CN108143719B

CN108143719B - Polypeptide-carrying nanoliposome and preparation method and application thereof

Info

Publication number: CN108143719B
Application number: CN201711448230.4A
Authority: CN
Inventors: 骆清铭; 张智红; 卢利森; 骆海明
Original assignee: Huazhong University of Science and Technology; Ezhou Institute of Industrial Technology Huazhong University of Science and Technology
Current assignee: Huazhong University of Science and Technology; Ezhou Institute of Industrial Technology Huazhong University of Science and Technology
Priority date: 2017-12-27
Filing date: 2017-12-27
Publication date: 2022-01-25
Anticipated expiration: 2037-12-27
Also published as: CN108143719A

Abstract

The invention discloses a polypeptide-carried nanoliposome, which contains phospholipid, cholesterol ester and polypeptide R4F in a molar ratio of 30 (0.5-1) to 8; and a nano liposome carrying curcumin, which contains curcumin, phospholipid, cholesterol ester and R4F in a molar ratio of (5-10): 30, (0.5-1): 8. Discloses a preparation method of the nano liposome, which adopts a film dispersion method of the existing liposome. Also discloses application of the nano-liposome in preparing a medicament for treating experimental spontaneous encephalomyelitis. The nano liposome has high targeting property, excellent physicochemical property, good biocompatibility, high encapsulation rate and low toxic and side effects, and can effectively treat experimental spontaneous encephalomyelitis.

Description

Polypeptide-carrying nanoliposome and preparation method and application thereof

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a polypeptide-carrying nanoliposome, and a preparation method and application thereof.

Background

The nano liposome is an artificial membrane, the hydrophilic head of phospholipid molecules in water is inserted into the water, the hydrophobic tail of the liposome extends to the air, and a spherical liposome with double-layer lipid molecules is formed after stirring, wherein the diameter of the spherical liposome is different from 25-1000 nm. The nano liposome can be used for transgenosis or carrying prepared medicines, particularly the latter, as a novel administration mode, the nano liposome can be fused with cell membranes to deliver the medicines into cells, and the nano liposome carrying the functions of the medicines is gradually concerned by people. The mode of carrying the drug by the nanoliposome changes the existing drug delivery mode, and solves the problems of insolubility, low utilization rate and the like of most hydrophobic drugs. At present, the use of PLGA nano liposome-encapsulated drugs for treating different diseases has been reported to achieve certain curative effect. In the treatment of inflammatory diseases such as encephalitis, nanoliposome encapsulated drugs have also been used. However, due to the lack of targeting specificity of the nanoliposome, the passive targeting uptake rate is low, and due to the large particle size of the nanocarrier, the nanocarrier cannot be effectively distributed on the whole affected part, and thus the treatment effect is not ideal enough. There is a need for more drug-loaded nanoliposomes with targeted specificity for the treatment of encephalitis.

Curcumin is a polyphenol compound, is mainly extracted from plant turmeric, and is a dietetic therapy medicine. Curcumin has a specific inhibitory effect on intracellular molecules NF-kp β, and thus effectively inhibits NF-kp-related inflammatory signaling molecular pathways, and is capable of greatly inhibiting the occurrence of inflammation, and has been receiving attention for a long time in the past. However, the clinical application is limited due to three major disadvantages of curcumin; a first disadvantage of curcumin is its extremely low water solubility, with the maximum water solubility of curcumin being 30nM, while the working curcumin concentrations are generally in the μ M range; a second disadvantage of curcumin is its chemical instability in aqueous solution, being easily metabolised by oxidation; a third disadvantage of curcumin is its extremely low cellular uptake, most of which is able to cross the cell membrane, but the amount of infiltration into the cytoplasm is very small. If these obstacles of curcumin can be overcome, it will help curcumin play a role in clinical treatment.

In conclusion, the nano liposome capable of effectively targeting the brain inflammation diseases is developed, and the application of the nano liposome in clinical disease diagnosis, particularly brain inflammation disease diagnosis, can be improved; the development of the nano-liposome carrying curcumin, which has ultra-small particle size and water solubility and can effectively target and treat brain inflammatory diseases, can greatly promote the application of curcumin in the treatment of clinical diseases, particularly brain inflammatory diseases.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: overcomes the defect of poor effect of the nanometer liposome targeting the brain inflammation diseases in the prior art, and provides the nanometer liposome targeting the brain inflammation diseases; preferably, the defects that curcumin is difficult to dissolve in water, unstable, low in cell uptake rate and not ideal in clinical treatment effect in the prior art are overcome, and the curcumin-carrying nano liposome capable of effectively treating brain inflammation diseases and the preparation method and application thereof are provided.

