CN115487148B - Ginsenoside mitoxantrone liposome, and preparation method and application thereof - Google Patents

Ginsenoside mitoxantrone liposome, and preparation method and application thereof Download PDF

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CN115487148B
CN115487148B CN202110678285.4A CN202110678285A CN115487148B CN 115487148 B CN115487148 B CN 115487148B CN 202110678285 A CN202110678285 A CN 202110678285A CN 115487148 B CN115487148 B CN 115487148B
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liposome
ginsenoside
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mitoxantrone
concentration
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CN115487148A (en
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李翀
王建新
王丹
陈颖江
詹华杏
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Xiamen Ginposome Pharmaceutical Co ltd
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    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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Abstract

The invention discloses a ginsenoside mitoxantrone liposome, a preparation method and application thereof. The invention provides a ginsenoside mitoxantrone liposome which comprises the following components in parts by mass: 5-15 parts of phospholipid, 0.1-4 parts of ginsenoside and 1 part of mitoxantrone salt; the ginsenoside mitoxantrone liposome does not comprise cholesterol. The ginsenoside mitoxantrone liposome has targeting effect and synergy and attenuation on tumor cells.

Description

Ginsenoside mitoxantrone liposome, and preparation method and application thereof
Technical Field
The invention relates to a ginsenoside mitoxantrone liposome, a preparation method and application thereof.
Background
The liposome is a directional drug-carrying system, belongs to a special dosage form of a targeted drug-carrying system, and can embed drugs in nano-sized particles, wherein the particles are similar to double-molecular layer microvesicles in a biological membrane structure, enter a human body to be mainly phagocytized by reticuloendothelial systems, and change the in-vivo distribution of the encapsulated drugs, so that the drugs are mainly accumulated in targeted tissues, thereby improving the therapeutic index of the drugs, reducing the therapeutic dose of the drugs and reducing the toxicity of the drugs.
Three application patents of CN201610693884.2, CN201811447245.3, CN201811447243.4 and the like all disclose the technical advantages that the liposome prepared by taking ginsenoside as a membrane material and mainly taking a passive drug carrying method, namely a thin film evaporation method, has stable quality, obvious drug effect and the like of the related liposome after the liposome is coated with chemotherapeutic drugs such as paclitaxel and the like.
CN200380104235.5, CN200380104175.7 and other patents disclose a preparation method of liposome active drug-carrying with phospholipid and cholesterol as membrane materials and ammonium sulfate as gradient.
CN201811532448.2, CN201811552395.0 and other patents disclose a liposome active drug-loading method using phospholipid and cholesterol as membrane materials and sucrose octasulfate triethylamine as gradient.
CN201811305299.6 discloses a method for preparing a membrane material from phospholipid and cholesterol, and from ammonium methylsulfonate, ammonium 4-hydroxybenzenesulfonate, triethylamine methylsulfonate, and triethylamine 4-hydroxybenzenesulfonate; the active liposome medicine carrying method with ammonium ethanedisulfonate, ammonium propanedisulfonate, triethylamine ethanedisulfonate, triethylamine propanedisulfonate, etc. as gradient.
In the prior art, the ginsenoside liposome can be prepared into a co-carried liposome of a poorly soluble drug by adopting a thin film evaporation method, but a water-soluble drug is generally prepared by adopting an active drug carrying method, wherein a bilayer membrane consists of phospholipid and cholesterol, an ionic salt solution such as ammonium sulfate, sucrose octasulfate triethylamine and the like is used as an inner water phase, and the water-soluble drug is loaded into the inner cavity of the liposome by adopting principles such as pH gradient and the like.
Therefore, how to select an optimal compound medicine compatibility and how to formulate an optimal preparation process so as to produce the ginsenoside mitoxantrone liposome with better medicine effect, lower toxicity, quality and other indexes which can meet the medicine requirements so as to meet the medicine reporting requirements, and a great deal of research work and technical attack are needed.
Disclosure of Invention
The invention aims to solve the technical problem of the existing mitoxantrone liposome, and provides a ginsenoside mitoxantrone liposome, a preparation method and application thereof. The mitoxantrone liposome disclosed by the invention has the advantages of stable property, small particle size, high drug encapsulation efficiency, good in-vivo compatibility, good in-vivo drug release, better drug effect and lower toxicity; the preparation method has a good preparation process, and the preparation conditions are easy to realize, so that the industrialization is facilitated; the optimization of the combination of the preparation process and the product performance is realized.
The invention provides a ginsenoside mitoxantrone liposome (Ginposome-MIT for short), which comprises the following components in percentage by mass: 5-15 parts of phospholipid, 0.1-4 parts of ginsenoside and 1 part of mitoxantrone salt; the ginsenoside mitoxantrone liposome does not contain cholesterol.
In one embodiment of the present invention, the ginsenoside mitoxantrone liposome further comprises 0.1-2 parts of PEG-DSPE (all called polyethylene glycol-distearoyl phosphatidylethanolamine), preferably, the PEG-DSPE is PEG2000-DSPE.
In one embodiment of the present invention, the mitoxantrone salt is a mitoxantrone salt obtained by ion-exchanging mitoxantrone hydrochloride with a salt solution by a pH gradient method (wherein mitoxantrone in the mitoxantrone salt and an anion in the salt solution form the mitoxantrone salt); the salt solution is sulfate aqueous solution, sulfonate aqueous solution or sucrose octasulfate aqueous solution; preferably, the salt solution is an ammonium sulfate aqueous solution, a sucrose octasulfate triethylamine aqueous solution, an ammonium methylsulfonate aqueous solution, a methyl sulfonic acid triethylamine aqueous solution, an ammonium ethanedisulfonate aqueous solution, an ammonium propanedisulfonate aqueous solution, an ethylene disulfonate triethylamine aqueous solution or an ethylene disulfonate triethylamine aqueous solution; more preferably, the salt solution is an aqueous solution of ammonium sulfate, an aqueous solution of sucrose octasulfate triethylamine, an aqueous solution of ammonium methylsulfonate or an aqueous solution of ethylene disulfonate triethylamine; such as an aqueous ammonium sulfate solution.
In one embodiment of the invention, the mitoxantrone salt is mitoxantrone sulfate, sucrose octasulfuric acid, mitoxantrone mesylate, mitoxantrone methylsulfonic acid, mitoxantrone ethyldisulfonic acid, mitoxantrone propyldisulfonic acid, or mitoxantrone propyldisulfonic acid; preferably, the mitoxantrone salt is mitoxantrone sulfate, sucrose octasulfate mitoxantrone, or mitoxantrone methylsulfonate; such as mitoxantrone sulfate.
In one embodiment of the present invention, the mitoxantrone hydrochloride is an aqueous solution of mitoxantrone hydrochloride, preferably, the concentration of the aqueous solution of mitoxantrone hydrochloride is 10mg/mL.
In one embodiment of the present invention, in the ginsenoside mitoxantrone liposome, the ginsenoside and the phospholipid form a phospholipid membrane, and preferably, the phospholipid membrane further comprises PEG-DSPE.
In one embodiment of the present invention, the inner side of the phospholipid membrane is an inner aqueous phase, the outer side of the phospholipid membrane is an outer aqueous phase, and the mitoxantrone salt is encapsulated in the inner aqueous phase; the mitoxantrone salt is mitoxantrone salt insoluble salt.
In one aspect of the invention, the inner aqueous phase is the salt solution, and the outer aqueous phase is a physiological isotonic solution; for example, the physiological isotonic solution is a 5% aqueous dextrose solution or a 10% aqueous sucrose solution.
In one embodiment of the invention, the salt solution has a concentration of 0.05M to 0.975M; for example 0.05M, 0.1M, 0.2M, 0.3M, 0.325M, 0.65M, 0.975M or 0.16M.
In one embodiment of the invention, when the salt solution is an aqueous solution of sucrose octasulfate triethylamine, the salt solution has a concentration of 0.05M to 0.3M, for example, 0.1M, 0.2M or 0.3M.
In one embodiment of the present invention, when the salt solution is an aqueous solution of triethylamine ethanedisulfonate, the salt solution has a concentration of 0.16M to 0.325M.
In one embodiment of the present invention, when the salt solution is an aqueous solution of ammonium methylsulfonate, the concentration of the salt solution is 0.325M to 0.975M.
In one embodiment of the invention, when the salt solution is an aqueous ammonium sulfate solution, the salt solution has a concentration of 0.16M to 0.325M, for example, 0.325.
In one embodiment of the present invention, the phospholipid is selected from one or more of hydrogenated phospholipid, egg yolk lecithin, soybean phospholipid and cephalin; preferably, the phospholipid is hydrogenated phospholipid or egg yolk lecithin.
In one scheme of the invention, the mass ratio of the mitoxantrone hydrochloride to the phospholipid is 1 (5-15); for example, the mass ratio of mitoxantrone hydrochloride to hydrogenated phospholipid is 1:10.
In one embodiment of the present invention, the ginsenoside is selected from one or more of 20 (S) -ginsenoside Rg3, ginsenoside pseudo-Rg 3, 20 (S) -ginsenoside Rh2, ginsenoside Rg5, ginsenoside Rk1 and ginsenoside Rp1, preferably, the ginsenoside is 20 (S) -ginsenoside Rg3 and/or 20 (S) -ginsenoside Rh2.
In one scheme of the invention, the mass ratio of the mitoxantrone salt to the ginsenoside is 1 (0.1-4); for example, the mass ratio of the mitoxantrone salt to the ginsenoside is 1:1 or 1:1.5.
In one embodiment of the invention, the HPLC purity of the ginsenoside is greater than or equal to 99%.
In one embodiment of the present invention, the mass fraction of the phospholipid is 10 parts.
In one embodiment of the invention, the mass fraction of the PEG-DSPE is 0.5 parts.
In one scheme of the invention, the mass fraction of the ginsenoside is 1 part.
In one embodiment of the invention, the mitoxantrone salt is 1 part by mass.
