Cholesterol derivatives and liposomes with mitochondrial targeting function and preparation methods and applications thereof

Abstract

The present invention relates to the field of biomaterials, and discloses a cholesterol derivative and liposome with mitochondrial targeting function, and a preparation method and application thereof. The structural formula of the cholesterol derivative is shown in the following formula (1). The cholesterol derivative can be assembled into liposomes with phospholipids for targeted delivery of drugs, and can promote the enrichment of drugs in mitochondria.

Description

Technical Field

[0001] The invention relates to the field of biomaterials, and in particular to cholesterol derivatives and liposomes, and preparation methods and applications thereof. Background Art

[0002] Mitochondria are the energy supply factory for cell life activities, and are involved in many physiological activities such as the synthesis of adenosine triphosphate (ATP), the generation of reactive oxygen species (ROS), and cell apoptosis. A large number of studies have shown that mitochondria play a very important role in the occurrence and development of tumors. Therefore, targeting mitochondria is a new strategy for cancer treatment.

[0003] Triphenylphosphine (TPP) has a lipophilic ion with a delocalized positive charge, which can generate electrostatic attraction with the hyperpolarized negative inner membrane of mitochondria in cancer cells to drive them through the membrane, and can also use its own lipophilicity to easily pass through the hydrophobic barrier of the mitochondrial membrane, effectively enter the mitochondria and accumulate in the mitochondria. Therefore, TPP is widely used as a mitochondrial targeting group. Summary of the invention

[0004] The purpose of the present invention is to overcome the problems of the prior art, such as the complex structure, long synthesis route, and single function of mitochondrial targeting materials, and to provide a cholesterol derivative and liposome with mitochondrial targeting function and a preparation method and application thereof. The cholesterol derivative is prepared by a one-step esterification reaction of (5-carboxypentyl) triphenylphosphine bromide and cholesterol, and has the mitochondrial targeting function of triphenylphosphine, and can utilize the characteristics of cholesterol as a liposome membrane stabilizer and phospholipids to assemble into liposomes for mitochondrial targeted delivery of anticancer drugs.

[0005] In order to achieve the above object, the present invention provides a cholesterol derivative having mitochondrial targeting function, wherein the structural formula of the cholesterol derivative is shown in the following formula (1):

[0006]

[0007] The second aspect of the present invention provides a method for preparing the above-mentioned cholesterol derivative having mitochondrial targeting function, the method comprising the following steps:

[0008] A1: In an inert atmosphere, (5-carboxypentyl)triphenylphosphine bromide, N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and organic solvent A are mixed to obtain a mixed solution;

[0009] A2: In an inert atmosphere, a solution containing cholesterol is added dropwise to the mixed solution obtained in step (1), followed by mixing.

[0010] Preferably, the molar ratio of (5-carboxypentyl)triphenylphosphonium bromide, N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and cholesterol is 1:1-2:1-2:1.

[0011] Preferably, in step A1, the mixing time is 1-24h;

[0012] Preferably, in step A2, the mixing time is 1-24h;

[0013] Preferably, in step A2, the dripping time is 5-10 min.

[0014] The third aspect of the present invention provides the use of the above-mentioned cholesterol derivative with mitochondrial targeting function in the preparation of targeted drug carriers.

[0015] The fourth aspect of the present invention provides a liposome with mitochondrial targeting function, wherein the liposome is prepared from a raw material containing a phospholipid, a drug and a cholesterol derivative represented by formula (1);

[0016] Preferably, the weight ratio of the phospholipid, the drug and the cholesterol derivative represented by formula (1) is 10-50:1:1-5;

[0017] Preferably, the drug is selected from at least one of tripterygium wilfordii, paclitaxel, doxorubicin and doxorubicin.

[0018] The fifth aspect of the present invention provides a method for preparing the above-mentioned liposome with mitochondrial targeting function, the method comprising the following steps:

[0019] B1: mixing phospholipids, drugs, cholesterol derivatives represented by formula (1) and organic solvent B, and then performing rotary evaporation to obtain a lipid film;

[0020] B2: Add water to the lipid film for hydration, followed by sonication.