One of the technical solutions adopted to solve the above technical problems of the present invention is:

a nanoliposome carrying polypeptide comprises phospholipid, cholesterol ester and targeting polypeptide in a molar ratio of 30 (0.5-1) to 8, wherein the targeting polypeptide is R4F with an amino acid sequence shown as SEQ ID NO. 1.

Preferably, the nanoliposome further comprises a lipophilic carbocyanine dye; preferably, the lipophilic carbocyanine dye, phospholipid, cholesterol ester and R4F are in a molar ratio of 2:30:1: 8.

the nanoliposome carrying the polypeptide is characterized by further comprising curcumin, wherein the molar ratio of the curcumin to phospholipid, cholesterol ester and R4F is (5-10): 30, (0.5-1): 8. Preferably, the nanoliposome is composed of curcumin, phospholipids, cholesterol esters and R4F.

The components of liposomes, according to common general knowledge in the art, mainly comprise phospholipids forming the bilayer structure of the liposomes and cholesterol/cholesterol ester substances which modify the membrane fluidity. Wherein, the lower the proportion of the active ingredient relative to the phospholipid and cholesterol ester substances, the better the encapsulation efficiency, but the drug loading is low; conversely, the drug loading is high, but encapsulation efficiency may decrease. The components, content and preparation method of the liposome can affect the encapsulation efficiency, drug loading capacity, nano particle size and the like of the final product.

Wherein, if the curcumin proportion is too low, the drug loading rate and the curative effect of the liposome are affected. Therefore, more preferably, the mass ratio of the curcumin, the phospholipid, the cholesterol ester and the R4F is 10:30:1: 8.

Even more preferably, the phospholipid is dimyristoyl phosphatidylcholine; the cholesterol ester is cholesterol oleate.

the preparation method of the nano liposome adopts the film dispersion method of the existing liposome.

According to the present invention, the nanoliposome can be prepared by conventional liposome preparation methods, such as thin film method, reverse phase evaporation method, solvent injection method, and multiple emulsion method. The present invention employs the most primitive but by far the most basic and widely used thin film dispersion method, preferably, the preparation method of nanoliposomes comprises the following steps:

1) dissolving phospholipid and cholesterol ester in a container containing organic solvent, and mixing to obtain a mixture;

2) forming a thin film of said mixture on the bottom of said container;

3) adding a buffer solution, and fully resuspending the drug film to form a uniform suspension;

4) and (4) performing ultrasonic treatment until the suspension becomes clear, adding the polypeptide, and uniformly mixing to obtain the polypeptide.

Preferably, the phospholipid is dimyristoyl phosphatidylcholine, and the cholesterol ester is cholesterol oleate.

Preferably, the step 1) further comprises adding curcumin, wherein the molar ratio of the curcumin to the phospholipids, the cholesterol ester and the R4F is (5-10): 30, (0.5-1): 8; preferably, the molar ratio of the curcumin to the phospholipid to the cholesterol ester to R4F is 10:30:1: 8; preferably, the steps further comprise 5) ultrafiltration to remove free curcumin and polypeptides; more preferably, the ultrafiltration is performed using a 30KD ultrafiltration tube.

Even more preferably, the organic solvent is chloroform; preferably, the film is formed by drying nitrogen or is prepared by evaporating an organic solvent by a rotary evaporator under reduced pressure; more preferably, the buffer solution is phosphate buffer solution with pH of 6.5-7.5.

the application of the nano-liposome in the preparation of the medicine for diagnosing or treating experimental spontaneous encephalomyelitis.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

