CN115400218A - Lipid composition for preparing liposome and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of liposome, in particular to a lipid composition for preparing liposome and a preparation method thereof. The lipid composition is a lyophilized powder comprising phospholipids and cholesterol in a liposome preparation ratio, and wherein at least a portion of the cholesterol is present in an amorphous form. The lipid composition has excellent hydration capability, so that the preparation process of the liposome can be effectively simplified, and the preparation time is shortened; because the lipid composition can be fully hydrated, the formed liposome can be ensured to have a correct bilayer structure, so that the reliable drug encapsulation rate is ensured, and the stability and the reliability of the process are improved.

Description

Lipid composition for preparing liposome and preparation method thereof

Technical Field

The invention relates to the technical field of liposome, in particular to a lipid composition for preparing liposome and a preparation method thereof.

Background

The formulations of liposomes and lipid nanoparticles typically contain a number of lipids, including phosphatidylcholine, cholesterol, phosphatidylethanolamine, and pegylated phosphatidylcholine or pegylated phosphatidylglycerol. The existing liposome preparation technology is that the lipids are respectively weighed and then added into an organic solvent (such as absolute ethyl alcohol) which is miscible with water for dissolving, and then an aqueous solution is added for hydration to form the liposome, and the method is called as a solvent injection method; or an organic solution dissolved as a lipid mixture by adding to a water-immiscible organic solvent (e.g., chloroform or methylene chloride), removing the organic solvent by a method such as pressure reduction, and then adding an aqueous solution to hydrate to form liposomes, which is called a "thin film dispersion method".

The pharmaceutical properties of the liposomes prepared by the two methods, including particle size, uniformity, lipid bilayer structure and permeability, depend on whether each lipid in the prescription can be fully hydrated in the hydration process and can be uniformly dispersed at the molecular level according to the input proportion.

There are still some problems with both of the above methods.

For the solvent injection method: cholesterol is a crystal which has low solubility in organic solvents such as ethanol. When the dosage of the organic solvent is too large, the liposome cannot be formed, and in order to ensure that the cholesterol can be fully dissolved in less organic solvent, the organic solvent needs to be heated to a temperature higher than the boiling point (for example, when the organic solvent is ethanol, the temperature needs to be heated to 80 ℃), then the mixed system of the cholesterol and the organic solvent is cooled, and other lipids are added for dissolution. The temperature of dissolution is generally required to be around 70 ℃. The disadvantages of this method are: the preparation method needs to be divided into steps and dissolved at different temperatures, so that the production efficiency is low; it is difficult to ensure that each lipid is uniformly dispersed at the molecular level, so that the concentration and proportion of each lipid are consistent with the input value; under high temperature conditions, the hydrolysis rate of phospholipid is greatly accelerated, the contents of hydrolyzed phospholipid and free fatty acid in the prepared liposome can be influenced, and the products of hydrolysis of the two phospholipids can be inserted into the lipid membrane of the liposome, so that the arrangement structure and permeability of the membrane are changed, and the medicine is leaked.

For the thin film dispersion method: a large volume of hazardous organic solvent is required, which brings environmental hazards; the residual organic solvent content of the liposomes must be determined; compared with the ethanol injection method, process scale-up is difficult.

Disclosure of Invention

The present invention relates to a lipid composition which is a lyophilized powder comprising phospholipids and cholesterol in a liposome preparation ratio, and wherein at least a portion of the cholesterol is present in an amorphous form.

According to a further aspect of the present invention, it also relates to a process for the preparation of the lipid composition as described above, comprising:

1) Dissolving said phospholipid and said cholesterol in a solvent;

the solvent comprises a pharmaceutically acceptable organic solvent and optional water, and the dielectric constant is 14-19;

2) Freeze drying to remove the solvent.

According to still another aspect of the present invention, there is also provided a method for preparing liposomes, comprising:

dispersing the lipid composition as described above into a hydration medium and optionally loading with an active drug; wherein the hydration medium is water or an aqueous solution free of organic solvents.

At least part of cholesterol in the lipid composition (in other parts of the invention, the expression of 'preformed lipid' can be adopted) prepared by the invention is amorphous, so that the solubility of the lipid composition in ethanol is greatly improved, high-temperature dissolution can be avoided when a solvent injection method is adopted, and blank liposomes with high concentration can be obtained, thereby being beneficial to improving the production efficiency and batch consistency; meanwhile, the lipid composition has excellent hydration capability, so that the preparation process of the liposome can be effectively simplified, and the preparation time is shortened; because the lipid composition can be fully hydrated, the formed liposome can be ensured to have a correct bilayer structure, so that the reliable drug encapsulation rate is ensured, and the stability and the reliability of the process are improved.

In addition, for the lipid component with low content, the concentration of the low-content lipid component in the blank liposome and the drug-loaded liposome can be better maintained by the preformed lipid, and the proportion of the individual lipid components in the prescription is ensured to be in line with expectation. The preformed lipid auxiliary material can realize full hydration and uniform dispersion of lipid components, so that the preformed lipid auxiliary material can be directly added into an aqueous solution in a dry powder form without using an organic solvent to prepare the liposome. For the drugs of which the proteins and the polypeptides are sensitive to organic solvents, the preformed lipid auxiliary material provides a brand-new preparation method of the liposome which completely removes the organic solvents, and is suitable for loading the drugs. This is not achieved with simple mixtures of lipids (i.e. simply mixing the individual lipids).

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is an example of an HPLC-CAD plot (A) of preformed lipids and working curves (B-D) for three lipids; wherein B is HSPC: y =0.6737X +0.7842 ² =0.995; c is cholesterol, Y =0.2208X +0.4256 ² =0.998; d is DSPE-PEG2000: y =0.3364X-0.1718 ² ＝0.9995；

FIG. 2 is an infrared spectrum overlay of preformed lipid S17 (number 4 in Table 2) and a simple mixture of lipids;

FIG. 3 is an infrared spectrum of two preformed lipids prepared using different solvent formulations;

figure 4 is an XRD diffractogram of single lipid, simple mixture and preformed lipid (S17);

figure 5 is an XRD pattern of simple mixture of lipids and preformed lipids (S17);

figure 6 is an XRD pattern of two preformed lipids;

FIG. 7 is the appearance of a simple mixture of preformed lipids and lipids vortexed after addition to an ammonium sulfate solution;

FIG. 8 is a graph showing the residual lipid on the surface of a polycarbonate membrane during extrusion of blank liposomes prepared from preformed lipids and a simple mixture;

FIG. 9 is a particle size of blank liposomes prepared by direct hydration method using a simple mixture of preformed lipids and lipids;

FIG. 10 is a graph of particle size of blank liposomes prepared from preformed lipids and simple mixtures using ethanol injection;

