CN115487148A - Ginsenoside mitoxantrone liposome, preparation method and application thereof - Google Patents

Ginsenoside mitoxantrone liposome, preparation method and application thereof Download PDF

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CN115487148A
CN115487148A CN202110678285.4A CN202110678285A CN115487148A CN 115487148 A CN115487148 A CN 115487148A CN 202110678285 A CN202110678285 A CN 202110678285A CN 115487148 A CN115487148 A CN 115487148A
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mitoxantrone
ginsenoside
solution
liposome
salt
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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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    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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Abstract

The invention discloses ginsenoside mitoxantrone liposome, a preparation method and application thereof. The invention provides ginsenoside mitoxantrone liposome which comprises the following components in parts by weight: 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. The ginsenoside mitoxantrone liposome has targeting effect, synergism and attenuation on tumor cells.

Description

Ginsenoside mitoxantrone liposome, preparation method and application thereof
Technical Field
The invention relates to ginsenoside mitoxantrone liposome, a preparation method and application thereof.
Background
The liposome is a directional medicine-carrying system, belonging to a special dosage form of a targeted medicine-feeding system, and can embed the medicine into the particles with the diameter of nanometer grade, and the particles are similar to bilayer micro-vesicles in a biological membrane structure, enter the human body and are mainly phagocytized by a reticuloendothelial system, and change the in vivo distribution of the encapsulated medicine, so that the medicine is mainly accumulated in the targeted tissue, thereby improving the therapeutic index of the medicine, reducing the therapeutic dose of the medicine and reducing the toxicity of the medicine.
Three application patents, CN201610693884.2, CN201811447245.3 and CN201811447243.4, all disclose the technical advantages that after a liposome taking ginsenoside as a membrane material is loaded with paclitaxel and other therapeutic drugs, which is mainly prepared by "passive drug loading", namely "thin film evaporation method", the related liposome has stable quality, remarkable drug effect and the like.
Patents CN200380104235.5 and CN200380104175.7 disclose a method for preparing active drug-loaded liposome using phospholipid and cholesterol as membrane material and ammonium sulfate as gradient.
Patents CN201811532448.2 and CN201811552395.0 disclose an active drug loading method for liposomes, which uses phospholipid and cholesterol as membrane materials and sucrose octasulfate triethylamine as a gradient.
CN201811305299.6 discloses a membrane material prepared from phospholipid and cholesterol, ammonium methylsulfonate, ammonium 4-hydroxybenzenesulfonate, triethylamine methylsulfonate and triethylamine 4-hydroxybenzenesulfonate; the liposome active drug loading method using ethanedisulfonic acid ammonium, propanedisulfonic acid ammonium, ethanedisulfonic acid triethylamine and propanedisulfonic acid triethylamine as gradient.
In the prior art, the ginsenoside liposome can be used for preparing a co-loaded liposome of an insoluble drug by adopting a film evaporation method, but the water-soluble drug is generally prepared by adopting an active drug loading method, wherein a bimolecular membrane is composed of phospholipid and cholesterol, ionic salt solution such as ammonium sulfate, sucrose octasulfate triethylamine and the like is used as an internal water phase, the water-soluble drug is loaded by the principles of pH gradient and the like, and the drug is loaded in an inner cavity of the liposome.
Therefore, how to select an optimal compound medicine compatibility and how to formulate an optimal preparation process are to produce the ginsenoside mitoxantrone liposome which has better drug effect, lower toxicity and can meet the requirements of medicines on quality and other indexes so as to meet the requirements of medicine application, and a great deal of research work and technical clearance 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 has the advantages of stable property, small particle size, high drug encapsulation rate, good in vivo compatibility, good in vivo drug release, better drug effect and lower toxicity; the preparation method has a good preparation process, the preparation conditions are easy to realize, and industrialization is facilitated; the optimization of the combination of the preparation process and the product performance is realized.
The invention provides ginsenoside mitoxantrone liposome (named as 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 phosphatidyl ethanolamine), preferably, the PEG-DSPE is PEG2000-DSPE.
In one embodiment of the present invention, the mitoxantrone salt is mitoxantrone salt obtained by ion exchange of mitoxantrone hydrochloride with a salt solution by a pH gradient method (wherein mitoxantrone in the mitoxantrone salt forms the mitoxantrone salt with an anion in the salt solution); 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, a methylsulphonate ammonium aqueous solution, a methylsulphonate triethylamine aqueous solution, an ethanedisulfonate ammonium aqueous solution, a propanedisulfonate ammonium aqueous solution, a ethanedisulfonate triethylamine aqueous solution or a propanedisulfonate triethylamine aqueous solution; more preferably, the salt solution is an ammonium sulfate aqueous solution, a sucrose octasulfate triethylamine aqueous solution, a methyl ammonium sulfonate aqueous solution or a ethanedisulfonic acid triethylamine aqueous solution; such as an aqueous ammonium sulfate solution.
In one embodiment of the present invention, the mitoxantrone salt is mitoxantrone sulfate, mitoxantrone octasulfate, mitoxantrone methanesulfonate, mitoxantrone methanesulfonic acid, mitoxantrone ethanedisulfonic acid, mitoxantrone propanedisulfonic acid, mitoxantrone ethanedisulfonic acid, or mitoxantrone propanedisulfonic acid; preferably, the mitoxantrone salt is mitoxantrone sulfate, sucrose octasulfate mitoxantrone, or mitoxantrone methanesulfonate; such as mitoxantrone sulfate.
In one embodiment of the present invention, the mitoxantrone hydrochloride is an aqueous mitoxantrone hydrochloride solution, and preferably, the concentration of the aqueous mitoxantrone hydrochloride solution is 10mg/mL.
In one embodiment of the present invention, the liposome of ginsenoside mitoxantrone comprises ginsenoside and phospholipid forming phospholipid membrane, preferably, the phospholipid membrane further comprises PEG-DSPE.
In one embodiment of the present invention, the phospholipid membrane has an inner aqueous phase on the inside and an outer aqueous phase on the outside, and the mitoxantrone salt is encapsulated in the inner aqueous phase; the mitoxantrone salt is a mitoxantrone salt insoluble salt.
In one embodiment of the present invention, the inner aqueous phase is the saline solution, and the outer aqueous phase is a physiological isotonic solution; for example, a physiologically isotonic solution is a 5% aqueous glucose solution or a 10% aqueous sucrose solution.
In one embodiment of the present invention, the concentration of the salt solution is 0.05M to 0.975M; such as 0.05M, 0.1M, 0.2M, 0.3M, 0.325M, 0.65M, 0.975M, or 0.16M.
In one aspect of the invention, when the salt solution is an aqueous solution of sucrose octasulfate triethylamine, the concentration of the salt solution is 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 concentration of the salt solution is 0.16M to 0.325M.
In one embodiment of the present invention, when the salt solution is an aqueous ammonium methanesulfonate solution, the concentration of the salt solution is 0.325M to 0.975M.
In one aspect of the invention, when the salt solution is an aqueous ammonium sulfate solution, the concentration of the salt solution is 0.16M to 0.325M, for example 0.325M.
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 embodiment of the present invention, the mass ratio of mitoxantrone hydrochloride to the phospholipid is 1 (5-15); for example, the mass ratio of mitoxantrone hydrochloride to hydrogenated phospholipid is 1.
In one embodiment of the present invention, the ginsenoside is one or more selected from the group consisting of 20 (S) -ginsenoside Rg3, ginsenoside pseudo Rg3, 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.
In a certain scheme of the invention, the HPLC purity of the ginsenoside is more than or equal to 99%.
In one embodiment of the present invention, the phospholipid is present in an amount of 10 parts by mass.
In one embodiment of the present invention, the PEG-DSPE is 0.5 part by mass.
In one embodiment of the present invention, the ginsenoside is 1 part by mass.
In one embodiment of the present 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 a certain 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;
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;
step 2, which is scheme 1 or scheme 2;
scheme 1 (high pressure homogenization) comprises the following steps:
and (2) 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 a liposome solution A2a.
Scheme 2 (extrusion process) comprises the following steps:
respectively sequentially extruding the liposome solution A1 obtained in the step 1 through extrusion plates with different apertures, and controlling the particle size D90 to be less than 100nm to obtain a liposome solution A2b;
step 3, putting the liposome solution A2a or A2b obtained in the step 2 into a dialysis bag, and dialyzing by taking isotonic solution as a dialysis medium; obtaining liposome solution A3;
step 4, mixing the solution A3 obtained in the step 3 with a mitoxantrone salt solution to obtain a liposome solution A4;
step 5, mixing the liposome solution A4 obtained in the step 4 with a ginsenoside solution, placing the mixture in a dialysis bag, and dialyzing by using the same isotonic solution obtained in the step 3 as a dialysis medium; to obtain the ginsenoside mitoxantrone liposome solution A5.
In a certain embodiment of the present invention, the preparation method of the ginsenoside mitoxantrone liposome further comprises one or more steps of the following steps:
and 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 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 embodiment 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.
In one embodiment of the present invention, in the preparation method of the ginsenoside mitoxantrone liposome, the ginsenoside is one or more selected from the group consisting of 20 (S) -ginsenoside Rg3, ginsenoside pseudo Rg3, 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 embodiment 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.