1) the targeting property is good: can be specifically targeted to the affected part of brain inflammation; 2) excellent physical and chemical properties: the average particle size of the nano-liposome is measured to be about 15nm by using a dynamic laser light scattering method; 3) the biocompatibility is good: the raw materials used for preparing the nano liposome are respectively used for clinical or clinical tests, and have good biocompatibility; 4) the preparation process is simple and is convenient for large-scale production; 5) the drug encapsulation rate is more than 60 percent, which meets the requirements of pharmacopoeia of the people's republic of China on the microcapsule preparation; by incubating curcumin with a certain dosage and the components for synthesizing the nanoliposome, the water solubility of the curcumin can be obviously improved, and the curcumin is changed from opaque suspension liquid into clear suspension liquid; 6) the treatment effect is good: the curcumin-carrying nano-liposome has a good treatment effect in experimental spontaneous encephalomyelitis, can efficiently target related inflammatory cells in blood by using the polypeptide R4F, and plays a role by blocking the related inflammatory cells from crossing a blood brain barrier; 7) the toxic and side effects are low: all materials of the nano liposome carrying curcumin are common in clinical application. Animal experiment research results show that the nano liposome treatment group carrying curcumin can obviously inhibit the generation of experimental spontaneous encephalomyelitis and promote the recovery of the body weight of a mouse; 8) the functions can be expanded: the nano liposome carrier carrying curcumin can be used for highlighting clinical application of curcumin through carrying polypeptide targeting related diseases, and simultaneously can be used for imaging by loading related dye molecules in the core or achieving the purpose of synergistic treatment by cooperatively loading other related medicaments, thereby realizing the efficacy of synergistic targeting or synergistic treatment of diseases.

Drawings

FIG. 1 is a general structural diagram of a curcumin-loaded nanoliposome;

FIG. 2 shows statistics of the nanoparticle size distribution obtained by dynamic light scattering of curcumin loaded in nanoliposomes;

fig. 3 is a stability test of nanoliposomes carrying curcumin at different incubation conditions, wherein No.1 is a normal 4 ℃ environment; no. 2 is a 3-hour environment at 37 ℃; no. 3 is placed in 10% serum at 37 ℃ for 3 hours; no. 4 is placed in 10% serum at 37 ℃ for 6 hours; no. 5 is placed in 10% serum at 37 ℃ for 6 hours; number 6 is 10 hours in 10% serum at 37 ℃; no. 7 is placed in 10% serum at 37 ℃ for 24 hours;

figure 4 is a graph of the effect of curcumin-loaded nanoliposomes at different injected doses on EAE incidence;

fig. 5 is statistics of treatment effect of curcumin-carrying nanoliposomes and corresponding control group treatment method after tail vein injection induction of EAE model mice, including statistics of body weight change (a) and statistics of mouse morbidity (B);

FIG. 6 shows that HE staining is performed on mouse brain after treatment of EAE model mice with nanoliposomes carrying curcumin to judge the infiltration of immune cells;

FIG. 7 shows LFB staining of spinal cords of mice treated with curcumin-loaded nanoliposomes in EAE model mice to determine myelin damage;

FIG. 8 shows the results of 24h imaging of nanoliposomes carrying the fluorescent dye DIRBOA injected via tail vein into EAE model mice (no obvious clinical symptoms) to evaluate the diagnostic effect on disease;

fig. 9 is the brain cryosection immunofluorescence results of C57 mice 24h post tail vein injection induced EAE model with nanoliposomes carrying curcumin.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Example 1 preparation of nanoliposomes carrying polypeptides

The nanoliposome in the embodiment mainly comprises targeting polypeptide R4F, phospholipid and cholesterol oleate, wherein the sequence of the targeting peptide R4F is FAEKFKEAVKDYFA KFWDGSG (SEQ ID No.1, derived from C57 strain mouse myelin sheath antigen polypeptide), and the preparation method of the nanoliposome carrying the polypeptide comprises the following steps:

1) 2mg of dimyristoyl phosphatidylcholine (DMPC) and 0.065mg of cholesterol oleate were mixed well in a glass test tube, and the mouth of the test tube was sealed with a sealing film;

2) blowing the chloroform in the test tube to dry in a stable nitrogen flow, so that the mixture in the step 1) can form a layer of thin film at the bottom of the test tube;

3) putting the test tube into a vacuum drier for vacuum drying for 1 h;

4) adding 2ml of phosphate buffer (pH 7.2) into the test tube, filling nitrogen, sealing, and fully suspending the product film at the bottom of the test tube by using a vortex shaking instrument to form uniform suspension;

5) placing the test tube at 42 ℃ and carrying out ultrasonic treatment until the suspension solution becomes clear;

6) a PBS solution (pH9.0) containing 2mg of the polypeptide R4F was added to the sealed tube by using a syringe, mixed well, sealed, and left overnight at 4 ℃.

7) The next day, the sample was concentrated using an ultrafiltration tube (30KD, Merck), centrifuged at 2500rpm in a 4 ℃ environment to remove free polypeptide, resulting in a monolayer nanoliposome (HPPS) with a 30:1:8 molar ratio of phospholipid, cholesterol ester and polypeptide, having a small particle size and good permeability.