FIG. 11 is a DSC of blank liposomes;

FIG. 12 is a cryo-electron micrograph of the prepared liposomal doxorubicin;

FIG. 13 is a DSC of the prepared liposome of doxorubicin;

FIG. 14 is an in vitro release profile of doxorubicin liposomes prepared using different lipid materials and methods of preparation;

fig. 15 is a cryo-transmission electron micrograph of irinotecan liposomes prepared;

figure 16 is an in vitro release profile of irinotecan liposomes prepared with different lipid starting materials.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

Unless otherwise defined, all terms (including technical and scientific terms) used in disclosing the invention are to be interpreted as commonly understood by one of ordinary skill in the art to which this invention belongs. The following definitions serve to better understand the teachings of the present invention by way of further guidance. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

As used herein, the term "and/or", "and/or" is selected to encompass any of two or more of the associated listed items, as well as any and all combinations of the associated listed items, including any two of the associated listed items, any more of the associated listed items, or all combinations of the associated listed items. It should be noted that when at least three items are connected by at least two conjunctive combinations selected from "and/or", "or" and/or ", it should be understood that in this application, the technical solutions unquestionably include the technical solutions all connected by" logical and ", and also unquestionably include the technical solutions all connected by" logical or ". For example, "A and/or B" includes three parallel schemes of A, B and A + B. For example, a reference to "a, and/or, B, and/or, C, and/or, D" includes any one of a, B, C, and D (i.e., all connected by "logical or"), any and all combinations of a, B, C, and D (i.e., any two or any three of a, B, C, and D), and any four combinations of a, B, C, and D (i.e., all connected by "logical and").

As used herein, the terms "comprising," "including," and "comprising" are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.

The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

The present invention relates to concentration values, which are intended to include fluctuations within a certain range. For example, it may fluctuate within a corresponding accuracy range. For example, 2%, may be allowed to fluctuate within 0.1%. For values that are larger or do not require more than fine control, the meaning is also allowed to include greater fluctuations. For example, 100mM, may allow fluctuations within the range of. + -. 1%, + -2%, + -5%, etc. The molecular weight is referred to, allowing the meaning to include fluctuations of ± 10%.

In the present invention, the terms "plurality" and "a plurality" mean, unless otherwise specified, 2 or more in number.

In the present invention, the technical features described in the open type include a closed technical solution composed of the listed features, and also include an open technical solution including the listed features.

In the present invention, "preferably", "better" and "preferable" are only embodiments or examples with better description, and it should be understood that the scope of the present invention is not limited by them. In the present invention, "optionally", "optional" and "optional" refer to the presence or absence, i.e., to any one of two juxtapositions selected from "present" and "absent". If multiple optional parts appear in one technical scheme, if no special description exists, and no contradiction or mutual constraint relation exists, each optional part is independent.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. The citation of the present application is incorporated by reference herein in its entirety for all purposes unless otherwise in conflict with the present objectives and/or technical disclosure of the present application. Where a citation is referred to herein, the definition of a reference in the document, including features, terms, nouns, phrases, etc., that is relevant, is also incorporated by reference. In the present invention, when the citation is referred to, the cited examples and preferred embodiments of the related art are also incorporated by reference in the present application, but the present invention is not limited to the embodiments. It should be understood that where the citation conflicts with the description herein, the application will control or be adapted in accordance with the description herein.

The present invention relates in a first aspect to a lipid composition which is a lyophilized powder comprising phospholipids and cholesterol in a liposome preparation ratio, and wherein at least part of the cholesterol is present in an amorphous form.

In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% of the cholesterol in the lipid composition is present in amorphous form.

According to the invention, "in amorphous form" means that cholesterol is present in an amorphous state, substantially without aggregation and recrystallization.

An important feature of the lipid composition is that there is no chemical interaction between the lipid components, but the dispersibility and solubility of the lipid in water and organic solvents are improved by physical effects such as intermolecular forces.

The lipid composition has excellent hydration capability, so that the preparation process of the liposome can be effectively simplified, and the preparation time is shortened; because the lipid composition can be fully hydrated, the formed liposome can be ensured to have a correct bilayer structure, so that the reliable drug encapsulation rate is ensured, and the stability and the reliability of the process are improved.

In some embodiments, the cholesterol comprises 38mol% to 42mol%, e.g., 39mol%, 40mol%, 41mol% of the lipid composition.

The phospholipid is usually a hydrophilic and lipophilic substance having a hydrophobic group composed of a long-chain alkyl group and a hydrophilic group composed of a phosphate group or the like in the molecule. Examples of the phospholipid include phosphatidylcholine (lecithin), phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, glycerolipids such as phosphatidylserine and phosphatidylinositol, sphingomyelin (sphingomyelin) such as sphingomyelin, natural or synthetic diphosphatidylphosphatidylcholine such as cardiolipin, derivatives thereof, and Hydrogenated Soybean Phosphatidylcholine (HSPC) according to a conventional method. The amount of the phospholipid is usually 20mol% or more, preferably 40mol% or more, preferably 62mol% or less, and preferably 60mol% or less of the entire liposome membrane component.

In some embodiments, the phospholipid comprises phosphatidylcholine. In some embodiments, the phosphatidylcholine includes one or more of Dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), hydrogenated Soybean Phospholipid (HSPC), hydrogenated lecithin (HEPC), dimyristoylphosphatidylcholine (DMPC), dilauroylphosphatidylcholine (DLPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1-myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), and 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC). Of these, phospholipids and sphingomyelin obtained by hydrogenation of HSPC and the like are preferable.

In some embodiments, the phospholipid further comprises a pegylated phospholipid.

In some embodiments, the amount of pegylated phospholipid is less than 8mol%, e.g., 7mol%, 6mol%, 5mol%, 4mol%, 3mol%, 2mol%, 1mol%, 0.5mol%, 0.1mol%.

In some embodiments, the pegylated phospholipid is polyethylene glycol-distearoylphosphatidylethanolamine (DSPE-PEG).

The DSPE-PEG can be selected from DSPE-PEG2000 or DSPE-PEG 5000.

Examples of the liposome membrane constituent include phospholipids, cholesterol, and the like, as well as anionic substances and zwitterionic substances used in liposomes. The following description is provided.

Examples of the anionic substance include diacylglycerol hemisuccinate, diacylglycerol hemimalonate, diacylglycerol hemiglutarate, diacylglycerol hemiadipate, diacylglycerol hemicyclohexane-1, 4-diacid, and fatty acids such as oleic acid, myristic acid, palmitic acid, stearic acid, nervonic acid, behenic acid, but are not limited thereto. Particularly preferred are saturated fatty acids which are solid at room temperature, and particularly preferred are palmitic acid and stearic acid. In the present specification, the normal temperature means 10 to 30 ℃. The content ratio of the anionic substance to the entire amount of the liposome constituent components is 0 to 20mol%, preferably 1mol% or more, and more preferably 5mol% or more.

