Lipid composition for preparing liposomes and preparation method thereof

Abstract

The present invention relates to the field of liposome technology, and more specifically, to a lipid composition for preparing liposomes and a method for preparing the same. The lipid composition is a lyophilized powder comprising phospholipids and cholesterol in a proportion consistent with liposome preparation, wherein at least a portion of the cholesterol is present in an amorphous form. The lipid composition has excellent hydration capacity, thereby effectively simplifying the liposome preparation process and shortening the preparation time. Since the lipid composition can be fully hydrated, the formed liposomes can be ensured to have a correct bilayer structure, thereby ensuring a reliable drug encapsulation rate and improving the stability and reliability of the process.

Description

Technical Field

[0001] The present invention relates to the technical field of liposomes, and in particular to a lipid composition for preparing liposomes and a preparation method thereof. Background Art

[0002] The formulation of liposomes and lipid nanoparticles typically contains multiple lipids, including phosphatidylcholine, cholesterol, phosphatidylethanolamine, and PEGylated phosphatidylcholine or PEGylated phosphatidylglycerol. Existing liposome preparation techniques involve weighing these lipids separately, dissolving them in a water-miscible organic solvent (such as anhydrous ethanol), and then adding an aqueous solution for hydration to form liposomes. This method is called the "solvent injection method." Alternatively, these lipids are added to a water-immiscible organic solvent (such as chloroform or dichloromethane) to form an organic solution of a lipid mixture. The organic solvent is then removed by methods such as decompression, and then an aqueous solution is added for hydration to form liposomes. This method is called the "thin film dispersion method."

[0003] The pharmaceutical properties of liposomes prepared by the above two methods, including particle size, uniformity, lipid bilayer structure and permeability, all depend on whether the individual lipids in the formulation can be fully hydrated during the hydration process and can be uniformly dispersed at the molecular level according to the input ratio.

[0004] There are still some problems with the above two methods.

[0005] Regarding the solvent injection method, cholesterol is a crystalline substance with low solubility in organic solvents such as ethanol. Liposomes cannot form if the amount of organic solvent used is too large. To ensure that cholesterol can fully dissolve in a small amount of organic solvent, the organic solvent must be heated to a temperature above its boiling point (for example, to 80 degrees Celsius when the organic solvent is ethanol). The cholesterol-organic solvent mixture is then cooled and the other lipids are added for dissolution. The dissolution temperature is generally required to be around 70 degrees Celsius. The disadvantages of this method are that it requires dissolution in separate steps and at different temperatures, resulting in low production efficiency; it is difficult to ensure uniform molecular dispersion of the individual lipids, thus maintaining the concentration and ratio of the individual lipids consistent with the input values; high temperatures greatly accelerate the hydrolysis rate of phospholipids, which may affect the content of hydrolyzed phospholipids and free fatty acids in the prepared liposomes. Furthermore, the hydrolysis products of these two phospholipids can insert into the lipid membrane of the liposome, altering the membrane's structure and permeability, leading to drug leakage.

[0006] For the thin film dispersion method: a large volume of hazardous organic solvents is required, which poses an environmental hazard; the residual amount of organic solvent in the liposomes must be measured; and compared with the ethanol injection method, process scale-up is difficult. Summary of the Invention

[0007] The present invention relates to a lipid composition, which is a freeze-dried powder comprising phospholipid and cholesterol in a proportion consistent with liposome preparation, wherein at least part of the cholesterol exists in an amorphous form.

[0008] According to yet another aspect of the present invention, it also relates to a method for preparing the lipid composition as described above, comprising:

[0009] 1) fully dissolving the phospholipid and the cholesterol in a solvent;

[0010] The solvent comprises a pharmaceutically acceptable organic solvent and optionally water, and has a dielectric constant of 14 to 19;

[0011] 2) Freeze-drying to remove the solvent.

[0012] According to another aspect of the present invention, it also relates to a method for preparing liposomes, comprising:

[0013] The lipid composition as described above is dispersed in a hydrating medium and optionally loaded with an active drug; wherein the hydrating medium is water or an aqueous solution without an organic solvent.

