CN113876711A - Preparation process of doxorubicin hydrochloride liposome - Google Patents

Preparation process of doxorubicin hydrochloride liposome Download PDF

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Publication number
CN113876711A
CN113876711A CN202010630933.4A CN202010630933A CN113876711A CN 113876711 A CN113876711 A CN 113876711A CN 202010630933 A CN202010630933 A CN 202010630933A CN 113876711 A CN113876711 A CN 113876711A
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liposome
ammonium sulfate
phase
drug
doxorubicin hydrochloride
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蒋志君
孙薇
曹青日
秦飞
陆小娟
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Jiang Su Pharmamaxcorp Co ltd
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Jiang Su Pharmamaxcorp Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A61K9/1278—Post-loading, e.g. by ion or pH gradient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • 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
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Abstract

The invention relates to the field of pharmaceutical preparations, in particular to a preparation process of doxorubicin hydrochloride liposome, which comprises the following steps: (a) adjusting the pH value of the ammonium sulfate aqueous solution to prepare blank liposome; (b) removing the blank liposome external aqueous phase ammonium sulfate, and adding a pH penetrating agent; (c) preparing a drug-loaded liposome by active drug loading; (d) adjusting the pH value of the external water phase of the drug-loaded liposome. The invention further enlarges the gradient difference of the pH of the internal phase and the external phase of the liposome by adjusting the pH of the internal phase and the external phase of the liposome and adding the pH penetrant, improves the encapsulation efficiency of the liposome, and simultaneously ensures the stability of the medicament and the phospholipid by the pH of the neutral external phase of the liposome.

Description

Preparation process of doxorubicin hydrochloride liposome
Technical Field
The invention relates to the field of pharmaceutical preparations, in particular to a preparation process of doxorubicin hydrochloride liposome.
Technical Field
Doxorubicin hydrochloride is a wide-spectrum antitumor agent containing anthrax rings. The doxorubicin hydrochloride can generate wide biochemical effects on organisms and has strong cytotoxicity. The traditional Chinese medicine composition is clinically used for acute leukemia, Hodgkin's disease, malignant lymphoma, breast cancer, rhabdomyosarcoma, Ewing sarcoma, nephroblastoma, neuroblastoma, bladder cancer and the like, and is a first-line medicine for liver cancer chemotherapy.
The liposome serving as a drug carrier has the advantages of targeting the drug to a reticuloendothelial system, prolonging the drug effect, reducing the drug toxicity, improving the curative effect and the like. The research of using liposome as the carrier of anthracycline anticancer drugs is started at the end of the last 70 th century, and clinical tests are started at the end of the 80 th century, and preliminary results show that after doxorubicin is encapsulated by the liposome, the dosage can be increased, the cardiotoxicity effect is reduced, the half-life period is obviously prolonged, and the anticancer effect is enhanced or not weakened. The first anticancer liposome in the world-aclarubicin liposome
Figure BDA0002565041110000011
FDA approval was obtained in the united states by the end of 1995.
The pH gradient method is a method in which, during the operation, phospholipids are first hydrated with a lower or higher pH buffer to prepare liposomes, and then the above-mentioned liposome external aqueous phase is adjusted or replaced with a higher or lower pH buffer to a desired pH value, thereby causing a difference in pH value between the liposome internal and external aqueous phases to generate a pH gradient. The liposomes are generally prepared by hydrating phospholipids with 0.3mol/L citrate buffer, pH4.0, using NaOH or Na2CO3Adjusting the pH value to 7.0-7.8.
The ammonium sulfate gradient method comprises preparing blank liposome with aqueous solution of ammonium sulfate as water phase and phospholipid, wherein ammonium sulfate exists in the liposome bilayer membrane, and ammonium ion (NH) is formed by dissociation of ammonium sulfate molecule4+) Can be further dissociated into ammonia molecules (NH)3) And hydrogen ion (H)+). Due to NH3The transmembrane rate and permeability coefficient are much greater than those of SO4 2-Therefore, it is only necessary to remove ammonium sulfate from the aqueous phase outside the liposomeCan form ammonium sulfate concentration gradient of inner and outer water phases of liposome, and NH is generated by using the concentration gradient as driving force3The molecules can diffuse out of the liposome internal aqueous phase into the liposome external aqueous phase. At the same time, with NH3The molecules diffuse outward, and a corresponding large amount of H begins to accumulate in the internal water phase of the liposome+Thereby establishing a pH gradient outside the liposome. According to the principle of ionic equilibrium between the inside and outside of the membrane, the presence of two gradients promotes the preferential loading of weakly basic drugs present in the external aqueous phase into the lipophilic liposomes in a reversed gradient. Compared with the common pH gradient method and the passive drug loading method, the ammonium sulfate gradient method can prepare the liposome of the alkalescent drug such as the anthracycline drug with higher entrapment rate. The principle of the ammonium sulfate gradient method can find that the pH gradient difference of the liposome internal and external phases has very important influence on the liposome encapsulation efficiency. However, most of the prior patents simply use a pH gradient method or an ammonium sulfate gradient method to prepare doxorubicin hydrochloride liposome, and few of the prior patents specially aim at accurately adjusting the pH of the liposome in internal and external phases during the preparation of the doxorubicin hydrochloride liposome by the ammonium sulfate gradient method.
Disclosure of Invention
The invention provides a preparation process of doxorubicin hydrochloride liposome, which not only utilizes an ammonium sulfate gradient method to ensure the encapsulation efficiency of the liposome, but also further improves the encapsulation efficiency of the liposome and ensures the stability of the liposome by accurately adjusting the pH of the internal phase and the external phase of the liposome and adding a pH penetrating agent.
A preparation process of doxorubicin hydrochloride liposome comprises the following steps:
(a) adjusting the pH of the ammonium sulfate aqueous solution to prepare blank liposome:
weighing a prescription amount of liposome material, and adding an organic solvent to dissolve the liposome material to obtain an oil phase; preparing an acidic ammonium sulfate aqueous solution as a water phase; mixing water phase and oil phase to prepare blank liposome, removing organic solvent under normal pressure or reduced pressure, and performing incubation and intermittent ultrasound on the liposome;
(b) removing the blank liposome external water phase ammonium sulfate, adding a pH penetrating agent:
removing the blank liposome external aqueous phase ammonium sulfate by dialysis or ultrafiltration; then adding a pH penetrant into the blank liposome according to a certain proportion;
(c) preparing a drug-loaded liposome by active drug loading:
adding a drug aqueous solution according to a certain drug-to-lipid ratio to carry out active drug loading;
(d) adjusting the pH of the external water phase of the drug-loaded liposome:
adjusting the pH of the external water phase of the drug-loaded liposome and incubating.
The research shows that the smaller the pH value of the aqueous phase in the liposome, the higher the entrapment rate; the closer the pH of the liposome external aqueous phase is to neutrality, the more stable the drug and phospholipid, and the smaller the liposome particle size. We have also found that the effect of the concentration of aqueous ammonium sulphate solution on the encapsulation efficiency is significant, basically the higher the concentration of aqueous ammonium sulphate solution, the higher the encapsulation efficiency.