In one scheme of the invention, the particle size D90 of the ginsenoside mitoxantrone liposome is less than or equal to 150nm.
In one scheme of the invention, the ginsenoside mitoxantrone liposome comprises the following components in percentage by mass: 10 parts of phospholipid, 0.5 part of PEG-DSPE, 1 part of ginsenoside and 1 part of mitoxantrone sulfate.
The invention also provides a preparation method of the ginsenoside mitoxantrone liposome, which comprises the following steps of;
step 1, dissolving phospholipid in an organic solvent to obtain a mixture A1, and then hydrating the mixture A1 with a salt solution to obtain a liposome solution A1;
Step2, which is scheme 1 or scheme 2;
Scheme 1 (high pressure homogenization) includes the steps of:
Homogenizing the liposome solution A1 obtained in the step 1 under high pressure, and controlling the particle size D90 to be less than 100nm to obtain liposome solution A2a.
Scheme 2 (extrusion process) includes the steps of:
Extruding the liposome solution A1 obtained in the step 1 through all pore-diameter extrusion plates in sequence, and controlling the particle diameter D90 to be below 100nm to obtain liposome solution A2b;
Step 3, placing the liposome solution A2a or A2b obtained in the step2 into a dialysis bag, and dialyzing by taking the isotonic solution as a dialysis medium; obtaining liposome solution A3;
Step 4, mixing the solution A3 obtained in the step 3 with mitoxantrone salt solution to obtain liposome solution A4;
Step 5, mixing the liposome solution A4 obtained in the step 4 with the ginsenoside solution, placing the mixture in a dialysis bag, and dialyzing by taking the isotonic solution same as that in the step 3 as a dialysis medium; obtaining the ginsenoside mitoxantrone liposome solution A5.
In one embodiment of the present invention, the method for preparing the ginsenoside mitoxantrone liposome further comprises one or more of the following steps:
And step 6, mixing the liposome solution A5 obtained in the step 5 with the PEG-DSPE physiological isotonic solution to obtain a liposome solution A6.
And 7, sterilizing, filtering and filling the liposome solution A5 obtained in the step 5 or the liposome solution A6 obtained in the step 6.
In one embodiment of the present invention, in the preparation method of the ginsenoside mitoxantrone liposome, the phospholipid is one or more selected from hydrogenated phospholipid, egg yolk lecithin, soybean phospholipid and cephalin; preferably, the phospholipid is hydrogenated phospholipid or egg yolk lecithin.
In one scheme of the invention, in the preparation method of the ginsenoside mitoxantrone liposome, the mass ratio of the mitoxantrone hydrochloride to the phospholipid is 1 (5-15); for example, the mass ratio of mitoxantrone hydrochloride to hydrogenated phospholipid is 1:10.
In one embodiment of the present invention, in the method for preparing the ginsenoside mitoxantrone liposome, the ginsenoside is selected from one or more of 20 (S) -ginsenoside Rg3, ginsenoside pseudo-Rg 3, 20 (S) -ginsenoside Rh2, ginsenoside Rg5, ginsenoside Rk1 and ginsenoside Rp1, preferably, the ginsenoside is 20 (S) -ginsenoside Rg3 and/or 20 (S) -ginsenoside Rh2.
In one scheme of the invention, in the preparation method of the ginsenoside mitoxantrone liposome, the mass ratio of the mitoxantrone hydrochloride to the ginsenoside is 1 (0.1-4); for example, the mass ratio of the mitoxantrone hydrochloride to the ginsenoside is 1:1 or 1:1.5.
In one embodiment of the present invention, in the method for preparing the ginsenoside mitoxantrone liposome, the HPLC purity of the ginsenoside is not less than 99%.
In one embodiment of the present invention, in the preparation method of the ginsenoside mitoxantrone liposome, the salt solution is ammonium sulfate, sucrose octasulfate triethylamine, ammonium methylsulfonate, and triethylamine methylsulfonate; ammonium ethanedisulfonate, ammonium propanedisulfonate, triethylamine ethanedisulfonate, triethylamine propanedisulfonate, and the like. For example, an aqueous ammonium sulfate solution.
In one embodiment of the present invention, in the method for preparing the ginsenoside mitoxantrone liposome, the concentration of the salt solution is 0.05M-0.975M; for example 0.05M, 0.1M, 0.2M, 0.3M, 0.325M, 0.65M, 0.975M or 0.16M.
In one embodiment of the present invention, in the method for preparing the ginsenoside mitoxantrone liposome, when the salt solution is sucrose octasulfate triethylamine aqueous solution, the concentration of the salt solution is 0.05M-0.3M, for example, 0.1M, 0.2M or 0.3M.
In one embodiment of the present invention, in the preparation method of the ginsenoside mitoxantrone liposome, when the salt solution is an aqueous solution of triethylamine ethanedisulfonate, the concentration of the salt solution is 0.16M-0.325M.
In one embodiment of the present invention, in the method for preparing the ginsenoside mitoxantrone liposome, when the salt solution is an aqueous solution of ammonium methylsulfonate, the concentration of the salt solution is 0.325M-0.975M.
In one embodiment of the present invention, in the method for preparing the ginsenoside mitoxantrone liposome, when the salt solution is an aqueous solution of ammonium sulfate, the concentration of the salt solution is 0.16M-0.325M, for example, 0.325.
In one embodiment of the present invention, in the step 1, the solvent is a conventional solvent for such reactions in the art; preferably, the solvent is ethanol; such as absolute ethanol.
In one embodiment of the present invention, in the step 1, the mass-to-volume ratio of the phospholipid to the organic solvent is 1 g/1-10 mL, for example, 1g/2mL.
In one embodiment of the present invention, in the step 1, the mixture A1 is obtained by heating and dissolving the phospholipid in an organic solvent; for example, the heating may be in a water bath to 55-65 ℃, such as 60 ℃.
In one embodiment of the present invention, the hydration temperature in step 1 may be 55-65deg.C, for example, 60deg.C.
In one embodiment of the present invention, in the step 1, the hydration is performed in a rotary evaporator, and the rotation speed is 40-60 rp/min, for example, 50rp/min.
In one embodiment of the present invention, in the step 1, the hydration time is related to the reaction scale, so that the solution is uniform, for example, 2 to 4 hours.
In one embodiment of the present invention, in the embodiment 1 of the step 2, the high-pressure homogenization is performed by using a freezing water cutting cycle at 0 to 10 ℃ in a homogenizer; preferably, the temperature of the liposome solution is ensured at 5-10 ℃.
In one embodiment of the present invention, in embodiment 1 of step2, the high-pressure homogenizing pressure is between 800 and 1400bar, for example 1200bar.
In one embodiment of the present invention, in embodiment 1 of step 2, the number of times of high-pressure homogenization is 3 to 4, for example, 4.
In one embodiment of the present invention, in embodiment 2 of step 2, the extrusion temperature is 35-45 ℃, for example 40 ℃.
In one embodiment of the present invention, in embodiment 2 of the step 2, the aperture of the extruded plate is 800nm,400nm,200nm,100nm.
In one aspect of the invention, in aspect 2 of step 2, the extrusion pressure is 600 to 800psi; such as 800psi.
In a certain embodiment of the present invention, in the embodiment 2 of the step 2, the number of extrusion times may be 4 to 10, for example, 4 times.
In one embodiment of the present invention, in the embodiment 2 of the step 2, the solution A1 sequentially passes through a polycarbonate membrane filter plate having a pore diameter of 800nm, 400nm, 200nm or 100nm, respectively.
In one embodiment of the present invention, in the step 3, the molecular weight cut-off of the dialysis bag is 8000 to 15000, for example, 10000.
In one embodiment of the present invention, in the step 3, the isotonic solution is 5% glucose or 10% sucrose aqueous solution.
In one embodiment of the present invention, in the step 3, the volume ratio of the solution A2a or A2b to the isotonic solution is 1:1000.
In one embodiment of the invention, in step 3, the dialysis temperature is 0-10deg.C, such as 4deg.C.
In one embodiment of the invention, in step 3, the dialysis is performed for a period of time to completely remove the salt solution in the outer aqueous phase of the solution A2a or A2b liposome, preferably, the dialysis is performed for a period of time ranging from 10 to 18 hours, for example, 12 hours.
In one embodiment of the present invention, in the step 3, in order to place the liposome solution A2a or A2b obtained in the step 2 in a dialysis bag, an isotonic solution is used as a dialysis medium, and the volume ratio of the sample to the dialysis medium is 1:1000, changing the dialyzate for 1 time every 4 hours during the dialysis, completely removing acid radical ions in the outer water phase of the blank liposome, and obtaining the blank liposome with the outer water phase consisting of the isotonic solution and the inner water phase consisting of the acid radical salt solution.
In one embodiment of the present invention, in the step 4, the concentration of the mitoxantrone hydrochloride aqueous solution is 5-20 mg/mL, for example, 1mg/mL, 5mg/mL, 10mg/mL, 15mg/mL or 20mg/mL; preferably 10 to 15mg/mL.
In a certain scheme of the invention, in the step 4, the volume ratio of the solution A3 obtained in the step 3 to the mitoxantrone hydrochloride aqueous solution is 1:1, and incubating in a water bath at 50-60 ℃ for 40 minutes to obtain the ginsenoside mitoxantrone liposome. Specifically, the aqueous phase in the liposome is acid radical mitoxantrone insoluble salt, and the aqueous phase outside the liposome is isotonic solution.
In one embodiment of the present invention, in the step 5, the concentration of the ginsenoside solution is 5-20 mg/mL, for example, 10mg/mL.
In one embodiment of the present invention, in the step 5, the solvent of the ginsenoside solution is the same as the solvent in the step 1.
In one embodiment of the invention, in the step 5, the mixing is performed by stirring, preferably for 30 to 60 minutes, for example 45 minutes.
In one embodiment of the present invention, in the step 5, the molecular weight cut-off of the dialysis bag is 8000 to 15000, for example, 10000.
