Blank liposome with ginsenoside Rg3 or its analog as membrane materials and preparations and uses thereof

Abstract

The present invention provides a blank liposome with ginsenoside Rg3 or its analog as the membrane material, preparations and uses thereof. The disclosed blank liposome has a membrane comprising a lipid and a ginsenoside analog of Formula I, presenting remarkable advantages in film formation, encapsulation efficiency, targeted drug delivery, blood circulation time, stability, safety and homogeneity. It can also be used to load active substances of drugs and cosmetics, biological agents, polynucleotides or oligonucleotides, and the preparation process is convenient. ##STR00001##

Claims

1. A blank liposome having a membrane, wherein the membrane comprises a lipid and a ginsenoside of Formula I: ##STR00005## wherein, * represents a chiral carbon; R.sup.1 is H, R.sup.10, R.sup.11 or hydroxy (OH); R.sup.10 is selected from the group consisting of: O-Glc, O-Rha, O-Lyx, O-Xyl, O-Ara(p), O-Ara(f), O-Glc(2.fwdarw.1)Glc, O-Glc(6.fwdarw.1)Glc, O-Glc(2.fwdarw.1)Rha, O-Glc(2.fwdarw.1)Xyl, O-Glc(6.fwdarw.1)Xyl, O-Glc(6.fwdarw.1)Rha, O-Glc(2.fwdarw.1)Ara(p), O-Glc(6.fwdarw.1)Ara(p), O-Glc(2.fwdarw.1)Ara(f), O-Glc(6.fwdarw.1)Ara(f), O-Glc(2.fwdarw.1)Glc(2.fwdarw.1)Glc, O-Glc(2.fwdarw.1)Glc(2.fwdarw.1)Xyl, O-Glc(6.fwdarw.1)Glc(6.fwdarw.1)Xyl, O-Glc(2.fwdarw.1)Glc(4.fwdarw.1)Xyl, O-Glc(2.fwdarw.1)Lyx, O-Glc(6.fwdarw.1)Lyx, O-Glc(2.fwdarw.1)Glc(2.fwdarw.1)Rha, O-Glc(2.fwdarw.1)Glc(2.fwdarw.1)Lyx, O-Glc(2.fwdarw.1)Glc(2.fwdarw.1)Ara(f), O-Glc(2.fwdarw.1)Glc(2.fwdarw.1)Ara(p), O-Glc(2.fwdarw.1)Glc(6.fwdarw.1)Glc, O-Glc(2.fwdarw.1)Glc(6.fwdarw.1)Rha, O-Glc(2.fwdarw.1)Glc(6.fwdarw.1)Xyl, O-Glc(2.fwdarw.1)Glc(6.fwdarw.1)Lyx, O-Glc(2.fwdarw.1)Glc(6.fwdarw.1)Ara(f), O-Glc(2.fwdarw.1)Glc(6.fwdarw.1)Ara(p), O-Glc(6.fwdarw.1)Glc(2.fwdarw.1)Glc, O-Glc(6.fwdarw.1)Glc(2.fwdarw.1)Rha, O-Glc(6.fwdarw.1)Glc(2.fwdarw.1)Xyl, O-Glc(6.fwdarw.1)Glc(2.fwdarw.1)Lyx, O-Glc(6.fwdarw.1)Glc(2.fwdarw.1)Ara(f), O-Glc(6.fwdarw.1)Glc(2.fwdarw.1)Ara(p), O-Glc(6.fwdarw.1)Glc(6.fwdarw.1)Glc, O-Glc(6.fwdarw.1)Glc(6.fwdarw.1)Rha, O-Glc(6.fwdarw.1)Glc(6.fwdarw.1)Lyx, O-Glc(6.fwdarw.1)Glc(6.fwdarw.1)Ara(f) and O-Glc(6.fwdarw.1)Glc(6.fwdarw.1)Ara(p); wherein Glc is glucopyranosyl, Xyl is xylopyranosyl, Rha is Rhamnopyranosyl, Ara(p) is arabinopyranosyl, Ara(f) is arabinofuranosyl, Lyx is Lyxosyl; number indicates carbon position, arrow (4) indicates the connection relationship, and the same hereinafter; R.sup.11 is a group formed by replacing one or more OH groups in R.sup.10 with R.sup.10, and each of the one or more than one R.sup.10 groups is independently the same as or different from each other.

2. The blank liposome of claim 1, wherein, R.sup.1 is OH or ##STR00006## wherein the carbon marked with * is in S-configuration.

3. The blank liposome of claim 1, wherein the ginsenoside of Formula I is Ginsenoside Rg3 or Rh2.

4. The blank liposome of claim 1, wherein the lipid is a phospholipid and the phospholipid is natural phospholipid, semi-synthetic phospholipid, or fully synthetic phospholipid; the mass ratio of the phospholipid to the ginsenoside of Formula I is in a range of 0.5:1-100:1, or 2:1-20:1, or 3:1-10:1.

5. The blank liposome of claim 1, wherein the membrane further comprises cholesterol; the mass ratio of the cholesterol to the ginsenoside is in a range of 0.01:1-100:1, or 0.1:1-10:1, or 0.5:1-2:1.

6. The blank liposome of claim 1, wherein the membrane further comprises a long-circulating material; the mass ratio of the long-circulating material to the ginsenoside of Formula I is in a range of 0.01:1-10:1, or 0.1:1-5:1, or 0.1:1-1:1.

7. The blank liposome of claim 1, further comprising a cryoprotectant, wherein the mass percentage of the cryoprotectant to the total mass of the blank liposome is in a range of 0.5-70%, or 5-60%, or 30-60%.

8. The blank liposome of claim 1, further comprising an antioxidant, wherein the mass percentage of the antioxidant to the total mass of the blank liposome is in a range of 0.001-15%, or 0.01-10%, or 0.01-5%.

9. The blank liposome of claim 1, further comprising a soybean oil, a sodium oleate, or both, wherein the mass percentage of either or both of the soybean oil and sodium oleate to the total mass of the blank liposome is in a range of 1-30%, or 1-20%, or 1-10%; wherein the mass ratio of the soybean oil or sodium oleate to the lipid is in a range of 0.1:1-10:1, or 0.1:1-5:1.

10. The blank liposome of claim 1, further comprising a surfactant, a heat-sensitive excipient, a pH sensitive material, or an ionic additive.

11. The blank liposome of claim 4, wherein the phospholipid is egg lecithin, soybean lecithin, hydrogenated soy lecithin or Lipoid S100.

12. The blank liposome of claim 6, wherein the long-circulating material is selected from the group consisting of dimyristoyl phosphatidylethanolamine-PEG (DMPE-PEG), dipalmitoyl phosphatidylethanolamine-PEG (DPPE-PEG), distearoyl phosphatidylethanolamine-PEG (DSPE-PEG), dioleoyl phosphatidylethanolamine-PEG (DOPE-PEG), C8 PEG ceramide (C8 ceramide-PEG), C16 PEG ceramide (C16 ceramide-PEG), distearoyl phosphatidylethanolamine-PEG-succinyl (DSPE-PEG succinyl), distearoyl phosphatidylethanolamine-PEG-carboxyl (DSPE-PEG carboxylic acid), distearoyl phosphatidylethanolamine-PEG-maleimide(DSPE-PEG maleimide), distearoyl phosphatidylethanolamine-PEG-propionamide bis-mercaptopyridine (DSPE-PEG PDP), distearoyl phosphatidylethanolamine-PEG-cyanuric chloride (DSPE-PEG cyanur), distearoyl phosphatidylethanolamine-PEG-amino(DSPE-PEG amine), distearoyl phosphatidylethanolamine-PEG-biotin (DSPE-PEG biotin), distearoyl phosphatidylethanolamine-PEG-folate (DSPE-PEG folate), dilauroyl phosphatidylethanolamine-PEG (DLPE-PEG), distearoyl phosphatidylethanolamine-PEG-active succinimidyl ester (DSPE-PEG-NHS), phosphatidylethanolamine-PEG-active succinimidyl ester (DMPE-PEG-NHS), dipalmitoyl phosphatidylethanolamine-PEG-active succinimidyl ester (DPPE-PEG-NHS), dilauroyl phosphatidylethanolamine-PEG-active succinimidyl ester (DLPE-PEG-NHS), distearoyl phosphatidylethanolamine-PEG-maleimide(DSPE-PEG-maleimide), dimyristoyl phosphatidylethanolamine-PEG-maleimide (DMPE-PEG-maleimide), dipalmitoyl phosphatidylethanolamine-PEG-maleimide (DPPE-PEG-maleimide), dilauroyl phosphatidylethanolamine-PEG-maleimide (DLPE-PEG-maleimide), distearoyl phosphatidylethanolamine-PEG-biotin (DSPE-PEG-biotin), distearoyl phosphatidylethanolamine-PEG-fluorescein (DSPE-PEG-FITC), distearoyl phosphatidylethanolamine-PEG-hydroxyl (DSPE-PEG-OH), distearoyl phosphatidylethanolamine-PEG-amino(DSPE-PEG-NH2), phosphatidylethanolamine-PEG-amino (DMPE-PEG-NH2)dipalmitoyl phosphatidylethanolamine-PEG-amino (DPPE-PEG-NH2), dilauroyl phosphatidylethanolamine-PEG-amino (DLPE-PEG-NH2), distearoyl phosphatidylethanolamine-PEG-carboxyl (DSPE-PEG-COOH), dimyristoyl phosphatidylethanolamine-PEG-carboxyl (DMPE-PEG-COOH), dipalmitoyl phosphatidylethanolamine-PEG-carboxyl (DPPE-PEG-COOH), dilauroyl phosphatidylethanolamine-PEG-carboxyl (DLPE-PEG-COOH), distearoyl phosphatidylethanolamine-PEG-thiol (DSPE-PEG-SH), distearoyl phosphatidylethanolamine-PEG-silane (DSPE-PEG-silane), distearoyl phosphatidylethanolamine-PEG-azide (DSPE-PEG-N3), cholesterol-PEG (cholesterol PEG), methoxyl-PEG-cholesterol (mPEG-CLS), cholesterol-PEG-active succinimidyl ester (cholesterol PEG NHS ester), cholesterol-PEG-maleimide (CLS-PEG-Mal), cholesterol-PEG-biotin (cholesterol PEG biotin), cholesterol-PEG-fluorescein (cholesterol PEG fluorescein), cholesterol-PEG-carboxyl (cholesterol PEG COOH), cholesterol-PEG-amino (cholesterol-PEG-N.sub.H2) and cholesterol-PEG-thiol Cholesterol-PEG-SH) preferably, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG2000.

13. The blank liposome of claim 7, wherein the cryoprotectant comprises a sugar, a polyol, an amino acid or a buffer reagent.

14. The blank liposome of claim 8, wherein the antioxidant is selected from the group consisting of sodium metabisulfite, sodium thiosulfate, propyl gallate, ascorbic acid, -tocopherol, -hydroxyl acid, flavonoid, phenylpropanoid, vitamin E, vitamin C, fumaric acid, cysteine, methionine, butylhydroxy anisole, butylated hydroxytoluene, thiodipropionic acid, sulfites, hydrosulphite, dithioaminobenzoic acid, citric acid, malic acid, sorbitol, glycerol, propylene glycol, hydroquinone, hydroxycoumarin, ethanolamine, phosphoric acid and phosphorous acid.

