized, 10mL of physiological saline is perfused, and brain, heart, liver, spleen, lung and kidney are dissected, weighed and stored in a refrigerator at-80 ℃.

Biological samples were processed according to the "3.4 sample processing method" and analyzed by sample introduction according to the "3.2 chromatographic conditions" and "3.3 mass spectrometry conditions" and data were processed using MultiQuant software.

5. Statistical analysis

Statistical analysis statistical differences were considered when p <0.05, < p <0.01, < p <0.001 were determined using Unpaired parameter t-test (un ai red ttest) of Graphpad software.

Experimental results:

1. modeling and evaluation of MPTP mice

1.1 autonomous Activity count

The experimental results are shown in fig. 8, and the mice in the blank group and the model mice have no significant difference in the autonomic activity count.

1.2 pole climbing experiments

Experimental results as shown in fig. 9, the time required for the model mice to climb from the top of the rod to the bottom of the rod was significantly increased compared to the blank.

1.3Nissl staining

As shown in FIG. 10, the model mice had small volumes and significantly reduced numbers of Nissl bodies in the substantia nigra compacts, compared to the placebo group

1.4 immunohistochemical staining

As a result, as shown in FIG. 11, compared with the blank group, the model group mice had a dense brain portion TH ⁺ The number of positive cells was significantly reduced. By combining the damage condition of Nissl corpuscles, the number of dopaminergic neurons of the substantia nigra compacta of the brain in an MPTP-treated model mouse can be obviously reduced, and the MPTP model mouse is successfully prepared.

2. In vivo fluorescence imaging in mice

2.2 in vivo fluorescence imaging of mice

DiR can be encapsulated in phospholipid bilayer thereof by liposome, so that the distribution of the liposome in the mouse can be intuitively reflected by observing the fluorescence distribution in the mouse. The results are shown in FIG. 12A), the fluorescence enriched in the brain of the mice is strongest, and the in vivo fluorescence intensity of the LRP1 targeted liposome group (DiR@ANG-Lip) mice is obviously stronger than that of the DiR@lip group. The fluorescence in the mice can maintain higher intensity within 5 hours, and the fluorescence intensity is rapidly attenuated after 5 hours. The difference in vivo fluorescence imaging between normal mice and the group of PD model mice was not apparent.

To observe the differences in the distribution of liposomes in each organ, the ex vivo tissue (treated with anesthetic and cardiac perfusion saline) of 3 mice was collected in each group 2h after dosing, as shown in fig. 12 (B and C), the fluorescence intensity of dir@ang-Lip in the ex vivo brain tissue was significantly stronger than that of dir@lip group, and also exhibited a stronger enrichment in the lungs. The DiR@lip group has weaker fluorescence intensity in each organ, and the reason is considered that the PEG long chain on the surface of the liposome obviously reduces the uptake of the liposome by each tissue. The in vitro tissue fluorescence imaging results of the DiR@ANG-Lip group and the DiR@ANG-Lip group show that the fluorescence distribution of the PD model mouse and the normal mouse in each organ is not obviously different, which indicates that MPTP modeling does not obviously influence the structural integrity of the blood brain barrier of the mouse.

To further observe the distribution of the areas of the fluorogenic liposomes in the brain tissue, the brain tissue was immersed in 10% formalin for 10s and cut into 2mm thick sections with the mouse meninges. As shown in FIG. 13, the fluorescence imaging result shows that the DiR@ANG-Lip group is obviously stronger than the DiR@lip group, and the fluorescence is mainly enriched in c 3-c 5, namely the thalamus region of the midbrain.

3.1 Selectivity

As shown in FIG. 14, the response of endogenous substances in the blank brain tissue was low, and there was no interference with the measurement of the tissue sample.

3.2 residual Rate

As a result, as shown in fig. 15, after injection of the high concentration ECH control, the residuals of the ECH and internal standard in the blank sample were below 20% of the lower limit of quantitation and no more than 5% of the internal standard, indicating that there was no residual interference in the continuous measurement of the sample.

3.3 Standard Curve and quantitative Limit

Echinacoside linear regression equation Y=2749X-38106, R ² =0.9980, the limit of quantification is S/n.gtoreq.10, the lower limit of quantification is 10.5 ng.ml ^-1 ECH is 10.5-2100 ng.mL ^-1 Good linearity over the concentration range (fig. 16).

3.4 precision and accuracy

The results are shown in Table 5, and the ECH has less than 15% of daily/daytime precision at the lower limit, low, medium and high 4 mass concentrations, and the accuracy is more than 85%, which indicates that the measuring method has good precision and accuracy.