[0021] Preferably, in step B1, the hydration conditions include: hydration temperature of 30-40° C., and hydration time of 30-120 min.

[0022] Preferably, in step B2, the sonication is performed in an ultrasonic cell disruptor;

[0023] Preferably, the ultrasonic power is 150-225 W, and the ultrasonic time is 10-20 min.

[0024] A sixth aspect of the present invention provides use of the above liposome in preparing a drug delivery system for treating tumor diseases.

[0025] Preferably, the tumor disease is selected from lung cancer, breast cancer or liver cancer.

[0026] The present invention uses cholesterol as a matrix and (5-carboxypentyl)triphenylphosphonium bromide as a targeting group to synthesize a cholesterol derivative (Chol-TPP) with mitochondrial targeting function through an esterification reaction; the cholesterol derivative can be assembled into liposomes with phospholipids for targeted drug delivery, and can promote drug enrichment in mitochondria. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the result of H NMR spectrum detection of the product prepared in Example 1;

[0028] Figure 2 is the result of carbon NMR spectrum test of the product prepared in Example 1;

[0029] Figure 3 is the mass spectrometry result of the product prepared in Example 1;

[0030] Figure 4 is an observation photograph of the product prepared in Example 2;

[0031] Figure 5 is the TEM characterization result of the product prepared in Example 2;

[0032] Figure 6 This is the result of observing mitochondrial targeting under a laser confocal microscope in Test Example 3. DETAILED DESCRIPTION

[0033] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0034] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0035] In one aspect, the present invention provides a cholesterol derivative having mitochondrial targeting function, wherein the structure of the cholesterol derivative is shown in the following formula (1):

[0036]

[0037] The second aspect of the present invention provides a method for preparing the above-mentioned cholesterol derivative having mitochondrial targeting function, the method comprising the following steps:

[0038] A1: In an inert atmosphere, (5-carboxypentyl)triphenylphosphine bromide, N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and organic solvent A are mixed to obtain a mixed solution;

[0039] A2: In an inert atmosphere, a solution containing cholesterol is added dropwise to the mixed solution obtained in step (1), followed by mixing.

[0040] In the method of the present invention, in order to improve the reaction efficiency and reduce the generation of by-products, the amount of the reactants can be reasonably controlled. Preferably, the molar ratio of (5-carboxypentyl) triphenylphosphine bromide, N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and cholesterol is 1:1-2:1-2:1. Specifically, it can be 1:1.1:1.1:1, 1:1.2:1.2:1, 1:1.3:1.3:1, 1:1.4:1.4:1, 1:5.1:1.5:1, 1:1.6:1.6:1, 1:1.6:1.6:1, 1:1.7:1.7:1, 1:1.8:1.8:1 or 1:1.9:1.9:1.

[0041] In the method of the present invention, the mixing in step A1 can be stirring mixing, and the mixing process can activate the carboxyl group, which is beneficial to the subsequent reaction.

[0042] Preferably, in step A1, the mixing time is 1-24 hours. Specifically, it can be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 15 hours, 20 hours, 22 hours or 24 hours.

[0043] In the method of the present invention, in step A1, the organic solvent A is any organic solvent commonly used in the art, preferably dichloromethane or chloroform.

[0044] In a preferred embodiment, in order to better promote the reaction between cholesterol and (5-carboxypentyl)triphenylphosphonium bromide, in step A2, the solution containing cholesterol is added dropwise to the solution obtained in step A1. Furthermore, the time of the addition is 5-10 minutes.

[0045] In the method of the present invention, the solution containing cholesterol is obtained by mixing cholesterol and an organic solvent. Preferably, the solvent mixed with cholesterol is the same organic solvent as the organic solvent A in step A1.

[0046] In a preferred embodiment, in step A2, the mixing time is 1-24 h; specifically, it can be 1 h, 2 h, 3 h, 4 h, 5 h, 10 h, 15 h, 20 h, 22 h or 24 h.