15. The blank liposome of claim 10, wherein the surfactant comprises polyethylene glycol or polysorbate.

16. The blank liposome of claim 10, wherein the heat-sensitive excipient comprises a heat-sensitive polymer and/or a heat-sensitive surfactant; wherein the heat-sensitive polymer comprises polypropylene acrylamide, polypropylene acrylic acid, polyphoester, or poly(ester amide) copolymer; wherein the heat-sensitive surfactant comprises a Tween surfactant or Brij surfactant.

17. The blank liposome of claim 10, wherein the ionic additive comprises a cationic additive or an anionic additive.

18. The blank liposome of claim 17, wherein the cationic additive is octadecylamine; wherein the anionic additive is phosphatidic acid or phosphatidylserine.

19. A process for preparing the blank liposome of claim 1, comprising: step (1): mixing the lipid and the ginsenoside of Formula I together in an organic solvent to obtain a clear solution, optionally, with one or more agents selected from a cholesterol, a long-circulating material, a hydrophobic antioxidant, a soybean oil, a sodium oleate, a hydrophobic surfactant, a hydrophobic heat-sensitive excipient, a hydrophobic pH sensitive material and a hydrophobic ionic additive; wherein the organic solvent is one or more solvents selected from alcohols, halogenated hydrocarbon solvents, and nitrile solvents; wherein the ginsenoside of Formula I is micronized into ultrafine powder with an average particle size no more than 50 m; or no more than 20 m, or no more than 10 m; and step (2): removing the organic solvent from the clear solution obtained from step (1) to form a film, mixing the film with an aqueous solution comprising a cryoprotectant, optionally, with one or more agents selected from a hydrophilic antioxidant, a hydrophilic surfactant, a hydrophilic heat-sensitive excipient, a hydrophilic pH sensitive material, and a hydrophilic ionic additive to give a mixture; performing an operation of sonication or high pressure homogenization, filtering the mixture to obtain an aqueous solution containing the blank liposome, drying the aqueous solution to obtain the blank liposome of claim 1; wherein the lipid and the ginsenoside of Formula I are the same as those in claim 1.

20. The process of claim 19, wherein in step (1), the halogenated hydrocarbon solvent is C.sub.1-4 halogenated hydrocarbon solvent, C.sub.1-2 halogenated hydrocarbon solvent, chloroform, dichloromethane or dichloroethane; wherein the alcohol is C.sub.1-4 alcohol solvent, C.sub.1-3 alcohol solvent, methanol, ethanol, n-propanol, isopropyl alcohol or n-butanol; wherein the nitrile solvent is acetonitrile.

21. An active substance-loaded liposome comprising a blank liposome of claim 1 and an active substance.

22. The active substance-loaded liposome of claim 21, wherein the active substance is an anti-cancer drug; wherein the mass ratio of the active substance to the ginsenoside of Formula I is in a range of 0.1:1-10:1, or 0.5:1-2:1.

23. The active substance-loaded liposome of claim 21, wherein the anticancer drug is one or more drugs selected from the group consisting of paclitaxel, docetaxel, cabazitaxel, tesetaxel, ortataxel, larotaxel, simotaxel, irinotecan hydrochloride, hydroxycamptothecin, aminocamptothecin, 7-ethyl-10-hydroxy camptothecin, cisplatin, carboplatin, oxaliplatin, harringtonine, homoharringtonine, triptolide, cytarabine, etoposide phosphate, desoxy-podophyllotoxin, huperzine-A, vinorelbine tartrate, vincristine sulfate, vinblastine sulfate, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, decitabine, arsenic trioxide (As.sub.2O.sub.3), all-trans retinoic acid, Azithromycin, daunorubicin, pingyangmycin, doxorubicin hydrochloride and idarubicin hydrochloride.

24. A process for preparing the active substance-loaded liposome of claim 21, comprising: step (1): mixing the lipid, the ginsenoside of Formula I and the active substance in an organic solvent to obtain a clear solution, optionally, with one or more agents selected from a cholesterol, a long-circulating material, a hydrophobic antioxidant, a soybean oil, sodium oleate, a hydrophobic surfactant, a hydrophobic heat-sensitive excipient, a hydrophobic pH sensitive material, and a hydrophobic ionic additive; wherein the solvent is one or more solvents selected from alcohols, halogenated hydrocarbon solvents and nitrile solvents; wherein the ginsenoside of Formula I is micronized into ultrafine powder and the average particle size is no more than 50 m; or no more than 20 m, or no more than 10 m; and step (2): removing the organic solvent from the clear solution obtained in step (1) to form a film, mixing the film with an aqueous solution comprising a cryoprotectant, and optionally one or more agents selected from a hydrophilic antioxidant, a hydrophilic surfactant, a hydrophilic heat-sensitive excipient, a hydrophilic pH sensitive material, and a hydrophilic ionic additive to give a mixture; performing an operation of sonification or high pressure homogenization, filtering the mixture to obtain an aqueous solution containing the active substance-loaded liposome, drying the aqueous solution to obtain the active substance-loaded liposome of claim 21.

25. The process of claim 24, wherein in step (1), the organic solvent, the lipid and the ginsenoside of Formula I are the same as those in claim 19; wherein in step (2), the cryoprotectant is added after the aqueous solution of the active substance-loaded liposome is prepared.

26. The blank liposome of claim 13, wherein the cryoprotectant is selected from the group consisting of trehalose, glucose, sucrose, propylene glycol, glycerol, xylitol and ammonium sulfate.

27. The blank liposome of claim 14, wherein the antioxidant is selected from the group consisting of vitamin E, vitamin C, sodium thiosulfate and sodium sulfite.

28. The active substance-loaded liposome of claim 23, wherein the anticancer drug is paclitaxel, docetaxel, irinotecan, doxorubicin or cisplatin.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is the particle size distribution of the liposome loaded with paclitaxel and cholesterol, wherein A is the electron microscope image showing the particle size of the liposome loaded with paclitaxel and cholesterol.

(2) FIG. 2 is the particle size distribution of the paclitaxel-loaded liposome with Rg3 as membrane material, wherein B is the electron microscopy image showing the particle size of the liposome loaded with paclitaxel and cholesterol.

(3) FIG. 3 is the results of leakage of Paclitaxel in Paclitaxel cholesterol-liposome (PTX-Cho-Lipo) and Paclitaxel Rg3-liposome (PTX-Rg3-Gipo).

(4) FIG. 4 is the long-circulation effects of the blank cholesterol-liposome (Cho-Blank), blank mPEG-DSPE-Cholesterol-Liposome (PEG-Blank), blank Rg5-Liposome (Rg5-blank), blank Rg3-Liposome (Rg3-blank) and blank Rh2-Liposome (Rh2-blank).

(5) FIG. 5 is in vivo IR783 fluorescence distribution of Control group(IR783-Cho-Lipo), IR783-Rg5-Gipo group, IR783-Rg3-Gipo group and IR783-Rh2-Gipo group at the 2.sup.nd, 4.sup.th, 8.sup.th, 12.sup.th and 24.sup.th hour after administration; wherein, FIGS. 5-A1-A5 are respectively the fluorescence distribution of the Control group at the 2.sup.nd, 4.sup.th, 8.sup.th, 12.sup.th and 24.sup.th hour; FIG. 5S is a fluorescence ruler, wherein color is red, yellow, green and blue in a sequence indicating the fluorescence intensity from the strongest to the weakest; FIGS. 5-B1-B5, 5-C1-C5, 5-D1-D5 are respectively the fluorescence distribution of the corresponding groups at the 2.sup.nd, 4.sup.th, 8.sup.th, 12.sup.th and 24.sup.th hour, and FIG. 5-B1-B5 are IR-783-Rg5-Gipo group, FIG. 5-C1-C5 are IR-783-Rh2-Gipo group, FIG. 5-D1-D5 are IR-783-Rg3-Gipo group.

(6) FIG. 6 is the in vivo IR783 fluorescence distribution that recorded at 24.sup.th hour; FIG. 6-S is a fluorescence ruler, wherein color is red, yellow, green and blue in a sequence indicating the fluorescence intensity from the strongest to the weakest and FIG. 6-A, FIG. 6-B, FIG. 6-C and FIG. 6-D are respectively the control group, IR-783-Rg5-Gipo group, IR-783-Rg3-Gipo group and IR-783-Rh2-Gipo group.

(7) FIG. 7 is the statistical analysis of fluorescence intensity in tumor-bearing mice of Control group, IR-783-Rh2-Gipo group, IR-783-Rg3-Gipo group and IR-783-Rg5-Gipo group.

(8) FIG. 8 is the cell survival rate of human breast cancer cell line (4T1) with addition of Rh2 group, Rh2-blank group, PTX group, PTX-Cho-Lipo group, PTX-Rh2-Gipo group

(9) FIG. 9 is the relative tumor volume of Control group, Rh2 group, Rh2-blank group, PTX-Cho-Lipo group, PTX-Rh2-Gipo group in human breast cancer cell line (4T1).

(10) FIG. 10 is the cell survival rate of human breast cancer cell line (4T1) with addition of DTX group, DTX-Cho-Lipo group, DTX-Rg3-Gipo group

(11) FIG. 11 is the relative tumor volume of Control group, Taxotere group, Nanoxel-PM group, DTX-Rg5-Gipo group and DTX-Rg3-Gipo group against human breast cancer cell line (4T1).

(12) FIG. 12 is the cell survival rate of rat C6 glioma cells with addition of Rg3 Group, Rg3-Blank group, PTX Group, PTX+Rg3 group, PTX-Cho-Lipo group and PTX-Rg3-Gipo group.

(13) FIG. 13 is the cell survival rate of in-situ glioma model (C6 cells) with addition of Control Group, PTX group, Rg3 Group, Rg3-Blank group, PTX+Rg3 group, PTX-Cho-Lipo group and PTX-Rg3-Gipo group

(14) FIG. 14 is the cell survival rate of human gastric cancer cells (BGC-823) with addition of Rg5 group, Rg3 group, Rh2 group, Rg5-blank group, Rg3-blank group, Rh2-blank group, PTX group, PTX-Cho-Lipo group, PTX-Rg5-Gipo group, PTX-Rg3-Gipo group and PTX-Rh2-Gipo group in.

(15) FIG. 15 is the relative tumor volume of control group, Rg3 Group, Rg3-Blank group, PTX-Cho-Lipo group, Abraxane group, PTX-Rg5-Gipo group, PTX-Rg3-Gipo group and PTX-Rh2-Gipo group against human gastric cancer cells (BGC-823).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(16) The following examples further illustrate the present invention, but the present invention is not limited thereto.

(17) Below presents preferred embodiments of the present invention based on the drawings in order to illustrate the technical schemes of the present invention in detail.

(18) 1. Experimental drugs: 20(S)-ginsenoside Rg3, 20(R)-ginsenoside Rg3, 20(S)-ginsenoside Rh2, 20(R)-ginsenoside Rh2 are commercially available in this field, such as Shanghai Ginposome PharmaTech Co., Ltd., Suzhou Star Ocean Ginseng Bio-pharmaceutical Co., Ltd., and/or Shanghai Yuanye Bio-Technology Co., Ltd.

(19) 2. Experimental Instruments: The instruments used in the following embodiments are self-owned by Shanghai Ginposome PharmaTech Co., Ltd., the model and supply information of the instruments are listed as follows:

(20) Ultra-Micro Pulverizer (ZD-10S, Shanghai Lvyi Machinery Manufacturing Co., Ltd.)

(21) High performance liquid chromatography (Agilent 1100), Alltech 3300ELSD detector, Anjielun Technology China Co., Ltd.