Table 5 within day, day precision and accuracy

3.5 dilution reliability

The results are shown in Table 6, where the dilution precision is less than 15% and the accuracy is greater than 85%, indicating that the 10-fold dilution reliability is good.

TABLE 6 dilution reliability

3.6 matric effect and extraction recovery

The results are shown in Table 7, and the RSD of blank brain tissue samples from different sources on ECH and internal standard matrix effects is less than 15%, and the absolute recovery rate is greater than 50%, so that the quantitative requirements of biological samples are met.

TABLE 7 matrix effect and extraction recovery

3.7 stability

The results are shown in Table 8, the samples are placed for 8 hours at room temperature, the samples are placed in a sample tray for 24 hours, the samples are frozen at-80 ℃ and thawed at room temperature for 3 times, and after the samples are frozen at-80 ℃ for 1 month, the deviation between the average value and the standard value of the samples with low and high concentrations is less than 15%, so that the stability of the samples is good.

Table 8 stability

4. Biological distribution of echinacoside in mice

The results are shown in FIG. 17, where the drug concentration of ECH@ANG-Lip in the brain of mice is significantly increased compared to the ECH and ECH@lip groups. Interestingly, long circulating liposomes (ech@lip) were also able to penetrate the BBB and achieved a strong enrichment in the brain, which might be related to the smaller particle size distribution of the liposomes and their phospholipid membrane properties. Compared with direct injection of free drug ECH, the concentration of the drug in each tissue of the liposome group is obviously increased for 1h, which indicates that the liposome has the effects of delaying drug metabolism and maintaining drug stability. In addition, the drug concentration of liposomes in spleen, lung and kidney is also high; the drug concentration detected in the liver was the lowest.

The application characterizes the brain enrichment characteristic of the LRP1 targeted liposome through fluorescence living imaging, in-vitro tissue and brain slice fluorescence imaging, and the brain enrichment of the LRP1 targeted liposome is obviously stronger than that of the long circulating liposome. In addition, a UPLC-MS/MS quantitative analysis method of echinacoside in brain tissues is established, and the distribution of ECH, ECH@Lip and ECH@ANG-Lip tail veins in the brain, heart, liver, spleen, lung and kidney of mice after the tail veins are dosed is determined, so that the brain targeting property of the LRP1 targeted liposome is further verified.

Example 5: therapeutic effect of LRP1 targeted echinacoside-loaded liposome on PD mice

The experimental method comprises the following steps:

1 laboratory animals and groups

40 SPF-class C57BL/6J male mice, 8 weeks old, 20-25 g in mass, provided by Beijing vitamin Toril Hua laboratory animal technology Co., ltd., animal quality eligibility certificate: 110011231103266568, license number: SCXK (Beijing) 2021-0006. The raising environment is SPF-level animal raising room with temperature of 20-25 deg.c, humidity of 40-70% and 12 hr light and shade circulation, and the ventilation times are maintained for 15 times/hr. The compositions are randomly divided into 5 groups according to the mass, 8 groups are respectively a normal control group, an MPTP model group, a levodopa positive drug group (L-Dopa), a echinacoside Free drug group (Free ECH) and an ECH@ANG-Lip group. Except normal control group injected with normal saline, each group was intraperitoneally injected with MPTP at a dose of 30mg/kg for 5 consecutive days, and each group of animals was given a treatment regimen as shown in Table 9 and FIG. 18.MPTP is formulated with physiological saline, and the injection volume is 0.25mL at 25g, and normal group is intraperitoneally injected with an equal amount of physiological saline. Animal handling methods were approved by the animal ethics committee of the chinese traditional medicine institute, academy of chinese medicine (2021B 130).

Table 9 treatment of experimental animals for drug administration

Note that: the L-dopa physiological saline solution is prepared (L-dopa is dissolved in a trace amount of concentrated hydrochloric acid, physiological saline containing 2% vitamin C is diluted, and then the pH is adjusted to be neutral by sodium hydroxide, so that the L-dopa is prepared for use at present)

2 mine test

And (3) building a 50x50x50cm square lattice by using an acrylic material plate as a mine test device, building a camera right above the mine test device, and recording a movement video of the mouse within 10 minutes. And analyzing the video data by using ANY-size software (glabell biotechnology limited company), and comparing the movement track, the total movement path, the average movement rate and the central area movement time ratio of the mice in the test time.