Further, in the preparation process of the doxorubicin hydrochloride liposome, the liposome material in the step (a) is hydrogenated soybean lecithin, cholesterol and DSPE-mPEG 2000.
Further, in the preparation process of the doxorubicin hydrochloride liposome, the concentration of the ammonium sulfate aqueous solution in the step (a) is 1.6-33 mg/ml, and the pH value of the adjusted ammonium sulfate aqueous solution is 5.0-6.5.
Further, in the above preparation process of doxorubicin hydrochloride liposome, the mixing ratio (v/v) of the water phase and the oil phase in step (a) is 5: 1-1: and 5, mixing the water phase and the oil phase at the temperature of 50-70 ℃.
Further, in the preparation process of the doxorubicin hydrochloride liposome, the intermittent ultrasonic time in the step (a) is 0-120 min.
Further, in the preparation process of the doxorubicin hydrochloride liposome, the cut-off molecular weight of the dialysis bag in the step (b) is 3.5-14 kd.
Further, in the preparation process of the doxorubicin hydrochloride liposome, the molecular weight cut-off of the ultrafiltration tube in the step (b) is 3-100 kd, and the rotation speed of a centrifugal machine is 7000-20000 rpm/min.
Further, in the above preparation process of doxorubicin hydrochloride liposome, the pH penetrant in step (b) is selected from one or more of disodium hydrogen phosphate, histidine and sucrose, and the mixing ratio (v/v) of the blank liposome and the pH penetrant is 2: 1-1: 2.
further, in the above process for preparing doxorubicin hydrochloride liposome, the ratio of drug to lipid in step (c), i.e., the ratio (v/v) of the drug aqueous solution to the blank liposome after the addition of the pH penetrant, is 5: 1-1: 9.
further, in the preparation process of the doxorubicin hydrochloride liposome, the pH of the external water phase of the adjusted drug-loaded liposome in the step (d) is 6.0-9.0.
Further, the preparation process of the doxorubicin hydrochloride liposome comprises the following specific steps:
(a) adjusting the pH of the ammonium sulfate aqueous solution to prepare blank liposome:
weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml absolute ethyl alcohol, heating at 60 deg.C to dissolve to obtain oil phase; preparing 2ml of 33mg/ml ammonium sulfate buffer solution (pH5.0) as a water phase; mixing the water phase and the oil phase, evaporating under reduced pressure to remove anhydrous ethanol, incubating in 60 deg.C water bath for 20min, and performing intermittent ultrasonic treatment for 35min to obtain blank liposome;
(b) removing the blank liposome external water phase ammonium sulfate, adding a pH penetrating agent:
removing the external aqueous phase ammonium sulfate of the liposome by ultrafiltration in a 50kd ultrafiltration tube at 15000 rpm; 50mmol/L histidine +7% sucrose as pH penetrant, according to a 1: 1(v/v) adding a pH penetrant into the blank liposome;
(c) preparing a drug-loaded liposome by active drug loading:
according to the medicine-fat ratio of 1: 3(v/v) dropwise adding 8mg/ml DOX & HCl aqueous solution to carry out active drug loading;
(d) adjusting the pH of the external water phase of the drug-loaded liposome:
adjusting pH of the liposome external water phase to 7.0 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain doxorubicin hydrochloride liposome, and storing in refrigerator at 4 deg.C in dark place.
Furthermore, the doxorubicin hydrochloride liposome is prepared by the preparation process of the doxorubicin hydrochloride liposome.
Furthermore, the preparation process of the doxorubicin hydrochloride liposome is applied to preparation of the doxorubicin hydrochloride liposome injection.
According to the scheme, the invention discloses a preparation process of doxorubicin hydrochloride liposome, which at least has the following beneficial effects: the preparation method further enlarges the gradient difference of the pH of the internal phase and the external phase of the liposome by adjusting the pH of the internal phase and the external phase of the liposome and adding the pH penetrant, improves the encapsulation efficiency of the liposome, ensures the stability of the medicament and the phospholipid by the pH of the external phase of the neutral liposome, has reasonable process steps, mild and controllable conditions, low production cost and good repeatability, can finally prepare the doxorubicin hydrochloride liposome with the encapsulation efficiency of 98 percent, the particle size of about 120nm and far exceeding the quality standard of pharmacopoeia, can be widely applied to the pharmaceutical field, and is particularly applied to the research, production and preparation of the doxorubicin hydrochloride liposome injection.
Drawings
FIG. 1 is a graph of particle size and encapsulation efficiency profiles for examples 28, 29 and 30;
FIG. 2 is a graph showing particle size and encapsulation efficiency profiles of examples 31, 32 and 33;
FIG. 3 is a graph of particle size and encapsulation efficiency profiles for examples 48, 49 and 50;
FIG. 4 is a diagram of the finished product of doxorubicin hydrochloride liposome.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Example 1
HSPC 38.3mg, CHO-HP 12.7mg and DSPE-mPEG200012.7mg are weighed respectively, 1ml of absolute ethyl alcohol is added, the mixture is heated to 40 ℃ for dissolution, and then the weight ratio of the mixture is determined according to the following formula 1: adding 1ml 250mmol/L ammonium sulfate buffer solution (pH5.5) at a ratio of 1(v/v), ventilating to volatilize and remove organic solvent, incubating in 60 deg.C water bath for 20min, and intermittently performing ultrasonic treatmentAnd (3) 30 min. According to the medicine-fat ratio of 3: 1(v/v) 0.5ml of the above blank liposome was added dropwise to 1.5ml of 2.7mg/ml DOX. HCl solution and Na was added2HPO4Adjusting pH of external water phase of liposome to 7.0, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 2
HSPC 38.3mg, CHO-HP 12.7mg and DSPE-mPEG200012.7mg are weighed respectively, 1ml of absolute ethyl alcohol is added, the mixture is heated to 40 ℃ for dissolution, and then the weight ratio of the mixture is determined according to the following formula 1: 1ml of 250mmol/L ammonium sulfate buffer solution (pH5.5) was added at a ratio of 1(v/v), the organic solvent was removed by evaporation under ventilation, and the mixture was incubated in a water bath at 60 ℃ for 20min and subjected to intermittent sonication for 35 min. According to the medicine-fat ratio of 3: 1(v/v) 0.5ml of the above blank liposome was added dropwise to 1.5ml of 2.7mg/ml DOX. HCl solution and Na was added2HPO4Adjusting pH of external water phase of liposome to 7.0, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 3
HSPC 38.3mg, CHO-HP 12.7mg and DSPE-mPEG200012.7mg are weighed respectively, 1ml of absolute ethyl alcohol is added, the mixture is heated to 40 ℃ for dissolution, and then the weight ratio of the mixture is determined according to the following formula 1: 1ml of 250mmol/L ammonium sulfate buffer solution (pH5.5) was added at a ratio of 1(v/v), the organic solvent was removed by evaporation under ventilation, and the mixture was incubated in a water bath at 60 ℃ for 20min with intermittent sonication for 0 min. According to the medicine-fat ratio of 3: 1(v/v) 0.5ml of the above blank liposome was added dropwise to 1.5ml of 2.7mg/ml DOX. HCl solution and Na was added2HPO4Adjusting pH of external water phase of liposome to 7.0, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 4
Respectively weighing 76.6mg of HSPC, 25.4mg of CHO-HP and 200025.4mg of DSPE-mPEG, adding 2ml of absolute ethyl alcohol, heating to 40 ℃ for dissolution, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) 2ml of 2.4mg/ml ammonium sulfate buffer (pH5.5) was added, the organic solvent was removed by evaporation under ventilation, incubated in a water bath at 60 ℃ for 20min and sonicated intermittently for 35 min. According to the medicine-fat ratio of 3: 1(v/v) 1ml of the above blank liposome was added dropwise to 3ml of 2.7mg/ml DOX. HCl solution and Na was added2HPO4Adjusting pH of external water phase of liposome to 7.0, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark placeAnd (4) storing.