(22) Rotary evaporator (ZX98-1 5L), Shanghai Looyesh Instrument Co., Ltd.;

(23) 20 L Rotary evaporator (R5002K), Shanghai Xiafeng Instrument Factory;

(24) Lyophilizer (FD-1D-80), Shanghai Bilang Instrument Manufacturing Co. Ltd.;

(25) Lyophilizer (PDFD GLZ-1B), Shanghai Pudong Freeze dryer Equipment Co., Ltd.)

(26) Precision weighing balance (CPA2250 0.00001 g Readability), Sartorius (Shanghai) Trade Co., Ltd.;

(27) Electronic balance (JY3003 0.001 g Readability), Shanghai Shunyu Hengping Science Instrument Co. Ltd.).

(28) 3. The present invention is further explained by the following embodiments, but not limited to the following embodiments. The experimental methods without giving specific conditions, are carried out by conventional methods and conditions used in this field, or according to commodity specifications. The temperature and pressure preferably refer to room temperature of 10 to 30 C. and standard atmosphere pressure if not specified. Reflux temperature, if not specified, is defined by the solvent used.

(29) Ultrafine Powder Process

(30) To get the ginsenoside Rg3 ultrafine powder, 500 g ginsenoside Rg3 is dried to water content less than 1% and crushed by Ultra-Micro Pulverizer ZD-10S for 30 min. During the process, the inside temperature of pulverizer chamber is maintained at 20-30 C. with a cooled circulating water. The average size of more than 90% particles is less than 10 m measured by electron microscope.

(31) The Preparation of the Liposomes

Embodiment 1

The Preparation of a Conventional Rg3 Liposome

(32) A mixture of Egg lecithin 1 g, cholesterol 0.1 g and ginsenoside 20(S)-Rg3 (without ultra-micro pulverization) 0.1 g were added to 20 mL anhydrous ethanol and stirred at room temperature to form a clear solution. Then the organic solvent was removed by a rotary evaporator in a thermostatic water bath at 40 to 50 C. The formed thin film was hydrated with 20 mL 5% trehalose aqueous solution (the percentage refers to the ratio of the mass of the trehalose to the total mass of the trehalose aqueous solution). The suspension was then sonicated until the particle size of the liposome was between 0.1 and 0.3 micron. After sonication, the liposome suspension was passed through a 0.22-micron microporous membrane to obtain an aqueous solution of ginsenoside Rg3 liposome. Then the aqueous solution was aliquoted into vials and placed in a freeze-dryer to lyophilization for 72 hours. The conventional Rg3 liposome was obtained and sealed in the vial by a protective gas (argon or nitrogen). By calculation, D10 of the liposome was 75 nm, D50 was 118 nm, D90 was 131 nm. As hereinafter, D10, D50, and D90 describe diameter, where 10%, 50%, and 90% of particle size distribution were under the reported particle size.

Embodiment 2

The Preparation of Rg3 Blank Liposome

(33) Egg lecithin 1 g and ginsenoside 20(S)-Rg3 ultrafine powder 0.1 g were added to 200 mL chloroform and stirred to form a clear solution at room temperature. The organic solvent was removed by a rotary evaporator in a thermostatic water bath at 40 to 50 C. to form a film. The formed thin film was hydrated with 20 mL 5% trehalose aqueous solution (the percentage refers to the ratio of the mass of the trehalose to the total mass of the trehalose aqueous solution). The liposome suspension was sonicated until the particle size of the liposome was between 0.1 and 0.3 micron. Then the suspension was passed through a 0.22-micron microporous membrane to obtain an aqueous solution of ginsenoside Rg3 liposome. Then the aqueous solution was aliquoted into vials and placed in a freeze-dryer to for 72 hours. After lyophilization, the obtained Rg3 blank liposome was sealed in the vial by a protective gas (argon or nitrogen). By calculation, the D10 of the liposome was 66 nm, D50 was 90 nm, D90 was 105 nm.

Embodiment 3

The Preparation of Rg5 Blank Liposome

(34) In accordance with the method in embodiment 2, the Rg5 Blank liposome was prepared by replacing Rg3 with Rg5. After evaluation, the D10 of the liposome was 70 nm, D50 was 96 nm and D90 was 111 nm.

Embodiment 4

The Preparation of Rg3 Blank Liposome

(35) Egg lecithin 0.5 g, ginsenoside 20(R)-Rg3 ultrafine powder 0.1 g and Vitamin E 0.1 g were added into 200 mL dichloromethane and stirred to form a clear solution at room temperature. The organic solvent was removed by a rotary evaporator in a thermostatic water bath at 40 to 50 C. to form a film. The formed thin film was hydrated with 20 mL 5% glucose aqueous solution (the percentage refers to the ratio of the mass of the glucose to the total mass of the glucose aqueous solution). The liposome suspension was sonicated until the particle size of the liposome was between 0.1 and 0.3 micron. Then the suspension was passed through a 0.22-micron microporous membrane to obtain an aqueous solution containing ginsenoside Rg3 liposome. Then the aqueous solution was aliquoted into vials and placed in a freeze-dryer to for 72 hours. After lypholization, the obtained Rg3 blank liposome was sealed in the vial by a protective gas (argon or nitrogen). By calculation, the D10 diameter of the liposome was 88 nm, D50 was 116 nm, D90 was 153 nm.

Embodiment 5

The Preparation of Rg3 Blank Liposome

(36) Soybean lecithin 0.6 g and ginsenoside 20(S)-Rg3 ultrafine powder 0.2 g were added into 200 mL chloroform/methanol (1:1, v/v) and stirred to form a clear solution at room temperature. The organic solvent was removed by a rotary evaporator in a thermostatic water bath at 50 to 60 C. to form a film. The formed thin film was then hydrated with 20 mL 5% sucrose aqueous solution (the percentage refers to the ratio of the mass of the sucrose to the total mass of the sucrose aqueous solution) and then sonicated until the particle size of the liposome was between 0.1 and 0.3 micron. The liposome suspension was passed through a 0.22-micron microporous membrane to obtain an aqueous solution containing ginsenoside Rg3 blank liposome. Then the aqueous solution was aliquoted into vials and placed in a freeze-dryer for 72 hours. After lyophilization, the obtained Rg3 blank liposome was then sealed by a protective gas (argon or nitrogen). By calculation, the D10 of the liposome was 60 nm, D50 was 84 nm, D90 was 102 nm.

Embodiment 6

The Preparation of Rh2 Blank Liposome

(37) Hydrogenated soybean lecithin (HSPC) 0.7 g, ginsenoside 20(S)-Rh2 ultrafine powder 0.1 g and cholesterol 0.2 g were added into 200 mL chloroform and stirred to form a clear solution at room temperature. The organic solvent was removed by rotary evaporation in a thermostatic water bath at 60 C. to 65 C. to form a film. The formed film was hydrated using 20 mL 5% mannitol aqueous solution (the percentage refers to the ratio of the mass of the mannitol to the total mass of the mannitol aqueous solution) and then sonicated until the particle size of the liposome was between 0.1 and 0.3 micron to obtain an aqueous solution of ginsenoside Rh2 blank liposome. Then the obtained aqueous solution was aliquoted into vials and placed in a freeze-dryer to lyophilization for 72 hours. Then the obtained Rh2 blank liposome was sealed in the vial by a protective gas (argon or nitrogen). By calculation, the D10 of the liposome was 94 nm, D50 was 120 nm, D90 was 133 nm.

Embodiment 7

The Preparation of Rh2 Blank Liposome

(38) Egg lecithin 0.4 g, ginsenoside 20(R)-Rh2 ultra-fine powder 0.1 g, soybean oil 0.2 g and vitamin C 0.1 g were added into 200 mL chloroform/isopropyl alcohol(9:1 v/v) and stirred to form a clear solution at room temperature. The organic solvent was removed by rotary evaporation in a thermostatic water bath at 60 C. to 65 C. to form a film. The formed film was hydrated with 20 mL 5% propanediol aqueous solution (the percentage refers to the ratio of the mass of the propanediol to the total mass of the propanediol aqueous solution) and sonicated until the particle size of the liposome was between 0.1 and 0.3 micron. After sonication, the liposome suspension was passed through a 0.22-micron microporousmembrane to obtain an aqueous solution of ginsenoside Rh2 blank liposome. Then the aqueous solution was aliquoted into vials and placed in a freeze-dryer to lyophilization for 72 hours. Then the obtained Rh2 blank liposome was sealed in the vial by a protective gas (argon or nitrogen). By evaluation, the D10 diameter of the liposome was 124 nm, D50 was 157 nm, D90 was 189 nm.

Embodiment 8

The Preparation of Rg3 Blank Liposome

(39) Egg lecithin 0.9 g, ginsenoside 20(S)-Rg3 ultrafine powder 0.2 g and PEG2000-DSPE 0.05 g were mixed with 200 mL chloroform/methanol (1:1, v/v) and stirred to form a clear solution at room temperature. The organic solvent was removed by rotary evaporation in a thermostatic water bath at 55 to 65 C. to form a film. The formed film was hydrated with 20 mL 5% glycerol aqueous solution (the percentage refers to the ratio of the mass of the glycerol to the total mass of the glycerol aqueous solution) and sonicated until the particle size of the liposome was between 0.1 and 0.3 micron. After sonication, the liposome suspension was passed through a 0.45-micron microporous membrane filter to obtain an aqueous solution of ginsenoside Rg3 blank liposome. Then the aqueous solution was aliquoted into vials and placed in a freeze-dryer to lyophilization for 72 hours. Then the obtained Rg3 blank liposome was sealed in the vial by protective gas (argon or nitrogen). By calculation, the D10 diameter of the liposome was 62 nm, D50 was 71 nm, D90 was 85 nm.

Embodiment 9

The Preparation of Rg3 Blank Liposomes

(40) Soybean lecithin S100 0.9 g, ginsenoside 20(S)-Rg3 ultrafine powder 0.2 g, Vitamin E 0.01 g, cholesterol 0.1 g and mPEG2000-DSPE 0.05 g were mixed with 20 mL chloroform/acetone (1:1 v/v) and stirred to form a clear solution at room temperature. The organic solvent was removed by rotary evaporation in a thermostatic water bath at 45 C. to 55 C. to form a film. The formed film was hydrated with 20 mL 5% galactose aqueous solution (the percentage refers to the ratio of the mass of the galactose to the total mass of the galactose aqueous solution) and sonicated until the particle size of the liposome was between 0.1 and 0.3 micron. After sonication, the liposome suspension was passed through a 1-micron microporous membrane to obtain an aqueous solution of ginsenoside Rg3 blank liposome. Then the aqueous solution was aliquoted into vials and placed in a freeze-dryer for 72 hours. After lyophilization, Then the obtained Rg3 blank liposome was sealed in the vial and protected by argon gas or nitrogen gas. By calculation the D10 diameter of the liposome was 65 nm, D50 was 130 nm, D90 was 143 nm.

Embodiment 10

The Preparation of Paclitaxel Rg3 Liposome

(41) Egg lecithin 0.8 g, ginsenoside 20(S)-Rg3 ultrafine powder 0.2 g and Paclitaxel 0.1 g were mixed with 200 mL chloroform and stirred to form a clear solution at room temperature. The organic solvent was removed by rotary evaporation in a water bath thermostatically controlled at 40 C. to 50 C. to form a film. The formed film was hydrated with 20 mL 5% trehalose aqueous solution (the percentage refers to the ratio of the mass of the trehalose to the total mass of the trehalose aqueous solution) and sonicated until the particle size of the liposome was between 0.1 and 0.3 micron. Thus, an aqueous solution of Paclitaxel Rg3 liposome was obtained. Then the aqueous solution was aliquoted into vials making 30 mg Paclitaxel in each vial. The aqueous solution was placed in a freeze-dryer for 72 hours. After lyophilization, the obtained Paclitaxel Rg3 liposome was sealed in the vial and protected by argon gas or nitrogen gas. By evaluation, the D10 diameter of the liposome was 76 nm, D50 was 90 nm, D90 was 105 nm, the encapsulation efficiency was more than 95%.