In one embodiment of the present invention, in the step 5, in order to slowly add the ginsenoside solution into the liposome solution A4 obtained in the step 4, stirring, volatilizing to remove most of the ethanol, and then placing in a dialysis bag, using the same isotonic solution in the step 3 as a dialysis medium, and dialyzing at 4 ℃ for 12 hours, wherein the volume ratio of the sample to the dialysis medium is 1:1000, changing the dialysate for 1 time every 4 hours during dialysis, and completely removing ethanol solvent, inorganic salt, unwrapped mitoxantrone hydrochloride and ginsenoside to obtain liposome solution A5.
In one embodiment of the present invention, in the step 6, the mass ratio of the PEG-DSPE to the phospholipid is (0.025-0.15): 1, for example, 0.05:1.
In one embodiment of the invention, in step 6, the PEG-DSPE concentration is 1-20mg/mL, e.g., 10mg/mL.
In one embodiment of the present invention, in the step 6, a certain amount of PEG-DSPE is accurately weighed, dissolved in the same isotonic solution as in the step 3, and then added to the liposome solution A5 obtained in the step 5.
In one embodiment of the present invention, in the step 7, the conditions and operations of the sterilization filtration and the filling may be conditions and operations conventional in the art, for example, in the sterilization filtration step, the liposome is filtered by using a 0.22 μm filter; in the filling step, filling in a 10mL or 20mL penicillin bottle, capping and packaging.
In one scheme of the invention, in the preparation method of the ginsenoside mitoxantrone liposome, the particle size D90 of the ginsenoside mitoxantrone liposome is less than or equal to 150nm, and the encapsulation rate is more than or equal to 80%.
The invention also provides a ginsenoside mitoxantrone liposome, which is prepared by the preparation method of the ginsenoside mitoxantrone liposome.
The invention also provides a ginsenoside mitoxantrone liposome, which comprises the following raw materials in percentage by mass: 5-15 parts of phospholipid, 0.1-4 parts of ginsenoside and 1 part of mitoxantrone salt; but does not contain cholesterol.
In one embodiment of the present invention, the raw materials of the ginsenoside mitoxantrone liposome further comprise 0.1-2 parts of PEG-DSPE (all called polyethylene glycol-distearoyl phosphatidylethanolamine), preferably, the PEG-DSPE is PEG2000-DSPE.
In one embodiment of the present invention, the mitoxantrone salt is a mitoxantrone salt obtained by ion-exchanging mitoxantrone hydrochloride with a salt solution by a pH gradient method (wherein mitoxantrone in the mitoxantrone salt and anions in the salt solution form the mitoxantrone salt); the salt solution is sulfate aqueous solution, sulfonate aqueous solution or sucrose octasulfate aqueous solution; preferably, the salt solution is an ammonium sulfate aqueous solution, a sucrose octasulfate triethylamine aqueous solution, an ammonium methylsulfonate aqueous solution, a methyl sulfonic acid triethylamine aqueous solution, an ammonium ethanedisulfonate aqueous solution, an ammonium propanedisulfonate aqueous solution, an ethylene disulfonate triethylamine aqueous solution or an ethylene disulfonate triethylamine aqueous solution; more preferably, the salt solution is an aqueous solution of ammonium sulfate, an aqueous solution of sucrose octasulfate triethylamine, an aqueous solution of ammonium methylsulfonate or an aqueous solution of ethylene disulfonate triethylamine; such as an aqueous ammonium sulfate solution.
In one embodiment of the present invention, the raw materials of the ginsenoside mitoxantrone liposome are mitoxantrone sulfate, sucrose octa-mitoxantrone sulfate, mitoxantrone methylsulfonate, mitoxantrone ethyldisulfonate, mitoxantrone propyldisulfonate, mitoxantrone ethyldisulfonate, or mitoxantrone propyldisulfonate; preferably, the mitoxantrone salt is mitoxantrone sulfate, sucrose octasulfate mitoxantrone, or mitoxantrone methylsulfonate; such as mitoxantrone sulfate.
In one embodiment of the present invention, the raw material of the ginsenoside mitoxantrone liposome is mitoxantrone hydrochloride aqueous solution, preferably, the concentration of the mitoxantrone hydrochloride aqueous solution is 10mg/mL.
In one embodiment of the present invention, the concentration of the salt solution in the raw material of the ginsenoside mitoxantrone liposome is 0.05M-0.975M; for example 0.05M, 0.1M, 0.2M, 0.3M, 0.325M, 0.65M, 0.975M or 0.16M.
In one embodiment of the present invention, in the raw material of the ginsenoside mitoxantrone liposome, when the salt solution is sucrose octasulfate triethylamine aqueous solution, the concentration of the salt solution is 0.05M-0.3M, for example, 0.1M, 0.2M or 0.3M.
In one embodiment of the present invention, in the raw material of the ginsenoside mitoxantrone liposome, when the salt solution is an aqueous solution of triethylamine ethanedisulfonate, the concentration of the salt solution is 0.16M-0.325M.
In one embodiment of the present invention, in the raw material of the ginsenoside mitoxantrone liposome, when the salt solution is an aqueous solution of ammonium methylsulfonate, the concentration of the salt solution is 0.325M-0.975M.
In one embodiment of the present invention, in the raw material of the ginsenoside mitoxantrone liposome, when the salt solution is an ammonium sulfate aqueous solution, the concentration of the salt solution is 0.16M-0.325M, for example, 0.325.
In one embodiment of the present invention, the ginsenoside mitoxantrone liposome is prepared from one or more of hydrogenated phospholipid, egg yolk lecithin, soybean phospholipid and cephalin; preferably, the phospholipid is hydrogenated phospholipid or egg yolk lecithin.
In one scheme of the invention, in the raw materials of the ginsenoside mitoxantrone liposome, the mass ratio of the mitoxantrone hydrochloride to the phospholipid is 1 (5-15); for example, the mass ratio of mitoxantrone hydrochloride to hydrogenated phospholipid is 1:10.
In one embodiment of the present invention, the ginsenoside is selected from one or more of 20 (S) -ginsenoside Rg3, ginsenoside pseudo-Rg 3, 20 (S) -ginsenoside Rh2, ginsenoside Rg5, ginsenoside Rk1 and ginsenoside Rp1, preferably, the ginsenoside is 20 (S) -ginsenoside Rg3 and/or 20 (S) -ginsenoside Rh2.
In one scheme of the invention, in the raw materials of the ginsenoside mitoxantrone liposome, the mass ratio of the mitoxantrone salt to the ginsenoside is 1 (0.1-4); for example, the mass ratio of the mitoxantrone salt to the ginsenoside is 1:1 or 1:1.5.
In one embodiment of the present invention, the ginsenoside mitoxantrone liposome has an HPLC purity of 99% or more.
In one embodiment of the invention, the mass fraction of the phospholipid in the raw material of the ginsenoside mitoxantrone liposome is 10 parts.
In one embodiment of the invention, the mass fraction of the PEG-DSPE in the raw material of the ginsenoside mitoxantrone liposome is 0.5 part.
In one embodiment of the present invention, the ginsenoside mitoxantrone liposome is prepared from 1 part by mass of ginsenoside.
In one embodiment of the present invention, the mass fraction of the mitoxantrone salt in the raw material of the ginsenoside mitoxantrone liposome is 1 part.
In one scheme of the invention, the raw materials of the ginsenoside mitoxantrone liposome comprise the following components in percentage by mass: 10 parts of phospholipid, 0.5 part of PEG-DSPE, 1 part of ginsenoside and 1 part of mitoxantrone hydrochloride.
The invention also provides a ginsenoside mitoxantrone liposome composition, which comprises glucose aqueous solution and the ginsenoside mitoxantrone liposome.
In one embodiment of the present invention, the aqueous glucose solution in the ginsenoside mitoxantrone liposome composition is a 5% aqueous glucose solution.
In one embodiment of the invention, the encapsulation efficiency of the ginsenoside mitoxantrone liposome in the ginsenoside mitoxantrone liposome composition is more than or equal to 80%.
In one embodiment of the present invention, the mass fractions of the phospholipid, the PEG-DSPE, the ginsenoside and the mitoxantrone in the ginsenoside mitoxantrone liposome composition have about 10% error due to the loss of the preparation process and the difference of the process.
The invention also provides application of the substance X in preparing a medicament for treating and/or preventing cancers; the substance X is ginsenoside mitoxantrone liposome as described above or ginsenoside mitoxantrone liposome composition as described above.
In one embodiment of the present invention, the particle size d90 of the ginsenoside mitoxantrone liposome or the ginsenoside mitoxantrone liposome composition in the application is less than or equal to 150nm.
In one embodiment of the present invention, the encapsulation efficiency of the ginsenoside mitoxantrone liposome or the ginsenoside mitoxantrone liposome composition in the application is not less than 80%.
In one embodiment of the present invention, the purity of the ginsenoside in the ginsenoside mitoxantrone liposome or the ginsenoside mitoxantrone liposome composition in the application is greater than or equal to 99%.
In one embodiment of the invention, the cancer may be breast cancer, colorectal cancer, breast cancer, primary liver cancer, gastric cancer, bladder cancer or brain tumor.
In any of the above aspects of the present invention, the method for measuring the encapsulation efficiency is a centrifugation method.
The term "particle size D90" refers to the particle size corresponding to a sample having a cumulative particle size distribution percentage of 90%. Its physical meaning is that its particle size is less than 90% of its particle size.
The above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present invention can be obtained.