Example 5
Respectively weighing 76.6mg of HSPC, 25.4mg of CHO-HP and 200025.4mg of DSPE-mPEG, adding 2ml of absolute ethyl alcohol, heating to 40 ℃ for dissolution, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) 2ml of 2.4mg/ml ammonium sulfate buffer (pH5.5) was added, the organic solvent was removed by evaporation under ventilation, incubated in a water bath at 60 ℃ for 20min and sonicated intermittently for 35 min. According to the medicine-fat ratio of 3: 1(v/v) 1ml of the above blank liposome was added dropwise to 3ml of 2.7mg/ml DOX. HCl solution and Na was added2HPO4Adjusting pH of external water phase of liposome to 7.0, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 6
Respectively weighing 76.6mg of HSPC, 25.4mg of CHO-HP and 200025.4mg of DSPE-mPEG, adding 2ml of absolute ethyl alcohol, heating to 40 ℃ for dissolution, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) 2ml of 2.4mg/ml ammonium sulfate buffer (pH5.5) was added, the organic solvent was removed by evaporation under ventilation, incubated in a water bath at 60 ℃ for 20min and sonicated intermittently for 35 min. Taking 10mmol/L histidine + 5% sucrose as a solvent of a DOX & HCl drug solution, and mixing the components according to a drug-to-lipid ratio of 3: 1(v/v) taking 1ml of the blank liposome, dropwise adding 3ml of 2.7mg/ml DOX & HCl solution, adjusting the pH value of the external water phase of the liposome to 7.0 by using hydrochloric acid and sodium hydroxide, incubating for 20min at 55 ℃ in a water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in a dark place.
Example 7
Respectively weighing 76.6mg of HSPC, 25.4mg of CHO-HP and 200025.4mg of DSPE-mPEG, adding 2ml of absolute ethyl alcohol, heating to 40 ℃ for dissolution, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) 2ml of 2.4mg/ml ammonium sulfate buffer (pH5.5) was added, the organic solvent was removed by evaporation under ventilation, incubated in a water bath at 60 ℃ for 20min and sonicated intermittently for 35 min. Taking 50mmol/L histidine +7% sucrose as a solvent of a DOX & HCl drug solution, and mixing the components according to a drug-to-lipid ratio of 3: 1(v/v) taking 1ml of the blank liposome, dropwise adding 3ml of 2.7mg/ml DOX & HCl solution, adjusting the pH value of the external water phase of the liposome to 7.0 by using hydrochloric acid and sodium hydroxide, incubating for 20min at 55 ℃ in a water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in a dark place.
Example 8
Respectively weighing 76.6mg of HSPC, 25.4mg of CHO-HP and 200025.4mg of DSPE-mPEG, adding 2ml of absolute ethyl alcohol, heating to 40 ℃ for dissolution, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) 2ml of 2.4mg/ml ammonium sulfate buffer (pH5.5) was added, the organic solvent was removed by evaporation under ventilation, incubated in a water bath at 60 ℃ for 20min and sonicated intermittently for 35 min. Taking 2mmol/L histidine +7% sucrose as a solvent of a DOX & HCl drug solution, and mixing the components according to a drug-to-lipid ratio of 3: 1(v/v) taking 1ml of the blank liposome, dropwise adding 3ml of 2.7mg/ml DOX & HCl solution, adjusting the pH value of the external water phase of the liposome to 7.0 by using hydrochloric acid and sodium hydroxide, incubating for 20min at 55 ℃ in a water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in a dark place.
Example 9
Respectively weighing HSPC 115.0mg, CHO-HP38.3 mg and DSPE-mPEG200038.3mg, adding 1ml of absolute ethyl alcohol, heating to 40 ℃ for dissolution, and then mixing the materials according to a water-oil ratio of 3: 1(v/v) 3ml of 2.4mg/ml ammonium sulfate buffer (pH5.5) was added, the organic solvent was removed by evaporation under ventilation, incubated in a water bath at 60 ℃ for 20min and sonicated intermittently for 35 min. Taking 10mmol/L histidine +7% sucrose as a solvent of a DOX & HCl drug solution, and mixing the components according to a drug-to-lipid ratio of 3: 1(v/v) taking 1ml of the blank liposome, dropwise adding 3ml of 2.7mg/ml DOX & HCl solution, adjusting the pH value of the external water phase of the liposome to 7.0 by using hydrochloric acid and sodium hydroxide, incubating for 20min at 55 ℃ in a water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in a dark place.
Example 10
Respectively weighing 191.6mg of HSPC, 63.8mg of CHO-HP and 8mg of DSPE-mPEG200063.8mg, adding 1ml of absolute ethyl alcohol, heating to 40 ℃ for dissolution, and then, according to the water-oil ratio of 5: 1(v/v) 5ml of 2.4mg/ml ammonium sulfate buffer (pH5.5) was added, the organic solvent was removed by evaporation under ventilation, incubated in a water bath at 60 ℃ for 20min and sonicated intermittently for 35 min. Taking 10mmol/L histidine +7% sucrose as a solvent of a DOX & HCl drug solution, and mixing the components according to a drug-to-lipid ratio of 3: 1(v/v) taking 1ml of the blank liposome, dropwise adding 3ml of 2.7mg/ml DOX & HCl solution, adjusting the pH value of the external water phase of the liposome to 7.0 by using hydrochloric acid and sodium hydroxide, incubating for 20min at 55 ℃ in a water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in a dark place.