Examples of the zwitterionic substance include N-alkyl-N, N-dimethylamino betaine such as lauryl betaine (lauryl dimethylamino acetic acid betaine); fatty acid amide alkyl-N, N-dimethyl amino acid betaines such as cocamidopropyl betaine and lauramidopropyl betaine; imidazoline type betaines such as sodium cocoamphoacetate, sodium lauroamphoacetate and the like; alkyl sulfobetaines such as alkyldimethyltaurines; sulfuric acid type betaines such as alkyldimethylaminoethanol sulfate; phosphoric acid type betaines such as alkyldimethylaminoethanol phosphate. The content ratio of the zwitterionic substance is 5 to 20mol%, preferably 1mol% or more, for example 5mol%, or for example 7mol%, based on the entire amount of the liposome-constituting components.

The liposome may contain other known additives, and examples of the antioxidant include vitamin E, which is a tocopherol homologue. The lipid derivative of the hydrophilic polymer for modifying the surface of the liposome is not particularly limited as long as the structural stability of the liposome is not impaired, and examples thereof include polyethylene glycol, dextran, pullulan, polysucrose, polyvinyl alcohol, synthetic polyamino acid, amylose, amylopectin, mannan, cyclodextrin, pectin, carrageenan, and derivatives thereof. Among them, polyethylene glycol and polyethylene glycol derivatives are preferable. The molecular weight of the hydrophilic polymer lipid derivative is preferably about 200 to 5 ten thousand, and more preferably about 1000 to 1 ten thousand.

A second aspect of the present invention relates to a process for the preparation of a lipid composition as described above, comprising:

1) Dissolving said phospholipid and said cholesterol in a solvent;

the solvent comprises a pharmaceutically acceptable organic solvent and optional water, and the dielectric constant is 14-19;

2) Freeze drying to remove the solvent.

Dissolution generally involves two processes: the structure of the solute molecule leaving its solid aggregate, and the solvation of the solute molecule. Among these, the solvation process is generally an exothermic process (enthalpy less than zero), and therefore, the main factor affecting the solute solubility and the speed of dissolution should be the speed at which the solute molecules gain energy and leave their solid structure. The internal energy of crystals with regular ordered structures is much higher than that of amorphous structures, and thus, solute molecules need higher energy to leave the crystalline structure, and the solubility of crystals is less than that of amorphous. Of the components of the preformed lipid, cholesterol is a relatively high melting point crystal. Thus, we believe that the preformed lipid achieves high solubility in organic solvents without the need for heating to high temperatures and without the need for stepwise dissolution, primarily by changing the physical state of cholesterol (and possibly other phospholipid components) to convert its crystalline form to amorphous form.

The dielectric constant is a macroscopic parameter that reflects the ability of a solvent to shield electrostatic interactions. The larger the dielectric constant, the more effectively this solvent is able to shield the attraction and repulsion of ions and dipoles across, the greater the solvation. The inventors have surprisingly found that a solvent having a dielectric constant between 14 and 19 (e.g. 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5) and incorporating freeze-drying, while sufficiently solubilizing the phospholipids and cholesterol, allows the preformed lipid to reach the amorphous state described above and improve its solubility properties.

Because the solid or powder prepared by the freeze-drying process usually has a loose structure, particularly the freezing point of the tertiary butanol is very low, a large number of needle-shaped pore canals are formed in the freeze-drying process. These channels further increase the surface area of the preformed lipid and thus can be added directly to water, quickly and fully hydrated. The solubility in organic solvents is also greatly increased, and if the tert-butanol content is too low, a good proportion of preformed lipids cannot be maintained. Thus, in some embodiments, the solvent comprises greater than 60v/v% t-butanol, e.g., 61v/v%, 62v/v%, 63v/v%, 64v/v%, 65v/v%, 66v/v%, 67v/v%, 68v/v%, 69v/v%, 70v/v%, 75v/v% or more.

In some embodiments, the organic solvent is a mixed system comprising acetone, t-butanol, and ethanol.

In some embodiments, the solvent comprises 20v/v% to 25v/v% acetone, e.g., 21v/v%, 22v/v%, 23v/v%, 24v/v%.

In some embodiments, the solvent comprises 10v/v% to 25v/v% ethanol, e.g., 15v/v%, 20v/v%.

In some embodiments, the temperature of the freeze-drying is from-30 ℃ to-50 ℃, such as-35 ℃, -40 ℃, -45 ℃.

In some embodiments, the freeze-drying time is 8h to 48h, such as 10h, 12h, 16h, 18h, 20h, 22h, 24h, 28h, 32h, 36h, 40h, 44h.

In some embodiments, the freeze-dried lipid is pulverized and/or portioned.

The third aspect of the present invention relates to a method for preparing a liposome, comprising:

the lipid composition as described above is dispersed into a hydration medium and optionally loaded with an active drug.

The liposomes can be blank liposomes or drug loaded liposomes.

In the present invention, the aqueous medium is an aqueous medium containing no organic solvent, and means a medium capable of dispersing the components constituting the liposome membrane, and is not particularly limited, and examples thereof include water, preferably distilled water for injection, physiological saline, an aqueous glucose solution, and ion-exchanged water, and isotonic agents, buffers, and the like, for example, a triethylamine sucrose octasulfate solution, an ammonium sulfate solution, a calcium acetate solution, and the like, may be added to these solutions. Alternatively, a physiologically active substance may be contained as a liposome-containing substance.

In some embodiments, the lipid composition is dispersed directly into the hydration medium.

The preformed lipid can realize full hydration and uniform dispersion of lipid components, so that the preformed lipid can be directly added into an aqueous solution in a dry powder form without using an organic solvent to prepare the liposome.

In some embodiments, the lipid composition is first dissolved by injecting it into a water-miscible organic dispersion, and the resulting solution is then dispersed into a hydration medium.

When the liposome is prepared by adopting an organic solvent injection method, the liposome does not need to be dissolved step by step, and all lipid components can be added into the organic solvent at one time in the form of prefabricated lipid, so that the process steps are greatly simplified; and the preformed lipid can ensure that all lipid components are basically and completely dissolved in an organic solvent, and the prepared lipid is fully hydrated after a hydration medium is added, so that the concentration and the proportion which are similar to the input value are obtained, the lipid membrane of the prepared liposome is ensured to have a stable and controllable structure, the loading capacity of the prepared liposome to medicines is ensured, and the stability and the reliability of the process are improved.