In a certain scheme of the invention, in the preparation method of the ginsenoside mitoxantrone liposome, the HPLC purity of the ginsenoside is more than or equal to 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, triethylamine methylsulfonate; ammonium ethanedisulfonate, ammonium propanedisulfonate, triethylamine ethanedisulfonate, triethylamine propanedisulfonate and the like. For example, aqueous ammonium sulfate.
In one embodiment of the present invention, in the preparation method of the ginsenoside mitoxantrone liposome, the concentration of the salt solution is 0.05M-0.975M; such as 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 ginsenoside mitoxantrone liposome, when the salt solution is sucrose octasulfate triethylamine aqueous solution, the concentration of the salt solution is 0.05M to 0.3M, such as 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 ethanedisulfonic acid triethylamine, the concentration of the salt solution is 0.16M to 0.325M.
In one embodiment of the present invention, in the method for preparing ginsenoside mitoxantrone liposome, when the salt solution is an ammonium methanesulfonate aqueous solution, the concentration of the salt solution is 0.325M to 0.975M.
In one embodiment of the present invention, in the method for preparing the ginsenoside mitoxantrone liposome, when the salt solution is an ammonium sulfate aqueous solution, the concentration of the salt solution is 0.16M to 0.325M, for example, 0.325M.
In one embodiment of the present invention, in 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/volume ratio of the phospholipid to the organic solvent is 1g/1 to 10mL, for example, 1g/2mL.
In one embodiment of the present invention, in the step 1, the phospholipid is dissolved in an organic solvent by heating to obtain the mixture A1; for example, the heating may be in a water bath to 55-65 deg.C, such as 60 deg.C.
In one embodiment of the present invention, in step 1, the hydration temperature may be 55-65 ℃, for example, 60 ℃.
In one embodiment of the present invention, in the step 1, the hydration is performed in a rotary evaporation bottle at a rotation speed of 40 to 60rp/min, for example, 50rp/min.
In one embodiment of the present invention, in the step 1, the hydration time is related to the scale of the reaction, 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 a cooling and cutting cycle using chilled water at a temperature of 0 to 10 ℃ in a homogenizer; preferably, the temperature of the liposome solution is ensured to be between 5-10 ℃.
In one embodiment of the present invention, in the embodiment 1 of step 2, the high-pressure homogenizing pressure is between 800 and 1400bar, for example 1200bar.
In one embodiment of the present invention, in the embodiment 1 of step 2, the number of times of the high pressure homogenization is 3 to 4, for example, 4.
In one embodiment of the present invention, in embodiment 2 of step 2, the temperature of the extrusion is 35-45 ℃, for example 40 ℃.
In a certain embodiment of the present invention, in embodiment 2 of step 2, the pore diameter of the extrusion plate is 800nm,400nm,200nm,100nm.
In one embodiment of the present invention, in embodiment 2 of step 2, the extrusion pressure is 600 to 800psi; such as 800psi.
In one embodiment of the present invention, in embodiment 2 of the step 2, the number of the extrusion times may be 4 to 10, for example 4.
In one embodiment of the present invention, in embodiment 2 of the step 2, the solution A1 is sequentially filtered through polycarbonate membrane filters having respective pore diameters of 800nm,400nm,200nm, or 100nm.
In one embodiment of the present invention, in the step 3, the cut-off molecular weight 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.
In one embodiment of the present invention, in the step 3, the dialysis temperature is 0-10 ℃, for example, 4 ℃.
In one embodiment of the present invention, in the step 3, the dialysis is performed for completely removing the salt solution in the external aqueous phase of the liposome of the solution A2a or A2b, and preferably, the dialysis is performed for 10 to 18 hours, such as 12 hours.
In one embodiment of the present invention, in the step 3, in order to put the liposome solution A2a or A2b obtained in the step 2 into a dialysis bag, and dialyze the liposome solution at 4 ℃ for 12 hours by using an isotonic solution as a dialysis medium, a volume ratio of the sample to the dialysis medium is 1:1000, changing the dialyzate every 4 hours during dialysis, completely removing acid radical ions in the external water phase of the blank liposome to obtain an external water phase consisting of isotonic solution, and using acid radical salt solution as the blank liposome of the internal water phase.
In one embodiment of the present invention, in the step 4, the concentration of the mitoxantrone hydrochloride aqueous solution is 5 to 20mg/mL, for example, 1mg/mL, 5mg/mL, 10mg/mL, 15mg/mL or 20mg/mL; preferably 10 to 15mg/mL.
In one embodiment of the present invention, in the step 4, the solution A3 obtained in the step 3 and the aqueous solution of mitoxantrone hydrochloride are mixed in a volume ratio of 1:1, and incubating in a water bath at 50-60 ℃ for 40 minutes to obtain the ginsenoside mitoxantrone liposome. Specifically, the liposome internal water phase is acid radical mitoxantrone insoluble salt, and the liposome external water phase is isotonic solution.
In one embodiment of the present invention, in the step 5, the concentration of the ginsenoside solution is 5 to 20mg/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 that in the step 1.
In one embodiment of the present invention, in the step 5, the mixing is stirring, preferably, the stirring time is 30 to 60 minutes, for example, 45 minutes.
In one embodiment of the present invention, in the step 5, the cut-off molecular weight 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 to the liposome solution A4 obtained in the step 4, stirring, volatilizing to remove most of the ethanol, and then placing in a dialysis bag, taking the same isotonic solution as the step 3 as a dialysis medium, dialyzing at 4 ℃ for 12 hours, wherein the volume ratio of the sample to the dialysis medium is 1:1000, changing the dialysis solution every 4 hr for 1 time during dialysis, and completely removing ethanol solvent, inorganic salts, uncoated 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 to 0.15): 1, for example, 0.05.
In one embodiment of the present invention, in step 6, the concentration of PEG-DSPE is 1-20mg/mL, for example 10mg/mL.
In a certain embodiment of the present invention, in step 6, a certain amount of PEG-DSPE is accurately weighed, dissolved in the same isotonic solution as in step 3, and then added to the liposome solution A5 obtained in step 5.
In one embodiment of the present invention, the conditions and operations of the sterile filtration and the filling in step 7 can be those conventional in the art, for example, in the sterile filtration step, the liposomes are filtered with a 0.22 μm filter membrane; in the filling step, the mixture is filled into a penicillin bottle with 10mL or 20mL, capped and packaged.
In a certain 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 entrapment rate is more than or equal to 80%.
The invention also provides the 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 material of the ginsenoside mitoxantrone liposome further comprises 0.1-2 parts of PEG-DSPE (all called polyethylene glycol-distearoyl phosphatidyl ethanolamine), preferably, the PEG-DSPE is PEG2000-DSPE.
In one embodiment of the present invention, in the raw material of the ginsenoside mitoxantrone liposome, the mitoxantrone salt is mitoxantrone salt obtained by ion exchange of 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 methanesulfonate aqueous solution, a triethylamine methanesulfonate aqueous solution, an ammonium ethanedisulfonate aqueous solution, an ammonium propanedisulfonate aqueous solution, a triethylamine ethanedisulfonate aqueous solution or a triethylamine propanedisulfonate aqueous solution; more preferably, the salt solution is an ammonium sulfate aqueous solution, a sucrose octasulfate triethylamine aqueous solution, an ammonium methylsulfonate aqueous solution or an ethanedisulfonic acid triethylamine aqueous solution; such as an aqueous ammonium sulfate solution.
In one embodiment of the present invention, in the raw material of the ginsenoside mitoxantrone liposome, the mitoxantrone salt is mitoxantrone sulfate, mitoxantrone octasulfate, mitoxantrone methanesulfonate, mitoxantrone mesylate, mitoxantrone ethanedisulfonic acid, mitoxantrone propanedisulfonic acid, mitoxantrone ethanedisulfonic acid, or mitoxantrone propanedisulfonic acid; preferably, the mitoxantrone salt is mitoxantrone sulfate, sucrose octasulfate mitoxantrone, or mitoxantrone methanesulfonate; such as mitoxantrone sulfate.
In one embodiment of the present invention, in the raw material of the ginsenoside mitoxantrone liposome, the mitoxantrone hydrochloride is a mitoxantrone hydrochloride aqueous solution, and 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; such as 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 starting 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 to 0.3M, such as 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 ethanedisulfonic acid triethylamine, the concentration of the salt solution is 0.16M to 0.325M.
In one embodiment of the present invention, in the raw material of the ginsenoside mitoxantrone liposome, when the salt solution is an ammonium methanesulfonate aqueous solution, the concentration of the salt solution is 0.325M to 0.975M.
In one embodiment of the present invention, in the starting material of the ginsenoside mitoxantrone liposome, when the salt solution is an aqueous ammonium sulfate solution, the concentration of the salt solution is 0.16M to 0.325M, for example, 0.325M.
In one embodiment of the invention, in the raw material of the ginsenoside mitoxantrone liposome, 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 embodiment of the invention, in the raw material 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.
In one embodiment of the present invention, in the raw material of the ginsenoside mitoxantrone liposome, the ginsenoside is one or more selected from the group consisting of 20 (S) -ginsenoside Rg3, ginsenoside pseudo Rg3, 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.
In a certain scheme of the invention, in the raw material of the ginsenoside mitoxantrone liposome, the HPLC purity of the ginsenoside is more than or equal to 99%.
In one embodiment of the present invention, the mass fraction of the phospholipid in the raw material of the ginsenoside mitoxantrone liposome is 10 parts.