Embodiment 11

The Preparation of Paclitaxel Rg5 Liposome

(42) In accordance with the method in embodiment 10, the Paclitaxel Rg5 liposome were prepared by replacing Rg3 with Rg5. By evaluation, the D10 of the liposome was 92 nm, D50 was 128 nm, D90 was 158 nm, the encapsulation efficiency was more than 95%.

Embodiment 12

The Preparation of Paclitaxel Rh2 Liposome

(43) Soybean lecithin 0.7 g, ginsenoside 20(S)-Rh2 ultrafine powder 0.2 g, Paclitaxel 0.1 g, cholesterol 0.1 g, soybean oil 0.1 g and vitamin C 0.1 g were mixed with 200 mL chloroform/acetonitrile (1:1, v/v) and stirred to form a clear solution at room temperature. The organic solvent was removed by rotary evaporation in a water bath thermostatically controlled at 50-60 C. to form a film. The formed film was hydrated with 20 mL 10% treassose aqueous solution (the percentage refers to the ratio of the mass of the trehalose to the total mass of the trehalose aqueous solution) and sonicated until the particle size of the liposome was between 0.1 and 0.3 micron. After sonication, an aqueous solution of paclitaxel Rh2 liposome was obtained. Then the aqueous solution was aliquoted into vials making 30 mg paclitaxel in each vial. The aqueous solution was placed in a freeze-dryer for 72 hours. After lyophilization, the obtained paclitaxel Rh2 liposome was sealed in the vial and protected by argon gas or nitrogen gas. By evaluation, the D10 diameter of the liposome was 79 nm, D50 was 118 nm, D90 was 130 nm, the encapsulation efficiency is more than 95%.

Embodiment 13

The Preparation of Docetaxel Rg3 Liposome

(44) Egg lecithin 0.9 g, ginsenoside 20(S)-Rg3 ultrafine powder 0.18 g, Docetaxel 0.1 g and cholesterol 0.225 g were mixed with 200 mL chloroform/methanol (1:1, v/v) and stirred in a water bath thermostatically controlled at 40-50 C. to form a clear solution. The organic solvent was removed by a membrane evaporator at 50 C. to 60 C. to form a film. The formed film was hydrated with 20 mL 5% sucrose aqueous solution (the percentage refers to the ratio of the mass of the sucrose to the total mass of the sucrose aqueous solution) and homogenized by a high-pressure homogenizer until the particle size of the liposome was between 0.1 and 0.3 micron. After homogenization, the liposome suspension was passed through a 0.22-micron microporous membrane to obtain an aqueous solution of docetaxel Rg3 liposome. Then the aqueous solution was aliquoted into vials making 20 mg docetaxel in each vial. The aqueous solution was placed in a freeze-dryer for 72 hours. After lyophilization, the obtained docetaxel Rg3 liposome was sealed in the vial and protected by argon gas or nitrogen gas. By evaluation, the D10 diameter of the liposome was 70 nm, D50 was 109 nm, D90 was 122 nm, the encapsulation efficiency was more than 95%.

Embodiment 14

The Preparation of Docetaxel Rg5 Liposome

(45) Egg lecithin 0.9 g, ginsenoside Rg5 ultra-fine powder 0.18 g, Docetaxel 0.1 g and cholesterol 0.225 g were mixed with 20 mL chloroform/methanol (1:1, v/v) and stirred in a water bath thermostatically controlled at 40-50 C. to form a clear solution. The organic solvent was removed by a membrane evaporator at 50 C. to 60 C. to form a film. The formed film was hydrated with 20 mL 5% sucrose aqueous solution (the percentage refers to the ratio of the mass of the sucrose to the total mass of the sucrose aqueous solution) and homogenized with a high-pressure homogenizer until the particle size of the liposome was between 0.1 and 0.3 micron. After homogenization, the liposome suspension is filtered by a 0.22-micron microporousmembrane to give an aqueous solution of docetaxel Rg5 liposome. Then the aqueous solution is aliquoted into vials making that each vial contains docetaxel 20 mg, then placed in a freeze-dryer to freeze dry for 72 hours. After lyophilization, the obtained docetaxel Rg5 liposome was sealed in the vial and protected by argon gas or nitrogen gas. By calculation, the D10 of the liposome was 73 nm, D50 was 101 nm, D90 was 118 nm, the encapsulation efficiency was more than 95%.

Embodiment 15

The Preparation of Docetaxel Rh2 Liposome

(46) Soybean lecithin 300 mg, ginsenoside 20(S)-Rh2 ultrafine powder 60 mg, Docetaxel 30 mg, cholesterol 75 mg and mPEG-DSPE 10 mg were mixed with 200 mL chloroform/methanol (1:1, v/v) and stirred to form a clear solution in a water bath thermostatically controlled at 40-50 C. The organic solvent was removed by a membrane evaporator at 50 C. to 60 C. to form a film. The formed film was hydrated with 20 mL 5% sucrose aqueous solution (the percentage refers to the ratio of the mass of the sucrose to the total mass of the sucrose aqueous solution) and homogenized with a high-pressure homogenizer until the particle size of the liposome was between 0.1 and 0.3 micron. After homogenization, the liposome suspension was passed through a 0.22-micron microporous membrane to give an aqueous solution of docetaxel Rh2 liposome. Then the aqueous solution was aliquoted into vials making that each vial contains docetaxel 20 mg, then placed in a freeze-dryer to freeze dry for 72 hours. After lyophilization, the obtained docetaxel Rh2 liposome was sealed in the vial and protected by argon gas or nitrogen gas. By calculation, the D10 diameter of the liposome was 81 nm, D50 was 129 nm, D90 was 148 nm, and the encapsulation efficiency was 95%.

Embodiment 16

The Preparation of Rg3 Irinotecan Liposomes

(47) Egg lecithin 0.9 g, ginsenoside 20(S)-Rg3 ultrafine powder 0.3 g and cholesterol 0.1 g were mixed with 200 mL dichloromethane/ethanol (1:1, V/V) and stirred to form a clear solution at room temperature. The organic solvent was removed by a rotary evaporator in a water bath thermostatically controlled at 50 C. to 60 C. to form a film. The formed film was hydrated with 20 mL 6.6% ammonium sulfate aqueous solution (the percentage refers to the ratio of mass of the ammonium sulfate to the total mass of the ammonium sulfate aqueous solution) and sonicated until the particle size of the blank liposome was between 0.1 and 0.3 micron to give an aqueous solution of Rg3 blank liposome. The solution of the blank liposome was dialyzed against 0.15 mol/L trehalose solution for 12 hours. After dialyzation, a certain amount of trehalose was added according to the volume of the dialyzed blank liposome solution to make the mass percentage of trehalose in the blank liposome solution reach 10% (the mass percentage refers to the mass of the trehalose relative to the total mass of the blank liposome solution). Then, 1 mL irinotecan hydrochloride aqueous solution (containing irinotecan hydrochloride 0.2 g with a mass percentage of 20%) was added and kept for 30 minutes in a water bath at 37 C. to give an aqueous solution of ginsenoside Rg3 irinotecan hydrochloride liposome. The aqueous solution was aliquoted into vials making that each vial contains 40 mg irinotecan hydrochloride, and then placed in a freeze-dryer to freeze dry for 72 hours. The obtained ginsenoside Rg3 irinotecan hydrochloride liposome was sealed in the vial filled with protective gas (argon or nitrogen). By calculation, the D10 diameter of the liposome was 92 nm, D50 was 139 nm, D90 was 165 nm. The encapsulation efficiency was more than 95%.

Embodiment 17

The Preparation of Rg3 Cisplatin Liposome

(48) Egg lecithin 0.8 g, ginsenoside 20(S)-Rg3 ultra-fine powder 0.2 g, cisplatin 0.1 g and soybean oil 0.1 g were mixed with 200 mL chloroform/methanol (1:1, v/v) and stirred to form a clear solution at room temperature. The organic solvent was removed by a rotary evaporator in a water bath thermostatically controlled at 40 C. to 50 C. to form a film. The formed film was hydrated with 20 mL 5% lactose aqueous solution (the percentage refers to the ratio of the mass of the lactose to the total mass of the lactose aqueous solution) and sonicated until the particle size of the liposome was between 0.1 and 0.3 micron. After sonication, the liposome suspension was passed through 1-micron microporous membrane to give an aqueous solution of cisplatin Rg3 liposome. Then the aqueous solution was aliquoted into vials making that each vial contains cisplatin 10 mg, and then placed in a freeze-dryer to freeze dry for 72 hours. After lyophilization, the obtained cisplatin Rg3 liposome was sealed in the vial filled with protective gas (argon or nitrogen). By calculation, the D10 of the liposome was 69 nm, D50 was 109 nm, D90 was 126 nm, and the encapsulation efficiency was more than 95%.

Embodiment 18

The Preparation of Rg3 Doxorubicin Liposome

(49) Soybean lecithin S100 0.9 g, ginsenoside 20(S)-Rg3 ultrafine powder 0.3 g and vitamin E 0.1 g were mixed with 200 mL chloroform/methanol (9:1, v/v) and stirred to form a clear solution in a water bath thermostatically controlled at 40 C.-50 C. The organic solvent was removed by a membrane evaporator at 50 C.-55 C. to form a film. The formed film was hydrated with 20 mL phosphoric acid buffer salt (PBS), stirred to form a clear solution. The clear solution is homogenized by a high-pressure homogenizer until the particle size of the liposome was between 0.1 and 0.3 micron to give an aqueous solution of Rg3 blank liposome. Then the aqueous solution was mixed with 1 mL doxorubicin hydrochloride aqueous solution with a mass percentage of 20% (doxorubicin hydrochloride 0.2 g) and 6 mL disodium hydrogen phosphate aqueous solution with a mass percentage of 7.1%, and purified water was added to adjust pH to 7.30. The mixture was kept in a water bath at 60 C. for 30 minutes to give an aqueous solution of ginsenoside Rg3 doxorubicin hydrochloride liposome. Then the aqueous solution was aliquoted into vials making that each vial contains 20 mg doxorubicin hydrochloride, and placed in a freeze-dryer for 72 hours. After lyophilzation, the obtained ginsenoside Rg3 doxorubicin hydrochloride liposome was sealed in a vial filled with protective gas (argon or nitrogen). By calculation, the D10 diameter of the liposome was 76 nm, D50 was 101 nm, D90 was 125 nm. The encapsulation efficiency was more than 95%.

Application Embodiments

(50) 1. Experimental Drugs

(51) Ginsenoside 20(S)-Rg3 (Rg3), paclitaxel, docetaxel, irrinotecan hydrochloride, doxorubicin and cisplatin are commercially available in this field.

(52) If without giving specific instructions, the conventional Rg3 liposomes were carried out according to embodiment 1, the Rg3 or Rh2 blank liposomes were carried out according to embodiment 2, Rg5 blank liposomes were carried out according to embodiment 3, Paclitaxel Rg3 liposomes were carried out according to embodiment 10, Paclitaxel Rg5 liposomes were carried out according to embodiment 11, Docetaxel Rg3 liposomes were carried out according to embodiment 13, Docetaxel Rg5 liposomes were carried out according to embodiment 14.