By "organic dispersion which is miscible with water" is meant that the organic dispersion is capable of being sparingly soluble, soluble or readily soluble in water.

In some embodiments, the water-miscible organic dispersion comprises ethanol and/or diethyl ether, preferably ethanol.

In some embodiments, the temperature of the mixing system of the lipid composition and ethanol is 40 ℃ or less and t or less than 70 ℃, or 40 ℃ or less and t or less than 60 ℃, or 50 ℃ or less and t or less than 60 ℃, and 50 ℃ or less and t or less than 70 ℃, and specifically, 45 ℃, 55 ℃ or 65 ℃ can also be selected.

When the concentration of the prepared liposome is lower than 50mg/ml, the preformed lipid can be completely dissolved in ethanol at the temperature as low as about 40 ℃. Hydrolysis of phospholipids follows a quasi-first order kinetic process. The hydrolysis rate constant is affected by the pH and temperature of the solution. Generally at the same pH, the rate of phospholipid hydrolysis at 70 ℃ is about 10 times that at 40 ℃. Therefore, when the blank liposome is prepared by adopting an organic solvent injection method, the lower temperature is favorable for maintaining the chemical stability of the phospholipid, reducing the generation of lysoPC and fatty acid and ensuring the storage stability of the liposome. When the temperature of the organic solvent is 70 ℃, the solubility of the preformed lipid can reach above about 3g/ml, and the concentration requirement of liposome preparation in industrial mass production can be completely met.

In some embodiments, the method further comprises mechanically modulating the mean particle size of the liposomes.

The particle size of the liposomes prepared according to the present invention can be adjusted as desired by those skilled in the art. The particle size is typically adjusted using mechanical methods, which in some embodiments include one or more of ball milling, jet milling, high-speed shearing, high-pressure homogenization, and high-pressure extrusion. The particle size (diameter) may be, for example, 50nm to 300nm, such as 250nm, 200nm, 150nm, 120nm, 110nm, 100nm, 90nm, 80nm, 70nm, etc.

The carried active medicine comprises one or more of taxanes, camptothecins, vinblastines, adriamycins, cyclosporins, flavonoids, dihydropyridines, tretinoins, anthraquinones, volatile oils, phalloidins, purine antagonists, pyrimidine antagonists, folic acid antagonists, gambogic acids, photosensitizers, proteins and nucleic acids or derivatives thereof.

Proteinaceous drugs such as antibodies or antibody-derived drugs (e.g. ADC, antibody nucleic acid conjugate drugs, etc.), polypeptide hormones (e.g. insulin, growth hormone, follicle stimulating hormone), cytokine-based drugs (e.g. interferon, granulocyte colony stimulating factor, erythropoietin, thrombopoietin, interleukin), enzymes (e.g. human prourokinase, human alpha glucosidase), and bone morphogenic protein 2, hirudin or other types of recombinant or native proteins. Nucleic acid drugs such as mRNA, shRNA, miRNA, siRNA, aptamer drugs, antisense nucleic Acid (ASO), activating RNA (saRNA), or certain plasmids, or vectors carrying gene editing systems such as CRISPR-Cas9 systems.

In some embodiments, the active drug carried is a vaccine active ingredient.

In some embodiments, the liposomes are prepared in the form of a composition. Therefore, the preparation process also comprises the processes of assembling/adding pharmaceutically acceptable carriers, diluents and adjuvants of the medicine. Acceptable carriers, diluents and adjuvants are non-toxic to recipients and are preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate or other organic acids; antioxidants, such as ascorbic acid; a low molecular weight polypeptide; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol, xylitol, erythritol, maltitol (maltotol) or sorbitol; starch, gum arabic (acacia), rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate.

Embodiments of the present invention will be described in detail with reference to examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures for the conditions not specified in the following examples, preferably with reference to the guidelines given in the present invention, may also be performed according to the experimental manual or the conventional conditions in the art, and may also be performed according to other experimental procedures known in the art, or according to the conditions suggested by the manufacturer.

In the following specific examples, the measurement parameters relating to the components of the raw materials, if not specified otherwise, may be subject to slight deviations within the accuracy of the weighing. Temperature and time parameters are involved to allow for acceptable deviation of the instrument test accuracy or operational accuracy.

Example 1 preparation of preformed lipids

1. According to the solvent ratio in table 1, an organic solvent mixture was prepared. According to the proportion of 3:1:1, hydrogenated Soybean Phospholipid (HSPC), cholesterol and polyethylene glycol 2000-distearoylphosphatidylethanolamine (PEG 2000-distearylphosphatidylethanolamine, PEG 2000-DSPE) were added to the solvent mixture at a concentration of 100mg/ml.

2. And (3) placing each lipid solution in a water bath at 40 ℃, heating for 3-5 minutes, taking out, and observing whether the solution is clear or not. Then, each lipid solution was placed in a-40 ℃ refrigerator and frozen overnight.

3. And (4) placing the frozen sample in a freeze dryer, freeze-drying for 12 hours, taking out, and placing in a refrigerator with the temperature of-20 ℃ for sealed storage.

4. Content determination of preformed lipid: quantitatively weighing the prepared preformed lipid prepared by freeze-drying, adding absolute ethyl alcohol, and preparing into a sample with the concentration of 1 mg/ml. Ethanol solutions of HSPC, cholesterol, and PEG2000-DSPE at graded concentrations, and mixed solutions of three lipids at 3. The method comprises respectively measuring the concentrations of three lipids in the ethanol solution of preformed lipid and the reference solution by high performance liquid Chromatography (CAD) and calculating the mass ratio of the three lipids. The results are shown in Table 2. FIG. 1 is an HPLC-CAD profile of three lipids.

TABLE 1 organic solvent mixture (vol/vol) for preformed lipid preparation

Solvent numbering	Acetone (II)	Tert-butyl alcohol	Anhydrous ethanol	Water (W)	Dielectric constant of mixed solvent	Clarification of lipid solution after heating
							S1
	5	90	5	0	12.07	Whether or not
							S2	10	85	5	0	12.56	Whether or not
S3	20	75	5	0	13.54	Whether or not
							S4	15	75	10	0	13.73	Whether or not
S5	20	70	10	0	14.22	Is that
							S6	20	60	20	0	15.58	Is that
S7	20	55	15	10	21.82	Whether or not
							S8	20	50	15	15	25.28	Whether or not
S9	20	45	15	20	28.74	Whether or not
							S10	25	45	15	15	25.77	Whether or not
S11	25	50	15	10	22.31	Whether or not
							S12	25	55	15	5	18.85	Whether or not
S13	30	50	10	10	22.12	Whether or not
							S14	30	45	10	15	25.58	Whether or not
S15	30	40	10	20	29.04	Whether or not
							S16	20	55	20	5	19.04	Whether or not
S17	20	60	15	5	18.36	Is that
							S18	20	65	15	0	14.9	Is that
S19	20	55	25	0	16.26	Is that
							S20	25	55	20	0	16.07	Is that
S21	25	60	15	0	15.39	Is that

TABLE 2 concentration and ratio of three lipids in preformed lipids prepared from different organic solvent mixtures

The proportion of preformed lipids numbered 5,6,8 and 9 in table 2 was not acceptable according to the requirements of the liposome formulation for the mass ratio of the three lipids HSPC, cholesterol and DSPE-PEG2000 (58-62%, 19-21% and 19-21%, respectively). The rest meet the preset requirements and are consistent with the contrast.