In one embodiment of the present invention, the ginsenoside mitoxantrone liposome is prepared from 0.5 part by mass of PEG-DSPE.
In one embodiment of the invention, the ginsenoside accounts for 1 part by mass of the raw materials of the ginsenoside mitoxantrone liposome.
In one embodiment of the present invention, the raw material of the ginsenoside mitoxantrone liposome comprises 1 part of mitoxantrone salt by mass.
In a certain scheme of the invention, the ginsenoside mitoxantrone liposome is prepared from 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 a glucose aqueous solution and the ginsenoside mitoxantrone liposome.
In one embodiment of the present invention, in the ginsenoside mitoxantrone liposome composition, the aqueous glucose solution is a 5% aqueous glucose solution.
In one embodiment of the invention, the entrapment rate 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 liposome composition of ginsenoside mitoxantrone has an error of about 10% in the mass fractions of the phospholipid, the PEG-DSPE, the ginsenoside and the mitoxantrone due to loss during the preparation process and process variation.
The invention also provides the application of the substance X in preparing the medicine for treating and/or preventing cancer; the substance X is the ginsenoside mitoxantrone liposome or the ginsenoside mitoxantrone liposome composition.
In a certain aspect of the present invention, in the application, the particle size D90 of the ginsenoside mitoxantrone liposome or the ginsenoside mitoxantrone liposome composition in the ginsenoside mitoxantrone liposome composition is not more than 150nm.
In a certain aspect of the present invention, in the application, the entrapment rate of the ginsenoside mitoxantrone liposome or the ginsenoside mitoxantrone liposome composition in the ginsenoside mitoxantrone liposome composition is not less than 80%.
In a certain aspect of the present invention, in the application, the ginsenoside mitoxantrone liposome or the ginsenoside mitoxantrone liposome composition has a ginsenoside purity of not less than 99%.
In one embodiment of the present invention, in the application, 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 embodiments of the present invention, the method for determining the encapsulation efficiency is centrifugation.
The term "particle size D90" refers to the particle size corresponding to 90% of the cumulative percent particle size distribution for a sample. Its physical meaning is that the particles have a size of less than 90% of its particle size.
The above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention without departing from the common general knowledge in the art.
The reagents and starting materials used in the present invention are commercially available.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: the positive progress effects of the invention are as follows: the ginsenoside mitoxantrone liposome provided by the invention has targeting effect on tumor cells, synergy and attenuation and drug synergism. Taking the ginsenoside Rg3 mitoxantrone liposome in the embodiment as an example, the drug effect is obviously superior to that of cholesterol mitoxantrone liposome; it is proved that Rg3 plays a plurality of better effects of 'medicament, auxiliary material, membrane material, target head' and the like in the ginsenoside Rg3 mitoxantrone liposome and plays a good medicament synergistic effect. Specifically, the method comprises the following steps:
(1) The drug effect is obviously improved. Especially 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, wherein the Rg3 (1.0) -MIT-PEG/Lp, rg3 (1.5) -MIT-PEG/Lp and Rg3 (2.0) -MIT-PEG/Lp high dose group (2 mg/kg) has the optimal drug effect, and the tumor completely disappears at the 28 th day, and has remarkable excellent effect compared with the common cholesterol mitoxantrone liposome group (C-MIT-PEG/LP group) and the cholesterol Rg3 mitoxantrone liposome group (C-MIT 3 (1.0) -MIT-PEG/Lp). Meanwhile, the tumor inhibition rate of the medium dose group (1 mg/kg) in the three experimental groups at day 28 is 9-11%, which is better than the tumor inhibition rate (11-20%) of the common cholesterol liposome group (C-MIT-PEG/LP group) and the high dose group (C-Rg 3 (1.0) -MIT-PEG/LP) of the cholesterol Rg3 mitoxantrone liposome group (C-Rg 3 (1.0) -MIT-PEG/LP) at day 28, and shows that the Rg3 mitoxantrone liposome of the invention has significant advantages on the pharmacodynamics of the traditional mitoxantrone liposome.
(2) The Glut1 targeting property is obviously improved. In the Glut1 targeting experiment of tumor-bearing mice, the Glut1 targeting of the ginsenoside liposome is improved by more than 4 times compared with the targeting of a common cholesterol liposome.
(3) The toxic and side effects are obviously reduced. Liposomes prepared according to the formulation of the present invention, rg3 mitoxantrone liposomes (Rg 3 (1.0) -MIT-PEG/Lp and Rg3 (2.0) -MIT-PEG/Lp group) and Rh2 mitoxantrone liposomes (Rh 2 (1.0) -MIT-PEG/Lp and Rh2 (2.0) -MIT-PEG/Lp group) died at 6mg/kg and 9mg/kg, 0/6 or 1/6 of 12mg/kg death, 3/6 or 4/6 of death at 18 mg/kg; while the cholesterol liposome group (C-MIT-PEG/LP group) died at 6mg/kg 1/6,9mg/kg 4/6. The results show that the LD50 of the Rg3 mitoxantrone liposome and the LD50 of the Rh2 mitoxantrone liposome are between 12 and 18mg/kg, and the LD50 of the cholesterol mitoxantrone liposome is between 6 and 9mg/kg, and the ginsenoside liposome has significantly reduced acute toxicity compared with the cholesterol liposome.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Experimental drugs and devices
Experimental drugs: 20 (S) -ginsenoside Rg3 (Rg 3), ginsenoside Rp1 (Rp 1), ginsenoside GQ (GQ), ginsenoside Rk1 (Rk 1), ginsenoside Rg5 (Rg 5), 20 (S) -ginsenoside Rh2 (Rh 2), ginsenoside Rk2 (Rk 2), 20 (S) -ginsenoside Rg2 (Rg 2), 20 (S) -ginsenoside Rh1 (Rh 1), 20 (S) -protopanaxadiol (PPD), 20 (S) -protopanaxatriol (PPT), mitoxantrone hydrochloride, etc. are commercially available in the art, such as Shanghai Ben medicine technology, shanghai gold and biological pharmacy, inc., shanghai leaf biology, etc.
The ginsenoside has the following molecular structural formula:
Figure BDA0003121731450000091
Figure BDA0003121731450000101
the test instrument: the instruments used in the following examples are owned instruments of Shanghai Benghai pharmaceutical science and technology Limited, the college of pharmacy of the university of Fudan, the model numbers and source information of which are as follows:
agilent liquid chromatography: agilent 1100, autai 3300ELSD, agilent technologies (China) Inc.;
and (3) rotary evaporation of an evaporator: ZX98-1 5L, shanghai Luyi Industrial trade, inc.;
ultrasonic cleaning machine (SB 3200DT, ningbo Xinzhi Biotech GmbH);
nitrogen blower (HGC-12A, constant Olympic technology development Co., ltd., tianjin);
probe ultrasound apparatus (JYD-650, shanghai wisdom communication instruments, inc., china);
high pressure homogenisers (B15, AVESTIN, canada);
micro-extruders (Mini-extruder, avanti Polar Lipids Inc);
laser particle size analyzer (Nano ZS, marvin, uk);
malvern Nanosizer ZS90 (Malvern, uk);
microplate reader (Thermo Scientific, waltham, MA, USA);
enzyme-labeling instruments (Infinitie 200, tecan tracing co., switzerland, ltd);
flow cytometry (BD Biosciences, USA);
flow cytometry (CytoFlex S, beckman Coulter, inc., USA);
inverted fluorescence microscope (Leica, DMI 4000d, germany);
fluorescence microscopy (Zeiss LSM 710, oberkochen, germany);
confocal laser microscopy (Leica, DMI 4000d, germany);
confocal In Vivo Microscopy (IVM);
an upright two-photon microscope (DM 5500Q; nikon);
small animal in vivo optical imaging system (IVIS) (PerkinElmer, USA);
biomacromolecule interaction instrument BiaCore T200 instrument (GE, USA);
clean bench (SW-CJ-1 FD, air technologies, inc., antai, suzhou);
20L rotary evaporator: R5002K, shanghai xiafeng industries ltd;
a freeze dryer: FD-1D-80, shanghai Bilang instruments manufacturing Co., ltd;
a freeze dryer: PDFD GLZ-1B, pudong Freeze drying Equipment, inc., shanghai;
an electronic balance: CPA2250 (precision 0.00001 g), sartorius trade ltd;
an electronic balance: JY3003 (precision 0.001 g), shanghai Shunhui constant-level scientific instruments, inc.;
photoelectric microscopes (XDS-1B, chongqing photoelectric instruments, inc.);
cell culture incubator (CCL-170B-8, singapore ESCO).
Animals and cell lines
Animals: BALB/c nude mice, age of mice 3-4 weeks, produced by Shanghai pharmaceutical research institute of Chinese academy of sciences.
Tumor cell lines:
breast carcinoma orthotopic tumor 4T1 cell line, provided by the college of pharmacy of Sudan university
Human colon cancer C-26 cell line, purchased from Kyoho Kai Biotech Co., ltd
Human pancreatic cancer Capan-1 cell line, purchased from Kyosu Kaiki Biotechnology Ltd
MCF-7 cell line for breast cancer, purchased from Kyosu Kai Biotech GmbH
The method for detecting the content of mitoxantrone hydrochloride comprises the following steps: according to the 'mitoxantrone hydrochloride' detection method in the second part of 2020 edition of Chinese pharmacopoeia.