(53) Each ginsenoside blank liposome was either prepared according to the above-mentioned method in the present invention, or according to embodiment 1 and making corresponding changes according to the needs.

(54) 2. Instruments

(55) The instruments used in the following embodiments and the application embodiments are self-owned by the School of Pharmacy, Fudan University, and the model and other information of the instruments are listed as follows: High performance liquid chromatography (HPLC), (Agilent 1100), Electronic balance (TB-215, Denver Instrument, USA); Ultrasonic cleaning machine (SB3200DT, Ningbo Xinzhi Biotechnology Co., Ltd.); Terbovap Sample Concentrator (HGC-12A, Tianjin Hengao Technology Development Co., Ltd.) Rotary evaporator (RE-2000A, Shanghai Yarong Biochemical Instrument Factory); Ultrapure water system (ULUP-IV-10T, Sichuan U & P Ultra Technology Co., Ltd.) Thermostatic oscillator (SHA-C, Changzhou Aohua Instrument Co., Ltd.) Ultrasonic cell crusher (JY92-II, Ningbo Xinzhi Biotechnology Co., Ltd.); High pressure homogenizer (EmulsiFlex-B15, AVESTIN Inc., Canada); Laser particle size analyzer (Zetasizer Nano ZS, Malvern Panalytical Ltd. UK); Mini-extruder Equipment (Avanti Polar Lipids Inc); Photoelectric Microscope (XDS-1B, Chongqing Optical Instrument Co., Ltd.); Clean bench (SW-CJ-1FD, Suzhou Antai air Technology Co., Ltd.); Cell incubator (CCL-170B-8, ESCO, Singapore); Fluorescence inverted microscope (IX-73, Olympus, Japan); Laser granulometer (Mastersizer 2000, Malvern Panalytical Ltd., UK); In-vivo Small animal imaging system (In-vivo Multispectral FX PRO, Bruker Corporation, US).

(56) 3. Experimental Cell Lines: 4T1 human breast cancer cell line (Nanjing KeyGEN Biotech Co., Ltd) A549 human lung cancer cell line (Nanjing KeyGEN Biotech Co., Ltd) BGC-823 human gastric adenocarcinoma cancer cell line (Nanjing KeyGEN Biotech Co., Ltd) In-situ glioma model in C6 cells (Nanjing KeyGEN Biotech Co., Ltd) Rat C6 glioma cell line (Nanjing KeyGEN Biotech Co., Ltd)

(57) 4. In Vitro Hemolysis Test

(58) Preparation of 2% red blood cell suspension: The blood from a healthy rabbit was collected into a conical flask containing glass beads and shook for 10 minutes, or the blood was agitated using a glass rod to remove the fibrinogen from blood and make defibrinated blood. Then, about 10 times volum of 0.9% sodium chloride solution was added to wash the cell. After centrifugation for 15 minutes at 1000-1500 RPM, the supernatant was discarded and and red blood cells were collected in the precipitation. Then, the red blood cell was obtained after washing the precipitation using 0.9% sodium chloride solution for 2-3 times according to the method above until the supernatant was clear. To obtain a 2% cell suspension, the obtained red blood cells were suspended in 0.9% sodium chloride solution.

(59) Hemolysis Test: 5 clean glass tubes were labelled with numbers. Tube number 1, 2 were used for test samples, tube number 3 was used for negative control, tube number 4 was used for positive control, tube number 5 was used for the contrast sample. As shown in table 5, 2% red blood cell suspension, 0.9% Sodium Chloride Solution, and purified water were added to the tube. After mixing, the tubes were incubated at 370.5 C. for 3 h. Results of hemolysis and aggregation were observed and recorded as shown in Table 5.

(60) TABLE-US-00001 TABLE 5 Test tube No. 1 2 3 4 5 2% red cell suspensions/mL 2.5 2.5 2.5 2.5 / 0.9% sodium chloride solution/mL 2.2 2.2 2.5 / 4.7 Purified water/mL / / / 2.5 / The test solution/mL 0.3 0.3 / / 0.3

(61) If it gave a clear and red solution in the tube, and no cells were settled at the bottom of the tube, it suggested hemolysis occurred. If it gave a colorless or clear solution and red blood cells were all settled at the bottom of the tube, or the supernatant was lightly colored, but no significant differences were observed between tube 1 or 2 and tube 5, it suggested no hemolysis occurred.

(62) If there was red/brown cloudy precipitate in the solution, thoroughly mixed the sample by gently inverting the tube 3 times. If the precipitate was still there, it indicated red blood cell aggregation. The sample should be further observed under microscope to confirm if red blood cell aggregation occurred.

(63) Results Analysis: If no hemolysis or aggregation occurs in the tube of negative control, but hemolysis occurred in the tube of positive control, and no hemolysis and aggregation occurs in the two tubes of test samples within 3 hours, the test sample meet the regulations. If hemolysis and aggregation occurs in one of the tubes with test sample within 3 hours, four more sample tests should be performed to confirm. Only when no hemolysis and aggregation occurs within 3 hours in all the four sample tubes, the test sample can be conformed that it meets the requirements, otherwise the test sample does not meet the requirements.

(64) In a specific experiment, concentration of the test sample (ginsenoside) can be adjusted according to the needs.

(65) 5. Experimental Animals

(66) Experimental animals: Kunming mice (or normal mice) are purchased from the Animal Center of the Third Military Medical University,

(67) BALB/C-nu/nu mice (or nude mice) are purchased from Shanghai SlACK Laboratory Animal Co., Ltd.

(68) 6. Cell Culture Method

(69) Cell lines were incubated at 37 C. in a humidified incubator with 5% CO.sub.2, and cultured in DMEM or RPMI1640 complete culture-medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 g/mL streptomycin. A solution of 0.25 trypsin-EDTA was used for sub-culturing cells, which was performed 2 to 3 times per week.

(70) 7. Drug Administration

(71) A negative control group (e.g. PBS group), a positive control group and a sample group (ginsenoside liposome loaded with a drug) were set up for each experiment. A total of 3-6 concentration gradients were set up, including half dilution or 5 times dilution. Each concentration repeated 3 times.

(72) 8. Determination of the Half-Maximal Inhibitory Concentration (IC.sub.50) of Tumor Cell

(73) Tumor cells in logarithmic growth phase were digested with trypsin and centrifuged, collected the cell pellet and resuspended it in a buffer. Then cells in the suspension solution were counted and seeded into a 96-well culture plate with 5000 cells per well by placing 100 l cell suspension solution in each well. On the next day, 100 l fresh culture medium containing different concentrations of samples or solvent as control were added to each well respectively (with a final concentration of DMSO<0.5%). For each sample, 10 different dose groups were set up, and each group repeated 3 times parallelly. After 72-hour incubation at 37 C., the supernatant was discarded and 100 l PBS and 10 l CCK-8 were added to each well. Then the plate was well shaked using a micro oscillator for uniform and continually cultured for 3 h. Absorbance is determined by a microplate reader at a reference wavelength of 630 nm and a detection wavelength of 450 nm. Tumor cells treated with a solvent were used as a control, IC.sub.50 is computed from the median-effect equation.

(74) 9. Determination of Cell Viability In Vitro

(75) Logarithmically growing tumor cells were collected and resuspended in DMEM complete medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 g/ml streptomycin to a final cell density of 410.sup.4 cells/ml. Then, 200 l cell suspension solution was seeded into each well of a 96-well plate (with a concentration of 810.sup.3 cells/well) and the plate was cultured in a CO.sub.2 cell culture incubator at 37 C. After 48 h, DMEM complete medium was removed and respectively replaced with 200 L different concentrations of anti-cancer drug, at least 6 different concentration groups. The group without replacing DMEM complete medium by anti-cancer drug solution was used as negative control. For each concentration group, 4 replicates were set up. The whole experiment was independently repeated 3 times. The cells were continuously cultured in a CO.sub.2 cell culture incubator at 37 C. After 72 h, 20 l 5 mg/mL MTT solution was added into each well and the plate was continuously cultured for 4 h. Then discarded the supernatant, added 150 l DMSO into each well, and shaked the plate for 10 min. The absorbance was measured at 490 nm using a microplate reader (Tecan infinite M 200 TECAN, Switzerland). The cell survival rate is calculated according to the following formula:

(76) $\mathrm{Cell}\mathrm{Survival}\mathrm{Rate}\left(\%\right)=\frac{{\mathrm{Abs}}_{490\left(\mathrm{sample}\right)}}{{\mathrm{Abs}}_{490\left(\mathrm{control}\right)}}100$

(77) Wherein Abs.sub.490(sample) is the absorbance of the experimental sample, Abs.sub.490(control) is the absorbance of the negative control.

(78) Small Animal Imaging In Vivo

(79) As shown in the embodiments.

(80) 11. In-Vivo Drug efficacy Test

(81) 100 uL logarithmically growing tumor cells with a density of 110.sup.7 to 1010.sup.7 cells/mL was injected subcutaneously into the right armpit of an 18 to 20 g nude mouse slowly using a 1 mL syringe. The growth of the tumor was observed. When the tumor volume was about 100 mm.sup.3, animals were randomized to groups and administered with different drugs. All mice were weighed, and the longest diameter and the shortest diameter of the tumor was measured with vernier calipers every two days. At the end of the experiment, the nude mice were sacrificed and the volumes of tumors were calculated. Then, the relative tumor volume (RTV), T/C ratio (the ratio of tumor volume in control versus treated mice) and the percent tumor growth inhibition (TGI) were calculated and statistically analyzed.

(82) Tumor volume was calculated according to the following formula: V=(LWH)/2, wherein V is tumor volume, L is tumor length, W is tumor width, H is tumor height.

(83) Relative tumor volume was calculated according to the following formula: RTV=TV.sub.n/TV.sub.0, wherein TV.sub.n is the tumor volume at day n, TV.sub.0 is the tumor volume at day zero (the administration day).

(84) The T/C ratio was determined by calculating RTV: T/C(%)=TRTV/CRTV100%, wherein TRTV is the RTV of the treatment group, CRTV is the RTV of the control group.

(85) The percent tumor growth inhibition (TGI) was calculated according to the following formula:
TGI(%)=((MTVcontrolMTVtreated/MTVcontrol))100, wherein MTVcontrol is the median tumor volume of control group, MTVtreated is the median tumor volume of the drug treatment group.

(86) Curative effect was evaluated based on the T/C ratio: T/C (%)>60 means the treatment has no effect; T/C (%)60 and the differences between the treatment group and the control group are statistically significant (P<0.05) means the treatment is effective.

(87) In the following application embodiments, C(M) means concentration, wherein the concentration of Taxol+Rg3 refers to the concentration of paclitaxel and ginsenoside Rg3 in the ginsenoside Rg3 paclitaxel liposome, for example, 5+30 means that in ginsenoside Rg3 paclitaxel liposome, the concentration of the paclitaxel is 5 M and the concentration of the ginsenoside Rg3 is 30 M. Time (d) is calculated by days.

(88) 12. Analysis Method of Paclitaxel

(89) Analysis of paclitaxel is according to the Paclitaxel analysis method in the United States Pharmacopeia (USP 34).

Application Embodiments

Embodiment 1

Hemolysis Test

(90) Experimental results are listed in table 1. HD50 is 50% of the maximum haemolysis.