Example 11
Respectively weighing HSPC 287.4mg, CHO-HP 95.7mg and DSPE-mPEG200095.7mg, adding 1ml absolute ethyl alcohol, heating to 65 deg.C for dissolving, and mixing according to a water-oil ratio of 5: 1(v/v) 5ml of 3.6mg/ml ammonium sulfate buffer (pH5.5) preheated to 65 ℃ was added, the organic solvent was removed by evaporation under ventilation, incubated in a water bath at 60 ℃ for 20min, and subjected to intermittent sonication for 60 min. Taking 10mmol/L histidine as a solvent of a DOX & HCl drug solution, and mixing the raw materials according to a drug-to-lipid ratio of 5: 1(v/v) taking 1ml of the blank liposome, dropwise adding 5ml of 2.4mg/ml DOX & HCl solution, adjusting the pH value of the external water phase of the liposome to 7.0 by using hydrochloric acid and sodium hydroxide, incubating for 20min at 55 ℃ in a water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in a dark place.
Example 12
Respectively weighing HSPC 287.4mg, CHO-HP 95.7mg and DSPE-mPEG200095.7mg, adding 1ml absolute ethyl alcohol, heating to 40 deg.C for dissolving, and mixing at a water-oil ratio of 5: 1(v/v) 5ml of 3.6mg/ml ammonium sulfate buffer (pH5.5) was added, the organic solvent was removed by evaporation under ventilation, incubated in a water bath at 60 ℃ for 20min and sonicated intermittently for 35 min. 1mol/L disodium hydrogen phosphate with the pH adjusted to 7.0 is used as a solvent of DOX & HCl medicine solution, and the weight ratio of medicine to fat is 5: 1(v/v) taking 1ml of the blank liposome, dropwise adding 5ml of 2.4mg/ml DOX & HCl solution, adjusting the pH value of the external water phase of the liposome to 7.0 by using hydrochloric acid and sodium hydroxide, incubating for 20min at 55 ℃ in a water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in a dark place.
Example 13
Respectively weighing HSPC 115.0mg, CHO-HP38.3 mg and DSPE-mPEG200038.3mg, adding 1ml of absolute ethyl alcohol, heating to 40 ℃ for dissolution, and then mixing the materials according to a water-oil ratio of 3: 1(v/v) 3ml of 2.4mg/ml ammonium sulfate buffer (pH5.5) was added, the organic solvent was removed by evaporation under ventilation, incubated in a water bath at 60 ℃ for 20min and sonicated intermittently for 35 min. Taking 10mmol/L histidine +7% sucrose as a solvent of a DOX & HCl drug solution, and mixing the components according to a drug-to-lipid ratio of 3: 1(v/v) taking 1ml of the blank liposome, dropwise adding 3ml of 2.7mg/ml DOX & HCl solution, adjusting the pH value of the external water phase of the liposome to 7.0 by using hydrochloric acid and sodium hydroxide, incubating for 20min at 55 ℃ in a water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in a dark place.
Example 14
Respectively weighing 191.6mg of HSPC, 63.8mg of CHO-HP and 8mg of DSPE-mPEG200063.8mg, adding 1ml of absolute ethyl alcohol, heating to 40 ℃ for dissolution, and then, according to the water-oil ratio of 5: 1(v/v) 5ml of 2.4mg/ml ammonium sulfate buffer (pH5.5) was added, the organic solvent was removed by evaporation under ventilation, incubated in a water bath at 60 ℃ for 20min and sonicated intermittently for 35 min. Taking 10mmol/L histidine +7% sucrose as a solvent of a DOX & HCl drug solution, and mixing the components according to a drug-to-lipid ratio of 3: 1(v/v) taking 1ml of the blank liposome, dropwise adding 3ml of 2.7mg/ml DOX & HCl solution, adjusting the pH value of the external water phase of the liposome to 7.0 by using hydrochloric acid and sodium hydroxide, incubating for 20min at 55 ℃ in a water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in a dark place.
Example 15
Respectively weighing HSPC 57.5mg, CHO-HP 19.1mg and DSPE-mPEG200019.1mg, adding 1ml of absolute ethyl alcohol, heating to 40 ℃ for dissolution, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) 1ml of 3.6mg/ml ammonium sulfate buffer (pH5.5) was added, the organic solvent was removed by evaporation under ventilation, incubated in a water bath at 60 ℃ for 20min and sonicated intermittently for 35 min. Taking 10mmol/L histidine +7% sucrose as a solvent of a DOX & HCl drug solution, and mixing the components according to a drug-to-lipid ratio of 5: 1(v/v) taking 1ml of the blank liposome, dropwise adding 5ml of 2.4mg/ml DOX & HCl solution, adjusting the pH value of the external water phase of the liposome to 7.0 by using hydrochloric acid and sodium hydroxide, incubating for 20min at 55 ℃ in a water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in a dark place.
Example 16
Respectively weighing HSPC 38.3mg, CHO-HP 12.7mg and DSPE-mPEG200012.7mg, adding 1ml absolute ethyl alcohol, heating to 40 ℃ for dissolution, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) 1ml of 2.4mg/ml ammonium sulfate buffer (pH5.5) was added, the organic solvent was removed by evaporation under ventilation, incubated in a water bath at 60 ℃ for 20min and sonicated intermittently for 35 min. Taking 2mmol/L histidine +7% sucrose as a solvent of a DOX & HCl drug solution, and mixing the components according to a drug-to-lipid ratio of 3: 1(v/v) taking 1ml of the blank liposome, dropwise adding 3ml of 2.7mg/ml DOX & HCl solution, adjusting the pH value of the external water phase of the liposome to 7.0 by using hydrochloric acid and sodium hydroxide, incubating for 20min at 55 ℃ in a water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in a dark place.
Example 17
Respectively weighing HSPC 38.3mg, CHO-HP 12.7mg and DSPE-mPEG200012.7mg, adding 1ml absolute ethyl alcohol, heating to 65 ℃ for dissolution, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) 1ml of 2.4mg/ml ammonium sulphate buffer (pH5.5) was added, the organic solvent was removed by rotary evaporation under ventilation, incubated in a water bath at 65 ℃ for 20min and sonicated intermittently for 35 min. Taking 2mmol/L histidine +7% sucrose as a solvent of a DOX & HCl drug solution, and mixing the components according to a drug-to-lipid ratio of 3: 1(v/v) taking 1ml of the blank liposome, dropwise adding 3ml of 2.7mg/ml DOX & HCl solution, adjusting the pH value of the external water phase of the liposome to 7.0 by using hydrochloric acid and sodium hydroxide, incubating for 20min in a water bath at 65 ℃ to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in a dark place.
Example 18
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, carrying out ultrasonic treatment until the mixture is dissolved, and then carrying out ultrasonic treatment according to the water-oil ratio of 1: 1(v/v) 2ml of 1.6mg/ml ammonium sulfate buffer (pH5.7) was added, the organic solvent was removed by rotary evaporation, incubated in a water bath at 60 ℃ for 20min and sonicated intermittently for 35 min. 1mol/L disodium hydrogen phosphate is taken as a pH penetrant, and the pH penetrant is prepared according to the weight ratio of 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) adding 8mg/ml DOX & HCl aqueous solution dropwise, incubating for 20min at 55 ℃ in water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in the dark.