1) Chromatographic conditions are as follows: c18 column (Kromasil C18, 250X 4.6mm,5 μm)
2) Mobile phase: dissolving with sodium heptanesulfonate solution (4.4 g sodium heptanesulfonate, adding appropriate amount of water for dissolution, adding 6.4mL glacial acetic acid, diluting with water to 730 mL): acetonitrile =70:30 The (volume ratio) is mobile phase.
3) Detection wavelength: 244nm, flow rate of 1.0mL/min, column temperature of 30 ℃, and sample injection amount of 20 μ L.
4) And (3) calculating: recording the chromatogram, and calculating the content of the mitoxantrone hydrochloride in the test solution by an external standard method.
The method for detecting the content of the ginsenoside comprises the following steps:
1) Chromatographic conditions are as follows: kromasil 100-3.5C4 150mm. Times.4.6 mm column.
2) Mobile phase: acetonitrile: water = 55.
3) Detection wavelength: 203nm, flow rate of 1mL/min, column temperature of 35 ℃, and sample injection amount of 10 μ L.
4) And (3) calculating: and recording the chromatogram, and calculating the content of Rg3 in the test solution by an external standard method.
The method for detecting the encapsulation rate of mitoxantrone (or ginsenoside) comprises the following steps:
taking 1mL of each of 2 liposome samples to be detected, centrifuging (18000 r/min,30min,3 times, each time with an interval of 30 minutes), respectively taking supernatant and liposome precipitate, washing the precipitated liposome with distilled water for 3 times, each time with 1mL of distilled water, combining the supernatants, placing in a 25mL volumetric flask, diluting with deionized water to a constant volume, and performing HPLC detection to obtain a drug concentration (the concentration of free mitoxantrone or ginsenoside in the ginsenoside mitoxantrone liposome) of V1; and the other part is put into a 25mL volumetric flask, the volume is determined by deionized water, and the concentration of the medicine is V0 by an HPLC method. Encapsulation efficiency = (V0-V1)/V0 × 100%.
For short: 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 HSPC and ginsenoside with the prescription amount, ultrasonically dissolving in 1mL chloroform, concentrating under reduced pressure to be dry, adding 10mL hydration solution, hydrating for 10 minutes, then ultrasonically treating for 25 times (600W, 5 seconds on, 5 seconds off) to obtain blank liposome of each experimental group, and detecting appearance and encapsulation rate.
3) The experimental results are as follows:
Figure BDA0003121731450000121
example 2 Effect of phospholipid dosage on mitoxantrone encapsulation efficiency in conventional active drug delivery method
Figure BDA0003121731450000122
Figure BDA0003121731450000131
And (4) analyzing results: by adopting an ethanol injection method in the traditional active drug loading method, the drug-lipid ratio (HSPC/drug) has a great influence on the encapsulation efficiency, and when the drug-lipid ratio is more than or equal to 10, the encapsulation efficiency has no obvious difference. Therefore, the present invention preferably has a phospholipid ratio of 5 to 15.
Example 3 Effect of Cholesterol dose on mitoxantrone encapsulation efficiency in conventional active drug Loading method
Figure BDA0003121731450000132
And (4) analyzing results: by adopting an ethanol injection method in the traditional active drug loading method, the cholesterol can improve the stability of the liposome and improve the encapsulation efficiency of the mitoxantrone. When the cholesterol/medicine is more than or equal to 0.5, the improvement is remarkable; when the cholesterol/medicine ratio is more than or equal to 1, the amount of the cholesterol has no obvious difference on the encapsulation efficiency.
Example 4 Effect of Rg3 dose on mitoxantrone encapsulation efficiency in conventional active drug delivery method
Figure BDA0003121731450000133
Figure BDA0003121731450000141
And (4) analyzing results: by adopting an ethanol injection method in the traditional active drug loading method, HSPC and Rg3 are synchronously formed into a film, and then ionic aqueous solution is hydrated, dialyzed by 5% glucose water and loaded with drugs, so that the qualified Rg3 mitoxantrone co-loaded liposome cannot be prepared.
Example 5 Effect of Cholesterol dose on Rg3 Liposome encapsulation efficiency in conventional active drug delivery method
Figure BDA0003121731450000142
And (4) analyzing results: by adopting an ethanol injection method in the traditional active drug loading method, the HSPC, the Rg3 and the cholesterol are synchronously formed into a film, then the ionic aqueous solution is hydrated and dialyzed by 5 percent glucose to obtain the Rg3 liposome, the Rg3 entrapment rate of the liposome prepared by the process is low, and Rg3 leakage is caused by the ionic aqueous solution.
Example 6 Effect of Cholesterol dose on Rg3 Liposome encapsulation efficiency in conventional active drug delivery method
Figure BDA0003121731450000143
And (4) analyzing results: by adopting an ethanol injection method in the traditional active drug loading method, when the external water phase is 5% glucose after dialysis, rg3 is loaded into the liposome as a drug, the encapsulation efficiency of Rg3 is qualified, and the influence of the dosage of cholesterol on the Rg3 encapsulation efficiency is not large. Effect example 1: the results of targeting experiments of the C6-C-Rg3 (post)/Lp group in the cell uptake experiment of Glut1 show that: glut1 mediated targeting of this group was poor, indicating that the glucose group of Rg3 was not exposed on the liposome surface, and therefore Rg3 should be entrapped in the lumen of the liposome.
Example 7 Rg3 and mitoxantrone simultaneous loading experiment in traditional active drug loading method
Figure BDA0003121731450000151
And (4) analyzing results: by adopting an ethanol injection method in a traditional active drug loading method, under the use levels of Rg3 and cholesterol in different proportions, the blank liposome has the inner water phase of ammonium sulfate solution, the outer water phase of 5% glucose solution, rg3 and mitoxantrone as drugs, and is synchronously loaded, and the encapsulation efficiency of the mitoxantrone and Rg3 is unqualified. The ionic aqueous solution affects the encapsulation efficiency of Rg3 and mitoxantrone hydrochloride at the same time.
Example 8 experiment of influence of first loading Rg3 and then loading mitoxantrone on encapsulation efficiency in traditional active drug loading method
Figure BDA0003121731450000152
Figure BDA0003121731450000161
And (4) analyzing results: the method is characterized in that an ethanol injection method in a traditional active drug loading method is adopted, rg3 and cholesterol in different proportions are adopted, after dialysis, the inner water phase of the blank liposome is an ammonium sulfate solution, the outer water phase of the blank liposome is a 5% glucose solution, rg3 is loaded firstly, then mitoxantrone hydrochloride is loaded, and the encapsulation rate of the prepared Rg3 mitoxantrone co-loaded liposome is unqualified.
Example 9 Effect of different preparation methods on the encapsulation efficiency of Rg3 mitoxantrone co-loaded liposomes
Figure BDA0003121731450000162
Figure BDA0003121731450000171
And (4) analyzing results:
1) The passive drug loading (thin film method) can not prepare the qualified Rg3 mitoxantrone co-loaded liposome;
2) The normal active drug-loading method can not prepare the qualified Rg3 mitoxantrone co-loaded liposome;
3) In a common active drug loading method, rg3 is loaded firstly and then mitoxantrone is loaded, or Rg3 and mitoxantrone are loaded synchronously, so that qualified Rg3 mitoxantrone co-loaded liposome cannot be prepared;
4) The normal active drug loading method is to load the mitoxantrone first and then Rg3, and can prepare the qualified Rg3 mitoxantrone co-loaded liposome. The application of the liposome prepared by the method of the invention is implemented in the following steps: the cell uptake experiment of Glut1 and the experiment results of the C6-Rg3 (post) -MIT/Lp group show that: the Rg3 liposome prepared by the method has a remarkable Glut1 mediated active targeting effect, and the Rg3 is proved to be embedded in a phospholipid bimolecular membrane, wherein glucosyl (Glc) in Rg3 molecules is exposed on the outer surface of the liposome.
Example 10 Effect of Rg3 dose on Rg3 mitoxantrone Co-entrapped Liposome encapsulation efficiency
Figure BDA0003121731450000172
Figure BDA0003121731450000181
And (4) analyzing results:
1) By adopting the ethanol injection method, the qualified Rg3 mitoxantrone co-carried liposome can be prepared, specifically, the internal aqueous phase of the liposome is mitoxantrone sulfate, the external aqueous phase of the liposome is 5% glucose isotonic solution, the bimolecular membrane of the liposome is hydrogenated phospholipid and Rg3, wherein glucose (Glc) in Rg3 molecules is exposed on the outer surface of the liposome, and the membrane material of the liposome does not contain cholesterol.
2) When the HSPC is Rg3: MIT =10, the encapsulation efficiency of Rg3 and mitoxantrone is good. With the increase of the dosage of Rg3, the encapsulation efficiency of Rg3 and mitoxantrone is sharply reduced.
3) The application range of the Rg3 is HSPC: rg3: MIT = 10.
Example 11 Effect of different salts on Rg3 mitoxantrone Co-loading Liposome encapsulation efficiency experiment
Figure BDA0003121731450000182
And (4) analyzing results: by adopting the ethanol injection method, the sucrose octasulfate triethylamine, the ammonium sulfate, the ammonium methanesulfonate, the triethylamine methanesulfonate, the ammonium ethanedisulfonate, the ammonium propanedisulfonate, the triethylamine ethanedisulfonate and the triethylamine propanedisulfonate can meet the preparation of the Rg3 mitoxantrone liposome, and the encapsulation efficiency is qualified.