(91) TABLE-US-00002 TABLE 1 Abbreviation Embodiment of Liposome Liposome Hemolysis No. Name Full Name (HD50) Embodiment 1 Rg3-Cho-Lipo conventional 20-50 g/mL Rg3 cholesterol liposome Embodiment 2 Rg3-blank Rg3 blank 650-700 g/mL liposome Embodiment 3 Rg5-blank Rg5 blank 450-500 g/mL liposome Embodiment 7 Rh2-blank Rh2 blank 400-500 g/mL liposome Embodiment 10 PTX-Rg3-Gipo Paclitaxel Rg3 650-700 g/mL Liposome Embodiment 11 PTX-Rg5-Gipo Paclitaxel Rg5 450-500 g/mL Liposome Embodiment 12 PTX-Rh2-Gipo Paclitaxel Rh2 400-500 g/mL Liposome Embodiment 13 DTX-Rg3-Gipo Docetaxel Rg3 650-700 g/mL Liposome Embodiment 14 DTX-Rg5-Gipo Docetaxell Rg5 450-500 g/mL Liposome Embodiment 15 DTX-Rh2-Gipo Docetaxel Rh2 400-500 g/mL Liposome

(92) As shown in Table 1, Rg3-Cho-Lipo showed severe hemolytic effect, whereas the hemolytic effects of Rg3-Blank, Rh2-Blank, PTX-Rg3-Gipo, PTX-Rh2-Gipo, DTX-Rg3-Gipo and DTX-Rh2-Gipo were similar to those of Rg5-Blank, PTX-Rg5-Gipo and DTX-Rg5-Gipo with HD50 value in the range of 400-700 g/mL, which can meet the safety standards of medicinal products.

(93) In addition, conventional Rg3-Cho-Lipo did not show hemolysis up to a concentration of 20-50 g/mL, mainly because that the encapsulation efficiency of the conventional Rg3 cholesterol liposome was low and Rg3 may leak more or less, thereby affecting the drug efficacy. Whereas, the encapsulation efficiency of the ginsenoside liposomes obtained from embodiment 2, embodiment 3, embodiment 7, embodiment 10, embodiment 12-13 and embodiment 15 in the present invention were high, similar to the encapsulation efficiency of Rg5-blank, PTX-Rg5-Gipo and DTX-Rg5-Gipo, thus, these drugs were all very efficient. Besides Rg3 and Rh2, it can further encapsulate drugs, such as Paclitaxel, indicating that Rg3 is used as membrane material in these liposomes.

Application Embodiment 2

Studies on the Effect of Mass Percentage of Ginsenoside in the Liposome on the Average Particle Size of the Liposome

(94) Sample test before lyophilization: 20 mL sample solution was diluted into 900 mL purified water at room temperature. The mixture was stirred for 1 min at 1700 rpm/min. Then, the sample was tested and the results were recordered.

(95) Sample test after lyophilization: A vial of lyophilized sample was hydrated with 20 mL purified water. Then, shaked the vial until the sample was fully dissolved. The sample solution was diluted into 900 mL purified water at room temperature and stirred for 1 min at 1700 rpm/min. Then, the sample was tested and the results were recorded.

(96) The experimental results are shown in Table 2.

(97) TABLE-US-00003 TABLE 2 Effects of mass percentage of ginsenoside in the liposome on the average particle size of the liposome Mass percentage Average Encapsu- Preparation of 20(S)-Rg3 particle lation Name method in liposomes size efficiency Rg3- According Egg lecithin:Rg3 = 147 nm 95% cholesterol to 10:0.1 liposome embodiment Egg lecithin:Rg3 = 438 nm 85% 1 10:1 Egg lecithin:Rg3 = 1 m 80% 10:2 Egg lecithin:Rg3 = 1 m 80% 10:5 Rg3-Blank ccording to Egg lecithin:Rg3 = 116 nm 95% liposome embodiment 10:0.1 2 Egg lecithin:Rg3 = 92 nm 95% 10:1 Egg lecithin:Rg3 = 126 nm 95% 10:2 Egg lecithin:Rg3 = 185 nm 95% 10:5 Paclitaxel- According Egg 103 nm 95% Rg3 to lecithin:Rg3:Paclitaxel = Liposome embodiment 10:0.1:0.05 10 Egg 85 nm 95% lecithin:Rg3:paclitaxel = 10:1:0.5 Egg 157 nm 95% lecithin:Rg3:Paclitaxel = 10:2:1 Egg 243 nm 95% lecithin:Rg3:paclitaxel = 10:5:2.5

(98) As showed in Table 2, the particle size increased and encapsulation efficiency decreased while increasing the mass percentage of Rg3 in Rg3-cholesterol liposome. Huan Yu, et al disclosed a Rg3-cholesterol liposome, which, in fact, is a conventional blank liposome loaded with Rg3 (See: International Journal of Pharmaceutics 450(2013)250-258). In the conventional Rg3-cholesterol liposomes, Rg3 is an active substance. With the increasing mass percentage of the Rg3, the encapsulation efficiency decreases and the particle size increases. Whereas, in the present invention, Rg3 is used as membrane material. With the increasing mass percentage of Rg3, particle size of the liposome becomes smaller and all the encapsulation efficiency are more than 95%. Therefore, Rg3 is used as membrane material in the present invention. Properties of the liposome also changes with the changes of the membrane material.

Application Embodiment 3

The Determination of Particle Size Distribution, Dispersion Coefficient and Electron Microscope Imaging of Paclitaxel Cholesterol Liposome and Paclitaxel Rg3 Liposome

(99) The determination of particle size distribution and dispersion coefficient: samples of PTX-Cho-Lipo and PTX-Rg3-Gipo were diluted 10 times. Then 1 mL diluted solution was added into the sample pool of Malvern laser particle size analyzer. Test results were recorded and analyzed.

(100) Morphology test of liposomes: 150 L PTX-Cho-Lipo solution and PTX-Rg3-Gipo solution were each diluted into 5 mL purified water. After dilution, a drop was placed on a carbon-coated copper grid and air dried for 10 minutes, then the sample was stained with 2% sodium acetate for 30 minutes. After removing the excess staining solution using a filter paper, the morphology of liposomes was observed and imaged using transmission electron microscope (TEM).

(101) The experimental results are listed in Table 3.

(102) TABLE-US-00004 TABLE 3 The particle sizes of PTX-Cho-Lipo and PTX-Rg3-Gipo Mean Distribution Particle size coefficient Zeta Name SD (nm) (PDI) Potential Paclitaxel cholesterol 114.4 5.18 0.27 0.004 8.7 2.128 liposome (PTX-Cho-Lipo) Paclitaxel Rg3 liposome 77.8 6.41 0.17 0.015 4.2 0.777 (PTX-Rg3-Gipo)

(103) As shown in FIGS. 1 and 2, FIG. 2 represents a normal distribution. As shown in Table 3, the distribution coefficient of PTX-Rg3-Gipo in the present invention is more optimal than that of PTX-Cho-Lipo, and the particle size of PTX-Rg3-Gipo is also smaller. The results suggest that PTX-Rg3-Gipo is better than PTX-Cho-Lipo in quality.

Application Embodiment 4

The Leakage Experiment of PTX-Cho-Lipo and PTX-Rg3-Gipo

(104) Freshly prepared PTX-Cho-Lipo and PTX-Rg3-Gipo were filtered using 0.22 micron membrane, and their encapsulation efficiency was determined and considered as a 100%. 3 mL each of the PTX-Cho-Lipo and PTX-Rg3-Gipo solutions were taken out and stored at 4 C. and their encapsulation efficiency were measured daily for 7 days. Plot a graph between the encapsulation efficiency and the time (days).

(105) The experimental results are shown in FIG. 3 and table 4.

(106) TABLE-US-00005 TABLE 4 The encapsulation efficency of paclitaxel in PTX-Cho-Lipo and PTX-Rg3-Gipo Encapsulation efficiency, % Time(days) PTX-Cho-Lipo PTX-Rg3-Gipo 1.00 100.00 100.00 2.00 62.90 94.32 3.00 44.95 89.67 4.00 40.61 85.85 5.00 36.57 83.37 6.00 33.25 81.68 7.00 35.85 80.32

(107) As shown in FIG. 3, there is a sharp drop in the encapsulation efficiency of PTX-Cho-Lipo from the beginning to the third day, however, few changes are observed in the encapsulation efficiency of PTX-Rg3-Gipo within 7 days.

(108) As shown in Table 4, under the same conditions, encapsulation efficiency of PTX-Rg3-Gipo in the present invention is higher than that of PTX-Cho-Lipo, which indicates that PTX-Rg3-Gipo is more stable in solution with less leakage. Thus, the quality of PTX-Rg3-Gipo is better than PTX-Cho-Lipo, and Rg3 is better than cholesterol as a liposome membrane material.

Application Embodiment 5

Effects of Liposome on Prolonged Circulation Time

(109) 30 nude mice (18-22 g) were randomly divided into 5 various groups (6 in each group), administered via mouse tail vein respectively with 0.3 mg/kg Cholesterol-blank liposome loaded with a fluorescent dye DID (DID-Cho-blank), mPEG-DSPE-Cholesterol blank liposome loaded with a fluorescent dye DID (DID-PEG-blank), Rg5-blank liposome loaded with loaded with a fluorescent dye DID (DID-Rg5-blank), Rg3-blank liposome loaded with a fluorescent dye DID (DID-Rg3-blank) and Rh2-blank liposome loaded with a fluorescent dye DID (DID-Rh2-blank). 0.2 mL blood samples were collected into heparinized centrifugal tubes via mice facial vein respectively after 2 min, 5 min, 15 min, 30 min, 1 hour, 3 hour, 6 hour, 12 hour and 24 hour. The DID fluorescence intensity of the collected blood sample was measured by a microplate reader. The fluorescence intensity of the first sample collected after 2 min was considered as 100% and other fluorescence intensity were calculated based on this value.

(110) Data Process and Analysis: The pharmacokinetic parameters of each liposome were calculated using pharmaceutical kinetics software 3p97, including Area under the Concentration-time Curve (AUC), half life of distribution (t1/2, t1/2) and half-life of elimination(t1/2), etc.

(111) The experimental results are listed in FIG. 4 and Table 5.

(112) TABLE-US-00006 TABLE 5 Characterization of liposome on prolonged ciruculation time Parameter DID-Cho-blank DID-PEG-blank DID-Rg5-blank DID-Rg3-blank DID-Rh2-blank t1/2/h 0.03 0.016 0.017 0.67 0.245 t1/2/h 0.798 0.917 0.47 1.603 1.892 t1/2/h 9.049 24.647 12.999 27.243 24.844 AUC(0-t)/mg .Math. L .Math. h 401.352 808.472 450.461 753.111 760.584 AUC(0-)/mg .Math. L .Math. h 455.227 1163.13 613.035 827.905 916.252

(113) As shown in Table 5, the values of AUC, half life of distribution (t1/2, t1/2) and half-life of elimination (t1/2) of DID-Rg3-blank and DID-Rh2-blank liposomes in the present invention are similar to the values of DID-PEG-blank, suggesting that they all have similar prolonged circulation time and similar therapeutic effect. Whereas, the circulation time and therapeutic effects of DID-Rg5-blank is shorter and weaker than DID-PEG-blank, only longer and stronger than the conventional DID-Cho-Blank.

Application Enbodiment 6

In Vivo Target Specificity Assay

(114) BALB/C-nu/nu mice bearing tumors in uniform size of 100 mm.sup.3 at right forelimbs without hemorrhagic necrosis, were intravenously injected via tail vein with liposomes in the present invention carrying 10% of near-infrared fluorescent probe (IR783) respectively (hereinafter named as the experimental group), which was obtained by encapsulating near-infrared fluorescent probe (IR783) into the present ginsenoside blank liposome, see embodiment 10 for details. A conventional blank lipsome caning near-infrared fluorescent probe (IR783) was hereinafter named as the control group which was obtained by encapsulating near-infrared fluorescent probe (IR783) into the blank liposome. The in vivo distributions of IR783 fluorescence were were recorded by in-vivo animal imaging system at the following time points, 2 h, 4 h, 8 h, 12 h and 24 h hour after administration, see FIG. 5.