Example 19
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, carrying out ultrasonic treatment until the mixture is dissolved, and then carrying out ultrasonic treatment according to the water-oil ratio of 1: 1(v/v) 2ml of 2.4mg/ml ammonium sulphate buffer (pH5.6) was added, the organic solvent was removed by rotary evaporation, incubated in a water bath at 60 ℃ for 20min and sonicated intermittently for 35 min. 10mmol/L histidine +7% sucrose as pH penetrant, according to a 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) adding 8mg/ml DOX & HCl aqueous solution dropwise, incubating for 20min at 55 ℃ in water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in the dark.
Example 20
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, carrying out ultrasonic treatment until the mixture is dissolved, and then carrying out ultrasonic treatment according to the water-oil ratio of 1: 1(v/v) 2ml of 2.4mg/ml ammonium sulphate buffer (pH5.6) was added, the organic solvent was removed by rotary evaporation, incubated in a water bath at 60 ℃ for 20min and sonicated intermittently for 35 min. 50mmol/L histidine +7% sucrose as pH penetrant, according to a 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) adding 8mg/ml DOX & HCl aqueous solution dropwise, incubating for 20min at 55 ℃ in water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in the dark.
Example 21
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, carrying out ultrasonic treatment until the mixture is dissolved, and then carrying out ultrasonic treatment according to the water-oil ratio of 1: 1(v/v) 2ml of 2.4mg/ml ammonium sulphate buffer (pH5.6) was added, the organic solvent was removed by rotary evaporation, incubated in a water bath at 60 ℃ for 20min and sonicated intermittently for 35 min. 100mmol/L histidine +7% sucrose as pH penetrant, according to a 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) adding 8mg/ml DOX & HCl aqueous solution dropwise, incubating for 20min at 55 ℃ in water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in the dark.
Example 22
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, carrying out ultrasonic treatment until the mixture is dissolved, and then carrying out ultrasonic treatment according to the water-oil ratio of 1: 1(v/v) 2ml of 2.4mg/ml ammonium sulphate buffer (pH5.6) was added, the organic solvent was removed by rotary evaporation, incubated in a water bath at 60 ℃ for 20min and sonicated intermittently for 35 min. 40mmol/L histidine +7% sucrose as pH penetrant, according to a 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) adding 8mg/ml DOX & HCl aqueous solution dropwise, incubating for 20min at 55 ℃ in water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in the dark.
Example 23
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, carrying out ultrasonic treatment until the mixture is dissolved, and then carrying out ultrasonic treatment according to the water-oil ratio of 1: 1(v/v) 2ml of 2.4mg/ml ammonium sulphate buffer (pH5.6) was added, the organic solvent was removed by rotary evaporation, incubated in a water bath at 60 ℃ for 20min and sonicated intermittently for 35 min. 50mmol/L histidine +7% sucrose as pH penetrant, according to a 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) adding 8mg/ml DOX & HCl aqueous solution dropwise, incubating for 20min at 55 ℃ in water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in the dark.
Example 24
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, carrying out ultrasonic treatment until the mixture is dissolved, and then carrying out ultrasonic treatment according to the water-oil ratio of 1: 1(v/v) 2ml of 2.4mg/ml ammonium sulphate buffer (pH5.6) was added, the organic solvent was removed by rotary evaporation, incubated in a water bath at 60 ℃ for 20min and sonicated intermittently for 35 min. 60mmol/L histidine +7% sucrose as pH penetrant, according to a 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) adding 8mg/ml DOX & HCl aqueous solution dropwise, incubating for 20min at 55 ℃ in water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in the dark.
Example 25
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, carrying out ultrasonic treatment until the mixture is dissolved, and then carrying out ultrasonic treatment according to the water-oil ratio of 1: 1(v/v) 2ml of 2.4mg/ml ammonium sulfate buffer (pH5.5) was added, the organic solvent was removed by evaporation under reduced pressure, incubated in a water bath at 60 ℃ for 20min and sonicated intermittently for 35 min. 50mmol/L histidine +7% sucrose as pH penetrant, according to a 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) adding 8mg/ml DOX & HCl aqueous solution dropwise, incubating for 20min at 55 ℃ in water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in the dark.
Example 26
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, carrying out ultrasonic treatment until the mixture is dissolved, and then carrying out ultrasonic treatment according to the water-oil ratio of 1: 1(v/v) adding 2ml 2.4mg/ml ammonium sulfate buffer solution (pH5.5), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and ultrafiltering to remove ammonium sulfate in liposome external water phase. 50mmol/L histidine +7% sucrose as pH penetrant, according to a 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) adding 8mg/ml DOX & HCl aqueous solution dropwise, incubating for 20min at 55 ℃ in water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in the dark.
Example 27
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml of 1.6mg/ml ammonium sulfate buffer solution (pH6.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and dialyzing to remove ammonium sulfate in liposome external water phase. Taking 50mmol/L histidine as a pH penetrating agent, and mixing the components in a ratio of 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) adding 8mg/ml DOX & HCl aqueous solution dropwise, incubating for 20min at 55 ℃ in water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in the dark.
Example 28
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml of 1.6mg/ml ammonium sulfate buffer solution (pH5.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and dialyzing to remove ammonium sulfate in liposome external water phase. Taking 50mmol/L histidine as a pH penetrating agent, and mixing the components in a ratio of 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) adding 8mg/ml DOX & HCl aqueous solution dropwise, incubating for 20min at 55 ℃ in water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in the dark.
Example 29
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml of 1.6mg/ml ammonium sulfate buffer solution (pH5.5), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and dialyzing to remove ammonium sulfate in liposome external water phase. Taking 50mmol/L histidine as a pH penetrating agent, and mixing the components in a ratio of 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) adding 8mg/ml DOX & HCl aqueous solution dropwise, incubating for 20min at 55 ℃ in water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in the dark.
Example 30
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml of 1.6mg/ml ammonium sulfate buffer solution (pH5.6), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and dialyzing to remove ammonium sulfate in liposome external water phase. Taking 50mmol/L histidine as a pH penetrating agent, and mixing the components in a ratio of 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) adding 8mg/ml DOX & HCl aqueous solution dropwise, incubating for 20min at 55 ℃ in water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in the dark.
Example 31
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml of 1.6mg/ml ammonium sulfate buffer solution (pH5.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and dialyzing to remove ammonium sulfate in liposome external water phase. Taking 50mmol/L histidine as a pH penetrating agent, and mixing the components in a ratio of 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) dropwise adding 8mg/ml DOX & HCl aqueous solution, adjusting pH of liposome external water phase to 7.0 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 32
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml of 1.6mg/ml ammonium sulfate buffer solution (pH5.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and dialyzing to remove ammonium sulfate in liposome external water phase. Taking 50mmol/L histidine as a pH penetrating agent, and mixing the components in a ratio of 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) dropwise adding 8mg/ml DOX & HCl aqueous solution, adjusting pH of liposome external water phase to 7.5 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 33
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml of 1.6mg/ml ammonium sulfate buffer solution (pH5.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and dialyzing to remove ammonium sulfate in liposome external water phase. Taking 50mmol/L histidine as a pH penetrating agent, and mixing the components in a ratio of 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) dropwise adding 8mg/ml DOX & HCl aqueous solution, adjusting pH of liposome external water phase to 7.9 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 34
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml of 1.6mg/ml ammonium sulfate buffer solution (pH5.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and dialyzing to remove ammonium sulfate in liposome external water phase. Taking 2mmol/L histidine as a pH penetrating agent, and performing reaction according to the following reaction conditions of 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) adding 8mg/ml DOX & HCl aqueous solution dropwise, incubating for 20min at 55 ℃ in water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in the dark.