Example 2 preparation of nanoliposomes carrying curcumin

In this embodiment, the nanoliposome carrying curcumin mainly comprises targeting polypeptide R4F, phospholipid, curcumin, and cholesterol oleate, wherein the sequence of the targeting peptide R4F is shown in SEQ ID No.1, and the step of preparing the nanocarrier carrying curcumin is:

1) mixing 2mg dimyristoyl phosphatidylcholine (DMPC), 0.065mg Cholesterol Oleate (CO), and 0.3mg curcumin in chloroform; mixing in glass test tube, and sealing the test tube opening with sealing film;

3) putting the test tube into a vacuum drier for vacuum drying for 1 h;

4) adding 2ml of phosphate buffer (pH 7.2) into the test tube, filling nitrogen, sealing, and fully suspending the drug film at the bottom of the test tube by using a vortex oscillation instrument to form uniform yellow suspension;

6) adding a PBS solution (pH9.0) containing 2mg of the targeting polypeptide R4F into the sealed test tube by using a syringe, uniformly mixing, sealing, and standing at 4 ℃ overnight;

7) the next day, the sample is concentrated by using an ultrafiltration tube (30KD, Merck), and centrifuged at 2500rpm in an environment of 4 ℃ to remove free polypeptide and curcumin, so that the monolayer nanoliposome (HPPS) with small particle size and good permeability, namely HPPS-curcumin carrying curcumin, of which the molar ratio of curcumin to phospholipid to cholesterol ester to polypeptide is 10:30:1:8 is obtained.

The free curcumin solution is insoluble in water and is suspended granular yellow opaque solution; the nano liposome solution coated with curcumin is a clear transparent yellow solution. The whole structure of the prepared nanoliposome is shown in figure 1, wherein phospholipid forms a monolayer membrane of the liposome, then R4F is embedded in the monolayer membrane of the phospholipid, and the core is loaded with curcumin and cholesterol ester. The average particle size was measured using a nanosized particle size potential analyzer Zetasizer Nano-ZS90, and as shown in FIG. 2, the average particle size was 15.2. + -. 2.3 nm. Envelope rate calculation formula: the encapsulation rate is the drug content in the actual carrier/the drug content added in the synthesized nano carrier, and the calculated encapsulation rate is 0.09-0.12 mg/0.15 mg-60% -80%. Fig. 3 is a stability test of the nanoliposome carrying curcumin under different incubation conditions, and results from nos. 1-6 show that the nanoliposome can be stable in 10% serum at 37 ℃ for 10 hours.

Example 3 preparation of DIRBOA-loaded nanoliposomes

In this embodiment, the nanoliposome carrying a fluorescent dye DIRBOA (lipophilic carbocyanine dye) mainly comprises targeting polypeptide R4F, phospholipid, DIRBOA, and cholesterol oleate, wherein the sequence of the targeting peptide R4F is shown as SEQ ID No.1, and the step of preparing the nanocarrier carrying DIRBOA is as follows:

1) dissolving 2mg dimyristoyl phosphatidylcholine (DMPC), 0.065mg Cholesterol Oleate (CO), 0.2mg DIRBOA in chloroform solution; mixing in glass test tube, and sealing the test tube opening with sealing film;

3) putting the test tube into a vacuum drier for vacuum drying for 1 h;

4) adding 2ml of phosphate buffer (pH 7.2) into the test tube, filling nitrogen, sealing, and fully suspending the film at the bottom of the test tube by using a vortex shaking instrument to form uniform suspension;

7) the next day, the sample was concentrated using an ultrafiltration tube (30KD, Merck), centrifuged at 2500rpm in a 4 ℃ environment to remove free polypeptide and curcumin, and unilamellar nanoliposome HPPS-DIRBOA with a molar ratio of DIRBOA, phospholipid, cholesterol ester and polypeptide of 2:30:1:8 was obtained.