(115) FIG. 5-A1-A5 are respectively in vivo distribution of IR783 fluorescence in the control group recorded at 2.sup.nd, 4.sup.th, 8.sup.th, 12.sup.th and 24.sup.th hour by in-vivo animal imaging system. FIG. 5-S is a fluorescence ruler, wherein the color is red, yellow, green and blue in sequence, indicating the fluorescence intensity, from the strongest to the weakest. FIG. 5-B1-B5, FIG. 5-C1-C5 and FIG. 5-D1-D5 are respectively the in vivo fluorescence distribution in the experimental group recorded at 2.sup.nd, 4.sup.th, 8.sup.th, 12.sup.th and 24.sup.th hour by in-vivo animal imaging system. FIG. 5-B1-B5 are respectively the fluorescence distribution of the Rg5-blank group; FIG. 5-C1-C5 are respectively the fluorescence distribution of the Rh2-blank group; FIG. 5-D1-D5 are respectively the fluorescence distribution of the Rg3-blank group.

(116) As shown in FIG. 5, the right forelimbs of the mice in the control group had no fluorescence, while the right forelimbs of the mice in the experimental groups have intensive fluorescence, indicating that ginsenoside blank liposomes can target tumor cells specifically.

(117) FIG. 6 is the in-vitro fluorescence distribution of IR783 after tumor removal imaged by in-vivo animal imaging system. FIG. 6-A is control group, and FIGS. 6-B, 6-C and 6-D are the experimental groups. After the in-vivo imaging, the tumors in the experimental group and control group are taken out and imaged in vitro. FIG. 6-S is a fluorescence ruler, wherein the color shows the relative fluorescence intensity, from strongest to weakest in a sequence of red, yellow, green and blue. FIG. 6-B, FIG. 6-C and FIG. 6-D respectively show the fluorescence intensity of Rg5-Gipo, Rg3-Gipo and Rh2-Gipo groups, suggesting that ginsenoside blank liposomes have very high specificity toward tumor cells.

(118) FIG. 7 is the comparison results between the fluorescence intensity of the control group and the experimental groups. It shows that the fluorescence intensity of Rg5-Gipo, Rg3-Gipo and Rh2-Gipo are significantly higher than that of the control group. Rg3-Gipo and Rh2-Gipo exhibit a significantly higher specificity to target than Rg5-Gipo group in BGC-823 human gastric cancer.

(119) In summary, the results suggest that Rg5-blank, Rg3-blank, and Rh2-blank have significantly higher specificity to target than the Cho-blank liposome. Moreover, Rg3-blank and Rh2-blank show a higher targeting specificity than Rg5-blank.

Application Embodiment 7

In Vivo and In Vitro Pharmacological Efficacy Assay

(120) 1. In Vitro Drug Efficacy Assay

(121) To test the drug efficacy in vitro, a total of 8 various concentrations were set up as shown in Table 6 and FIG. 8. FIG. 8 shows the cell survival rate of human breast cancer cell line (4T1) with addition of Rh2 group, Rh2-blank group, PTX group, PTX-Cho-Lipo group and PTX-Rh2-Gipo group respectively.

(122) TABLE-US-00007 TABLE 6 Concentration and viability of human breast cancer cells (4T1) with addition of Rh2 group, Rh2-blank group, PTX group, PTX-Cho-lipo group and PTX-Rh2-Gipo group C(M) Cell Viability PTX- PTX- PTX- PTX- Rh2- Cho- Rh2- Rh2- Cho- Rh2- Rh2 blank PTX Lipo Gipo Rh2 blank PTX Lipo Gipo 8.00000 8.00000 2.00000 2.00000 2.00000 100.73 100.25 52.00 36.52 23.07 2.66667 2.66667 0.66667 0.66667 0.66667 104.25 93.08 48.54 36.50 24.47 0.88889 0.88889 0.22222 0.22222 0.22222 96.04 95.03 48.48 39.00 26.36 0.29630 0.29630 0.07407 0.07407 0.07407 98.07 97.72 49.25 52.92 38.40 0.09877 0.09877 0.02469 0.02469 0.02469 99.90 95.43 55.83 71.95 44.87 0.03292 0.03292 0.00823 0.00823 0.00823 94.47 91.77 59.19 101.07 57.24 0.01097 0.01097 0.00274 0.00274 0.00274 92.85 93.55 94.70 96.12 71.48 0.00366 0.00366 0.00091 0.00091 0.00091 104.04 97.43 106.72 101.03 77.96

(123) As shown in Table 6 and FIG. 8, free Rh2 and Rh2-blank groups show low activity in vitro against human breast cancer cells (4T1). With low concentration, the cell viability of PTX-Cho-lipo group is lower than PTX group. While no matter the concentration is high or low, the cell viability of PTX-Rh2-Gipo group is much higher than the PTX group.

(124) 2. In Vivo Drug Efficacy Assay

(125) To evaluate the drug efficacy in vivo, 45 subcutaneous tumor-bearing nude mice were randomized into 5 treatment groups (9 in each group) and intravenously injected with PBS solution (control group), ginsenoside Rh2 (Rh2 group), ginsenoside Rh2 blank liposome (Rh2-Blank group), conventional paclitaxel cholesterol liposome (PTX-Cho-Lipo group) and ginsenoside Rh2 paclitaxel liposome (PTX-Rh2-Gipo group) via tail vein at a dose of 30 mg/kg. The changes of body weights of mice in each group were recorded every 2 days, and the longest diameter and the shortest diameter of tumors were measured with vernier calipers. The tumor volume was calculated by the following formula: V=(dmaxdmin.sup.2)/2, wherein dmin and dmax are respectively the shortest diameter and the longest diameter (mm) of the tumor; a relative tumor volume (RTV) was calculated according to the measurement results, by the formula: RTV=TVn/TV.sub.0, wherein TVn is the volume of the tumor measured every 2 days, TV.sub.0 is the volume of the tumor measured at day zero (the administration day).

(126) TABLE-US-00008 TABLE 7 Antitumor effects of control group, Rh2 group, Rh2-blank group, PTX-Cho-lipo group and PTX-Rh2-Gipo group in human breast cancer cell 4T1 4T1 Relative tumor volume time(d) Control Rh2 Rh2-blank PTX-Cho-Lipo PTX-Rh2-Gipo 0 100.00 100.00 100.00 100.00 100.00 3 273.99 200.94 214.73 199.01 95.23 6 249.60 316.69 193.95 229.01 166.89 9 290.21 276.04 273.64 289.80 162.85 12 555.41 400.20 310.41 317.20 168.02 15 507.64 473.53 403.28 435.89 167.88 18 700.78 510.20 400.30 449.06 178.55 21 965.30 898.52 603.59 511.90 245.27

(127) As shown in Table 7 and FIG. 9, after the same period of time, the volume of tumor in control group and Rh2 group are the maximum while in the PTX-Rh2-Gipo group is the minimum, followed by PTX-Cho-lipo group and Rh2-blank group. Results suggest that PTX-Rh2-Gipo group has better antitumor effects.

(128) 3. In Vitro Cytotoxicity Studies

(129) The in vitro cytotoxicity was evaluated using human breast cancer cell line (4T1). The cell survival rate of human breast cancer cell line (4T1) with addition of DTX group, DTX-Cho-Lipo group, DTX-Rg3-Gipo group and Nanoxel-PM group at various concentration were shown in Table 8 and FIG. 10.

(130) TABLE-US-00009 TABLE 8 The viability of human breast cancer cells (4T1) with addition of DTX group, DTX-Cho-Lipo group, DTX-Rg3-Gipo group and Nanoxel-PM group at various concentration Concentration Cell viability(%) (g/ml) DTX DTX-Cho-Lipo DTX-Rg3-Gipo Nanoxel-PM 3 51.85 39.85 40.67 40.54 0.6 48.89 40.96 43.98 44.78 0.12 46.26 45.56 42.60 37.62 0.024 49.86 55.45 48.52 44.52 0.0048 50.94 54.08 48.94 51.03 0.00096 61.65 59.35 50.33 61.96 0.00019 72.48 69.81 52.11 76.16 3.84E05 83.55 76.62 65.00 84.21 7.68E06 86.66 79.15 81.59 88.55

(131) As shown in Table 8 and FIG. 10, after the same period of time, the overall viability of human breast cancer cells 4T1 with addition of DTX-Rg3-Gipo group is significantly higher than DTX-Cho-Lipo group, especially in lower concentrations.

(132) 4. In Vivo Drug Efficacy Assay

(133) To evaluate the drug efficacy in vivo, 45 subcutaneous tumor-bearing nude mice were were randomerized into 5 groups (9 in each group), and intravenously injected with PBS solution (Control group,), Taxotere, Nanoxel-PM, DTX-Rg5-Gipo and DTX-Rg3-Gipovia tail vein at a dose of 10 mg.Math.kg.sup.1. The changes in mice body weights in each group were recorded every 2 days, and the longest diameter and the shortest diameter of tumors were measured with vernier calipers. The tumor volume is calculated by the following formula: V=(dmaxdmin.sup.2)/2, wherein dmin and dmax are respectively the shortest diameter and the longest diameter (mm) of the tumor; a relative tumor volume (RTV) is calculated according to the measurement results by the formula: RTV=TVn/TV.sub.0,

(134) wherein TVn is the volume of the tumor measured every 2 days, TV.sub.0 is the volume of the tumor measured at day zero (the administration day).

(135) TABLE-US-00010 TABLE 9 Antitumor effect of Control group, Taxotere group, Nanoxel-PM group, DTX-Rg5-Gipo group and DTX-Rg3-Gipo group in human breast cancer cell 4T1 Relative tumor volume 4T1 Nanoxel- DTX-Rg5- DTX-Rg3- time(d) Control Taxotere PM Gipo Gipo 0 100.00 100.00 100.00 100.00 100.00 3 273.99 206.85 192.51 168.40 115.34 6 249.60 254.61 140.99 141.67 84.92 9 290.21 198.66 172.59 203.55 125.33 12 555.41 224.67 134.94 155.11 89.09 15 507.64 231.03 181.05 150.37 86.65 18 700.78 361.50 175.55 197.97 65.99 21 764.79 322.15 184.46 151.56 86.11

(136) As shown in Table 9 and FIG. 11, after the same period of time, the volume of tumor in the PBS group is the maximum while in the DTX-Rg3-Gipo group is the minimum, followed by the DTX-Rg5-Gipo group and Nanoxel-PM group that are basically equivalent. The results suggest that DTX-Rg3-Gipo group has better anti-tumor activity.

Application Embodiment 8

In Vivo and In Vitra Pharmacological Efficacy Assay

(137) 8.1. In Vitro Drug Efficiency Assay

(138) A total of 10 different concentrations of each sample were set up as shown in Table 10. The survival rate of rat glioma C6 cells with addition of Rg3 group, Rg3-blank group, PTX group, PTX+Rg3 group, PTX-Cho-Lipo group and PTX-Rg3-Gipo group at various concentrations respectively are listed in Table 11 and FIG. 12.