3. Neurotransmitter content determination

3.1 Striatal material

The mice were given 0.1mL/10g of 0.6% pentobarbital sodium, the chest of the mice was cut, the heart was exposed, the right auricle was cut, the mice were heart perfused, a perfusion needle was inserted into the left ventricle from the apex, 10mL of physiological saline was injected, the striatum was rapidly taken, and the samples were weighed and placed in a 0.5mL EP tube.

3.2 sample pretreatment

Striatum was taken, by weight (mg): volume (μl) =2: 15, 10% trichloroacetic acid is added in proportion, the tissue refiner works for 3min at 60Hz frequency (6 steel balls with the diameter of 1.87mm are added), 3000rpm is carried out, centrifugation is carried out for 10min at 4 ℃, and all the upper layer solution is transferred into a 1.5mL centrifuge tube; again, centrifugation was carried out at 13000rpm at 4℃for 10min, the supernatant was transferred and analyzed by UPLC-MS/MS.

3.3 chromatographic conditions

Ultra performance liquid chromatography, ACQUITY UPLC CSH 130C 18 column (Waters, USA, 2.1X100mm,1.7 μm). Mobile phase a (0.1% formic acid in water with 10mM ammonium formate) and mobile phase B (acetonitrile). Gradient elution: 0-1 min,5% B; 1-6 min, 5-95% B; 6-8 min,95% of B; 8-8.10 min, 95-5% B; 8.10-10 min,5% B. The column temperature is 40 ℃, the flow rate is 0.2mL/min, and the sample injection amount is 2 mu L.

3.4 Mass Spectrometry Condition

Triple quadrupole tandem mass spectrometer 6500, electrospray ion source, parameters were as follows: air curtain gas (CUR) 40Arb, collision gas (CAD) 6Pa, spray voltage (IS) -4500V, auxiliary gas heating Temperature (TEM) 550 ℃, auxiliary gas 1 (GS 1) 55Arb, auxiliary gas 2 (GS 2) 55Arb, inlet voltage (EP) -10V. Positive ion mode, MRM mode, and ion pair information is shown in table 10.

Table 10 ion pair information

4 oxidative stress level determination

The extent of oxidative damage and the level of antioxidant of tissues are reflected by measuring the levels of Malondialdehyde (MDA), glutathione peroxidase (GSH-PX) and SOD in the striatum. The striatum tissue is taken, 200 mu L of 1% triton is added to 20mg of tissue, after tissue homogenization, centrifugation is carried out for 10min at 3000rpm and 4 ℃, and the supernatant is transferred to obtain 10% homogenized supernatant. The supernatants were used for BCA protein concentration assay, MDA assay, GSH-PX assay and SOD assay, respectively, and were assayed with reference to the kit instructions.

5 statistical analysis

Statistical analysis using One-way ANOVA of Graphpad software, p <0.05, p <0.01, p <0.001, #p <0.05, #p <0.01, #p <0.001 were considered statistically different.

Experimental results:

1 mine test

The difference between the motor behavior and motor ability of the mice can be detected in the mine test, and the test results are shown in fig. 19 (a), wherein the movement track of the mice in the MPTP model group is significantly reduced in the central area compared with the blank group. And the movement track of the positive medicine group (L-Dopa) and the ECH@ANG-Lip group in the central area is obviously increased compared with that of the model group. In addition, compared with a model group, the positive drug group and the ECH@ANG-Lip group mice have remarkable improvement on the total movement path, the average speed and the central area movement time ratio, which indicates that the ECH@ANG-Lip can remarkably improve the movement behavior of MPTP mice.

2 neurotransmitter content determination

The results are shown in fig. 19 (B) and table 11, and compared to the placebo group, the model group, the positive drug group, the echinacoside free drug group and the ech@ang-Lip group exhibited significant down-regulation of striatal dopamine levels. Compared with the model group, the positive medicine group and the ECH@ANG-Lip group have the effect of obviously up-regulating the average of dopamine, 3, 4-dihydroxyphenylacetic acid and homovanillic acid, which proves that the ECH@ANG-Lip can obviously restore the level of dopamine and metabolites thereof in the striatum region of the MPTP mouse.

TABLE 11 determination of striatal neurotransmitter contentn＝6～8)

3 oxidative stress level determination

As shown in fig. 20 and table 12, MDA levels in the striatum of MPTP mice were significantly increased, GSH-PX and SOD activities were significantly decreased, while positive drug group L-Dopa and treatment group ech@ang-Lip significantly decreased MDA levels in the striatum of MPTP mice, and significantly up-regulated GSH-PX and SOD levels, as compared to the placebo group. The ECH@ANG-Lip can obviously improve the level of oxidative stress in the striatum of an MPTP mouse and enhance the antioxidant capacity of the striatum of the mouse.