[0014] At least part of the cholesterol in the lipid composition prepared by the present invention (which may also be referred to as "preformed lipid" in other parts of the present invention) is amorphous, thereby greatly improving the solubility of the lipid composition in ethanol. When a solvent injection method is used, high-temperature dissolution can be avoided, and high-concentration blank liposomes can be obtained, which is beneficial to improving production efficiency and batch consistency; at the same time, the lipid composition has excellent hydration ability, thereby effectively simplifying the preparation process of liposomes and shortening the preparation time; since the lipid composition can be fully hydrated, it can be ensured that the formed liposomes have a correct bilayer structure, thereby ensuring a reliable drug encapsulation rate and improving the stability and reliability of the process.

[0015] In addition, for the lipid composition with low content, prefabricated lipids can better maintain the concentration of this low-content lipid composition in blank liposomes and drug-loaded liposomes, thereby ensuring that the ratio of individual lipid compositions in the prescription meets expectations. Because this prefabricated lipid adjuvant can realize the sufficient hydration and uniform dispersion of lipid composition, therefore, organic solvent can be not used at all, and directly added in the aqueous solution in the form of dry powder, and prepared as liposome. For the drugs that proteins and polypeptides are sensitive to organic solvents, this prefabricated lipid adjuvant provides a brand-new, completely removed liposome preparation method of organic solvent, which is applicable to the loading of such drugs. This is that simple mixture of lipids (i.e., simply mixing each lipid) cannot be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 HPLC-CAD spectrum (A) of preformed lipids and working curves (BD) of three lipids provided in one embodiment; wherein B is HSPC: Y=0.6737X+0.7842, R 2 =0.995; C is cholesterol, Y=0.2208X+0.4256, R 2 =0.998; D is DSPE-PEG2000: Y=0.3364X-0.1718, R 2 =0.9995;

[0018] Figure 2 The infrared spectra of the preformed lipid S17 (number 4 in Table 2 ) and the simple lipid mixture are superimposed;

[0019] Figure 3 IR spectra of two preformed lipids prepared using different solvent formulations;

[0020] Figure 4 XRD diffraction patterns of single lipid, simple mixture and preformed lipid (S17);

[0021] Figure 5 XRD patterns of simple lipid mixture and preformed lipid (S17);

[0022] Figure 6 are the XRD patterns of the two preformed lipids;

[0023] Figure 7 The appearance of preformed lipids and simple lipid mixtures after vortexing in ammonium sulfate solution;

[0024] Figure 8 The lipid residues on the polycarbonate membrane surface during the extrusion of blank liposomes prepared with preformed lipids and simple mixtures;

[0025] Figure 9 The particle size of blank liposomes prepared by direct hydration method using preformed lipids and simple lipid mixtures;

[0026] Figure 10 The particle size of blank liposomes prepared by ethanol injection method for preformed lipids and simple mixtures;

[0027] Figure 11 is the DSC graph of blank liposomes;

[0028] Figure 12 This is a cryo-electron microscopy image of the prepared doxorubicin liposomes;

[0029] Figure 13 is the DSC graph of the prepared doxorubicin liposomes;

[0030] Figure 14 The in vitro release curves of doxorubicin liposomes prepared using different lipid raw materials and preparation methods are shown;

[0031] Figure 15 This is a cryo-transmission electron microscopy image of the prepared irinotecan liposomes;

[0032] Figure 16 The in vitro release curves of irinotecan liposomes prepared using different lipid raw materials. DETAILED DESCRIPTION

[0033] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0034] Unless otherwise indicated, all terms (including technical and scientific terms) used to disclose the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are provided to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0035] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0036] As used herein, the terms "comprising," "including," and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0037] The recitation of numerical ranges herein by endpoints includes all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0038] Concentration values ​​used in this invention include fluctuations within a certain range. For example, fluctuations within a certain precision range are permitted. For example, for 2%, fluctuations within ±0.1% are permitted. For larger values ​​or values ​​that do not require overly precise control, greater fluctuations are permitted. For example, for 100 mM, fluctuations within ±1%, ±2%, ±5%, etc. are permitted. Regarding molecular weight, fluctuations within ±10% are permitted.

[0039] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0040] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0041] In the present invention, "preferably", "better", "more preferably", and "suitably" are merely descriptions of preferred implementation methods or examples, and should be understood to not limit the scope of protection of the present invention. In the present invention, "optionally", "optional", and "optional" refer to being optional, that is, to being selected from either of the two parallel options of "with" or "without". If multiple "options" appear in a technical solution, unless otherwise specified and without contradiction or mutual restriction, each "optional" is independent.