(139) TABLE-US-00011 TABLE 10 Concentrations of Rg3 group, Rg3-blank group, PTX group, PTX + Rg3 group, PTX-Cho-Lipo group and PTX-Rg3-Gipo group used to against rat glioma cells (C6) C (g/ml) Rg3 Rg3-blank PTX PTX + Rg3 PTX-Cho-Lipo PTX-Rg3-Gipo 20 20 10 10 10 10 6.67 6.67 3.333 3.333 3.333 3.333 2.22 2.22 1.111 1.111 1.111 1.111 0.74 0.74 0.370 0.370 0.370 0.370 0.25 0.25 0.1235 0.1235 0.1235 0.1235 0.08 0.08 0.0412 0.0412 0.0412 0.0412 0.03 0.03 0.0137 0.0137 0.0137 0.0137 0.01 0.01 0.0046 0.0046 0.0046 0.0046 0.003 0.003 0.0015 0.0015 0.0015 0.0015 0.001 0.001 0.0005 0.0005 0.0005 0.0005

(140) TABLE-US-00012 TABLE 11 The viability of rat C6 glioma cells with addition of Rg3, Rg3-blank, PTX, PTX + Rg3, PTX-Cho-Lipo and PTX-Rg3-Gipo Cell Viability PTX-Cho- PTX-Rg3- Rg3 Rg3-blank PTX PTX + Rg3 Lipo Gipo 89.02171 46.4009329 39.58829 31.71681 30.98532 22.91087 95.02171 65.10124 43.21006 33.91804 32.77745 23.9622 96.16015 78.86144 43.89464 34.35981 37.70992 24.9767 92.822 84.03477 45.97812 35.96694 38.48503 25.59589 95.42692 86.15499 49.90699 37.27702 45.13212 30.10103 94.23058 88.33881 60.48813 45.9984 50.08808 33.76731 92.03087 88.33881 65.36402 55.32691 59.15443 37.20547 92.41679 89.61094 73.2867 62.60873 72.80094 50.51328 98.82296 91.32832 79.49252 66.44756 85.5549 58.3184 100 100 100 100 100 100

(141) As shown in Table 11 and FIG. 12, PTX-Rg3-Gipo group show better cell activity than PTX-Cho-Lipo group and PTX+Rg3 group. The results suggest that the cell activity of PTX-Rg3-Gipo group has been greatly improved.

(142) 8.2. Survival Curve and Median Survival Day

(143) A total of 63 subcutaneous tumor-bearing nude mice were randomized into 7 groups (9 in each group), and intravenously injected with PBS solution (Control group,), Rg3, Rg3-blank, PTX, PTX+Rg3, PTX-Cho-Lipo and PTX-Rg3-Gipo via tail vein at a dose of 10 mg.Math.kg.sup.1. From the 12.sup.th day after injection, the numbers of survived nude mice were recorded daily until all nude mice die. Survival curves of nude mice in each group were plotted by GraphPad Prism-5 software, and median survival time was calculated.

(144) TABLE-US-00013 TABLE 12 The number of survived mice in each group at corresponding time against in-situ glioma Time(d) PBS PTX Rg3 PTX + Rg3 PTX-Cho-Lipo Rg3-blank PTX-Rg3-Gipo 12 9 10 10 10 10 10 10 14 9 10 10 10 10 10 10 16 8 9 9 9 10 10 10 18 6 8 9 9 10 10 10 20 5 7 8 7 9 9 10 22 4 6 7 6 8 9 10 24 3 6 7 6 8 9 9 26 3 5 6 5 7 9 9 28 2 4 5 3 6 7 8 30 0 4 4 3 5 5 8 32 0 4 4 3 5 5 8 34 0 3 3 1 4 4 7 36 0 1 3 1 3 3 7 38 0 1 2 0 3 3 7 40 0 0 1 0 3 2 7 42 0 0 0 0 3 2 7 44 0 0 0 0 3 2 6 46 0 0 0 0 3 2 6 48 0 0 0 0 2 2 6 50 0 0 0 0 2 2 5 52 0 0 0 0 2 1 5 54 0 0 0 0 1 1 5 56 0 0 0 0 1 1 5 58 0 0 0 0 0 1 3 60 0 0 0 0 0 0 1 62 0 0 0 0 0 0 0

(145) TABLE-US-00014 TABLE 13 The median survival days in each group at corresponding time against in-situ glioma PTX- PTX- PTX + Cho- Rg3- Groups PBS PTX Rg3 Rg3 Lip Rg3 Gipo Median 21 27 29 27 35 32 54 survial (day)

(146) As shown in Table 12, Table 13 and FIG. 13, the median survival time of PTX-Rg3-Gipo group is significantly longer than those of PTX-Cho-Lipo group and PTX+Rg3 group.

Application Embodiment 9

In Vivo and In Vivo Pharmacological Efficacy Assay

(147) 1. In Vitro Cell Viability Assay

(148) A total of 9 different concentrations were set up as shown in Table 12. The survival rate of human gastric cancer cells (BGC-823) with addition of Rg5 group, Rg3 group, Rh2 group, Rg5-blank group, Rg3-blank group, Rh2-blank group, PTX group, PTX-Cho-Lipo group, PTX-Rg5-Gipo group, PTX-Rg3-Gipo group and PTX-Rh2-Gipo group at various concentrations are shown in FIG. 12 and Table 14 respectively.

(149) TABLE-US-00015 TABLE 14 Concentrations of Rg5 group, Rg3 group, Rh2 group, Rg5-blank group, Rg3-blank group, Rh2-blank group, PTX group, PTX-Cho-Lipo group, PTX-Rg5-Gipo group, PTX-Rg3- Gipo group and PTX-Rh2-Gipo group used in human gastric cancer cells (BGC-823) C(g/mL) PTX PTX- PTX- PTX- Rg5- Rg3- Rh2- Cho- Rg5- Rg3- Rh2- Rg5 Rg3 Rh2 blank blank blank PTX Lipo Gipo Gipo Gipo 0.0109 0.0109 0.0109 0.0109 0.0109 0.0109 0.0027 0.0027 0.0027 0.0027 0.0027 0.0329 0.0329 0.0329 0.0329 0.0329 0.0329 0.0082 0.0082 0.0082 0.0082 0.0082 0.0987 0.0987 0.0987 0.0987 0.0987 0.0987 0.0246 0.0246 0.0246 0.0246 0.0246 0.2962 0.2962 0.2962 0.2962 0.2962 0.2962 0.0740 0.0740 0.0740 0.0740 0.0740 0.8888 0.8888 0.8888 0.8888 0.8888 0.8888 0.2222 0.2222 0.2222 0.2222 0.2222 2.6666 2.6666 2.6666 2.6666 2.6666 2.6666 0.6666 0.6666 0.6666 0.6666 0.6666 8 8 8 8 8 8 2 2 2 2 2 24 24 24 24 24 24 6 6 6 6 6

(150) TABLE-US-00016 TABLE 15 Cell viability of human gastric cancer cells (BGC-823) with addition of Rg5 group, Rg3 group, Rh2 group, Rg5-blank group, Rg3-blank group, Rh2-group, PTX group, PTX-Cho-Lipo group, PTX-Rg5-Gipo group, PTX-Rg3-Gipo group and PTX-Rh2-Gipo group Cell Viability Rg5blank Rg3blank Rh2blank PTX PTX-Cho-Lipo PTX-Rg5-Gipo PTX-Rg3-Gipo PTX-Rh2-Gipo 102.7 100.6 101.2 91.9 100.5 82.6 94.8 85.4 101.2 101.4 103.7 80.5 85.5 52.6 87.6 68.6 103.3 98.0 100.7 60.9 53.9 38.8 69.8 36.4 100.0 87.6 102.0 50.3 43.6 35.3 35.5 32.1 100.4 77.9 100.8 41.6 38.4 34.7 29.3 29.2 104.4 72.7 89.3 42.7 36.0 34.0 28.9 28.7 99.8 64.6 75.6 38.0 34.9 33.3 25.5 28.3 94.8 58.8 65.6 33.2 33.1 23.5 24.2 24.3 92.7 53.5 57.7 32.2 32.8 20.7 6.8 17.6

(151) As shown in Table 15 and FIG. 14, the cell viability in PTX-Rg5-Gipo group, PTX-Rh2-Gipo group and PTX-Rg3-Gipo group are the best, followed by PTX-Cho-Lipo group and PTX group.

(152) 2. In vivo pharmacological efficacy assay: A total of 72 subcutaneous tumor-bearing nude mice were randomerized into 8 groups (9 in each group), and intravenously injected with PBS solution (Control group,), Rg3, Rg3-blank, PTX-Cho-Lipo, Abraxane, PTX-Rg5-Gipo, PTX-Rg3-Gipo and PTX-Rh2-Gipo, via tail vein at a dose of 10 mg.Math.kg.sup.1. The changes of mice body weights in each group were recorded every 2 days, and the longest diameter and the shortest diameter of tumors were measured with vernier calipers. The tumor volume is calculated by the following formula: V=(dmaxdmin.sup.2)/2, wherein dmin and dmax are respectively the shortest diameter and the longest diameter (mm) of the tumor; a relative tumor volume (RTV) is calculated according to the measurement results by the formula: RTV=TVn/TV.sub.0,

(153) wherein TVn is the volume of the tumor measured every 2 days, TV.sub.0 is the volume of the tumor measured at day zero (the administration day).

(154) Experimental results are listed in Table 16 and FIG. 15.

(155) TABLE-US-00017 TABLE 16 Drug efficacy of Control group, Rg3 group, Rg3-blank group, PTX-Cho-Lipo group, Abraxane group, PTX-Rg5-Gipo group, PTX-Rg3-Gipo group and PTX-Rh2-Gipo group against human gastric cancer cell (BGC-823) Relative tumor volume BGC- PTX- PTX- PTX- PTX- 823 Cho- Rg5- Rg3- Rh2- time(d) PBS Rg3 Rg3-blank Lipo Abraxane Gipo Gipo Gipo 1 164.67 166.88 168.24 165.27 164.67 165.77 163.63 163.50 3 215.94 267.38 275.58 275.58 225.78 187.97 239.71 197.10 5 322.94 317.96 319.38 381.27 316.77 165.73 161.94 134.83 7 469.00 417.96 472.85 553.90 362.70 134.84 119.29 79.99 9 777.24 717.96 657.85 782.11 401.47 115.01 78.56 53.36 11 1273.61 1340.69 817.79 1044.41 541.84 119.72 31.50 22.67 13 1747.54 1636.89 901.41 1313.14 595.41 171.04 30.04 23.87 15 2039.45 2034.43 1107.42 1410.32 736.65 186.47 15.40 6.83 17 2039.45 2034.43 1118.43 1515.96 840.69 193.32 18.89 4.05 19 2039.45 2034.43 1207.42 1826.55 1187.35 439.52 19.96 1.71 21 2039.45 2034.43 1321.36 1982.04 1215.19 535.77 4.14 2.00 23 2039.45 2034.43 1525.74 2061.15 1351.30 560.69 13.96 3.24

(156) As shown in Table 16 and FIG. 15, after the same period of time, the tumor volume in Control group is the maximum while in the PTX-Rg3-Gipo group and the PTX-Rh2-Gipo group are the minimum, followed by the PTX-Rg5-Gipo group, the Abraxane group and PTX-Cho-Lipo group. The data suggest that PTX-Rg3-Gipo group and PTX-Rh2-Gipo group have significant better anti-tumor activity.

(157) It is to be understood that the foregoing description of two preferred embodiments is intended to be purely illustrative of the principles of the invention, rather than exhaustive thereof, and that changes and variations will be apparent to those skilled in the art, and that the present invention is not intended to be limited other than expressly set forth in the following claims.