The reagents and materials used in the present invention are commercially available.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that: the invention has the positive progress effects that: the ginsenoside mitoxantrone liposome provided by the invention has targeting effect on tumor cells, synergy and attenuation and drug synergy. Taking ginsenoside Rg3 mitoxantrone liposome as an example, the drug effect is obviously better than that of cholesterol mitoxantrone liposome; the Rg3 is proved to have better multiple functions of medicines, auxiliary materials, membrane materials, target heads and the like in the ginsenoside Rg3 mitoxantrone liposome, and has good medicine synergistic effect. Specifically:
(1) The drug effect is obviously improved. In particular, rg3 (1.0) -MIT-PEG/Lp group, rg3 (1.5) -MIT-PEG/Lp group, rg3 (2.0) -MIT-PEG/Lp and Rh2 (1.0) -CPT-PEG/Lp group were optimal in drug effect, wherein the high dose group (2 mg/kg) of Rg3 (1.0) -MIT-PEG/Lp, rg3 (1.5) -MIT-PEG/Lp and Rg3 (2.0) -MIT-PEG/Lp had been completely disappeared at day 28, and had significant advantages over the common cholesterol mitoxantrone liposome group (C-MIT-PEG/LP group) and cholesterol Rg3 mitoxantrone liposome group (C-Rg 3 (1.0) -MIT-PEG/Lp). Meanwhile, the tumor inhibition rate of the medium dose group (1 mg/kg) of the three experimental groups on day 28 is 9-11%, which is better than that of the common cholesterol liposome group (C-MIT-PEG/LP group) and the high dose group (2 mg/kg) of the cholesterol Rg3 mitoxantrone liposome group (C-Rg 3 (1.0) -MIT-PEG/LP) on day 28, and the tumor inhibition rate (11-20%) of the Rg3 mitoxantrone liposome of the invention on day 28 shows that the Rg3 mitoxantrone liposome has obvious advantage on pharmacodynamics of the traditional mitoxantrone liposome.
(2) Glut1 targeting is significantly improved. In Glut1 targeting experiments of tumor-bearing mice, glut1 targeting of the ginsenoside liposome is improved by more than 4 times compared with that of common cholesterol liposome.
(3) The toxic and side effects are obviously reduced. Liposomes prepared according to the formulations of the present invention, rg3 mitoxantrone liposomes (Rg 3 (1.0) -MIT-PEG/Lp group and Rg3 (2.0) -MIT-PEG/Lp group) and Rh2 mitoxantrone liposomes (Rh 2 (1.0) -MIT-PEG/Lp group and Rh2 (2.0) -MIT-PEG/Lp group) died at 6mg/kg and 9mg/kg, 12mg/kg died at 0/6 or 1/6, 18mg/kg died at 3/6 or 4/6; while cholesterol liposome group (C-MIT-PEG/LP group) died 1/6 at 6mg/kg, 9mg/kg died 4/6. The LD50 of Rg3 mitoxantrone liposome and Rh2 mitoxantrone liposome is between 12-18mg/kg, and the LD50 of cholesterol mitoxantrone liposome is between 6-9mg/kg, showing that the acute toxicity of ginsenoside liposome is obviously reduced compared with cholesterol liposome.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
Experimental drugs and devices
Experimental drugs: 20 (S) -ginsenoside Rg3 (abbreviated as Rg 3), ginsenoside pseudo Rg3 (abbreviated as pseudo Rg 3), ginsenoside Rp1 (abbreviated as Rp 1), ginsenoside pseudo GQ (abbreviated as pseudo GQ), ginsenoside Rk1 (abbreviated as Rk 1), ginsenoside Rg5 (abbreviated as Rg 5), 20 (S) -ginsenoside Rh2 (abbreviated as Rh 2), ginsenoside Rk2 (abbreviated as Rk 2), 20 (S) -ginsenoside Rg2 (abbreviated as Rg 2), 20 (S) -ginsenoside Rh1 (abbreviated as Rh 1), 20 (S) -protopanaxadiol (abbreviated as PPD), 20 (S) -protopanaxatriol (abbreviated as PPT), mitoxantrone hydrochloride and the like are commercially available in the field, such as Shanghai-main-medicine science and technology Co, shanghai-gold and biopharmaceutical Co, shanghai-derived biological technology Co, and the like.
The molecular structural formula of the ginsenoside is as follows:
Test instrument: the instruments used in the following examples were the instruments and equipment owned by Shanghai Bensu medical science and technology Co., ltd, university of double denier medical college, and the equipment model and source information were as follows:
agilent liquid chromatography: agilent 1100 set, autai 3300ELSD, agilent technologies (China) Inc.;
Spin-on evaporator: ZX98-1 5L, shanghai Lu Yi Goodyear Co., ltd;
Ultrasonic cleaning machine (SB 3200DT, ningbo Xinzhi biotechnology Co., ltd.);
Nitrogen blowing instrument (HGC-12A, heng ao technology development Co., tianjin);
Probe ultrasonic instrument (JYD-650, shanghai Zhi Xin instruments Co., ltd., china);
a high pressure homogenizer (B15, AVESTIN, canada);
Mini extruder (Mini-extruder, avanti Polar LIPIDS INC);
Laser particle size analyzer (Nano ZS, markov in the united kingdom);
a malvern particle sizer Malvern Nanosizer ZS (malvern, uk);
Microplate reader (Thermo Scientific, waltham, MA, USA);
Enzyme labeling apparatus (INFINITIE, TECAN TRADING co., ltd);
flow cytometry (BD Biosciences, USA);
flow cytometry (CytoFlex S, beckman Coulter, inc., USA);
inverted fluorescence microscopy (Leica, DMI 4000d, germany);
Fluorescence microscopy (Zeiss LSM 710, oberkochen, germany);
laser confocal microscopy (Leica, DMI 4000d, germany);
confocal living microscope (Confocal intravital microscopy, IVM);
a front two-photon microscope (DM 5500Q; nikon);
A small animal living body optical imaging system (in vivo IMAGING SYSTEM, IVIS) (PERKINELMER, USA);
biomacromolecule interactor BiaCore T200 instrument (GE, USA);
Clean bench (SW-CJ-1 FD, air technologies Co., ltd.);
20L rotary evaporator: R5002K, shanghai xiafeng real company limited;
Freeze dryer: FD-1D-80, shanghai Bilang instruments Co., ltd;
Freeze dryer: PDFD GLZ-1B, shanghai Pudong freeze drying Equipment Co., ltd;
An electronic balance: CPA2250 (precision 0.00001 g), sidoris (Shanghai) trade Co., ltd;
An electronic balance: JY3003 (precision 0.001 g), shanghai Shunyu Hengping scientific instruments Co., ltd;
photo-electric microscope (XDS-1B, chongqing photo-electric instruments Co., ltd.);
cell incubator (CCL-170B-8, singapore ESCO).
Animals and cell lines
Animals: BALB/c nude mice are 3-4 weeks old and produced by Shanghai pharmaceutical research institute, national academy of sciences.
Tumor cell lines:
Breast cancer in-situ tumor 4T1 cell line provided by university of double denier pharmacy
Human colon cancer C-26 cell line, available from Jiangsu Kaiki Biotechnology Co., ltd
Human pancreatic cancer Capan-1 cell line, available from Jiangsu Kaiki Biotechnology Co., ltd
Breast cancer MCF-7 cell line from Jiangsu Kaiki Biotechnology Co., ltd
The method for detecting the mitoxantrone hydrochloride content comprises the following steps: the "mitoxantrone hydrochloride" detection method is used in the "Chinese pharmacopoeia" 2020 edition.
1) Chromatographic conditions: c18 column (Kromasil C18, 250X 4.6mm,5 μm)
2) Mobile phase: sodium heptanesulfonate solution (sodium heptanesulfonate 4.4g, water was added in an appropriate amount to dissolve, glacial acetic acid 6.4mL, diluted with water to 730 mL): acetonitrile=70: 30 (volume ratio) is the mobile phase.
3) Detection wavelength: 244nm, flow rate of 1.0mL/min, column temperature of 30 ℃ and sample injection amount of 20 mu L.
4) And (3) calculating: recording the chromatogram, and calculating the content of mitoxantrone hydrochloride in the sample solution by an external standard method.
The method for detecting the ginsenoside content comprises the following steps:
1) Chromatographic conditions: kromasil 100-3.5C4150mm.times.4.6 mm column.
2) Mobile phase: acetonitrile: water=55:45.
3) Detection wavelength: 203nm, a flow rate of 1mL/min, a column temperature of 35 ℃ and a sample injection amount of 10 mu L.
4) And (3) calculating: and recording a chromatogram, and calculating the Rg3 content in the test sample solution by an external standard method.
The method for detecting the encapsulation efficiency of mitoxantrone (or ginsenoside) comprises the following steps:
Taking 2 liposome samples to be detected, respectively taking 1mL, centrifuging (18000 r/min,30min,3 times each for 30 min), respectively taking supernatant and liposome precipitate, washing the precipitated liposome with distilled water for 3 times each for 1mL, combining the supernatant, fixing the volume in a 25mL volumetric flask by deionized water, and detecting by HPLC to obtain the drug concentration (the concentration of free mitoxantrone or ginsenoside in the ginsenoside mitoxantrone liposome) as V1; the other part is placed in a 25mL volumetric flask, deionized water is used for volume determination, and the drug concentration is detected to be V0 by an HPLC method. Encapsulation efficiency= (V0-V1)/v0×100%.
Short for the sake of brevity: mitoxantrone hydrochloride (MIT), hydrogenated phospholipid (HSPC), cholesterol (Cho), 20 (S) -ginsenoside Rg3 (Rg 3), 20 (S) -ginsenoside Rh2 (Rh 2).
Example 1: stability study of ginsenoside blank liposome in various ionic aqueous solutions
Weighing the prescription amount of HSPC and ginsenoside, dissolving in 1mL of chloroform by ultrasonic, concentrating under reduced pressure to dryness, adding 10mL of hydration solution, hydrating for 10 minutes, then carrying out ultrasonic treatment for 25 times (600W, 5 seconds on and 5 seconds off), obtaining blank liposome of each experimental group, and detecting appearance and encapsulation efficiency.