[0042] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this invention are cited with all their contents and all their purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description in this application.

[0043] The first aspect of the present invention relates to a lipid composition, which is a lyophilized powder comprising phospholipids and cholesterol in a ratio consistent with liposome preparation, wherein at least part of the cholesterol is in an amorphous form.

[0044] 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 in amorphous form.

[0045] According to the present invention, "existing in an amorphous form" means that cholesterol exists in a non-crystalline form and does not substantially aggregate or recrystallize.

[0046] An important feature of the lipid composition is that there is no chemical interaction between the lipid components, but rather physical interactions such as intermolecular forces improve the dispersibility and solubility of the lipids in water and organic solvents.

[0047] The lipid composition has excellent hydration ability, thus effectively simplifying the preparation process of liposomes and shortening the preparation time; because the lipid composition can be fully hydrated, it can ensure that the formed liposomes have a correct bilayer structure, thereby ensuring a reliable drug encapsulation rate and improving the stability and reliability of the process.

[0048] In some embodiments, the cholesterol accounts for 38 mol% to 42 mol% of the lipid composition, such as 39 mol%, 40 mol%, or 41 mol%.

[0049] Phospholipids are generally hydrophilic and lipophilic substances having a hydrophobic group composed of a long-chain alkyl group and a hydrophilic group composed of a phosphate group etc. in the molecule. As phospholipids, for example, glycerolipids such as phosphatidylcholine (lecithin), phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol, sphingosine phospholipids such as sphingomyelin, natural or synthetic diphosphatidyl phospholipids such as cardiolipin and their derivatives, and substances obtained by hydrogenating these substances according to conventional methods (e.g., hydrogenated soybean phosphatidylcholine (HSPC)) etc. can be used. The amount of phospholipids is generally 20 mol% or more, preferably 40 mol% or more, preferably 62 mol% or less, and preferably 60 mol% or less in the overall liposome membrane constituents.

[0050] In some embodiments, the phospholipid includes phosphatidylcholine. In some embodiments, the phosphatidylcholine includes dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), hydrogenated soybean lecithin (HSPC), hydrogenated phosphatidylcholine (HEPC), dimyristoylphosphatidylcholine (DMPC), dilauroylphosphatidylcholine (DLPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1-myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl-2-myristoylphosphatidylcholine, 1-stearoyl-2-myristoylphosphatidylcholine, 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC) and 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC) in one or more. Among these substances, phospholipids, sphingomyelins etc. obtained by hydrogenation are preferably HSPC etc.

[0051] In some embodiments, the phospholipid further comprises a PEGylated phospholipid.

[0052] In some embodiments, the content of PEGylated phospholipid is less than 8 mol%, such as 7 mol%, 6 mol%, 5 mol%, 4 mol%, 3 mol%, 2 mol%, 1 mol%, 0.5 mol%, 0.1 mol%.

[0053] In some embodiments, the PEGylated phospholipid is polyethylene glycol-distearoylphosphatidylethanolamine (DSPE-PEG).

[0054] DSPE-PEG can be selected from, for example, DSPE-PEG2000 or DSPE-PEG5000.

[0055] As components constituting the liposome membrane, in addition to phospholipids and cholesterol, anionic substances and zwitterionic substances used for liposomes are mentioned, as described below.

[0056] Examples of anionic substances include, but are not limited to, diacylglycerol hemisuccinate, diacylglycerol hemimalonate, diacylglycerol hemiglutarate, diacylglycerol hemiadipate, diacylglycerol hemicyclohexane-1,4-dioic acid, and fatty acids such as oleic acid, myristic acid, palmitic acid, stearic acid, nervonic acid, and behenic acid. Saturated fatty acids that are solid at room temperature are particularly preferred, with palmitic acid and stearic acid being particularly preferred. In this specification, room temperature refers to 10°C to 30°C. The content of these anionic substances relative to the total amount of the liposome constituents is 0 to 20 mol%, preferably 1 mol% or greater, and more preferably 5 mol% or greater.