Example 35
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml of 1.6mg/ml ammonium sulfate buffer solution (pH5.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and dialyzing to remove ammonium sulfate in liposome external water phase. Taking 10mmol/L histidine as a pH penetrating agent, and mixing the components in a ratio of 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) adding 8mg/ml DOX & HCl aqueous solution dropwise, incubating for 20min at 55 ℃ in water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in the dark.
Example 36
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml of 1.6mg/ml ammonium sulfate buffer solution (pH5.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and dialyzing to remove ammonium sulfate in liposome external water phase. Taking 100mmol/L histidine as a pH penetrating agent, and mixing the components in a ratio of 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) adding 8mg/ml DOX & HCl aqueous solution dropwise, incubating for 20min at 55 ℃ in water bath to obtain DOX-Liposomes-PEG, and storing in a refrigerator at 4 ℃ in the dark.
Example 37
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, carrying out ultrasonic treatment until the mixture is dissolved, and then carrying out ultrasonic treatment according to the water-oil ratio of 1: 1(v/v) adding 2ml 2.4mg/ml ammonium sulfate buffer solution (pH5.5), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 50min, and ultrafiltering to remove ammonium sulfate in liposome external water phase. 50mmol/L histidine +7% sucrose as pH penetrant, according to a 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) dropwise adding 8mg/ml DOX & HCl aqueous solution, adjusting pH of liposome external water phase to 7.0 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 38
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml of 1.6mg/ml ammonium sulfate buffer solution (pH5.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and dialyzing to remove ammonium sulfate in liposome external water phase. Taking 2mmol/L histidine as a pH penetrating agent, and performing reaction according to the following reaction conditions of 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) dropwise adding 8mg/ml DOX & HCl aqueous solution, adjusting pH of liposome external water phase to 7.0 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 39
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml of 1.6mg/ml ammonium sulfate buffer solution (pH5.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and dialyzing to remove ammonium sulfate in liposome external water phase. Taking 10mmol/L histidine as a pH penetrating agent, and mixing the components in a ratio of 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) dropwise adding 8mg/ml DOX & HCl aqueous solution, adjusting pH of liposome external water phase to 7.0 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 40
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml of 1.6mg/ml ammonium sulfate buffer solution (pH5.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and dialyzing to remove ammonium sulfate in liposome external water phase. Taking 100mmol/L histidine as a pH penetrating agent, and mixing the components in a ratio of 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) dropwise adding 8mg/ml DOX & HCl aqueous solution, adjusting pH of liposome external water phase to 7.0 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
EXAMPLE 41
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, performing ultrasonic treatment at 60 ℃ until the mixture is dissolved, and then performing ultrasonic treatment according to a water-oil ratio of 1: 1(v/v) adding 2ml 2.4mg/ml ammonium sulfate buffer solution (pH5.5), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and dialyzing to remove ammonium sulfate in liposome external water phase. 100mmol/L histidine +7% sucrose as pH penetrant, according to a 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) dropwise adding 8mg/ml DOX & HCl aqueous solution, adjusting pH of liposome external water phase to 7.0 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 42
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml 2.4mg/ml ammonium sulfate buffer solution (pH5.5), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and dialyzing to remove ammonium sulfate in liposome external water phase. 50mmol/L histidine +7% sucrose as pH penetrant, according to a 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) dropwise adding 8mg/ml DOX & HCl aqueous solution, adjusting pH of liposome external water phase to 7.0 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 43
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml of 1.6mg/ml ammonium sulfate buffer solution (pH5.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and dialyzing to remove ammonium sulfate in liposome external water phase. Taking 200mmol/L histidine as a pH penetrating agent, and performing reaction according to the following steps of 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) dropwise adding 8mg/ml DOX & HCl aqueous solution, adjusting pH of liposome external water phase to 7.0 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 44
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml of 1.6mg/ml ammonium sulfate buffer solution (pH5.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and dialyzing to remove ammonium sulfate in liposome external water phase. Taking 300mmol/L histidine as a pH penetrating agent, and performing reaction according to the weight ratio of 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) dropwise adding 8mg/ml DOX & HCl aqueous solution, adjusting pH of liposome external water phase to 7.0 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 45
Respectively weighing 153.3mg of HSPC, 51.0mg of CHO-HP and 200051.0mg of DSPE-mPEG, adding 2ml of absolute ethyl alcohol, carrying out ultrasonic treatment at 60 ℃ until the mixture is dissolved, and then carrying out ultrasonic treatment according to the water-oil ratio of 1: 1(v/v) adding 2ml of 1.6mg/ml ammonium sulfate buffer solution (pH5.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and dialyzing to remove ammonium sulfate in liposome external water phase. Taking 100mmol/L histidine as a pH penetrating agent, and mixing the components in a ratio of 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 3: 1(v/v) dropwise adding 2.67mg/ml DOX & HCl aqueous solution, adjusting pH of liposome external water phase to 7.0 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain DOX-Liposomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 46
Respectively weighing HSPC 44.7mg, CHO-HP 14.9mg and DSPE-mPEG200014.9mg, adding 2ml absolute ethyl alcohol, performing ultrasonic treatment at 60 ℃ until the mixture is dissolved, and then performing ultrasonic treatment according to a water-oil ratio of 1: 1(v/v) adding 2ml of 1.6mg/ml ammonium sulfate buffer solution (pH5.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and dialyzing to remove ammonium sulfate in liposome external water phase. Taking 100mmol/L histidine as a pH penetrating agent, and mixing the components in a ratio of 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 6(v/v) adding 14mg/ml DOX & HCl aqueous solution drop by drop, adjusting pH of liposome external water phase to 7.0 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 47
Respectively weighing HSPC 42.6mg, CHO-HP 14.2mg and DSPE-mPEG200014.2mg, adding 2ml absolute ethyl alcohol, performing ultrasonic treatment at 60 ℃ until the mixture is dissolved, and then performing ultrasonic treatment according to a water-oil ratio of 1: 1(v/v) adding 2ml of 1.6mg/ml ammonium sulfate buffer solution (pH5.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and dialyzing to remove ammonium sulfate in liposome external water phase. Taking 100mmol/L histidine as a pH penetrating agent, and mixing the components in a ratio of 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 9(v/v) adding 20mg/ml DOX & HCl aqueous solution drop by drop, adjusting pH of liposome external water phase to 7.0 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 48