3) Experimental results:
example 2 experiments on the Effect of phospholipid usage on mitoxantrone encapsulation efficiency in traditional active drug delivery method
Analysis of results: the ethanol injection method in the traditional active drug loading method is adopted, so that the drug-to-lipid ratio (HSPC/drug) has a larger influence on the encapsulation efficiency, and when the drug-to-lipid ratio is more than or equal to 10, the encapsulation efficiency is not obviously different. Therefore, the phospholipid ratio of the drug to the lipid of 5-15 is preferable in the present invention.
Example 3 experiment of the Effect of cholesterol usage on mitoxantrone encapsulation efficiency in traditional active drug delivery method
Analysis of results: by adopting the ethanol injection method in the traditional active drug-loading method, the cholesterol can improve the stability of liposome and the encapsulation rate of mitoxantrone. When cholesterol/medicine is more than or equal to 0.5, the improvement is remarkable; when cholesterol/drug is not less than 1, there is no significant difference in the amount of cholesterol to the encapsulation efficiency.
Example 4 experiment of Effect of Rg3 usage on mitoxantrone encapsulation efficiency in traditional active drug delivery method
Analysis of results: by adopting an ethanol injection method in a traditional active drug-loading method, HSPC and Rg3 are synchronously formed into films, then an ionic aqueous solution is hydrated, 5% glucose is dialyzed and drug-loaded, and a qualified Rg3 mitoxantrone co-carrier liposome cannot be prepared.
Example 5 experiment of Effect of cholesterol usage on Rg3 Liposome encapsulation efficiency in conventional active drug delivery method
Analysis of results: by adopting an ethanol injection method in a traditional active drug-loading method, HSPC, rg3 and cholesterol are firstly synchronously formed into films, then an ionic aqueous solution is hydrated and 5% glucose is dialyzed to obtain Rg3 liposome, and the Rg3 encapsulation rate of the liposome prepared by the process is low, and Rg3 leakage is caused by the ionic aqueous solution.
Example 6 experiment of Effect of cholesterol usage on Rg3 Liposome encapsulation efficiency in conventional active drug delivery method
Analysis of results: when the external water phase is 5% glucose after dialysis by adopting an ethanol injection method in the traditional active drug loading method, rg3 is loaded into liposome as a drug, and the encapsulation rate of Rg3 is qualified, wherein the influence of the using amount of cholesterol on the encapsulation rate of Rg3 is small. Effect example 1: the results of the targeting experiments in the C6-C-Rg3 (post)/Lp group in the cell uptake experiment of Glut1 showed that: glut 1-mediated targeting was poor in this group, showing that the glucosyl group of Rg3 was not exposed at the liposome surface, and therefore Rg3 should be entrapped in the inner lumen of the liposome.
EXAMPLE 7 synchronous Loading experiment of Rg3 and mitoxantrone in traditional active drug delivery method
Analysis of results: the ethanol injection method in the traditional active drug loading method is adopted, under the condition that Rg3 and cholesterol are used in different proportions, the inner water phase of the blank liposome is an ammonium sulfate solution, the outer water phase is a 5% glucose solution, rg3 and mitoxantrone are drugs, synchronous loading is carried out, and the encapsulation rates of the mitoxantrone and Rg3 are unqualified. The ionic aqueous solution affects both the encapsulation efficiency of Rg3 and mitoxantrone hydrochloride.
Example 8 experiment of Effect of Rg 3-on-mitoxantrone on encapsulation efficiency in traditional active drug delivery method
Analysis of results: the method adopts an ethanol injection method in the traditional active drug-loading method, adopts Rg3 and cholesterol in different proportions, after dialysis, the inner water phase of the blank liposome is an ammonium sulfate solution, the outer water phase is a 5% glucose solution, rg3 is loaded firstly, mitoxantrone hydrochloride is loaded secondly, and the encapsulation rate of the prepared Rg3 mitoxantrone co-loaded liposome is unqualified.
Example 9 experiment of the Effect of different preparation methods on the encapsulation efficiency of Rg3 mitoxantrone co-encapsulation liposomes
Analysis of results:
1) Passive drug loading (thin film method) cannot prepare qualified Rg3 mitoxantrone co-loaded liposomes;
2) The common active drug-loading method can not prepare qualified Rg3 mitoxantrone co-loaded liposome;
3) The common active drug loading method is to load Rg3 and then mitoxantrone, or load Rg3 and mitoxantrone synchronously, so that qualified Rg3 mitoxantrone co-carrier liposome cannot be prepared;
4) The common active drug-loading method is to load mitoxantrone first and then Rg3, and can prepare qualified Rg3 mitoxantrone co-loaded liposome. The liposome prepared by the method is applied in the invention to implement 1: cell uptake assay of Glut1, C6-Rg3 (post) -MIT/Lp panel assay results showed: the Rg3 liposome prepared by the method has remarkable Glut1 mediated active targeting effect, and is proved to be embedded in a phospholipid bilayer membrane, wherein the glucose group (Glc) in the Rg3 molecule is exposed on the outer surface of the liposome.
Example 10 experiment of Effect of Rg3 amount on encapsulation efficiency of Rg3 mitoxantrone co-carrier liposomes
Analysis of results:
1) By adopting the ethanol injection method, qualified Rg3 mitoxantrone co-carrier liposome can be prepared, specifically, the inner water phase of the liposome is mitoxantrone sulfate, the outer water phase of the liposome is 5% glucose isotonic solution, the bilayer membrane of the liposome is hydrogenated phospholipid and Rg3, wherein the glucose radical (Glc) in Rg3 molecules is exposed on the outer surface of the liposome, and the liposome membrane material does not contain cholesterol.
2) The Rg3 mitoxantrone liposome has good encapsulation efficiency of Rg3 and mitoxantrone when HSPC comprises Rg3 and MIT=10:0.1-4:1. As the amount of Rg3 increases, the encapsulation efficiency of Rg3 and mitoxantrone decreases dramatically.
3) The application range of Rg3 of the invention is HSPC: rg3: mit=10:0.1 to 4:1.
EXAMPLE 11 Effect of different salts on encapsulation efficiency of Rg3 mitoxantrone co-carrier liposomes
Analysis of results: by adopting the ethanol injection method, the preparation of Rg3 mitoxantrone liposome can be satisfied by sucrose octasulfate triethylamine, ammonium sulfate, ammonium methylsulfonate, triethylamine methylsulfonate, ammonium ethyldisulfonate, ammonium propyldisulfonate, triethylamine ethyldisulfonate and triethylamine propyldisulfonate, and the encapsulation rate is qualified.
Example 12 experiment of the Effect of different salt concentrations on the encapsulation efficiency of Rg3 mitoxantrone co-carrier liposomes
Analysis of results: when the concentration of the sucrose octasulfate triethylamine is lower than 0.05M, the ethanol injection method provided by the invention can not meet the process requirements of the encapsulation rate; when the concentration is more than or equal to 0.1M, the encapsulation efficiency is not obviously different. 2) When the concentrations of the ammonium sulfate and the ethanedisulfonic acid triethylamine are lower than 0.16M, the encapsulation efficiency cannot meet the process requirements; at concentrations of 0.32M and 0.65M, there was no significant difference in encapsulation efficiency. 3) When the concentration of ammonium methylsulfonate is lower than 0.325M, the encapsulation efficiency can not meet the process requirement; at concentrations of 0.65M and 0.975M, there was no significant difference in encapsulation efficiency.
Example 13 experiments on the Effect of different ginsenosides on the encapsulation efficiency of the sapogenone co-carrier liposomes
Analysis of results: the encapsulation efficiency of the co-carried liposome prepared by the ethanol injection method of the invention, which is 20 (S) -Rg3, 20 (S) -Rh2, rg5, rk1, rp1, pseudo Rg3, pseudo GQ, PPD and other saponins, meets the quality requirements; and the encapsulation efficiency of the co-carried liposome prepared from 20 (R) -Rg3, PPT and other saponins does not meet the quality requirement.
Example 14 experiment of the Effect of different homogenization methods on the encapsulation efficiency of Rg3 mitoxantrone co-encapsulation liposomes
Analysis of results: the ethanol injection method of the invention can meet the process requirements in three common methods (an ultrasonic method, a high-pressure homogenizing method and a push-through membrane method) for controlling the particle size.
Example 15 experiment of the Effect of different phospholipids on the encapsulation efficiency of Rg3 mitoxantrone co-encapsulation liposomes
Analysis of results: the encapsulation efficiency of the Rg3 mitoxantrone co-carrier liposome prepared from hydrogenated phospholipid, egg yolk lecithin, soybean phospholipid and cephalin by adopting the ethanol injection method meets the requirements of drug application, and the PEG-DSPE does not meet the requirements.
EXAMPLE 16 experiments on the Effect of different mitoxantrone concentrations on the encapsulation efficiency of Rg3 mitoxantrone co-encapsulation liposomes
Analysis of results: the ethanol injection method of the invention is optimal in the case of drug concentration of 5-15 mg/mL, and particularly optimal in the case of drug concentration of 10 mg/mL. When the drug concentration is lower than 5mg/mL or higher than 20mg/mL, the encapsulation efficiency is not in compliance with the drug quality requirements.
EXAMPLE 17 Effect of different physiological isotonic solutions on encapsulation efficiency of Rg3 mitoxantrone co-entrapped liposomes
Analysis of results: by adopting the ethanol injection method, 5% glucose and 10% sucrose aqueous solution have no obvious difference on the encapsulation rate of Rg3 and mitoxantrone, and 0.9% physiological saline is not suitable for use.
EXAMPLE 18 preparation of Rg3 mitoxantrone liposomes
1. Prescription: 10g of HSPC, 1g of Rg3, 1g of mitoxantrone hydrochloride, a proper amount of absolute ethyl alcohol, a proper amount of 5% glucose injection, a proper amount of water for injection and a proper amount of 0.325M ammonium sulfate solution.