[0057] Examples of zwitterionic substances include N-alkyl-N,N-dimethylamino betaines such as lauryl betaine (lauryldimethylaminoacetic acid betaine); fatty acid amide alkyl-N,N-dimethylamino betaines such as cocamidopropyl betaine and lauroamidopropyl betaine; imidazoline-type betaines such as sodium cocoamphoacetate and sodium lauroamphoacetate; alkyl sulfobetaines such as alkyldimethyltaurine; sulfate-type betaines such as alkyldimethylaminoethanol sulfate; and phosphate-type betaines such as alkyldimethylaminoethanol phosphate. The proportion of these zwitterionic substances relative to the total amount of liposome components is 5 to 20 mol%, preferably 1 mol% or greater, for example 5 mol% or 7 mol%.

[0058] The liposomes may also contain other known additives, for example, as antioxidants, tocopherol homologues, i.e., vitamin E, etc. In addition, the hydrophilic polymer lipid derivatives used to modify the liposome surface are not particularly limited as long as they do not impair the structural stability of the liposome. Examples include polyethylene glycol, dextran, pullulan, polysucrose, polyvinyl alcohol, synthetic polyamino acids, amylose, amylopectin, mannan, cyclodextrin, pectin, carrageenan, and derivatives thereof. Among them, polyethylene glycol and polyethylene glycol derivatives are preferred. The molecular weight of the hydrophilic polymer lipid derivative is preferably about 200 to 50,000, more preferably about 1000 to 10,000.

[0059] A second aspect of the present invention relates to a method for preparing the lipid composition as described above, comprising:

[0060] 1) fully dissolving the phospholipid and the cholesterol in a solvent;

[0061] The solvent comprises a pharmaceutically acceptable organic solvent and optionally water, and has a dielectric constant of 14 to 19;

[0062] 2) Freeze-drying to remove the solvent.

[0063] Dissolution typically involves two processes: the departure of solute molecules from their solid aggregate structure and the solvation of solute molecules. The solvation process is typically an exothermic process (enthalpy is less than zero), so the primary factor influencing solute solubility and dissolution speed should be the rate at which solute molecules gain energy and leave their solid structure. Crystals with a regular, ordered structure have much higher internal energy than amorphous structures. Therefore, it takes a higher amount of energy for solute molecules to leave the crystalline structure, and the solubility of crystals is lower than that of amorphous structures. Among the components of preformed lipids, cholesterol is a crystalline form with a relatively high melting point. Therefore, we believe that the preformed lipids primarily transform the physical state of cholesterol (and possibly other phospholipid components) into an amorphous form, enabling them to achieve high solubility in organic solvents without the need for heating to high temperatures or dissolving in stages.

[0064] Dielectric constant is a macroscopic parameter that reflects the ability of a solvent to shield electrostatic effects. The greater the dielectric constant, the more effectively the solvent can shield the attraction and repulsion between ions and dipoles, and the greater the solvation effect. The inventors unexpectedly discovered that, under the premise of being able to fully dissolve phospholipids and cholesterol, the dielectric constant of the solvent is between 14 and 19 (e.g., 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5) and is paired with freeze-drying, which can make the prefabricated lipids reach the above-mentioned amorphous state and improve their solubility properties.

[0065] Because the solid or powder prepared by the freeze-drying process often has a loose structure, and in particular the freezing point of tert-butanol is very low, a large number of needle-like channels will be formed during the freeze-drying process. These channels further increase the surface area of ​​the preformed lipids, so they can be directly added to water for rapid and sufficient hydration. And the solubility in organic solvents is also greatly increased. If the tert-butanol content is too low, it is impossible to achieve a good ratio for maintaining the preformed lipids. Therefore, in some embodiments, the solvent contains tert-butanol more than 60v / v%, for example 61v / v%, 62v / v%, 63v / v%, 64v / v%, 65v / v%, 66v / v%, 67v / v%, 68v / v%, 69v / v%, 70v / v%, 75v / v% or higher.

[0066] In some embodiments, the organic solvent is a mixed system comprising acetone, tert-butanol, and ethanol.

[0067] In some embodiments, the solvent comprises 20 v / v% to 25 v / v% acetone, for example, 21 v / v%, 22 v / v%, 23 v / v%, or 24 v / v%.

[0068] In some embodiments, the solvent comprises 10 v / v% to 25 v / v% ethanol, for example, 15 v / v%, 20 v / v%.