Example 2 characterization of physical states and intermolecular interactions of preformed lipids

Taking 9 groups of prefabricated lipid powder and reference substances in the table 2, HSPC, cholesterol and DSPE-PEG2000 according to the mass ratio of 3:1: 1A small amount of a simple mixture was prepared and its IR spectrum was measured using Nicolet iS20 Fourier Infrared (Thermo, USA).

The infrared spectrum is shown in FIG. 2, in which 3400cm ^-1 Is the peak of a small amount of water contained in the sample. The main difference in the infrared spectra of preformed lipids and simple mixtures is 1700cm ^-1 ～600cm ^-1 Differences in fingerprint area (marked with light and dark circles, respectively). It is believed that the preformed lipid does not contain new chemicals, but has different forces between its molecules compared to a simple mixture, and thus is easily dissolved in organic solvents such as ethanol.

FIG. 3 is an IR spectrum of preformed lipids prepared using two different solvent formulations. Wherein the concentrations and ratios of the three lipids of preformed lipid S17 are as expected, but the lipid ratio of preformed lipid S12 is not as 3:1:1, and the content of DSPE-PEG2000 is lower. Comparing the infrared spectra of S17 and S12, the difference between the two is almost zero, which indicates that the lipid proportion in S12 is unqualified, and is not caused by forming chemical bonds in the preparation process or changing the molecular structure of the lipid.

The diffraction behaviour of each sample was measured using a D8 Advance X-ray diffractometer (Bruker, uk) at 5-70 degrees, taking HSPC, cholesterol, DSPE-PEG2000, a simple mixture of the three (HSPC/cholesterol/DSPE-PEG 2000= 3).

As shown in FIG. 4, cholesterol is a typical crystal, and has a high-intensity crystal diffraction peak, DSPE-PEG2000 also has a crystal structure, and the crystal structure of HSPC is weaker. After mixing the three and grinding into fine powder, the crystal diffraction peaks of cholesterol and DSPE-PEG2000 in the mixture are weakened but still exist. In comparison with the preformed lipid (S17), the diffraction peaks of cholesterol and DSPE-PEG2000 were found to almost disappear. FIG. 5 more clearly shows that the simple mixture still has more distinct crystal diffraction peaks (including cholesterol and DSPE-PEG 2000), while the preformed lipid (S17) is very weak. Comparing the XRD patterns of the two preformed lipids in fig. 6, it can be seen that some crystalline diffraction peaks of cholesterol and DSPE-PEG2000 still exist in S12, while S17 is almost amorphous. We believe this is the main reason why the ratio of the three lipids in the S12 preformed lipid is not as expected.

As described above, the process for producing a preformed lipid represented by S17 can change the physical state of a lipid (mainly cholesterol) to an amorphous state by changing the intermolecular force in a preformed lipid composition mainly with a mixed solvent having a specific dielectric constant, and can achieve solubility in an organic solvent such as ethanol.

Because the solid or powder prepared by the freeze-drying process often has a loose structure, particularly the freezing point of the tertiary butanol is very low, a large number of needle-shaped pore canals are formed in the freeze-drying process. These channels further increase the surface area of the preformed lipid and thus can be added directly to water, quickly and fully hydrated. The solubility in ethanol is also greatly increased, so that if the t-butanol content is too low, a good proportion of preformed lipid is not maintained.

EXAMPLE 3 preparation of blank liposomes

1. The blank liposomes (drug-free) were prepared from preformed lipids by direct hydration and compared to simple lipid mixtures

Weighing 150mg of preformed lipid (HSPC/cholesterol/DSPE-PEG 2000= 3; 90mg HSPC,30mg cholesterol and 30mg DSPE-PEG2000 (simple lipid mixture) were weighed; when added to 3ml of ammonium sulfate solution (250mM, pH 5.5) and vortexed for 1min, the pre-liposome group showed fewer bubbles, while the simple mixture group showed significantly more bubbles (FIG. 7).

Placing the two groups of lipid suspensions in a water bath at 60 ℃, and magnetically stirring for 20min; then, the film was gradually extruded through polycarbonate films of 200nm,100nm,80nm and 50nm, respectively, using an extruder (Lipex extruder, evonik). After the blank liposomes prepared from the simple mixture group were filmed, white particles remained clearly on the 200nm extruded film, and the crystals were carefully observed (fig. 8). Since both HSPC and DSPE-PEG2000 are amorphous, the precipitate on the extruded film should be primarily cholesterol. Since the precipitation blocked the extruded membrane and had to be replaced to continue extrusion, the preparation of blank liposomes from simple mixtures of lipids took more time in the extrusion step (Table 3).

TABLE 3 preparation of blank liposomes by direct hydration of preformed lipids and simple mixtures of lipids for extrusion

The two prepared blank liposomes were diluted with physiological saline and then measured for particle size and particle size distribution by dynamic light scattering (Zetasizer NanoZS90, malvern, uk) (fig. 9). It can be seen that the blank liposomes prepared with preformed lipids have a smaller average particle size and are significantly more uniform.

2. Blank liposomes (drug-free) were prepared from preformed lipids using ethanol injection and compared to simple lipid mixtures

Weighing 150mg of preformed lipid (HSPC/cholesterol/DSPE-PEG 2000= 3; 90mgHSPC,30mg cholesterol and 30mgDSPE-PEG2000 (simple lipid mixture) were weighed; firstly, adding cholesterol into absolute ethyl alcohol, heating and dissolving, then cooling, and adding the other two lipids for dissolving. Then 3ml ammonium sulfate solution (250mM, pH 5.5) was added, magnetically stirred in a water bath at 60 ℃ for 20min, and hydrated to prepare liposomes. Adopting the same method as 1 to extrude through a film; the particle sizes of the two groups of blank liposomes after extrusion were measured, and the results are shown in fig. 10.