2. The operation method comprises the following steps:
Step (1): film formation and hydration
Weighing HSPC with a prescription amount, dissolving the HSPC in 20mL of absolute ethyl alcohol, adding 100mL of 0.325M ammonium sulfate, hydrating for 10 minutes at 55-60 ℃, volatilizing to remove most of the ethanol, and preparing a blank liposome crude product with an internal water phase and an external water phase being ammonium sulfate solution;
Step (2): push through the membrane
And (3) allowing the blank liposome solution obtained in the step (1) to sequentially pass through a polycarbonate membrane filter plate with pore diameters of 800nm, 400nm, 200nm and 100nm for 4 times under the pressure of 600-800psi, and finally obtaining the blank liposome with the particle size smaller than 100nm and the inner and outer water phases being ammonium sulfate solutions.
Step (3): dialysis
Placing the blank liposome in the step (2) in a dialysis bag with the molecular weight cut-off of 10000, dialyzing for 12 hours at 4 ℃ by taking 5% glucose aqueous solution as a dialysis medium, wherein the volume ratio of the sample to the dialysis medium is 1:1000, 1 dialysis solution is changed every 4 hours during dialysis, and ammonium sulfate in the outer water phase of the blank liposome is completely removed, so that the blank liposome with the outer water phase consisting of 5% glucose and the ammonium sulfate as the inner water phase is obtained.
Step (4): loading mitoxantrone
Mixing the blank liposome in the step (3) with mitoxantrone hydrochloride aqueous solution with the concentration of 10mg/ml according to the volume ratio of 1:1, and incubating in a water bath at 50-60 ℃ for 40 minutes to obtain the mitoxantrone liposome. Specifically, the aqueous phase in the liposome is mitoxantrone sulfate insoluble salt, and the aqueous phase outside the liposome is 5% glucose aqueous solution.
Step (5): embedding Rg3
Slowly adding 100mL 10mg/mL of Rg3 ethanol solution into the mitoxantrone liposome solution in the step (4) at 20-30 ℃, stirring for 45 minutes, volatilizing to remove most of ethanol, then placing into a dialysis bag with the molecular weight cutoff of 10000, dialyzing for 12 hours at 4 ℃ by using 5% glucose aqueous solution as a dialysis medium, wherein the volume ratio of the sample to the dialysis medium is 1:1000, changing the dialysate for 1 time every 4 hours during dialysis, and completely removing ethanol solvent, inorganic salt, unwrapped mitoxantrone hydrochloride and Rg3 to obtain Rg3 mitoxantrone liposome.
Step (6): addition of PEG-DSPE
Accurately weighing 0.2g of PEG-DSPE, dissolving in 300mL of 5% glucose, and then adding into the Rg3 mitoxantrone liposome solution in the step (5), thereby obtaining Rg3 mitoxantrone liposome solution with the concentration of both mitoxantrone and Rg3 being about 2 mg/mL.
Step (7) of sterilizing and filtering
The Rg3 mitoxantrone liposome of step (6) was filtered through a 0.22 μm filter.
Step (8): filling
Filling the solution obtained in the step (7) into a 10mL or 20mL penicillin bottle, capping and packaging to obtain the penicillin bottle.
Through detection, the liposome has mitoxantrone concentration=4.88 mg/mL, rg3 concentration=4.91 mg/mL, particle size D90=101 nm, rg3 encapsulation rate=98.65%, and mitoxantrone encapsulation rate=96.79%.
Example 19 effect of PEG-DSPE amount on stability of Rg3 mitoxantrone co-Carrier liposomes
The preparation method comprises the following steps: taking the Rg3 mitoxantrone liposome solution in the step (5) of the example 18, adding PEG-DSPE water solutions with different concentrations according to the prescription of the example, carrying out other subsequent steps in the same way as in the example 18, and placing each prescription preparation in a refrigerator at 2-8 ℃ to examine the stability of the liposome solution.
Analysis of results:
1) PEG-DSPE is not added, after the Rg3 mitoxantrone liposome is stored for 3 months at the temperature of 2-8 ℃, the particle size is rapidly increased, and the leakage rate of Rg3 and mitoxantrone is obviously increased;
2) When PEG-DSPE/HSPC is less than or equal to 0.025, after the liposome is preserved for 3 months at 2-8 ℃, the particle size of Rg3 mitoxantrone liposome is obviously increased, the encapsulation efficiency is obviously reduced, and the quality requirement of stability is not qualified. Wherein PEG-DSPE/hspc= 0.025,3 months of stability data is acceptable.
3) When PEG-DSPE/HSPC is more than or equal to 0.025, after the liposome is preserved for 3 months at the temperature of 2-8 ℃, the particle size of Rg3 mitoxantrone liposome is stable, the encapsulation rate of Rg3 and mitoxantrone is stable, and the requirements of medicine declaration are met.
4) When PEG-DSPE/HSPC is more than or equal to 0.05, the particle size and the encapsulation rate are not obviously different.
Application example 1: cell uptake assay for Glut1
1) The purpose of the experiment is as follows: observing whether the Rg3 liposomes have more uptake on tumor cells by comparing uptake of the fluorescein-loaded Rg3 liposomes with that of cholesterol liposomes on 4T1 cells; the Glut1 targeting mechanism is proved by adding glucose inhibitors and the like; the ginsenosides of the invention were confirmed to be located in the phospholipid bilayer membrane by Glut1 targeting, and the glucosyl group was exposed on the outer surface of the liposome.
2) The experimental method comprises the following steps: to compare uptake of 4T1 into each experimental group, the uptake mechanism of liposomes was examined, 4T1 cells were seeded into 12-well plates at a cell density of 2×10 5, and for experimental group + glucose, experimental group + phlorizin and experimental group + quercetin group, 20mM glucose solution, phlorizin solution and quercetin solution were used, respectively, in place of the medium after 12 hours. The three solutes should be dissolved in glucose-free medium, after incubation for 1 hour, each experimental group of drugs (ultraviolet fluorescent developer concentration 100 ng/ml) was added, after incubation for 4 hours, digested, washed with fresh PBS solution and analyzed by flow cytometry.
To investigate the uptake mechanism of Rg3 liposomes, the substrates (glucose), the Glut1 competitive inhibitor phlorizin and quercetin were pre-incubated for 1 hour to saturate Glut1 before adding the formulation, and the fluorescence intensity of Rg3-Lp/C6 was reduced by 31%,43% and 74%, respectively. From this, the addition of Glut1 substrate and inhibitor prevents the cellular uptake of Rg3-Lp/C6, demonstrating that ginsenoside Rg3 liposome can enhance its uptake efficiency by interacting with Glut 1.
3) The preparation method of the experimental group comprises the following steps: the operating conditions are the same as those of the examples of the present invention.
Method 1 (passive drug delivery): the method comprises the steps of dissolving a prescription amount of HSPC, ginsenoside and/or cholesterol, fluorescent probe (coumarin) and/or medicine in a proper amount of mixed solvent of ethanol and chloroform (volume ratio is 1:1), concentrating under reduced pressure to dryness, hydrating purified water, performing ultrasound, and detecting fluorescence intensity according to an experimental method of an application example.
Method 2 (active drug delivery): the prescribed amounts of HSPC, rg3 and fluorescent probe were sonicated in an appropriate amount of ethanol, hydrated for 10 minutes with 0.325M ammonium sulfate solution, sonicated 25 times (5 seconds on and 5 seconds off), dialyzed with 5% glucose solution, sequentially loaded with (and/or) drug, dialyzed again to remove free drug, (and/or) an appropriate amount of PEG-DSPE to obtain liposome solutions for each experimental group, and then the fluorescence intensity was detected according to the experimental method of the present application example.
Method 3 (active drug delivery): the prescribed amounts of HSPC and fluorescent probe were sonicated in appropriate amounts of ethanol, hydrated for 10 minutes with 0.325M ammonium sulfate solution, sonicated 25 times (5 seconds on and 5 seconds off), dialyzed with 5% glucose solution, then sequentially loaded with drug or Rg3, dialyzed again to remove free drug, and/or an appropriate amount of PEG-DSPE was added to obtain liposome solutions for each experimental group, and then the fluorescence intensity was detected according to the experimental method of the application example.
Experimental results 1 are as follows:
The above comparison is
Conclusion of experiment:
1) The targeting experimental data prove that the traditional passive drug loading method (thin film evaporation method) is adopted: acceptable Rg3 mitoxantrone co-carrier liposomes cannot be prepared.
2) Adopts a traditional active drug loading method, in particular:
i. Addition of Rg3 prior to dialysis, the acid radical solution caused leakage of Rg3 in the liposomes, thereby causing failure of liposome preparation.
Rg3 is added after dialysis in two cases:
a) Rg3 is added before mitoxantrone hydrochloride, so that the preparation of the liposome fails because Rg3 in the liposome is seriously leaked due to ionic solution generated by the medicine;
b) Rg3 was added after mitoxantrone hydrochloride and the liposome preparation was successful.
The two conditions are basically the same, and the sequence of the two conditions is different, but ionic solutions exist, different results are generated, and the mechanism is not clear.
3) The addition of PEG-DSPE in an appropriate amount affected Glut 1-mediated targeting, suggesting that the amount of PEG-DSPE was limited.
4) This experiment suggests that the Rg3 mitoxantrone co-entrapped liposomes of the invention should be prepared in the same or similar manner as in example 18.
Experimental results 2 are as follows:
From the above results, it was found that the fluorescence intensity of C6-C/Lp was not significantly changed with the addition of Glut1 substrate and inhibitor, but cellular uptake of C6-Rg3/Lp was prevented, and that ginsenoside Rg3 liposome was enhanced in uptake efficiency by interaction with Glut1, thereby demonstrating that Rg3 was located on the membrane of liposome and that the glucosyl group (Glc) of Rg3 was exposed on the surface of liposome.