Example 4 Targeted Experimental spontaneous encephalomyelitis

Curcumin-loaded nanoliposomes were used to model experimental spontaneous encephalomyelitis (EAE) treatment:

constructing an experimental spontaneous encephalomyelitis model: freund's adjuvant (5mg/mL inactivated conjugated Mycobacteria) emulsified mouse self polypeptide MOG35-55 (mouse neuronal myelin sheath antigen polypeptide, for published sequences, see Ingunn M Stremmes, et al. active indication of experimental allogenic cytopaelitis, Nature protocol,2006) sequence MEVGWYRSPFSRVVHL YRNGK, synthesized by Shanghai Chu peptide Biotech, Inc., injected at four points subcutaneously, followed by injection of Pertussis Toxin (PTX) at 0 and 24 hours in the mouse tail vein. The inoculation date was defined as day 0, and the dates thereafter were designated as

days

1, 2, 3 and … … 19, respectively. Mice were stratified and randomly grouped on day 9 as needed, with no less than 5 per group (mice with experimental spontaneous encephalomyelitis were grouped).

Tail vein injection was started on day 9; a total of 5 injections; before injection, each mouse was labeled, and the mass of each mouse was weighed and recorded in a pre-prepared table.

When the Control group (blank Control group) was injected, only sterile PBS was injected into the tail vein, and the injection amount was 0.1ml/10 g.

V. therapy group (treatment group) curcumin nanoliposomes prepared in example 2 were injected in tail vein at injection doses of 1.8mg, 0.9mg and 0.18mg curcumin content/kg mouse body weight and injection volumes of 0.1ml/10g mouse body weight, i.e. the HPPS-100nM curcumin, HPPS-50nM curcumin and HPPS-10nM curcumin groups shown in table 1.

NP Control group (nanoliposome Control group) nanoliposomes carrying the polypeptide R4F of example 1 were used as controls, and the concentration injected was determined according to the concentration of the polypeptide, consistent with that of the Therapy group.

The injection period was 5 times every other day. And the body weight of the mice was measured and the clinical behavior was evaluated each time. Measurements were taken from day 9 and every other day, and the measurement data was recorded in the table. FIG. 4 is a graph of clinical scores, with each group's score increasing with increasing days of induction, with the HPPS-50nM and HPPS-100nM groups scoring significantly lower than the other groups.

Table 1 statistics of the therapeutic effect of different concentrations of curcumin-loaded nanoliposomes against EAE

The therapeutic effect of the nanocarrier carrying curcumin is shown in table 1, and the incidence rate of mice in the HPPS-50nM curcumin group is 60%; in the HPPS-100nM curcumin group, the mouse morbidity is reduced to 40%, which is better than the HPPS-10nM curcumin group and the control treatment effect.

As the physiological status of the mice was measured by using the body weight of the mice, it can be seen from FIG. 5 that 100nmol of HPPS-Curcumin (Curcumin) injected into tail vein is very effective, the body weight of the mice can be basically maintained to be constant (FIG. 5A), and the EAE model of the group of treated mice is also more effective (FIG. 5B).

After 5 rounds of treatment, the ratio of inhibiting inflammation of the nano-liposome carrying curcumin is 80%, which is remarkably improved relative to other control groups, and the morbidity score is reduced from 3.5 to 1.5 on average, and the results are shown in figures 6 and 7. FIG. 6 is HE staining of brains of mice treated with different groups for observing infiltration of peripheral blood immune cells, wherein the enlarged part of the image is the ventricular region and the site of infiltration of immune cells, and it can be seen that the brain of mice treated with HPPS-Curcumin 100nmol has less infiltration of immune cells. Fig. 7 shows LFB staining of spinal cord in mice treated in different groups to determine the damage degree of myelin sheath of spinal cord nerve, and the results in fig. 7 show that the nanoliposome added with curcumin can significantly inhibit damage of myelin sheath by immune system and maintain the integrity of neuron.

The judgment standard of the morbidity score is as follows:

0: the mice are normal without any abnormal behavior

1: mouse tail droop

2: drooping tail of mouse + gradual paralysis of hind limb of mouse

3: drooping tail of mouse + complete paralysis of hind limb of mouse

4: drooping mouse tail, complete paralysis of mouse hind limb, paralysis of mouse forelimb

5: death of mice

Example 5 early diagnosis of Experimental spontaneous encephalomyelitis

The nanoliposome carrying the fluorescent dye DIRBOA is used for an experimental spontaneous encephalomyelitis early diagnosis model:

constructing an experimental spontaneous encephalomyelitis model: freund's adjuvant (5mg/mL inactivated conjugated mycobacteria) emulsified mouse self polypeptide MOG35-55, injected four points subcutaneously, followed by mouse caudal vein injection of Pertussis Toxin (PTX) at 0 and 24 hours. The inoculation date was designated as day 0, and the dates thereafter were designated as

days

1, 2, 3 and … … 12, respectively.