Example 12 Effect of different salt concentrations on the encapsulation efficiency of Rg3 mitoxantrone Co-loaded liposomes
Figure BDA0003121731450000183
Figure BDA0003121731450000191
And (4) analyzing results: by adopting the ethanol injection method, 1) when the concentration of the sucrose octasulfate triethylamine is lower than 0.05M, the encapsulation efficiency cannot meet the process requirement; when the concentration is more than or equal to 0.1M, the encapsulation efficiency has no obvious difference. 2) When the concentration of ammonium sulfate and triethylamine ethanedisulfonate is lower than 0.16M, the encapsulation efficiency can not meet the process requirement; there was no significant difference in encapsulation efficiency between the concentrations of 0.32M and 0.65M. 3) When the concentration of the ammonium methanesulfonate is lower than 0.325M, the encapsulation efficiency cannot meet the process requirements; there was no significant difference in encapsulation efficiency between the concentrations of 0.65M and 0.975M.
Example 13 Effect of different ginsenosides on the encapsulation efficiency of the liposome with the saponin mitoxantrone
Figure BDA0003121731450000192
And (4) analyzing results: by adopting the ethanol injection method, the entrapment rate of the co-carried liposome prepared from 20 (S) -Rg3, 20 (S) -Rh2, rg5, rk1, rp1, pseudo Rg3, pseudo GQ, PPD and other saponins meets the quality requirement; the entrapment rate of the co-carried liposome prepared from 20 (R) -Rg3, PPT and other saponins does not meet the quality requirement.
Example 14 Effect of different homogenization methods on the encapsulation efficiency of Rg3 mitoxantrone co-loaded liposomes
Figure BDA0003121731450000193
Figure BDA0003121731450000201
And (4) analyzing results: the ethanol injection method of the invention can meet the process requirements in three common methods (ultrasonic method, high-pressure homogenization method and extrusion film-passing method) for controlling the particle size.
Example 15 Effect of different phospholipids on the encapsulation efficiency of Rg3 mitoxantrone co-loaded liposomes
Figure BDA0003121731450000202
And (4) analyzing results: by adopting the ethanol injection method, the encapsulation efficiency of the Rg3 mitoxantrone co-carried liposome prepared from hydrogenated phospholipid, egg yolk lecithin, soybean lecithin and cephalin meets the requirement of drug declaration, and PEG-DSPE does not meet the requirement.
Example 16 Effect of different mitoxantrone concentrations on the encapsulation efficiency of Rg3 mitoxantrone co-loaded liposomes
Figure BDA0003121731450000203
And (4) analyzing results: by adopting the ethanol injection method, the encapsulation efficiency is optimal when the drug concentration is 5-15 mg/mL, and particularly the drug concentration is optimal when the drug concentration is 10mg/mL. When the concentration of the medicine is lower than 5mg/mL or higher than 20mg/mL, the entrapment rate does not meet the quality requirement of the medicine.
EXAMPLE 17 Effect of different physiologically isotonic solutions on the encapsulation efficiency of Rg3 mitoxantrone co-loaded liposomes
Figure BDA0003121731450000204
Figure BDA0003121731450000211
And (4) analyzing results: by adopting the ethanol injection method, 5% glucose and 10% sucrose aqueous solution has no obvious difference on the encapsulation efficiency of Rg3 and mitoxantrone, and 0.9% normal saline is not suitable.
Example 18 preparation of Rg3 mitoxantrone liposomes
1. Prescription: 1g of HSPC 10g, 1g of Rg3 g, 1g of mitoxantrone hydrochloride, a proper amount of absolute ethyl alcohol, a proper amount of 5% glucose injection, a proper amount of injection water 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 the formula amount, dissolving the HSPC in 20mL of absolute ethyl alcohol, adding 100mL of 0.325M ammonium sulfate, hydrating at 55-60 ℃ for 10 minutes, volatilizing to remove most of ethanol, and preparing a blank liposome crude product of which the inner and outer water phases are ammonium sulfate solutions;
step (2): push through the membrane
And (2) sequentially passing the blank liposome solution obtained in the step (1) through polycarbonate membrane filter plates with the aperture of 800nm,400nm,200nm and 100nm respectively for 4 times at the pressure of 600-800psi, and finally obtaining the blank liposome with the particle size of less than 100nm and the internal and external aqueous phases of ammonium sulfate solution.
And (3): dialysis
Putting the blank liposome obtained in the step (2) into a dialysis bag with the molecular weight cutoff of 10000, taking 5% glucose aqueous solution as a dialysis medium, dialyzing for 12 hours at 4 ℃, wherein the volume ratio of the sample to the dialysis medium is 1:1000, changing the dialyzate every 4 hours during dialysis, completely removing ammonium sulfate from the external aqueous phase of the blank liposome to obtain blank liposome with 5% glucose as external aqueous phase and ammonium sulfate as internal aqueous phase.
And (4): loaded mitoxantrone
Mixing the blank liposome obtained in the step (3) with 10mg/ml mitoxantrone hydrochloride aqueous solution 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 liposome internal water phase is mitoxantrone sulfate insoluble salt, and the liposome external water phase is 5% glucose aqueous solution.
And (5): embedding Rg3
Slowly adding 100mL of 10mg/mL of Rg3 ethanol solution into the mitoxantrone liposome solution in the step (4) at the temperature of 20-30 ℃, stirring for 45 minutes, volatilizing to remove most ethanol, then placing into a dialysis bag with cut-off molecular weight of 10000, taking 5% glucose aqueous solution as a dialysis medium, dialyzing for 12 hours at the temperature of 4 ℃, wherein the volume ratio of the sample to the dialysis medium is 1:1000, changing the dialyzate every 4 hours during the dialysis, and completely removing the ethanol solvent, inorganic salt, uncoated mitoxantrone hydrochloride and Rg3 to obtain the Rg3 mitoxantrone liposome.
And (6): adding PEG-DSPE
Accurately weighing 0.2g of PEG-DSPE, dissolving in 300mL of 5% glucose, and adding into the Rg3 mitoxantrone liposome solution obtained in the step (5) to obtain the Rg3 mitoxantrone liposome solution with mitoxantrone and Rg3 concentration of about 2 mg/mL.
Step (7) of sterilizing filtration
And (3) filtering the Rg3 mitoxantrone liposome obtained in the step (6) through a 0.22 mu m filter membrane.
And (8): filling
And (4) filling the solution obtained in the step (7) into a 10mL or 20mL penicillin bottle, capping and packaging to obtain the penicillin bottle.
The liposome is detected to have mitoxantrone concentration =4.88mg/mL, rg3 concentration =4.91mg/mL, particle size D90=101nm, rg3 encapsulation rate =98.65%, and mitoxantrone encapsulation rate =96.79%.
Example 19 Effect of PEG-DSPE dosage on Rg3 mitoxantrone Co-Carrier Liposome stability
The preparation method comprises the following steps: the Rg3 mitoxantrone liposome solution obtained in the step (5) of the example 18 is taken, PEG-DSPE aqueous solutions with different concentrations are added according to the formula of the example, other subsequent steps are the same as the example 18, and the preparation of each formula is placed in a refrigerator at 2-8 ℃ to examine the stability of the liposome solution.
Figure BDA0003121731450000221
And (4) analyzing results:
1) PEG-DSPE is not added, the particle size of the Rg3 mitoxantrone liposome rapidly rises after being stored for 3 months at the temperature of 2-8 ℃, and the leakage rate of Rg3 and mitoxantrone obviously rises;
2) When PEG-DSPE/HSPC is less than or equal to 0.025, the particle size of the Rg3 mitoxantrone liposome is obviously increased after being stored for 3 months at the temperature of 2-8 ℃, the entrapment rate is obviously reduced, and the quality requirement of the stability is unqualified. Wherein, PEG-DSPE/HSPC =0.025,3 months of stability data is acceptable.
3) When the PEG-DSPE/HSPC is more than or equal to 0.025, the particle size of the Rg3 mitoxantrone liposome is stable and the encapsulation efficiency of Rg3 and mitoxantrone is stable after being stored for 3 months at the temperature of 2-8 ℃, thereby meeting the requirements of drug declaration.
4) When the PEG-DSPE/HSPC is more than or equal to 0.05, the particle size and the encapsulation efficiency have no significant difference.
Application example 1: cellular uptake assay for Glut1
1) Purpose of the experiment: observing whether the Rg3 liposome has more uptake on tumor cells by comparing the uptake of the fluorescein-loaded Rg3 liposome and the cholesterol liposome on 4T1 cells; the Glut1 targeting mechanism was demonstrated by the addition of glucose inhibitors and the like; the ginsenoside of the invention is positioned in a phospholipid bimolecular membrane through Glut1 targeting verification, and glucosyl is exposed on the outer surface of the liposome.
2) The experimental method comprises the following steps: to compare the uptake of 4T1 into each experimental group, the mechanism of uptake of liposomes was investigated, and 4T1 cells were aligned at 2X 10 5 The cell density of (2) was inoculated in 12-well plates, and for the experimental group + glucose, the experimental group + phlorizin, and the experimental group + quercetin, the culture medium was replaced with 20mM glucose solution, phlorizin solution, and quercetin solution, respectively, after 12 hours. The three solutes should be dissolved in a glucose-free medium, incubated for 1 hour, added with each experimental drug (concentration of ultraviolet fluorescent developer is 100 ng/ml), incubated for 4 hours, digested, washed with fresh PBS solution, and analyzed by flow cytometry.