Application example 2: in vivo pharmacodynamic study of human breast cancer (MCF-7)
1) The test method comprises the following steps: the tumor cell line (MCF-7) was injected subcutaneously into mice to establish a subcutaneous tumor model. When tumor volume reached 100mm 3 (7 d post inoculation), mice were treated in random groups (n=8 each), each group was injected with Blank solvent (5% glucose, blank), mitoxantrone liposome injection (C-MIT-PEG/LP group) and each experimental group, and the doses were three groups (2 mg, 1mg, 0.5 mg) high and low in mitoxantrone, once every 7 days, for up to day 28, and the length, width and recorded body weight of the tumor were measured while dosing. The formula for calculating the tumor volume (V) is V= (W 2 X L)/2. Length (L) is the longest diameter of a solid tumor and width (W) is the shortest diameter perpendicular to the length. At the end of the experiment at day 28, all animals were sacrificed and tumors were removed for imaging and histological examination.
Tumor inhibition rate t= (tumor weight of non-dosed group-test group)/tumor weight of non-dosed group
Remarks: mitoxantrone + rg3 = 2mg/kg +2mg/kg, indicating drug concentration, the same applies below.
2) The experimental groups were as follows:
3) The test results are as follows:
Conclusion:
1) Rg 3/mit=1.0, 1.5 and 2.0, there is no significant difference in pharmacodynamics.
2) The in vivo pharmacodynamics of Rg3 mitoxantrone liposomes were significantly better than that of the normal cholesterol mitoxantrone liposome group (C-MIT-PEG/LP group) and cholesterol Rg3 mitoxantrone liposome group (C-Rg 3 (1.0) -MIT-PEG/LP), wherein Rg3 (1.0) -MIT-PEG/LP, rg3 (1.5) -MIT-PEG/LP and Rg3 (2.0) -MIT-PEG/LP high dose group (12 mg/kg) had been completely eradicated at day 28, and the tumors were significantly better than that of the normal cholesterol mitoxantrone liposome group (C-MIT-PEG/LP group) and cholesterol Rg3 mitoxantrone liposome group (C-Rg 3 (1.0) -MIT-PEG/LP). Meanwhile, the tumor inhibition rate of the medium dose group (8 mg/kg) of the three experimental groups on day 28 is 9-11%, which is better than that of the common cholesterol liposome group (C-MIT-PEG/LP group) and the high dose group (12 mg/kg) of the cholesterol Rg3 mitoxantrone liposome group (C-Rg 3 (1.0) -MIT-PEG/LP) on day 28, and the tumor inhibition rate (11-20%) of the Rg3 mitoxantrone liposome of the invention on day 28 shows that the Rg3 mitoxantrone liposome has obvious advantage on pharmacodynamics of the traditional mitoxantrone liposome.
Human colon cancer C-26 cell line: according to the in vivo pharmacodynamics experimental method, the study data for the in vivo pharmacodynamics of human colon cancer (C-26) cells are as follows.
Project Blank C-MIT-PEG/LP group Rg3(1.0)-MIT-PEG/Lp Rh2(1.0)-MIT-PEG/Lp
Dosage for administration / 2mg/kg 2mg/kg+2mg/kg 2mg/kg+2mg/kg
Tumor inhibition rate for 7 days -27％ 46％ 38％ 35％
Tumor inhibition rate for 14 days -53％ 48％ 25％ 24％
Tumor inhibition rate of 21 days -72％ 37％ 11％ 9％
Tumor inhibition rate for 28 days -91％ 22％ Tumor disappearing Tumor disappearing
The results show that:
1) The pharmacodynamics of Rg3 mitoxantrone liposome and Rh2 mitoxantrone liposome have no obvious difference;
2) The pharmacodynamics of Rg3 mitoxantrone and Rh2 mitoxantrone liposomes were significantly better than those of the cholesterol liposome group (C-MIT-PEG/LP group).
Human pancreatic cancer Capan-1: according to the in vivo pharmacodynamics experimental method, the study data for the in vivo pharmacodynamics of human pancreatic cancer (Capan-1) cells are as follows.
The results show that:
1) The pharmacodynamics of Rg3 mitoxantrone liposome and Rh2 mitoxantrone liposome have no obvious difference;
2) The pharmacodynamics of Rg3 mitoxantrone and Rh2 mitoxantrone liposomes were significantly better than those of the cholesterol liposome group (C-MIT-PEG/LP group).
Application example 3: acute toxicity (LD 50) study (SD rat)
1) The experimental method comprises the following steps: rats 160-260 g, 6-9 weeks old, 6 per group, mode of administration: slow static push (about 1 mL/min), dosing frequency: 3 times per day.
The doses of mitoxantrone in the test samples were set to 6,9, 12 and 18 mg/kg/day, and Rg3 in the test samples was calculated based on the prescribed doses. A vehicle control group (5% glucose injection), a commercial positive control group (C-MIT-PEG/LP group), rg3 (1.0) -MIT-PEG/LP, rg3 (2.0) -MIT-PEG/LP, rh2 (1.0) -MIT-PEG/LP, rh2 (2.0) -MIT-PEG/LP were simultaneously set, and slow static push (about 1 mL/min) was performed for 3 times per day with at least 4h intervals per administration.
2) The preparation method of the experimental group comprises the following steps: prepared according to the procedure for example 18, according to the requirements of the recipe.
3) The experimental results are shown in the following table:
it was shown by the above experiments that,
1) There was no significant difference in acute toxicity of Rg3 mitoxantrone liposomes and Rh2 mitoxantrone liposomes;
2) Rg3 mitoxantrone liposomes (Rg 3 (1.0) -MIT-PEG/Lp group and Rg3 (2.0) -MIT-PEG/Lp group) and Rh2 mitoxantrone liposomes (Rh 2 (1.0) -MIT-PEG/Lp group and Rh2 (2.0) -MIT-PEG/Lp group) died at 6mg/kg and 9mg/kg, 12mg/kg died at 0/6 or 1/6, 18mg/kg died at 3/6 or 4/6; while cholesterol liposome group (C-MIT-PEG/LP group) died 1/6 at 6mg/kg, 9mg/kg died 4/6. Indicating that the LD50 of Rg3 mitoxantrone liposome and Rh2 mitoxantrone liposome is between 12-18mg/kg, and the LD50 of cholesterol mitoxantrone liposome is between 6-9mg/kg, showing a significant reduction in acute toxicity of ginsenoside liposome over cholesterol liposome (4/6, where the number 6 at the end indicates a total number of test mice of 6, and the number at the end indicates a number of death of mice of 4).

Claims (11)

1. A preparation method of a ginsenoside mitoxantrone liposome is characterized by comprising the following steps of;
step 1, dissolving phospholipid in an organic solvent to obtain a mixture A1, and then hydrating the mixture A1 with a salt solution to obtain a liposome solution A1;
Step2, which is scheme 1 or scheme 2;
Scheme 1: a high pressure homogenization method comprising the steps of:
homogenizing the liposome solution A1 obtained in the step 1 under high pressure, and controlling the particle diameter D90 to be less than 100nm to obtain liposome solution A2a;
Scheme 2: extrusion process comprising the steps of:
Extruding the liposome solution A1 obtained in the step 1 through all pore-diameter extrusion plates in sequence, and controlling the particle diameter D90 to be below 100nm to obtain liposome solution A2b;
Step 3, placing the liposome solution A2a or A2b obtained in the step2 into a dialysis bag, and dialyzing by taking the isotonic solution as a dialysis medium; obtaining liposome solution A3;
step 4, mixing the liposome solution A3 obtained in the step 3 with mitoxantrone hydrochloride aqueous solution to obtain liposome solution A4;
step 5, mixing the liposome solution A4 obtained in the step 4 with the ginsenoside solution, placing the mixture in a dialysis bag, and dialyzing by taking the isotonic solution same as that in the step 3 as a dialysis medium; obtaining a ginsenoside mitoxantrone liposome solution A5;
the mass ratio of the mitoxantrone hydrochloride to the phospholipid is 1:10;
The mass ratio of the mitoxantrone hydrochloride to the ginsenoside is 1 (0.1-4);
The ginsenoside is selected from one or more of 20 (S) -ginsenoside Rg3, ginsenoside pseudo Rg3, 20 (S) -ginsenoside Rh2, ginsenoside Rg5, ginsenoside Rk1 and ginsenoside Rp 1;
The phospholipid is selected from one or more of hydrogenated phospholipid, egg yolk lecithin, soybean phospholipid and cephalin;
the salt solution is an aqueous solution of ammonium sulfate, sucrose octasulfate triethylamine, ammonium methylsulfonate, triethylamine methylsulfonate, ammonium ethanedisulfonate, ammonium propanedisulfonate, triethylamine ethanedisulfonate or triethylamine disulfonate;
The concentration of the ammonium sulfate aqueous solution is 0.325M-0.65M;
the concentration of the sucrose octasulfate triethylamine aqueous solution is 0.1M-0.3M;
the concentration of the ammonium methylsulfonate aqueous solution is 0.65M-0.975M;
the concentration of the aqueous solution of the triethylamine methylsulfonate is 0.65M-0.975M;
the concentration of the ammonium ethanedisulfonate aqueous solution is 0.325M-0.65M;
The concentration of the ammonium malonate aqueous solution is 0.325-0.65M;
the concentration of the ethanedisulfonic acid triethylamine water solution is 0.325-0.65M;
the concentration of the aqueous solution of the trisethylamine propanedisulfonate is 0.325M-0.65M;
in the step3, the isotonic solution is 5% glucose or 10% sucrose aqueous solution; in the step 4, the concentration of the mitoxantrone hydrochloride aqueous solution is 5-15 mg/mL.
2. The method for preparing a ginsenoside mitoxantrone liposome according to claim 1, wherein the concentration of the aqueous ammonium sulfate solution is 0.325M or 0.65M;
the concentration of the sucrose octasulfate triethylamine aqueous solution is 0.1M, 0.2M or 0.3M;
the concentration of the ammonium methylsulfonate aqueous solution is 0.65M or 0.975M;
the concentration of the aqueous solution of the triethylamine methylsulfonate is 0.65M or 0.975M;
the concentration of the ammonium ethanedisulfonate aqueous solution is 0.325M or 0.65M;
the concentration of the ammonium malonate aqueous solution is 0.325M or 0.65M;
The concentration of the ethanedisulfonic acid triethylamine water solution is 0.325M or 0.65M;
The concentration of the aqueous solution of the trisethylamine propanedisulfonate is 0.325M or 0.65M.