TABLE 4 preparation of blank Liposome solubilized lipids and extrusion step with preformed lipids and simple mixture in ethanol injection method

3. Lipid concentration, ratio and thermodynamic characterization of blank liposomes prepared with preformed lipids, and comparison with blank liposomes prepared from simple mixtures of lipids

The blank liposomes prepared in steps 2 and 3 using the preformed lipid and simple mixture of lipids as adjuvants were dialyzed overnight against 10% sucrose solution to remove ammonium sulfate from the outer aqueous phase. Measuring the particle sizes of the four groups of blank liposomes after dialysis by a dynamic light scattering method; the lipid concentrations in the four blank liposomes were determined by HPLC-CAD method and the lipid ratios were calculated. The thermodynamic behaviour of four groups of blank liposomes was determined by differential thermal scanning (microcall DSC, marvens, uk) (heating scan at a rate of 1 ℃/min; each with 10% sucrose solution as reference solution).

The grain diameters of the four groups of blank liposomes after dialysis are all about 100-110 nm, and the grain diameters are uniformly distributed. Among them, the liposomes prepared by ethanol injection method for the preformed lipids had the smallest average particle size (table 5).

TABLE 5 particle size and PDI (mean. + -. SD, n = 3) after dialysis for four groups of blank liposomes

In terms of lipid concentration and ratio, the concentration of cholesterol was significantly lower and the concentration of DSPE-PEG2000 was also lower in liposomes prepared from simple mixtures of lipids by direct hydration, resulting in a significant deviation of the mass ratio of the three lipids in the liposomes from the input ratio of 3. In combination with the solid precipitates observed on the extruded film during extrusion, it is demonstrated that when liposomes are prepared by direct hydration using a simple mixture of lipids as the starting material, the lipids are not fully hydrated, especially cholesterol precipitates, thus affecting the lipid concentration and ratio of the liposomes prepared. The concentrations and the proportions of the three lipids in the blank liposome obtained by the two preparation methods of the preformed lipid are consistent with the proportions in the preformed lipid, which shows that the preformed lipid ensures the full hydration and uniform dispersion of each lipid component.

TABLE 6 lipid content and ratio of blank liposomes prepared from different starting materials and preparation methods

The phase transition behavior and thermodynamic parameters of the lipid membrane are closely related to the lipid concentration and the ratio of the three lipids. Therefore, we also characterized the phase transition behavior of each group of blank liposomes on the basis of quantitative determination of the concentration of each lipid in the lipid membrane. As shown in fig. 11, the phase transition temperature (Tm) of four liposomes was about 52 degrees, which is the phase transition temperature of HSPC. The DSC curves of the remaining three blank liposome samples, except for the liposomes prepared by direct hydration with a simple mixture, completely coincide. Wherein, the blank liposome prepared by the simple lipid mixture by the direct hydration method has obviously high and narrow phase transition peaks, and the phase transition peaks of the other three groups of blank liposomes are flat. This is mainly due to the fact that the ratio of cholesterol in liposomes prepared by the direct hydration method from simple mixtures is significantly lower, so that the phase transition of the lipid membrane from the colloidal phase (liquid ordered phase) to the liquid crystalline phase (liquid ordered phase) is more pronounced, exhibiting a high and narrow phase transition peak; while the remaining three groups of liposomes, which contain more cholesterol (about 40% molar), are in liquid-separated phase and the transition to liquid-crystalline phase is less pronounced.

The thermodynamic parameter values (table 7) obtained after quantitative fitting of the DSC curves of the four groups of blank liposomes also show that the simple mixture of liposomes prepared by the direct hydration method has a lipid membrane structure different from the remaining three groups and has a higher enthalpy value and consistency of phase transition (Δ T) _1/2 Smaller). And the thermodynamic parameters of the remaining three groups of liposomes are similar to those reported in the literature. To explain inThe content and the proportion of three lipids in a lipid membrane are kept at the ratio of 3.

TABLE 7 thermodynamic parameters of blank liposomes of each group

To summarize:

(1) The blank liposome is prepared by taking preformed lipid as a raw material and adopting a direct hydration method and an ethanol injection method. The concentration and the proportion of each lipid component of the obtained blank liposome and the structure of a lipid membrane are all in accordance with expectations.

(2) Compared with the simple lipid mixture, when the liposome is prepared by a direct hydration method, the preformed lipid can reduce the generation of bubbles in the stirring process, and ensure the correct concentration and proportion of the three lipids in the formula. And completely eliminates the problems of precipitation and solvent residue of lipid (especially cholesterol) in the extrusion process of mass production in the future.

(3) When the blank liposome is prepared by the ethanol injection method, the preformed lipid can be completely added into the ethanol and dissolved at a lower temperature, so that the method is easier to operate than a simple mixture, is favorable for simplifying the operation process and is favorable for the chemical stability of the lipid. Compared with the direct hydration method, the method can be the selection of fat-soluble drugs.

Example 4 preparation of preformed lipids for Doxiloids

Referring to the commercially available doxorubicin liposome (Doxil, yanson company, usa), a doxorubicin solution was added to each blank liposome provided in example 3, and the mixture was magnetically stirred at 60 ℃ for about 20min for drug loading to prepare a drug-loaded liposome having a target drug concentration of 2mg/ml and a total lipid concentration of about 16 mg/ml. The particle size and the particle size distribution of the drug-loaded liposome are measured by a dynamic light scattering method; the encapsulation efficiency of the liposomes was measured by a resin adsorption method.

As shown in table 8, the blank liposomes prepared from the simple mixture by direct hydration have very low encapsulation efficiency for doxorubicin (which is likely related to precipitation of cholesterol during hydration and extrusion, the lipid formulation changes after precipitation of cholesterol, increasing permeability, resulting in a lower drug loading gradient); the entrapment rate of the other three blank liposomes to doxorubicin is about 99%.

Table 8 particle size, particle size distribution (mean ± SD, n = 3) and encapsulation efficiency of four groups of drug-loaded liposomes

For three groups of drug-loaded liposomes with encapsulation efficiency of 99%, the morphology of the liposomes was further observed by a cryo-transmission electron microscope (fig. 12). The three groups of liposomes have similar appearance, are in coffee bean shape, and contain rod-shaped doxorubicin-ammonium sulfate nanocrystals inside.

The concentration of three lipids in the above three groups of drug-loaded liposomes with encapsulation efficiency of 99% was determined by HPLC-CAD method. The concentration of each lipid component and the total lipid concentration in the three drug-loaded liposomes were as expected and consistent with Doxil, a commercially available product (Table 9).