Tail vein injection started on day 11; the concentration of HPPS-DIRBOA prepared in injection example 3 was 20nmol (calculated as the concentration of DIRBOA); mice were placed for 24h, after which fluorescence imaging was performed using a laboratory self-contained whole fluorescence imaging system.

The Control group is set to carry out tail vein injection of HPPS-DIRBOA for normal mice, and other operations are carried out as an experimental group.

Imaging of the mice clearly shows that the signals of the nanocarriers are clearly observed in the early stage of the mice that induced diseases, and the signals are in the extracted immune cells, as shown in fig. 8. As can be seen from the in vivo imaging results in fig. 8, in comparison to the mice without induced disease, the simultaneous injection of nanoliposomes carrying the fluorescent dye DIRBOA induced only a large amount of fluorescent signals in the brains of EAE model mice, whereas normal mice did not contain DIRBOA signals; the brain and spinal cord of mice that alone induced the EAE disease model contained a large number of DIRBOA signals as can be seen by ex vivo imaging of the brain and spinal cord anatomy of mice and normal mice that induced EAE disease, and to confirm the origin of these signals, it was found that most of the fluorescent signals were mainly located among the extracted immune cells by isolation of immune cells of the spinal cord and brain of the mice followed by fluorescence signal acquisition at the cell level.

The CX3CR1-GFP hybrid mice for inducing the EAE model are subjected to brain cryosection 24h after the injection of HPPS-DIRBOA, and it can be seen from figure 9 that DIRBOA fluorescence signals are mainly concentrated in inflammatory monocytes and neutrophils, which indicates that the nanoliposome is mainly taken up by the inflammatory monocytes and the neutrophils, and also indicates that the efficacy of the nanoliposome is possibly related to the targeting of the two cells.

<110> university of science and technology in Huazhong;

institute of Industrial and technology, Ezhou, university of science and technology, Huazhong

<120> polypeptide-carried nanoliposome and preparation method and application thereof

<130> P1711487C

<140> 2017114482304

<141> 2017-12-27

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 21

<212> PRT

<213> Mus Musculus

<220>

<223> R4F

<400> 1

Phe Ala Glu Lys Phe Lys Glu Ala Val Lys Asp Tyr Phe Ala Lys Phe

1 5 10 15

Trp Asp Gly Ser Gly

20

Claims

1. The polypeptide-carried nanoliposome for treating encephalomyelitis is characterized by consisting of curcumin, phospholipid, cholesterol ester and targeting polypeptide, wherein the targeting polypeptide is R4F with an amino acid sequence shown as SEQ ID NO.1, the molar ratio of the curcumin to the phospholipid to the cholesterol ester to R4F is 10:30:1:8, the phospholipid is dimyristoyl phosphatidylcholine, and the cholesterol ester is cholesterol oleate.

2. The method for preparing nanoliposomes according to claim 1, wherein the method for preparing nanoliposomes uses a thin film dispersion method of existing liposomes.

3. The method of preparing nanoliposomes according to claim 2, comprising the steps of:

1) dissolving curcumin, phospholipid and cholesterol ester in a molar ratio of 10:30:1 in a container containing an organic solvent, and uniformly mixing to obtain a mixture;

2) forming a film of the mixture on the bottom of the container;

3) adding a phosphate buffer solution with the pH value of 6.5-7.5, and fully suspending the film to form a uniform suspension;

4) performing ultrasonic treatment until the suspension becomes clear, adding the targeting polypeptide, and uniformly mixing to obtain the polypeptide;

the phospholipid is dimyristoyl phosphatidylcholine, and the cholesterol ester is cholesterol oleate.

4. The method of preparing nanoliposomes according to claim 3, wherein the step further comprises 5) ultrafiltration to remove free curcumin and the targeting polypeptide.

5. The method for preparing nanoliposomes according to claim 4, wherein the ultrafiltration is performed using a 30KD ultrafiltration tube.

6. The method for preparing nanoliposomes according to any one of claims 3 to 5, wherein the organic solvent is chloroform.

7. The method of claim 6, wherein the film is formed by drying with nitrogen or evaporating the organic solvent under reduced pressure with a rotary evaporator.

8. The method for preparing nanoliposomes according to claim 7, wherein the phosphate buffer is a phosphate buffer at pH 7.2.

9. Use of nanoliposomes according to claim 1 in the preparation of a medicament for the treatment of experimental spontaneous encephalomyelitis.