3. The method of claim 1, wherein the method of preparing the ginsenoside mitoxantrone liposome satisfies one or more of the following conditions:
(1) The phospholipid is hydrogenated phospholipid or egg yolk lecithin;
(2) The ginsenoside is 20 (S) -ginsenoside Rg3 and/or 20 (S) -ginsenoside Rh2;
(3) The mass ratio of the mitoxantrone hydrochloride to the ginsenoside is 1:1 or 1:1.5;
(4) The HPLC purity of the ginsenoside is more than or equal to 99%;
(5) The salt solution is an ammonium sulfate aqueous solution;
(6) The preparation method of the ginsenoside mitoxantrone liposome further comprises one or more of the following steps:
Step 6, mixing the liposome solution A5 obtained in the step 5 with the PEG-DSPE physiological isotonic solution to obtain a liposome solution A6;
And 7, sterilizing, filtering and filling the liposome solution A5 obtained in the step 5 or the liposome solution A6 obtained in the step 6.
4. The method of claim 3, wherein the method of preparing the ginsenoside mitoxantrone liposome satisfies one or more of the following conditions:
(1) In the step1, the organic solvent is ethanol;
(2) In the step 1, the mass-volume ratio of the phospholipid to the organic solvent is 1 g/1-10 mL;
(3) In the step 1, the phospholipid is heated and dissolved in an organic solvent to obtain the mixture A1;
(4) In the step 1, the hydration temperature is 55-65 ℃;
(5) In the step 1, the hydration is carried out in a rotary steaming bottle, and the rotating speed is 40-60 rp/min;
(6) In the step 1, the hydration time is related to the reaction scale so as to make the solution uniform;
(7) In the scheme 1 of the step 2, the high-pressure homogenization is realized by using a freezing water cooling cutting cycle at 0-10 ℃ in a homogenizer;
(8) In the scheme 1 of the step 2, the high-pressure homogenizing pressure is 800-1400 bar;
(9) In the scheme 1 of the step 2, the number of times of high-pressure homogenization is 3-4 times;
(10) In the scheme 2 of the step 2, the extrusion temperature is 35-45 ℃;
(11) In the scheme 2 of the step 2, the aperture of the extrusion plate is 800nm,400nm,200nm or 100nm;
(12) In the scheme 2 of the step 2, the extrusion pressure is 600-800 psi;
(13) In the scheme 2 of the step 2, the extrusion times are 4-10 times;
(14) In the scheme 2 of the step 2, the solution A1 respectively passes through a polycarbonate membrane filter plate with the aperture of 800nm, 400nm, 200nm or 100nm in sequence;
(15) In the step 3, the molecular weight cut-off of the dialysis bag is 8000-15000;
(16) In the step 3, the volume ratio of the solution A2a or A2b to the isotonic solution is 1:1000;
(17) In the step 3, the dialysis temperature is 0-10 ℃;
(18) In the step 3, the dialysis is performed for a time to completely remove the salt solution in the outer aqueous phase of the solution A2a or A2b liposome;
(19) In the step 3, in order to place the liposome solution A2a or A2b obtained in the step 2 in a dialysis bag, an isotonic solution is used as a dialysis medium, and the solution is dialyzed for 12 hours at 4 ℃ with a volume ratio of the sample to the dialysis medium of 1:1000, changing the dialysate for 1 time every 4 hours during dialysis, completely removing acid radical ions in the outer water phase of blank liposome, and obtaining the blank liposome with the outer water phase consisting of isotonic solution and the inner water phase consisting of acid radical salt solution;
(20) In the step 4, the concentration of the mitoxantrone hydrochloride aqueous solution is 5mg/mL, 10mg/mL or 15mg/mL;
(21) In the step 4, the volume ratio of the solution A3 obtained in the step 3 to the mitoxantrone hydrochloride aqueous solution is 1:1, mixing, and incubating in a water bath at 50-60 ℃ for 40 minutes to obtain the ginsenoside mitoxantrone liposome; specifically, the aqueous phase in the liposome is acid radical mitoxantrone insoluble salt, and the aqueous phase outside the liposome is isotonic solution;
(22) In the step 5, the concentration of the ginsenoside solution is 5-20 mg/mL;
(23) In the step5, the solvent of the ginsenoside solution is the same as the solvent in the step 1;
(24) In the step 5, the mixing is stirring;
(25) In the step 5, the molecular weight cut-off of the dialysis bag is 8000-15000;
(26) In the step 5, in order to slowly add the ginsenoside solution into the liposome solution A4 obtained in the step 4, stir, volatilize and remove most of the ethanol, then place in a dialysis bag, dialyze for 12 hours at 4 ℃ with the same isotonic solution as the step 3 as a dialysis medium, wherein the volume ratio of the sample to the dialysis medium is 1:1000, changing the dialysate for 1 time every 4 hours during dialysis, and completely removing ethanol solvent, inorganic salt, unwrapped mitoxantrone hydrochloride and ginsenoside to obtain liposome solution A5;
(27) In the step 6, the mass ratio of the PEG-DSPE to the phospholipid is (0.025-0.15) 1;
(28) In the step 6, the concentration of the PEG-DSPE is 1-20mg/mL;
(29) In the step 6, a certain amount of PEG-DSPE is accurately weighed, dissolved in the same isotonic solution in the step3, and then added into the liposome solution A5 obtained in the step 5;
(30) In the step 7, in the sterilization and filtration step, a filter membrane with the size of 0.22 mu m is adopted to filter the liposome; in the filling step, filling in a 10mL or 20mL penicillin bottle, capping and packaging;
(31) The particle size D90 of the ginsenoside mitoxantrone liposome is less than or equal to 150nm, and the encapsulation rate is more than or equal to 80%.
5. The method of claim 3, wherein the method of preparing the ginsenoside mitoxantrone liposome satisfies one or more of the following conditions:
(1) In the step 1, the organic solvent is absolute ethyl alcohol;
(2) In the step 1, the mass-volume ratio of the phospholipid to the organic solvent is 1g/2mL;
(3) In the step 1, the phospholipid is heated and dissolved in an organic solvent to obtain the mixture A1; the heating is to heat to 55-65 ℃ in a water bath;
(4) In the step 1, the hydration temperature is 60 ℃;
(5) In the step 1, the hydration is carried out in a rotary steaming bottle, and the rotating speed is 50 rp/min;
(6) In the step 1, the hydration time is 2-4 hours;
(7) In the scheme 1 of the step 2, the high-pressure homogenization is carried out by using freezing water at 0-10 ℃ in a homogenizer for cutting circulation, so that the temperature of liposome solution is ensured to be 5-10 ℃;
(8) In the scheme 1 of the step2, the high-pressure homogenizing pressure is 1200bar;
(9) In the scheme 1 of the step 2, the number of times of high-pressure homogenization is 4;
(10) In the scheme 2 of the step 2, the extrusion temperature is 40 ℃;
(11) In the scheme 2 of the step 2, the extrusion pressure is 800 psi;
(12) In the scheme 2 of the step2, the number of times of extrusion is 4 times;
(13) In the step 3, the molecular weight cut-off of the dialysis bag is 10000;
(14) In the step 3, the dialysis temperature is 4 ℃;
(15) In the step 3, the dialysis time is 10-18 hours;
(16) In the step 4, the concentration of the mitoxantrone hydrochloride aqueous solution is 10-15 mg/mL;
(17) In the step5, the concentration of the ginsenoside solution is 10mg/mL;
(18) In the step 5, the mixing is stirring, and the stirring time is 30-60 minutes;
(19) In the step 5, the molecular weight cut-off of the dialysis bag is 10000;
(20) In the step 6, the mass ratio of the PEG-DSPE to the phospholipid is 0.05:1;
(21) In the step 6, the concentration of the PEG-DSPE is 10mg/mL; (22) When the salt solution is an ammonium sulfate aqueous solution, the concentration of the salt solution is 0.325M.
6. The method of claim 5, wherein in step 1, the mixture A1 is obtained by heating and dissolving the phospholipid in an organic solvent; the heating is water bath heating to 60 ℃;
and/or, in the step 3, the dialysis time is 12 hours;
And/or, in the step 5, the mixing is stirring, and the stirring time is 45 minutes.
7. A ginsenoside mitoxantrone liposome prepared by the preparation method of any one of claims 1-6.
8. A ginsenoside mitoxantrone liposome composition comprising an aqueous solution of glucose and a ginsenoside mitoxantrone liposome of claim 7.
9. The ginsenoside mitoxantrone liposome composition of claim 8, wherein the ginsenoside mitoxantrone liposome composition meets one or both of the following conditions:
(1) The glucose aqueous solution is 5% glucose aqueous solution;
(2) The encapsulation rate of the ginsenoside mitoxantrone liposome is more than or equal to 80 percent.
10. Use of a substance X in the preparation of a medicament for the treatment and/or prophylaxis of cancer, wherein the substance X is a ginsenoside mitoxantrone liposome according to claim 7 or a ginsenoside mitoxantrone liposome composition according to claim 8,
The cancer is breast cancer, colorectal cancer, primary liver cancer, gastric cancer, bladder cancer or brain tumor.
11. Use of substance X according to claim 10 for the preparation of a medicament for the treatment and/or prophylaxis of cancer, wherein said use satisfies one or more of the following conditions:
(1) The particle diameter D90 of the ginsenoside mitoxantrone liposome or the ginsenoside mitoxantrone liposome composition is less than or equal to 150nm;
(2) The encapsulation rate of the ginsenoside mitoxantrone liposome or the ginsenoside mitoxantrone liposome composition is more than or equal to 80 percent;
(3) The purity of the ginsenoside in the ginsenoside mitoxantrone liposome or the ginsenoside mitoxantrone liposome composition is more than or equal to 99 percent.
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