TABLE 12 determination of oxidative stress level in striatumn＝6～8)

In conclusion, ECH has good neuroprotection on dopaminergic neurons in vitro, but 0.83% of oral absolute bioavailability and extremely low blood brain barrier permeability may severely limit the clinical application of the ECH, and enrichment concentration of echinacoside in brain tissues can reach 230.22 +/-33.76 ng/g by constructing LRP1 targeted liposome, so that neuroprotection of ECH on MPTP mice is remarkably improved. The dopaminergic neurons in the substantia nigra region of the MPTP parkinsonism mouse model are severely lost, leading to reduced dopamine synthesis, and therefore the striatal region dopamine and its metabolite levels are significantly down-regulated. The ECH@ANG-Lip can obviously up-regulate the level of dopamine and metabolites thereof in the striatum area, which proves that the ECH can inhibit the loss of dopaminergic neurons in the substantia nigra area and plays a good role in neuroprotection. In the mine test, the more the movement of the mice in the central area, the higher the enthusiasm of the mice for exploring new environments is; the total movement path and average movement rate of the mice can reflect the difference in the motor ability of the mice. ECH@ANG-Lip significantly improved locomotor activity in MPTP mice.

The above examples only represent a few embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the present application. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. Accordingly, the scope of protection of the present application is to be determined by the claims appended hereto.

Claims

1. A method for preparing echinacoside liposome, comprising the steps of:

2. The method for preparing Echinacoside liposome according to claim 1, wherein the mass ratio of soybean lecithin to cholesterol is 1:5; the concentration of the soybean lecithin is 20mg.mL ^-1 The method comprises the steps of carrying out a first treatment on the surface of the The mass ratio of the echinacoside to the soybean lecithin is 1:40, a step of performing a; the phospholipid-polyethylene glycol and soybean lecithin areThe ratio of the amounts is 1:5.

3. The method for preparing Echinacoside liposome according to claim 1, comprising: dissolving soybean lecithin, cholesterol, phospholipid-polyethylene glycol and echinacoside in an organic solvent, volatilizing the organic solvent in a water bath, adding physiological saline to hydrate and dissolve the film, and performing ultrasonic treatment and filtration treatment to obtain the echinacoside liposome; the ultrasonic power is 30-90W; the water bath temperature is 40-60 ℃, and the hydration time is 10-60min.

4. The echinacoside liposome is characterized in that the echinacoside liposome is prepared by the preparation method of the echinacoside liposome in any one of claims 1-3.

5. A method for preparing LRP1 targeted echinacoside-loaded liposome, which is characterized by comprising the following steps:

6. The method for preparing LRP1 targeted echinacoside-loaded liposomes according to claim 5, wherein the mass ratio of soybean lecithin to cholesterol is 1:5; the concentration of the soybean lecithin is 20mg.mL ^-1 The method comprises the steps of carrying out a first treatment on the surface of the The mass ratio of the echinacoside to the soybean lecithin is 1:40, a step of performing a; the phospholipid-polyethylene glycolThe mass ratio of maleimide to soybean lecithin is 1:5.

7. The method for preparing LRP1 targeted echinacoside-loaded liposomes according to claim 5, wherein the organic solvent is a mixed solvent of ethanol and methanol or a solvent of chloroform and methanol.

8. The method for preparing LRP1 targeted echinacoside-loaded liposome according to claim 5, wherein said dissolving soybean lecithin, cholesterol, phospholipid-polyethylene glycol-maleimide, echinacoside in an organic solvent for film treatment, and dissolving the film by hydration, ultrasonic treatment, and filtration treatment comprises:

Dissolving soybean lecithin, cholesterol, phospholipid-polyethylene glycol-maleimide and echinacoside in an organic solvent, volatilizing the organic solvent in a water bath, adding physiological saline to hydrate and dissolve the film, and carrying out ultrasonic treatment and filtration treatment; the ultrasonic power is 30-90W; the water bath temperature is 40-60 ℃, and the hydration time is 10-60min.

9. The LRP1 targeted echinacoside-loaded liposome is characterized in that the LRP1 targeted echinacoside-loaded liposome is prepared by the preparation method of the LRP1 targeted echinacoside-loaded liposome in any one of claims 5-8.

10. Use of an echinacoside liposome according to claim 4 or an LRP 1-targeted echinacoside-loaded liposome according to claim 9 in the manufacture of a medicament for the treatment of parkinson's disease.