[0047] In a preferred embodiment, in step A2, a reaction is carried out during the mixing process to obtain the cholesterol derivative described in formula (1). The mixing process has no special requirements for temperature and can be carried out at room temperature.

[0048] In a preferred embodiment, step A2 further comprises separating and purifying the product obtained after the mixed reaction, and the separation and purification process comprises: extracting the reaction solution with dilute hydrochloric acid, collecting the organic solvent layer, performing rotary evaporation to obtain a small amount of yellow liquid, and then separating and purifying by silica gel column chromatography to obtain a colorless liquid, and drying to obtain Chol-TPP.

[0049] The third aspect of the present invention provides the use of the above-mentioned cholesterol derivative with mitochondrial targeting function in the preparation of targeted drug carriers.

[0050] The fourth aspect of the present invention provides a liposome with mitochondrial targeting function, wherein the liposome is prepared from raw materials containing phospholipids, drugs and a cholesterol derivative represented by formula (1).

[0051] In a preferred embodiment, the weight ratio of the phospholipid, the drug and the cholesterol derivative represented by formula (1) is 10-50:1:1-5; more preferably 20-30:1:2.5-3.5.

[0052] In a specific embodiment, the weight ratio of the phospholipid to the drug can be 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1.

[0053] In a specific embodiment, the weight ratio of the drug to the cholesterol derivative represented by formula (1) can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5.

[0054] In the present invention, the drug can be a fat-soluble drug or a water-soluble drug. By preparing liposomes with the drug, phospholipids and the cholesterol derivative shown in formula (1), mitochondrial targeted delivery of the drug is achieved, thereby improving the therapeutic effect of the drug on tumors.

[0055] In a preferred embodiment, the drug is selected from at least one of tripterygium wilfordii, paclitaxel, doxorubicin and doxorubicin.

[0056] In the present invention, the phospholipid may be selected from at least one of soybean lecithin, egg yolk lecithin, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

[0057] The fifth aspect of the present invention provides a method for preparing the above-mentioned liposome with mitochondrial targeting function, the method comprising the following steps:

[0058] B1: mixing phospholipids, drugs, cholesterol derivatives represented by formula (1) and organic solvent B, and then performing rotary evaporation to obtain a lipid film;

[0059] B2: Add water to the lipid film for hydration, followed by sonication.

[0060] In a preferred embodiment, the hydration conditions include: a hydration temperature of 30-40°C, and a hydration time of 30-120 min. Specifically, the hydration temperature can be 30°C, 35°C or 40°C; the hydration time can be 30 min, 35 min, 40 min, 50 min, 55 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min or 120 min.

[0061] In a preferred embodiment, the organic solvent B is selected from chloroform.

[0062] Further preferably, in step B2, the hydration process preferably uses deionized sterile water.

[0063] In a preferred embodiment, the ultrasound is performed in an ultrasonic cell disruptor. Preferably, the ultrasound power is 150-225W, and the ultrasound time is 10-20min. Specifically, the ultrasound power can be 150W, 160W, 170W, 180W, 190W, 200W, 210W, 220W or 225W; the ultrasound time can be 10min, 12min, 14min, 16min, 18min or 20min.

[0064] A sixth aspect of the present invention provides use of the above liposome in preparing a drug delivery system for treating tumor diseases.

[0065] Preferably, the tumor disease is selected from lung cancer, breast cancer or liver cancer.

[0066] The present invention will be described in detail below through examples, but the protection scope of the present invention is not limited thereto.