TABLE 9 concentration of three lipids and Total lipid concentration in drug-loaded liposomes

The thermodynamic behavior of the three groups of drug-loaded liposomes with high encapsulation efficiency is characterized by DSC. The test conditions were the same as for the blank liposome DSC. As shown in fig. 13, in the DSC of the three drug-loaded liposomes, two endothermic phase transition peaks can be observed: wherein, the flat phase transition peak of Tm at about 52 ℃ is the phase transition of the lipid membrane; the sharp narrow peak of Tm around 70 ℃ is the melting peak of the doxorubicin nanocrystal in the internal aqueous phase. This phase transition behavior is also consistent with that reported in the literature for Doxil, indicating that after two groups of blank liposomes prepared from preformed lipids are loaded with doxorubicin, the resulting drug-loaded liposomes have very similar structures (including lipid membrane structures and nanocrystal structures) to Doxil, and thus have similar thermodynamic parameters (table 10).

TABLE 10 thermodynamic parameters of doxorubicin liposomes prepared and reported in the literature for Doxil and Lipodox (Lipodox is a simulated doxorubicin liposome from Sun Pharma, india)

In vitro release rate study of three groups of drug-loaded liposomes:

physiological saline (+ -ammonium ion) with pH 7.4 as release medium, according to a ratio of 1: adding the prepared three groups of doxorubicin liposomes into a release medium according to the volume ratio of 50, adding Dowex resin to adsorb released medicines, and constructing a leak tank condition. Samples were taken at different time points, and the liposome-encapsulated drug concentration was determined, and the cumulative percent release was calculated and compared to the release behavior of Doxil.

As shown in fig. 14, in normal saline without ammonium ions, the drug release rates of the three groups of drug-loaded liposomes are very slow, and the 32hr cumulative release percentage is less than 10%, which indicates that the three groups of preparations all maintain stable drug-loaded performance; under the release promoting effect of ammonium ions, the release of the three groups of preparations is obviously increased, the trend and the cumulative release percentage of each time point are very close, and the release rate is very close to that of Doxil.

The in vitro release experiment result shows that the release behaviors of the prepared three groups of doxorubicin lipidosome are consistent and similar to Doxil.

To summarize:

compared with the current universal technology for preparing blank liposome by taking a simple lipid mixture as a raw material and adopting an ethanol injection method, the blank liposome prepared by the preformed lipid (comprising direct hydration and the ethanol injection method) can realize stable and effective drug loading, and the success rate of the technology is ensured. The prepared doxorubicin lipidosome has good consistency with the original research on the particle size, the appearance, the thermodynamic parameters and the in vitro release behavior.

Example 5 preformed lipids for preparation of irinotecan liposomes

1. Preparation of blank liposomes

Weighing 90mg of preformed lipid (DSPC/cholesterol/DSPE-PEG 2000=61.6, molar ratio) and adding all into a small amount of absolute ethanol, heating in a water bath at 40 ℃ to dissolve; 67mgDSPC,21.8mg cholesterol and 1.2mg DSPE-PEG2000 (lipid simple mixture) were weighed; firstly, adding cholesterol into absolute ethyl alcohol, heating to dissolve, then cooling, and adding the other two lipids to dissolve. Then adding 3ml of sucrose octasulfate triethylamine solution (81.25mM, pH 5.5), magnetically stirring in a water bath at 60 ℃ for 20min, and hydrating to prepare liposome; extruding through a liposome extruder; the particle sizes of the two groups of blank liposomes after extrusion were measured.

2. Measurement of lipid concentration and ratio of blank liposome prepared from preformed lipid, and comparison with blank liposome prepared from simple mixture of lipids

And (2) dialyzing the blank liposome prepared by taking the simple mixture of the preformed lipid and the lipid as the auxiliary material in the step (1) with normal saline overnight, and removing the sucrose octasulfate triethylamine in the external water phase. Measuring the particle size of blank liposome before and after dialysis by dynamic light scattering method; the lipid concentration in the blank liposomes was determined and the lipid ratio was calculated using HPLC-CAD quantification of lipids as described in the first set of examples.

The blank liposome prepared from the simple mixture of the preformed lipid and the lipid has the particle size of about 100-110 nm before and after dialysis, and the particle size is uniformly distributed. (Table 11).

TABLE 11 particle size and PDI of blank liposomes before and after dialysis (mean. + -. SD, n = 3)

In terms of lipid concentration and ratio, the blank liposome prepared from the simple mixture of lipids, due to the low input amount of DSPE-PEG2000 (3 ml of blank liposome was prepared, only 1mg was added), the measured concentration of DSPE-PEG2000 in the blank liposome was lower than the theoretical value due to the loss during the preparation process, and thus resulted in the change of the ratio of three lipid components. The concentrations and ratios of the three lipids in the blank liposomes prepared from the preformed lipids were as expected (table 12). The result shows that the loss of low-content lipid in the preparation process can be avoided by taking the preformed lipid as an auxiliary material, so that the proportion and the concentration of each lipid component in the blank liposome are ensured.

TABLE 12 lipid content and ratio of blank liposomes prepared from different starting materials and preparation methods

3. Blank liposome prepared from preformed lipid for Ilicazone loading capacity, and compared with blank liposome prepared from simple lipid mixture

According to a commercially available irinotecan liposome (Onvyde), irinotecan hydrochloride solution is added into each blank liposome, magnetic stirring is carried out at 55 ℃ for about 20min, and then drug loading is carried out, so that a drug-loaded liposome with the target drug concentration of 4.3mg/ml and the total lipid concentration of about 9.2mg/ml is prepared. The particle size and the particle size distribution of the drug-loaded liposome are measured by a dynamic light scattering method; the Zeta potential of the liposomes was determined electrophoretically (Zetasizer NanoZS90, malvern, UK); the encapsulation efficiency of the liposomes was measured by a resin adsorption method. The mean particle size and encapsulation efficiency of the liposomes were the same for both groups (table 13).

TABLE 13 particle size, particle size distribution, zeta potential (mean. + -. SD, n = 3) and encapsulation efficiency of Ilicism liposomes

Lipid material	Particle size (nm)	PDI	Zeta potential (mv)	Encapsulation efficiency (%)
					Simple mixture	108.5±4.61	0.053±0.014	-42.3±2.8	98.5
Preformed lipids	105.4±4.02	0.046±0.002	-34.3±3.2	99.8

The morphology of the two groups of irinotecan liposomes was essentially the same (fig. 15). The component with higher electron density is visible inside, but there is no well-defined fine structure.