To study the uptake mechanism of Rg3 liposomes, substrate (glucose), glut1 competitive inhibitors phlorizin and quercetin were incubated for 1 hour in advance to saturate Glut1 first, then formulation was added, and the fluorescence intensity of Rg3-Lp/C6 was reduced by 31%,43% and 74%, respectively. Therefore, due to the addition of the Glut1 substrate and the inhibitor, the cellular uptake of Rg3-Lp/C6 is prevented, and the fact that the ginsenoside Rg3 liposome can enhance the uptake efficiency through interaction with the Glut1 is proved.
3) The preparation method of the experimental group comprises the following steps: the operating conditions were the same as those of the examples of the present invention.
Method 1 (passive drug loading): the preparation method comprises the following steps of dissolving a prescribed amount of HSPC, ginsenoside and/or cholesterol, fluorescent probe (coumarin) and/or drug in a mixed solvent of a proper amount of ethanol and chloroform (volume ratio is 1.
Method 2 (active drug loading): the preparation method comprises the steps of ultrasonically dissolving HSPC, rg3 and a fluorescent probe in a proper amount of ethanol, adding 0.325M ammonium sulfate solution for hydration for 10 minutes, then ultrasonically treating for 25 times (starting 5 seconds and stopping 5 seconds), dialyzing with 5% glucose solution, sequentially loading (and/or) medicaments, dialyzing again to remove free medicaments, (and/or) proper amount of PEG-DSPE (polyethylene glycol-DSPE) to obtain liposome solutions of each experimental group, and detecting the fluorescence intensity according to the experimental method of the application example.
Method 3 (active drug loading): ultrasonically dissolving HSPC and a fluorescent probe with a prescription dose in a proper amount of ethanol, adding 0.325M ammonium sulfate solution for hydrating for 10 minutes, then ultrasonically treating for 25 times (starting 5 seconds and stopping 5 seconds), dialyzing with 5% glucose solution, sequentially loading the medicine or Rg3, dialyzing again to remove free medicine, (and/or) adding a proper amount of PEG-DSPE to obtain liposome solution of each experimental group, and then detecting the fluorescence intensity according to the experimental method of the application example.
Figure BDA0003121731450000231
Figure BDA0003121731450000241
Experimental result 1 is as follows:
Figure BDA0003121731450000242
Figure BDA0003121731450000251
above ratio is
And (4) experimental conclusion:
1) By adopting the traditional passive drug loading method (thin film evaporation method), the targeting experimental data prove that: can not prepare qualified Rg3 mitoxantrone co-carried liposome.
2) Adopts a traditional active drug loading method, specifically:
i. adding Rg3 before dialysis, the acid solution causes Rg3 to leak in the liposome, thereby causing failure of liposome preparation.
Adding Rg3 after dialysis, two cases can be distinguished:
a) Rg3 is added before mitoxantrone hydrochloride, and Rg3 in the liposome is seriously leaked due to ionic solution generated by the medicament, so that the liposome preparation fails;
b) Rg3 is added after mitoxantrone hydrochloride, and the liposome is successfully prepared.
The two conditions are basically the same, and the ionic solution exists in the two conditions although the sequence of the two conditions is different, but different results are produced, and the mechanism is not clear.
3) The addition of a proper amount of PEG-DSPE influences the Glut1 mediated targeting, which indicates that the dosage of PEG-DSPE is limited.
4) The experiment indicates that the Rg3 mitoxantrone co-carried liposome of the invention needs to be prepared by the same or similar method as the embodiment 18.
The experimental results 2 are as follows:
Figure BDA0003121731450000261
from the results, the fluorescence intensity of C6-C/Lp is not changed significantly with the addition of the Glut1 substrate and the inhibitor, but the cellular uptake of C6-Rg3/Lp is prevented, and the ginsenoside Rg3 liposome is proved to enhance the uptake efficiency through the interaction with Glut1, so that the Rg3 is proved to be positioned on the membrane of the liposome, and the glucose group (Glc) of the Rg3 is exposed on the surface of the liposome.
Application example 2: pharmacodynamic study of human Breast cancer (MCF-7) in vivo
1) The test method comprises the following steps: injecting the tumor cell strain (MCF-7) into the subcutaneous part of a mouse to establish a subcutaneous tumor model. When the tumor volume reaches 100mm 3 (7 d post-inoculation), mice were treated randomly in groups (n =8 groups), each group was injected in tail vein with Blank solvent (5% glucose, blank), mitoxantrone liposome injection (C-MIT-PEG/LP) and experimental groups at three high, medium and low groups (2 mg, 1mg, 0.5 mg) in terms of mitoxantrone, dosed once every 7 days for up to day 28, and tumor length, width and body weight were measured and recorded. The formula for calculating the tumor volume (V) is V = (W) 2 X L)/2. The length (L) is the longest diameter of a solid tumor and the width (W) is the shortest diameter perpendicular to the length. At the end of the experiment on day 28, all animals were sacrificed and tumors were removed for imaging and histological examination.
Tumor inhibition rate T = (tumor of non-administered group-tumor weight of test group)/tumor weight of non-administered group
Remarking: mitoxantrone + Rg3=2mg/kg +2mg/kg, indicating drug concentration, as follows.
2) The experimental groups were as follows:
Figure BDA0003121731450000271
3) The test results are as follows:
Figure BDA0003121731450000272
and (4) conclusion:
1) Rg3/MIT =1.0, 1.5 and 2.0, with no significant difference in pharmacodynamics.
2) The in vivo pharmacodynamics of the Rg3 mitoxantrone liposome has remarkable excellent effects compared with that of a common cholesterol mitoxantrone liposome group (C-MIT-PEG/LP group) and a cholesterol Rg3 mitoxantrone liposome group (C-Rg 3 (1.0) -MIT-PEG/LP), wherein the Rg3 (1.0) -MIT-PEG/LP, the Rg3 (1.5) -MIT-PEG/LP and the Rg3 (2.0) -MIT-PEG/LP high dose group (12 mg/kg) have the advantages that the tumor completely disappears at 28 days and the high-dose group has remarkable excellent effects compared with that of the common cholesterol mitoxantrone liposome group (C-MIT-PEG/LP group) and the cholesterol Rg3 mitoxantrone liposome group (C-MIT-PEG/LP). Meanwhile, the 28 th-day tumor inhibition rate of the medium-dose group (8 mg/kg) in the three experimental groups is 9-11%, and the three experimental groups are better than the 28 th-day tumor inhibition rate (11-20%) of the common cholesterol liposome group (C-MIT-PEG/LP group) and the cholesterol Rg3 mitoxantrone liposome group (C-Rg 3 (1.0) -MIT-PEG/LP) in the 28 th-day tumor inhibition rate (12 mg/kg), and the Rg3 mitoxantrone liposome has obvious pharmacodynamic advantages on the traditional mitoxantrone liposome.
Human colon cancer C-26 cell line: the data for the in vivo pharmacodynamic study of human colon cancer (C-26) cells according to the in vivo pharmacodynamic test method are as follows.
Item Blank C-MIT-PEG/LP panel Rg3(1.0)-MIT-PEG/Lp Rh2(1.0)-MIT-PEG/Lp
Administration dosage / 2mg/kg 2mg/kg+2mg/kg 2mg/kg+2mg/kg
Tumor inhibition rate in 7 days -27％ 46％ 38％ 35％
Tumor inhibition rate of 14 days -53％ 48％ 25％ 24％
Tumor inhibition rate in 21 days -72％ 37％ 11％ 9％
Tumor inhibition rate in 28 days -91％ 22％ Disappearance of tumor Disappearance of tumor
The results show that:
1) The pharmacodynamics of the Rg3 mitoxantrone liposome and the Rh2 mitoxantrone liposome are not obviously different;
2) The pharmacodynamics of the Rg3 mitoxantrone liposome and the Rh2 mitoxantrone liposome are remarkably more effective than that of the cholesterol liposome group (C-MIT-PEG/LP group).
Human pancreatic cancer, capan-1: according to the in vivo pharmacodynamic experimental approach, the data on the in vivo pharmacodynamics of human pancreatic cancer (Capan-1) cells are as follows.
Figure BDA0003121731450000281
The results show that:
1) The pharmacodynamics of the Rg3 mitoxantrone liposome and the Rh2 mitoxantrone liposome are not obviously different;
2) The pharmacodynamics of the Rg3 mitoxantrone liposome and the Rh2 mitoxantrone liposome are remarkably more effective than that of the cholesterol liposome group (C-MIT-PEG/LP group).
Application example 3: acute toxicity (LD 50) study (SD rats)
1) The experimental method comprises the following steps: rats 160-260g, 6-9 weeks old, 6 per group, administration mode: slow rest push (about 1 mL/min), dosing frequency: 3 times per day.
The mitoxantrone dosages of the test samples in the test are set to be 6,9, 12 and 18 mg/kg/day, and the Rg3 content in the test samples is calculated according to the prescription dosage. 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, a slow and static push (about 1 mL/min), 3 times/day, and at least 4h of dosing interval.
2) The preparation method of the experimental group comprises the following steps: prepared according to the recipe requirements by the method of example 18.