[0067] Example 1

[0068] The preparation of the cholesterol derivative (Chol-TPP) represented by formula (1) comprises the following steps:

[0069] A1; Under nitrogen protection, 1 mmol (5-carboxypentyl) triphenylphosphine bromide (TPP), 1.2 mmol N,N'-dicyclohexylcarbodiimide (DCC) and 1.2 mmol 4-dimethylaminopyridine (DMAP) were dissolved in 20 mL dichloromethane, and stirred at room temperature for 3 h to activate the carboxyl group to obtain a mixed solution;

[0070] A2: Dissolve 1 mmol of cholesterol (Chol) in 10 mL of dichloromethane, and slowly add it dropwise to the mixed solution obtained in step A1 under nitrogen protection for 5 min; continue to stir and react at room temperature for 24 h; extract the reaction solution with dilute hydrochloric acid solution, take the organic solvent layer, and rotary evaporate to obtain a small amount of yellow liquid, separate and purify it by silica gel column chromatography to obtain a colorless liquid, and dry it.

[0071] The prepared product was characterized by hydrogen nuclear magnetic resonance spectroscopy. Figure 1 As shown, the results are as follows:

[0072] 1 H NMR(400MHz,Chloroform-d)δ7.87-7.75(m,9H),7.72-7.62(m,6H),5.32(d,J= 5.6Hz,1H),3.89-3.71(m,2H),3.55-3.39(m,1H),2.27-2.20(m,2H),2.00-1.84 (m,4H),1.69-1.57(m,7H),1.51-1.41(m,4H),1.34-1.21(m,10H),1.15-1.05(m ,6H),1.02-0.94(m,5H),0.92-0.86(m,4H),0.84(d,J=6.6Hz,6H),0.65(s,3H).

[0073] HRMS (ESI) (m / z): [M+H] + calcd for, 745.51; found, 745.51136.

[0074] The prepared product was characterized by carbon nuclear magnetic resonance spectroscopy. Figure 2 The prepared product was characterized by mass spectrometry, and the results were as shown Figure 3 shown.

[0075] From the above test results, it can be seen that Example 1 successfully prepared the compound with the structural formula as described in formula (1).

[0076] Example 2

[0077] The preparation of liposome S1 comprises the following steps:

[0078] B1: 60 mg of soybean lecithin, 2 mg of tripterygium wilfordii and 6 mg of the cholesterol derivative of formula (1) prepared in Example 1 were dissolved in 5 mL of chloroform and transferred to an eggplant-shaped bottle. The chloroform was removed by rotary evaporation at 40°C for 10 min to form a lipid film;

[0079] B2: Add pure water to the lipid film prepared in step B1 and hydrate at 35°C for 90 minutes, then use an ultrasonic cell disruptor to sonicate for 10 minutes at an ultrasonic intensity of 225W.

[0080] Example 3

[0081] The preparation of liposome S2 comprises the following steps:

[0082] B1: 40 mg of soybean lecithin, 2 mg of tripterygium wilfordii and 6 mg of the cholesterol derivative represented by formula (1) prepared in Example 1 were dissolved in 5 mL of chloroform and transferred to an eggplant-shaped bottle. The chloroform was removed by rotary evaporation at 40°C for 10 min to form a lipid film;

[0083] B2: Add pure water to the lipid film prepared in step B1 and hydrate at 30°C for 90 minutes, and then use an ultrasonic cell disruptor to sonicate for 10 minutes at an ultrasonic intensity of 225W.

[0084] Example 4

[0085] B1: 50 mg of soybean lecithin, 2 mg of tripterygium wilfordii and 6 mg of the cholesterol derivative represented by formula (1) prepared in Example 1 were dissolved in 5 mL of chloroform and transferred to an eggplant-shaped bottle. The chloroform was removed by rotary evaporation at 40°C for 10 min to form a lipid film;

[0086] B2: Add pure water to the lipid film prepared in step B1 and hydrate at 35°C for 90 minutes, and then use an ultrasonic cell disruptor to sonicate for 20 minutes at an ultrasonic intensity of 150W.

[0087] Example 5

[0088] B1: 60 mg of soybean lecithin, 2 mg of tripterygium wilfordii and 7 mg of the cholesterol derivative represented by formula (1) prepared in Example 1 were dissolved in 10 mL of chloroform and transferred to an eggplant-shaped bottle. The chloroform was removed by rotary evaporation at 40° C. for 10 min to form a lipid film;

[0089] B2: Add pure water to the lipid film prepared in step B1 and hydrate at 35°C for 120 min, then use an ultrasonic cell disruptor to sonicate for 20 min at an ultrasonic intensity of 225 W.