The concentration of each lipid component in the two elitican drug-loaded liposomes was determined by HPLC-CAD method. The total lipid concentration of both liposomes was close to that of the commercial product (9.15 mg/ml), but, similar to the results for the concentrations and ratios of the individual lipid components in the blank liposomes, the concentrations and ratios of the three lipids in irinotecan liposomes prepared with preformed lipids as excipients were expected, particularly where the concentration and molar ratio of the low-content lipid DSPE-PEG2000 were consistent with those of the commercial product (0.12 mg/ml and 0.29%, respectively); the concentration and ratio of DSPE-PEG2000 in liposomes prepared with the simple lipid mixture as the adjuvant were lower than those of the commercially available products (Table 14).

TABLE 14 concentration of three lipids and Total lipid concentration in drug-loaded liposomes

In table 14, the difference in DSPE-PEG2000 concentration in the two irinotecan-loaded liposomes groups is also reflected in the difference in Zeta potential of the two liposomes (table 13). In the drug-loaded liposome prepared by the simple mixture, the concentration of DSPE-PEG2000 is low, so that the negative charge of a phosphate group at the head of DSPC in a lipid membrane is easier to expose, and the absolute value of Zeta potential is higher.

In vitro release rate studies of two groups of irinotecan drug-loaded liposomes:

physiological saline (+ -ammonium ion) with pH 7.4 as release medium, according to a ratio of 1: and (3) adding the prepared two groups of illitegam liposomes into a release medium according to the volume ratio of 50, adding Dowex resin to adsorb released medicines, and constructing a leak tank condition. Samples were taken at different time points, and the liposome-encapsulated drug concentration was determined, and the cumulative release percentage was calculated and compared to the release behavior of Onevyde.

As shown in fig. 16, in physiological saline without ammonium ions, the drug release rates of the two groups of drug-loaded liposomes are very slow, and the 24hr cumulative release percentage is lower than 10%, which indicates that the preparations both maintain stable drug-loaded performance; under the release promoting effect of ammonium ions, the release of the two groups of preparations is obviously increased, the trend and the cumulative release percentage of each time point are very close, and the release rate is very close to that of Onivyde.

To summarize:

(1) With DSPC/cholesterol/DSPE-PEG 2000=61.6:38.1:0.3 The blank liposome prepared by adopting the ethanol injection method and the blank liposome prepared by the simple mixture have similar particle size and distribution. However, the pre-formed lipid group can better maintain the concentration of each lipid component, especially the low-content lipid component (DSPE-PEG 2000) therein, which is beneficial to maintaining the proportion of the lipid components and is in line with the input proportion.

(2) Compared with the current general process for preparing the blank liposome by taking a simple lipid mixture as a raw material and adopting an ethanol injection method, the blank liposome prepared by the preformed lipid can stably and efficiently load irinotecan, and the particle size and the appearance of the obtained irinotecan liposome are similar to those of the irinotecan liposome prepared by the simple mixture. The lipid concentration, ratio and in vitro release rate of irinotecan liposomes prepared with preformed lipids are consistent with the commercial product.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims, and the description and the drawings can be used for explaining the contents of the claims.

Claims (22)

1. A lipid composition which is a lyophilized powder comprising phospholipids and cholesterol in proportions consistent with liposome preparation, and wherein at least a portion of the cholesterol is present in amorphous form.

2. The lipid composition of claim 1, wherein the cholesterol comprises 38mol% to 42mol%, or 38mol% to 40mol% of the lipid composition.

3. The lipid composition according to claim 1 or 2, said phospholipid comprising phosphatidylcholine.

4. The lipid composition of claim 3, the phosphatidylcholine comprising one or more of dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, hydrogenated soybean phospholipid, hydrogenated lecithin, dimyristoylphosphatidylcholine, dilauroylphosphatidylcholine, 1-stearoyl-2-palmitoylphosphatidylcholine, 1-myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl-2-myristoylphosphatidylcholine, 1-stearoyl-2-myristoylphosphatidylcholine, 1-palmitoyl-2-myristoylphosphatidylcholine, and 1-palmitoyl-2-stearoylphosphatidylcholine.

5. The lipid composition according to any one of claims 1, 2, 4, said phospholipids further comprising a pegylated phospholipid, preferably in an amount of less than 8mol%.

6. The lipid composition of claim 5, wherein the pegylated phospholipid is polyethylene glycol-distearoylphosphatidylethanolamine, preferably polyethylene glycol-distearoylphosphatidylethanolamine 2000.

7. A process for preparing a lipid composition according to any one of claims 1 to 6, comprising:

1) Dissolving said phospholipid and said cholesterol in a solvent;

the solvent comprises a pharmaceutically acceptable organic solvent and optional water, and the dielectric constant is 14-19;

2) Freeze drying to remove the solvent.

8. The method of claim 7, wherein the solvent comprises greater than 60v/v% t-butanol.

9. The method of claim 8, wherein the organic solvent is a mixed system comprising acetone, t-butanol, and ethanol.

10. The method of claim 9, wherein the solvent comprises 60 to 70v/v% t-butanol.

11. The method of claim 10, wherein the solvent comprises 20-25 v/v% acetone and 10-25 v/v% ethanol.

12. The method according to any one of claims 7 to 11, wherein the temperature of the freeze-drying is from-30 ℃ to-50 ℃.

13. The method of claim 12, wherein the freeze-drying time is 8 to 48 hours.

14. The method according to any one of claims 7 to 11 or 13, wherein the freeze-dried lipid is pulverized and/or packaged.

15. A method of preparing liposomes comprising:

dispersing the lipid composition according to any one of claims 1 to 6 in a hydration medium and optionally loading with an active drug; wherein the hydration medium is water or an aqueous solution free of organic solvents.

16. The method of claim 15, wherein the lipid composition is dispersed directly into the hydration medium.

17. The method of claim 15, wherein the lipid composition is dissolved by injecting the lipid composition into a water-miscible organic dispersion, and the resulting solution is dispersed in a hydration medium.

18. The method of claim 17, wherein the water-miscible organic dispersion comprises ethanol.

19. The method of claim 18, wherein the temperature of the mixing system of the lipid composition and ethanol is 40 ℃ or more and t or less than 70 ℃, or 40 ℃ or more and t or less than 60 ℃, or 50 ℃ or more and t or less than 70 ℃.

20. The method of any one of claims 15-19, further comprising mechanically manipulating the average particle size of the liposomes.

21. The method of claim 20, wherein the mechanical process comprises one or more of ball milling, jet milling, high shear, high pressure homogenization, and high pressure extrusion.

22. The preparation method according to any one of claims 15 to 19 and 21, wherein the carried active drug comprises one or more of taxanes, camptothecins, vincas, adriamycines, cyclosporins, flavones, dihydropyridines, tretinoins, anthraquinones, volatile oils, phalloidins, purine antagonists, pyrimidine antagonists, folic acid antagonists, gambogic acids, photosensitizers, proteins and nucleic acids.

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