Figure BDA0003121731450000291
3) The experimental results are given in the following table:
Figure BDA0003121731450000292
through the experiments shown above, it is possible to show that,
1) The acute toxicity of the Rg3 mitoxantrone liposome and the Rh2 mitoxantrone liposome is not obviously different;
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, 0/6 or 1/6 at 12mg/kg, 3/6 or 4/6 at 18 mg/kg; while the cholesterol liposome group (C-MIT-PEG/LP group) died at 6mg/kg 1/6,9mg/kg 4/6. Indicating that the LD50 of Rg3 mitoxantrone liposomes and Rh2 mitoxantrone liposomes is between 12-18mg/kg and the LD50 of cholesterol mitoxantrone liposomes is between 6-9mg/kg, showed a significant decrease in acute toxicity of ginsenoside liposomes over cholesterol liposomes (4/6, the number 6 at/after indicates 6 total rats tested and the number at/before indicates 4 dead rats).

Claims (11)

1. The ginsenoside mitoxantrone liposome is characterized by comprising 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.
2. A ginsenoside mitoxantrone liposome of claim 1, wherein the ginsenoside mitoxantrone liposome satisfies one or more of the following conditions:
(1) The ginsenoside mitoxantrone liposome also comprises 0.1-2 parts of PEG-DSPE, preferably PEG-DSPE is PEG2000-DSPE;
(2) The mitoxantrone salt is prepared by carrying out ion exchange on mitoxantrone hydrochloride and a salt solution by a pH gradient method, wherein the salt solution is a sulfate aqueous solution, a sulfonate aqueous solution or a sucrose octasulfate aqueous solution; preferably, the salt solution is an ammonium sulfate aqueous solution, a sucrose octasulfate triethylamine aqueous solution, an ammonium methanesulfonate aqueous solution, a triethylamine methanesulfonate aqueous solution, an ammonium ethanedisulfonate aqueous solution, an ammonium propanedisulfonate aqueous solution, a triethylamine ethanedisulfonate aqueous solution or a triethylamine propanedisulfonate aqueous solution; more preferably, the salt solution is an ammonium sulfate aqueous solution, a sucrose octasulfate triethylamine aqueous solution, an ammonium methylsulfonate aqueous solution or an ethanedisulfonic acid triethylamine aqueous solution; for example, an aqueous ammonium sulfate solution;
(3) The mitoxantrone salt is mitoxantrone sulfate, sucrose octasulfate, mitoxantrone methanesulfonate, mitoxantrone methanesulfonic acid, mitoxantrone ethanedisulfonic acid, mitoxantrone propanedisulfonic acid, mitoxantrone ethanedisulfonic acid, or mitoxantrone propanedisulfonic acid; preferably, the mitoxantrone salt is mitoxantrone sulfate, sucrose octasulfate mitoxantrone, or mitoxantrone methanesulfonate; such as mitoxantrone sulfate;
(4) In the ginsenoside mitoxantrone liposome, the ginsenoside and the phospholipid form a phospholipid membrane, and preferably, the phospholipid membrane further comprises PEG-DSPE;
(5) The concentration of the salt solution is 0.05M-0.975M; e.g., 0.05M, 0.1M, 0.2M, 0.3M, 0.325M, 0.65M, 0.975M, or 0.16M;
(6) 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;
(7) The ginsenoside is one or more selected from 20 (S) -ginsenoside Rg3, 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;
(8) 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;
(9) The HPLC purity of the ginsenoside is more than or equal to 99 percent;
(10) The particle size D90 of the ginsenoside mitoxantrone liposome is less than or equal to 150nm.
3. A ginsenoside mitoxantrone liposome of claim 2, wherein the ginsenoside mitoxantrone liposome satisfies one or more of the following conditions:
(1) The mitoxantrone hydrochloride is a mitoxantrone hydrochloride aqueous solution, and preferably, the concentration of the mitoxantrone hydrochloride aqueous solution is 10mg/mL;
(2) The mass ratio of the mitoxantrone hydrochloride to the phospholipid is 1 (5-15); for example, the mass ratio of the mitoxantrone hydrochloride to the hydrogenated phospholipid is 1;
(3) The inner side of the phospholipid membrane is an inner water phase, the outer side of the phospholipid membrane is an outer water phase, and the mitoxantrone salt is encapsulated in the inner water phase; the mitoxantrone salt is a mitoxantrone salt insoluble salt; preferably, the inner aqueous phase is the saline solution, and the outer aqueous phase is a physiological isotonic solution; for example, a physiologically isotonic solution is a 5% aqueous glucose solution or a 10% aqueous sucrose solution;
(4) When the salt solution is an aqueous solution of sucrose octasulfate triethylamine, the concentration of the salt solution is 0.05M to 0.3M, such as 0.1M, 0.2M, or 0.3M;
(5) When the salt solution is the triethylamine ethanedisulfonate aqueous solution, the concentration of the salt solution is 0.16M-0.325M;
(6) When the salt solution is an ammonium methanesulfonate aqueous solution, the concentration of the salt solution is 0.325M-0.975M;
(7) When the salt solution is an aqueous ammonium sulfate solution, the concentration of the salt solution is 0.16M to 0.325M, e.g., 0.325;
(8) The mass fraction of the phospholipid is 10 parts;
(9) The mass fraction of the PEG-DSPE is 0.5 part;
(10) The mass fraction of the ginsenoside is 1 part;
(11) The mass fraction of the mitoxantrone salt is 1 part;
(12) 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.
4. A method for preparing ginsenoside mitoxantrone liposome is characterized by comprising the following steps;
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;
step 2, which is scheme 1 or scheme 2;
scheme 1: a high pressure homogenization process comprising the steps of:
carrying out high-pressure homogenization on the liposome solution A1 obtained in the step 1, and controlling the particle size D90 to be less than 100nm to obtain a liposome solution A2a;
scheme 2: an extrusion process comprising the steps of:
respectively sequentially extruding the liposome solution A1 obtained in the step 1 through extrusion plates with different apertures, and controlling the particle size D90 to be less than 100nm to obtain a liposome solution A2b;
3, putting the liposome solution A2a or A2b obtained in the step 2 into a dialysis bag, and dialyzing by taking an isotonic solution as a dialysis medium; obtaining liposome solution A3;
step 4, mixing the liposome solution A3 obtained in the step 3 with a mitoxantrone hydrochloride aqueous solution to obtain a liposome solution A4;
step 5, mixing the liposome solution A4 obtained in the step 4 with a ginsenoside solution, placing the mixture in a dialysis bag, and dialyzing by using the same isotonic solution as the isotonic solution obtained in the step 3 as a dialysis medium; to obtain the ginsenoside mitoxantrone liposome solution A5.
5. The method of preparing a ginsenoside mitoxantrone liposome of claim 4, wherein the method of preparing a ginsenoside mitoxantrone liposome satisfies one or more of the following conditions:
(1) 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;
(2) The mass ratio of the mitoxantrone hydrochloride to the phospholipid is 1 (5-15); for example, the mass ratio of the mitoxantrone hydrochloride to the hydrogenated phospholipid is 1;
(3) The ginsenoside is one or more selected from 20 (S) -ginsenoside Rg3, 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;
(4) 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;
(5) The HPLC purity of the ginsenoside is more than or equal to 99 percent;
(6) The salt solution is ammonium sulfate, sucrose octasulfate triethylamine, ammonium methylsulfonate and triethylamine methylsulfonate; aqueous solutions such as ammonium ethanedisulfonate, ammonium propanedisulfonate, triethylamine ethanedisulfonate, triethylamine propanedisulfonate, for example, aqueous ammonium sulfate solutions;
(7) The concentration of the salt solution is 0.05M-0.975M; e.g., 0.05M, 0.1M, 0.2M, 0.3M, 0.325M, 0.65M, 0.975M, or 0.16M;
more preferably, when the salt solution is an aqueous solution of sucrose octasulfate triethylamine, the concentration of the salt solution is 0.05M to 0.3M, such as 0.1M, 0.2M or 0.3M;
more preferably, when the salt solution is an aqueous solution of triethylamine ethanedisulfonate, the concentration of the salt solution is 0.16M-0.325M;
more preferably, when the salt solution is an ammonium methanesulfonate aqueous solution, the concentration of the salt solution is 0.325M-0.975M;
more preferably, when the salt solution is an aqueous ammonium sulfate solution, the concentration of the salt solution is 0.16M to 0.325M, such as 0.325;
(8) 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 a 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.