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml 2.4mg/ml ammonium sulfate buffer solution (pH5.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and ultrafiltering to remove ammonium sulfate in liposome external water phase. 50mmol/L histidine +7% sucrose as pH penetrant, according to a 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) dropwise adding 8mg/ml DOX & HCl aqueous solution, adjusting pH of liposome external water phase to 7.0 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 49
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml of 18mg/ml ammonium sulfate buffer solution (pH5.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and ultrafiltering to remove ammonium sulfate in external aqueous phase of liposome. 50mmol/L histidine +7% sucrose as pH penetrant, according to a 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) dropwise adding 8mg/ml DOX & HCl aqueous solution, adjusting pH of liposome external water phase to 7.0 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 50
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml of 33mg/ml ammonium sulfate buffer solution (pH5.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and ultrafiltering to remove ammonium sulfate in external aqueous phase of liposome. 50mmol/L histidine +7% sucrose as pH penetrant, according to a 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) dropwise adding 8mg/ml DOX & HCl aqueous solution, adjusting pH of liposome external water phase to 7.0 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 51
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml of 33mg/ml ammonium sulfate buffer solution (pH5.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and ultrafiltering to remove ammonium sulfate in external aqueous phase of liposome. 50mmol/L histidine +7% sucrose as pH penetrant, according to a 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) dropwise adding 8mg/ml DOX & HCl aqueous solution, adjusting pH of liposome external water phase to 7.0 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 52
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml of 33mg/ml ammonium sulfate buffer solution (pH5.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and ultrafiltering to remove ammonium sulfate in external aqueous phase of liposome. Using 26.7mmol/L histidine + 26.7% sucrose as pH penetrant, according to the weight ratio of 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) dropwise adding 8mg/ml DOX & HCl aqueous solution, adjusting pH of liposome external water phase to 7.0 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Example 53
Respectively weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating to 60 ℃ to dissolve, and then mixing the materials according to a water-oil ratio of 1: 1(v/v) adding 2ml of 33mg/ml ammonium sulfate buffer solution (pH5.0), evaporating under reduced pressure to remove organic solvent, incubating in 60 deg.C water bath for 20min, intermittently performing ultrasonic treatment for 35min, and ultrafiltering to remove ammonium sulfate in external aqueous phase of liposome. 50mmol/L histidine +7% sucrose as pH penetrant, according to a 1: 1(v/v) adding a pH penetrating agent into blank liposome, and then mixing the mixture according to the medicine-lipid ratio of 1: 3(v/v) dropwise adding 8mg/ml DOX & HCl aqueous solution, adjusting pH of liposome external water phase to 7.0 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain DOX-lipomes-PEG, and storing in refrigerator at 4 deg.C in dark place.
Test example 1: particle size of liposomes
An appropriate amount of the liposomes prepared in examples 1 to 5, 8, 10 to 11, 13 to 14, 16 to 28, 30 to 35, and 37 to 53 was diluted with distilled water, and the particle size was measured by a laser particle size analyzer (ZLS). The above experiments were performed 3 times and the average value was taken.
TABLE 1 Liposome particle size of the different examples
Figure BDA0002565041110000301
Figure BDA0002565041110000311
Figure BDA0002565041110000321
Test example 2 encapsulation efficiency of liposomes
Method-ultracentrifugation method
1ml of the doxorubicin hydrochloride liposome prepared in example 4 was centrifuged at 12000rpm/min in an ep tube for 5min, and then the supernatant was subjected to HPLC to determine the content of free doxorubicin hydrochloride as Won. And (3) putting 1ml of doxorubicin hydrochloride liposome into a 100ml volumetric flask, performing constant volume with methanol, and performing HPLC analysis to obtain Wtotal. The envelope rate formula is:
EE ═ 100% W/W total
TABLE 2 encapsulation efficiency of liposomes for different examples
Examples Encapsulation efficiency (%)
4 2.9
Method two dialysis method (dialysis 24h)
2ml of doxorubicin hydrochloride liposome prepared in examples 5 to 11, 13 to 17, 19 to 24 and 27 are respectively put into a dialysis bag, 600ml of water is externally added, and after dialysis is carried out for 24 hours under the stirring condition, the content of doxorubicin hydrochloride is measured by HPLC (high performance liquid chromatography) from dialysate to be used as a W swimming pool. And (3) putting 1ml of doxorubicin hydrochloride liposome into a 100ml volumetric flask, performing constant volume with methanol, and performing HPLC analysis to obtain Wtotal.
The envelope rate formula is:
EE ═ 100% W/W total
TABLE 3 encapsulation efficiency of liposomes for different examples
Figure BDA0002565041110000331
Figure BDA0002565041110000341
Method three dialysis method (dialysis 6h)
2ml of doxorubicin hydrochloride liposome prepared in examples 25 to 26 and 28 to 53 were put in a dialysis bag, and 600ml of water was externally added, and after dialysis was performed for 6 hours under a stirring condition, the content of doxorubicin hydrochloride was measured by HPLC from the dialysate, and the dialysate was taken as Wye. And (3) putting 1ml of doxorubicin hydrochloride liposome into a 100ml volumetric flask, performing constant volume with methanol, and performing HPLC analysis to obtain Wtotal. The envelope rate formula is:
EE ═ 100% W/W total
TABLE 4 encapsulation efficiency of liposomes for different examples
Figure BDA0002565041110000342
Figure BDA0002565041110000351
Figure BDA0002565041110000361
Summary of test examples
The above examples 1-5 are a group of comparative examples, and compared with the technical solutions described in the claims, the examples in this group are mainly that no pH penetrant is added in the process, and ammonium sulfate in the aqueous phase outside the liposome is not removed.
The above examples 6-17 are another group of comparative examples, which are compared with the technical solutions described in the claims, and in this group of examples the pH penetrant is added simultaneously with the drug and the ammonium sulfate in the aqueous phase outside the liposome is not removed.
The above examples 18-26 are further comparative examples, which do not remove ammonium sulphate from the external aqueous phase of liposomes and do not adjust the pH of the external aqueous phase, compared to the technical solutions described in the claims.
The above examples 27-30 and 34-36 are another group of comparative examples, which do not adjust the pH of the external aqueous phase compared to the solutions described in the claims.
The above examples 31-33 and 37-53 are experimental examples of the invention according to the claimed method, wherein the ammonium sulfate concentration in examples 49, 50, 51, 53 is several times higher than in the other examples, and each is 50mmol/L histidine +7% sucrose as pH penetrant, and finally the pH of the liposome external aqueous phase is adjusted to 7.0.
FIG. 1 is a graph of particle size and encapsulation efficiency profiles for examples 28, 29 and 30; FIG. 2 is a graph showing particle size and encapsulation efficiency profiles of examples 31, 32 and 33; FIG. 3 is a graph showing the particle size and encapsulation efficiency profiles of examples 48, 49 and 50; FIG. 4 is a diagram of the liposome preparation of doxorubicin hydrochloride of example 51.