[0090] Example 6

[0091] The method described in Example 2 was followed, except that in step B1, the amount of the cholesterol derivative represented by formula (1) prepared in Example 1 was 2 mg.

[0092] Example 7

[0093] The method described in Example 2 was followed, except that in step B1, the amount of the cholesterol derivative represented by formula (1) prepared in Example 1 was 10 mg.

[0094] Test Example 1

[0095] The product prepared in Example 2 was observed using an opalescent pen, and the photographic results are as follows: Figure 4 As shown in the figure, it can be seen that the liposome solution has an obvious Tyndall effect.

[0096] The product prepared in Example 2 was characterized by transmission electron microscopy (TEM). Figure 5 As shown in the figure, the liposomes are approximately spherical in shape and have a relatively uniform shape.

[0097] Test Example 2

[0098] The particle size, potential and polydispersity index (PDI) of the products prepared in Examples 2-7 were measured using a Malven particle size analyzer. The results are shown in Table 1.

[0099] Table 1

[0100] Particle size / nm Zeta potential / mV PDI Example 2 83.77±1.04 28.57±0.53 0.20±0.01 Example 3 113.10±3.52 29.50±0.50 0.28±0.01 Example 4 101.65±2.09 30.80±0.95 0.32±0.00 Example 5 118.20±1.13 32.70±1.06 0.31±0.10 Example 6 114.60±2.43 10.50±1.62 0.32±0.20 Example 7 122.6±0.80 43.57±0.47 0.30±0.01

[0101] It can be seen from the above characterization results that compared with other embodiments, the mitochondrial targeted liposomes prepared in Example 2 have the smallest particle size and good particle size distribution uniformity, which is more conducive to the liposomes being enriched around tumor cells through the enhanced permeability and retention effect (EPR) of solid tumors, providing the prerequisite for the subsequent entry into cells and intracellular mitochondrial targeting. In addition, the potential of each embodiment is related to the amount of cholesterol derivatives used. The more the amount used, the greater the potential of the prepared liposomes, indicating that positively charged cholesterol derivatives can be assembled into liposomes with electrically neutral soybean lecithin.

[0102] Test Example 3

[0103] Targeting performance testing

[0104] Coumarin 6 liposomes (C6-TL) were prepared by the following process: the same method as in Example 2 was used, except that the same weight of fluorescent probe coumarin 6 was used to replace triptolide.

[0105] The mitochondrial localization ability of coumarin 6-loaded liposomes was studied by laser confocal microscopy. Human hepatoblastoma HepG2 cells were inoculated in a confocal dish. After the cells adhered to the wall, they were washed twice with 4°C PBS. Appropriate amounts of free C6 and C6-TL at a concentration of 1 μg / mL were added respectively, and the culture medium was discarded after culturing in a 37°C incubator for 4 hours. The cells were washed twice with 4°C PBS, fixed with paraformaldehyde, stained with serum-free culture medium containing MitoTrackerRed for 30 minutes, washed twice with 4°C PBS, stained with serum-free culture medium containing Hoechest for 10 minutes, and washed with 4°C PBS before observing mitochondrial targeting under a laser confocal microscope. The results are shown in Figure 6 As shown in the figure, when the green fluorescence of coumarin 6 itself and the red fluorescence of mitochondria are superimposed to yellow, it indicates that coumarin 6 is accumulated in mitochondria. From the comparison of the yellow fluorescence intensity of the C6-TL group and the free coumarin 6 group, it can be seen that coumarin 6 can be better enriched in mitochondria after being coated with mitochondrial targeting liposomes, while coumarin 6 not coated by liposomes is difficult to accumulate in mitochondria, indicating that liposomes constructed by cholesterol derivatives have mitochondrial targeting function, can deliver drugs to mitochondria, and are conducive to the drug exerting its efficacy in mitochondria.