6. A method of preparing a ginsenoside mitoxantrone liposome of claim 5, wherein the method of preparing a ginsenoside mitoxantrone liposome satisfies one or more of the following conditions:
(1) In the step 1, the organic solvent is ethanol; such as absolute ethanol;
(2) 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;
(3) In the step 1, the phospholipid is heated and dissolved in an organic solvent to obtain a mixture A1; for example, the heating may be in a water bath to 55-65 ℃, e.g., 60 ℃;
(4) In step 1, the temperature of hydration may be 55-65 ℃, for example, 60 ℃;
(5) In the step 1, the hydration is carried out in a rotary evaporation bottle, and the rotating speed is 40-60 rp/min, such as 50rp/min;
(6) 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;
(7) In the scheme 1 of the step 2, the high-pressure homogenization is a cooling and cutting cycle in a homogenizer by using chilled water at the temperature of 0-10 ℃; preferably, the temperature of the liposome solution is ensured to be 5-10 ℃;
(8) In the embodiment 1 of step 2, the high-pressure homogenizing pressure is between 800 and 1400bar, such as 1200bar;
(9) In the embodiment 1 of the step 2, the number of times of the high-pressure homogenization is 3 to 4, for example, 4;
(10) In scheme 2 of step 2, the temperature of the extrusion is 35-45 ℃, for example 40 ℃;
(11) In the scheme 2 of the step 2, the pore diameter of the extrusion plate is 800nm,400nm,200nm and 100nm;
(12) In the scheme 2 of the step 2, the extrusion pressure is 600-800 psi; such as 800psi;
(13) In scheme 2 of step 2, the number of times of extrusion may be 4 to 10, for example 4;
(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 turn;
(15) In the step 3, the cut-off molecular weight of the dialysis bag is 8000-15000, for example, the cut-off molecular weight is 10000;
(16) In the step 3, the isotonic solution is 5% glucose or 10% sucrose aqueous solution;
(17) In the step 3, the volume ratio of the solution A2a or A2b to the isotonic solution is 1;
(18) In the step 3, the dialysis temperature is 0-10 ℃, for example, 4 ℃;
(19) In step 3, the dialysis is performed for a time period sufficient to completely remove the salt solution in the external aqueous phase of the liposomes of the solution A2a or A2b, preferably for a time period of 10 to 18 hours, for example 12 hours;
(20) In the step 3, in order to put the liposome solution A2a or A2b obtained in the step 2 into a dialysis bag, an isotonic solution is used as a dialysis medium, and dialysis is carried out for 12 hours at 4 ℃, wherein the volume ratio of the sample to the dialysis medium is 1:1000, changing the dialysate every 4 hours during dialysis for 1 time, completely removing acid radical ions in the external aqueous phase of the blank liposome to obtain an external aqueous phase consisting of isotonic solution, and using acid radical salt solution as the blank liposome of the internal aqueous phase;
(21) In the step 4, the concentration of the mitoxantrone hydrochloride aqueous solution is 5-20 mg/mL, such as 1mg/mL, 5mg/mL, 10mg/mL, 15mg/mL or 20mg/mL; preferably 10 to 15mg/mL;
(22) 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 inner aqueous phase of the liposome is acid radical mitoxantrone insoluble salt, and the outer aqueous phase of the liposome is isotonic solution;
(23) In the step 5, the concentration of the ginsenoside solution is 5-20 mg/mL, for example 10mg/mL;
(24) In the step 5, the solvent of the ginsenoside solution is the same as that in the step 1;
(25) In the step 5, the mixing is stirring, preferably, the stirring time is 30 to 60 minutes, such as 45 minutes;
(26) In the step 5, the cut-off molecular weight of the dialysis bag is 8000-15000, for example, the cut-off molecular weight is 10000;
(27) 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 ethanol, and then putting into a dialysis bag, taking the same isotonic solution as the step 3 as a dialysis medium, dialyzing for 12 hours at 4 ℃, wherein the volume ratio of the sample to the dialysis medium is 1:1000, changing the dialyzate every 4 hours during dialysis for 1 time, and completely removing ethanol solvent, inorganic salt, uncoated mitoxantrone hydrochloride and ginsenoside to obtain liposome solution A5;
(28) In the step 6, the mass ratio of the PEG-DSPE to the phospholipid is (0.025-0.15) 1, for example, 0.05;
(29) In the step 6, the concentration of the PEG-DSPE is 1-20mg/mL, such as 10mg/mL;
(30) In the step 6, a certain amount of PEG-DSPE is accurately weighed and dissolved in the same isotonic solution in the step 3, and then the PEG-DSPE is added into the liposome solution A5 obtained in the step 5;
(31) In step 7, the conditions and operations of the sterilizing filtration and the filling can be the conditions and operations which are conventional in the processes in the field; for example, in the sterile filtration step, the liposomes are filtered using a 0.22 μm filter; in the filling step, filling the mixture into a 10mL or 20mL penicillin bottle, capping and packaging;
(32) The particle size D90 of the ginsenoside mitoxantrone liposome is less than or equal to 150nm, and the entrapment rate is more than or equal to 80%.
7. A ginsenoside mitoxantrone liposome characterized in that it is prepared by the preparation method of any one of claims 4 to 6.
8. The ginsenoside mitoxantrone liposome is characterized in that the ginsenoside mitoxantrone liposome 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.
9. The ginsenoside mitoxantrone liposome of claim 8, wherein the ginsenoside mitoxantrone liposome satisfies one or more of the following conditions:
(1) The ginsenoside mitoxantrone liposome raw material also comprises 0.1-2 parts of PEG-DSPE, preferably, the PEG-DSPE is PEG2000-DSPE;
(2) The mitoxantrone salt is mitoxantrone salt obtained by carrying out ion exchange on mitoxantrone hydrochloride and a salt solution by a pH gradient method; 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 methanesulfonate aqueous solution, a triethylamine methanesulfonate aqueous solution, an ammonium ethanedisulfonate aqueous solution, an ammonium propanedisulfonate aqueous solution, a triethylamine ethanedisulfonate aqueous solution or a triethylamine propanedisulfonate aqueous solution; more preferably, the salt solution is an ammonium sulfate aqueous solution, a sucrose octasulfate triethylamine aqueous solution, an ammonium methylsulfonate aqueous solution or an ethanedisulfonic acid triethylamine aqueous solution; for example, aqueous ammonium sulfate;
(3) The mitoxantrone salt is mitoxantrone sulfate, sucrose octasulfate, mitoxantrone methanesulfonate, mitoxantrone methanesulfonic acid, mitoxantrone ethanedisulfonic acid, mitoxantrone propanedisulfonic acid, mitoxantrone ethanedisulfonic acid, or mitoxantrone propanedisulfonic acid; preferably, the mitoxantrone salt is mitoxantrone sulfate, sucrose octasulfate mitoxantrone, or mitoxantrone methanesulfonate; such as mitoxantrone sulfate;
(4) The concentration of the salt solution is 0.05M-0.975M; e.g., 0.05M, 0.1M, 0.2M, 0.3M, 0.325M, 0.65M, 0.975M, or 0.16M;
more preferably, when the salt solution is an aqueous solution of sucrose octasulfate triethylamine, the concentration of the salt solution is 0.05M to 0.3M, such as 0.1M, 0.2M or 0.3M;
or, when the salt solution is the triethylamine ethanedisulfonate aqueous solution, the concentration of the salt solution is 0.16M-0.325M;
or, when the salt solution is an ammonium methanesulfonate aqueous solution, the concentration of the salt solution is 0.325M-0.975M;
or, when the salt solution is an aqueous ammonium sulfate solution, the concentration of the salt solution is 0.16M to 0.325M, e.g., 0.325;
(5) The concentration of the mitoxantrone hydrochloride aqueous solution is 10mg/mL;
(6) 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;
(7) The ginsenoside is one or more selected from 20 (S) -ginsenoside Rg3, 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;
(8) 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;
(9) The HPLC purity of the ginsenoside is more than or equal to 99 percent;
(10) The mass fraction of the phospholipid is 10 parts;
(11) The mass fraction of the PEG-DSPE is 0.5 part;
(12) The mass fraction of the ginsenoside is 1 part;
(13) The mass fraction of the mitoxantrone salt is 1 part;
(14) The ginsenoside mitoxantrone liposome is prepared from the following raw materials in percentage by mass: 10 parts of phospholipid, 0.5 part of PEG-DSPE, 1 part of ginsenoside and 1 part of mitoxantrone hydrochloride.
10. A ginsenoside mitoxantrone liposome composition comprising an aqueous glucose solution and a ginsenoside mitoxantrone liposome of any of claims 1-3 or claims 7-9;
preferably, the ginsenoside mitoxantrone liposome composition meets one or two of the following conditions:
(1) The glucose aqueous solution is 5% glucose aqueous solution;
(2) The entrapment rate of the ginsenoside mitoxantrone liposome is more than or equal to 80 percent.
11. Use of a substance X for the manufacture of a medicament for the treatment and/or prevention of cancer, wherein the substance X is a ginsenoside mitoxantrone liposome as defined in any of claims 1-3 or claims 7-9 or a ginsenoside mitoxantrone liposome composition as defined in claim 10;
preferably, the application satisfies one or more of the following conditions:
(1) The particle size D90 of the ginsenoside mitoxantrone liposome or the ginsenoside mitoxantrone liposome composition in the ginsenoside mitoxantrone liposome is less than or equal to 150nm;
(2) The entrapment rate of the ginsenoside mitoxantrone liposome or the ginsenoside mitoxantrone liposome composition in 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;
(4) The cancer is breast cancer, colorectal cancer, breast cancer, primary liver cancer, gastric cancer, bladder cancer or brain tumor.
CN202110678285.4A 2021-06-18 2021-06-18 Ginsenoside mitoxantrone liposome, preparation method and application thereof Pending CN115487148A (en)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
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US20170172920A1 (en) * 2015-08-19 2017-06-22 Shanghai Ginposome Pharmatech Co., Ltd. Liposomes with ginsenoside as membrane material and preparations and use thereof
CN109833298A (en) * 2017-11-29 2019-06-04 厦门本素药业有限公司 Using Hydrolizates as novel blank liposome, preparation method and the application of membrane material
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