From the results of the above tests in tables 1-4, the encapsulation efficiency of liposome and the particle size of liposome are shown to be higher when the pH of the aqueous phase in the liposome is lower in the process; the pH of the liposome external water phase is closer to neutral, the more stable the medicament and the phospholipid are, and the smaller the liposome particle size is; it has also been found that the effect of the concentration of the aqueous ammonium sulfate solution on the encapsulation efficiency is great, basically the higher the concentration of the aqueous ammonium sulfate solution, the higher the encapsulation efficiency. Compared with other examples, the encapsulation efficiency of the examples 49-53 is far higher than that of 80% in pharmacopeia, reaches more than 98%, and the particle size is about 120nm, so that the process disclosed by the invention is relatively stable, the quality standard of an injection below 200nm can be fully met, and the method is particularly obviously progressive compared with the prior art.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that modifications can be made by those skilled in the art without departing from the principle of the present invention, and these modifications should also be construed as the protection scope of the present invention.

Claims (10)

1. A preparation process of doxorubicin hydrochloride liposome is characterized by comprising the following steps:
(a) adjusting the pH of the ammonium sulfate aqueous solution to prepare blank liposome:
weighing a prescription amount of liposome material, and adding an organic solvent to dissolve the liposome material to obtain an oil phase; preparing an acidic ammonium sulfate aqueous solution as a water phase; mixing water phase and oil phase to prepare blank liposome, removing organic solvent under normal pressure or reduced pressure, and performing incubation and intermittent ultrasound on the liposome;
(b) removing the blank liposome external water phase ammonium sulfate, adding a pH penetrating agent:
removing the blank liposome external aqueous phase ammonium sulfate by dialysis or ultrafiltration; then adding a pH penetrant into the blank liposome according to a certain proportion;
(c) preparing a drug-loaded liposome by active drug loading:
adding a drug aqueous solution according to a certain drug-to-lipid ratio to carry out active drug loading;
(d) adjusting the pH of the external water phase of the drug-loaded liposome:
adjusting the pH of the external water phase of the drug-loaded liposome and incubating.
2. The process for preparing doxorubicin hydrochloride liposome according to claim 1, wherein in step (a) said liposome material is hydrogenated soybean lecithin and cholesterol, DSPE-mPEG 2000.
3. The process for preparing doxorubicin hydrochloride liposome according to claim 1, wherein in step (a), the concentration of the ammonium sulfate aqueous solution is 1.6-33 mg/ml, and the pH of the ammonium sulfate aqueous solution after adjustment is 5.0-6.5.
4. The process for preparing doxorubicin hydrochloride liposome according to claim 1, wherein in step (a), the mixing ratio (v/v) of the aqueous phase and the oil phase is 5: 1-1: 5, mixing the water phase and the oil phase at the temperature of 50-70 ℃; in the step (a), the intermittent ultrasonic time is 0-120 min.
5. The process for preparing doxorubicin hydrochloride liposome according to claim 1, wherein in step (b) the cut-off molecular weight of the dialysis bag is 3.5-14 kd; in the step (b), the molecular weight cut-off of the ultrafiltration tube is 3-100 kd, and the rotating speed of the centrifugal machine is 7000-20000 rpm/min.
6. The process for preparing doxorubicin hydrochloride liposome according to claim 1, wherein in step (b) said pH penetrant is selected from one or more of disodium hydrogen phosphate, histidine and sucrose, and the mixing ratio (v/v) of blank liposome and pH penetrant is 2: 1-1: 2.
7. the process for preparing doxorubicin hydrochloride liposome according to claim 1, wherein in step (c) the drug-to-lipid ratio (i.e. the ratio of drug aqueous solution to blank liposome after pH penetrant addition) (v/v) is 5: 1-1: 9; in the step (d), the pH of the adjusted external water phase of the drug-loaded liposome is 6.0-9.0.
8. The preparation process of doxorubicin hydrochloride liposome according to claim 1, comprising the steps of:
(a) adjusting the pH of the ammonium sulfate aqueous solution to prepare blank liposome:
weighing HSPC 51.1mg, CHO-HP 17.0mg and DSPE-mPEG200017.0mg, adding 2ml of absolute ethyl alcohol, heating at 60 ℃, and dissolving to obtain an oil phase; preparing 2ml of 33mg/ml ammonium sulfate buffer solution (pH5.0) as a water phase; mixing the water phase and the oil phase at a ratio of 1: 1(v/v), evaporating under reduced pressure to remove anhydrous ethanol, incubating in 60 deg.C water bath for 20min, and performing intermittent ultrasonic treatment for 35min to obtain blank liposome;
(b) removing the blank liposome external water phase ammonium sulfate, adding a pH penetrating agent:
removing the external aqueous phase ammonium sulfate of the liposome by ultrafiltration in a 50kd ultrafiltration tube at 15000 rpm; 50mmol/L histidine +7% sucrose as pH penetrant, according to a 1: 1(v/v) adding a pH penetrant into the blank liposome;
(c) preparing a drug-loaded liposome by active drug loading:
according to the medicine-fat ratio of 1: 3(v/v) dropwise adding 8mg/ml DOX & HCl aqueous solution to carry out active drug loading;
(d) adjusting the pH of the external water phase of the drug-loaded liposome:
adjusting pH of the liposome external water phase to 7.0 with hydrochloric acid and sodium hydroxide, incubating in water bath at 55 deg.C for 20min to obtain doxorubicin hydrochloride liposome, and storing in refrigerator at 4 deg.C in dark place.
9. Doxorubicin hydrochloride liposome prepared by the process for preparing doxorubicin hydrochloride liposome according to any one of claims 1 to 8.
10. Use of the doxorubicin hydrochloride liposome preparation process of any one of claims 1-8 in the preparation of a doxorubicin hydrochloride liposome injection.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101703471A (en) * 2009-11-18 2010-05-12 中国药科大学 Improved method for preparing liposome by using ammonium sulfate gradient method
CN101897667A (en) * 2009-05-26 2010-12-01 石药集团中奇制药技术（石家庄）有限公司 Doxorubicin hydrochloride liposome injection and preparation technology thereof
US20110002977A1 (en) * 2006-12-29 2011-01-06 Chunlei Li Liposomal pharmaceutical preparation and method for manufacturing the same
US20200016079A1 (en) * 2017-03-31 2020-01-16 Fujifilm Corporation Liposome composition and pharmaceutical composition

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110002977A1 (en) * 2006-12-29 2011-01-06 Chunlei Li Liposomal pharmaceutical preparation and method for manufacturing the same
CN101897667A (en) * 2009-05-26 2010-12-01 石药集团中奇制药技术（石家庄）有限公司 Doxorubicin hydrochloride liposome injection and preparation technology thereof
CN101703471A (en) * 2009-11-18 2010-05-12 中国药科大学 Improved method for preparing liposome by using ammonium sulfate gradient method
US20200016079A1 (en) * 2017-03-31 2020-01-16 Fujifilm Corporation Liposome composition and pharmaceutical composition

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