[0106] Test Example 4

[0107] The encapsulation efficiency of the drug was determined by high performance liquid chromatography. The test process was as follows: the drug-loaded liposomes were fixed to 5 mL, and the amount of tripterygium wilfordii in the drug-loaded liposome solution before and after filtration with a 0.22 μm microporous membrane was determined by high performance liquid chromatography. The encapsulation efficiency EE of tripterygium wilfordii was calculated according to the following formula:

[0108] EE(%)=W / W0×100%

[0109] Wherein W is the amount of tripterygium wilfordii in the liposome solution after passing through the membrane, and W0 is the amount of tripterygium wilfordii in the liposome solution before passing through the membrane.

[0110] The test results are shown in Table 2.

[0111] Table 2

[0112]

[0113]

[0114] As can be seen from the above table, the drug encapsulation rate is related to the dosage of phospholipids and cholesterol derivatives. When the weight ratio of phospholipids to tripterygium wilfordii is less than 30:1, the encapsulation rate of tripterygium wilfordii is significantly reduced, such as in Examples 3 and 4; when the dosage of cholesterol derivatives increases, the hydrophobic inner layer of the liposome is occupied by too many cholesterol derivatives, resulting in a decrease in the encapsulation rate of the drug, such as in Examples 5 and 7. In Example 2, under the optimal formulation ratio and preparation conditions, the encapsulation rate of tripterygium wilfordii is close to 100%, indicating that the liposomes prepared from cholesterol derivatives have the ability to efficiently encapsulate drugs.

[0115] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A cholesterol derivative with mitochondrial targeting function, characterized in that: The structural formula of the cholesterol derivative is shown in the following formula (1):

2. A method for preparing the cholesterol derivative with mitochondrial targeting function according to claim 1, characterized in that: The method comprises the following steps: A1: In an inert atmosphere, (5-carboxypentyl)triphenylphosphine bromide, N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and organic solvent A are mixed to obtain a mixed solution; A2: In an inert atmosphere, a solution containing cholesterol is added dropwise to the mixed solution obtained in step (1), followed by mixing.

3. The method according to claim 2, characterized in that The molar ratio of (5-carboxypentyl)triphenylphosphonium bromide, N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and cholesterol is 1:1-2:1-2:

1.

4. The method according to claim 2 or 3, characterized in that: In step A1, the mixing time is 1-24 hours.

5. The method according to claim 4, characterized in that In step A2, the mixing time is 1-24 hours.

6. The method according to claim 4, characterized in that In step A2, the dropping time is 5-10 min.

7. Use of the cholesterol derivative with mitochondrial targeting function according to claim 1 in the preparation of targeted drug carriers.

8. A liposome with mitochondrial targeting function, characterized in that: The liposome is prepared from raw materials containing phospholipids, drugs and a cholesterol derivative shown in formula (1).

9. The liposome with mitochondrial targeting function according to claim 8, characterized in that: The weight ratio of the phospholipid, the drug and the cholesterol derivative represented by formula (1) is 10-50:1:1-5.

10. The liposome with mitochondrial targeting function according to claim 8, characterized in that: The drug is selected from at least one of tripterygium wilfordii, paclitaxel, doxorubicin and adriamycin.

11. A method for preparing the liposome with mitochondrial targeting function according to claim 8, characterized in that: The method comprises the following steps: B1: mixing phospholipids, drugs, cholesterol derivatives represented by formula (1) and organic solvent B, and then performing rotary evaporation to obtain a lipid film; B2: Add water to the lipid film for hydration, followed by sonication.

12. The method according to claim 11, characterized in that In step B2, the hydration conditions include: hydration temperature of 30-40° C., and hydration time of 30-120 min.

13. The method according to claim 11 or 12, characterized in that: In step B2, the ultrasonication is performed in an ultrasonic cell disruptor.

14. The method according to claim 13, characterized in that The ultrasonic power is 150-225W, and the ultrasonic time is 10-20min.

15. Use of the liposome according to claim 8 in preparing a drug delivery system for treating lung cancer, breast cancer or liver cancer.

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