[0069] In some embodiments, the freeze-drying temperature is -30°C to -50°C, such as -35°C, -40°C, or -45°C.

[0070] In some embodiments, the freeze-drying time is 8 h to 48 h, for example, 10 h, 12 h, 16 h, 18 h, 20 h, 22 h, 24 h, 28 h, 32 h, 36 h, 40 h, or 44 h.

[0071] In some embodiments, the freeze-dried lipids are pulverized and / or packaged.

[0072] A third aspect of the present invention relates to a method for preparing liposomes, comprising:

[0073] The lipid composition as described above is dispersed in a hydration medium and optionally loaded with an active drug.

[0074] The liposomes can be blank liposomes or drug-loaded liposomes.

[0075] In the present invention, the hydration medium is an aqueous medium that does not contain an organic solvent and is capable of dispersing the components constituting the liposome membrane. It is not particularly limited and may be, for example, water, preferably distilled water for injection, physiological saline, aqueous glucose solution, or ion-exchanged water. These solutions may contain isotonic agents, buffers, and the like, such as sucrose octasulfate triethylamine solution, ammonium sulfate solution, and calcium acetate solution. Alternatively, the liposome may contain a physiologically active substance as a substance contained within the liposome.

[0076] In some embodiments, the lipid composition is dispersed directly into the hydration medium.

[0077] Since the prefabricated lipid can achieve full hydration and uniform dispersion of lipid components, it can be directly added to an aqueous solution in the form of dry powder without using any organic solvent to prepare liposomes.

[0078] In some embodiments, the lipid composition is first injected into a water-miscible organic dispersion to dissolve it, and then the resulting solution is dispersed into a hydration medium.

[0079] When preparing liposomes using the organic solvent injection method, there is no need to dissolve them in steps. All lipid components can be added to the organic solvent in the form of prefabricated lipids at one time, greatly simplifying the process steps. In addition, the prefabricated lipids can ensure that each lipid component is basically completely dissolved in the organic solvent and is fully hydrated after adding the hydration medium, thereby obtaining a concentration and ratio close to the input value, thereby ensuring that the lipid membrane of the prepared liposome has a stable and controllable structure, ensuring the drug loading capacity, and improving the stability and reliability of the process.

[0080] The “water-miscible organic dispersion” means that the organic dispersion is slightly soluble, soluble or readily soluble in water.

[0081] In some embodiments, the water-miscible organic dispersion comprises ethanol and / or diethyl ether, preferably ethanol.

[0082] In some embodiments, the temperature of the mixed system of the lipid composition and ethanol is 40℃≤t≤70℃, or 40℃≤t≤60℃, or 50℃≤t≤60℃, 50℃≤t≤70℃, and specific temperatures such as 45℃, 55℃, and 65℃ can also be selected.

[0083] When the concentration of the prepared liposomes is lower than 50 mg / ml, the preformed lipids can be completely dissolved in ethanol at temperatures as low as around 40 degrees Celsius. The hydrolysis of phospholipids follows a pseudo-first-order kinetic process. The hydrolysis rate constant is affected by the pH value and temperature of the solution. Generally, at the same pH value, the phospholipid hydrolysis rate at 70°C is about 10 times that at 40°C. Therefore, when using the organic solvent injection method to prepare blank liposomes, lower temperatures are beneficial for maintaining the chemical stability of phospholipids, reducing the production of LysoPC and fatty acids, and ensuring the storage stability of the liposomes. When the organic solvent temperature is 70 degrees Celsius, the solubility of the preformed lipids can reach above approximately 3 g / ml, which can fully meet the concentration requirements for liposome preparation in large-scale industrial production.

[0084] In some embodiments, the method further comprises regulating the average particle size of the liposomes by mechanical means.

[0085] The particle size of the liposomes prepared according to the present invention can be adjusted according to the needs of those skilled in the art. Particle size is usually adjusted by mechanical methods. In some embodiments, the mechanical methods include one or more of ball milling, air flow milling, high-speed shearing, high-pressure homogenization, and high-pressure extrusion. The particle size (diameter) can be, for example, 50 nm to 300 nm, such as 250 nm, 200 nm, 150 nm, 120 nm, 110 nm, 100 nm, 90 nm, 80 nm, 70 nm, etc.

[0086] The active drugs contained include any one or more substances or their derivatives of taxanes, camptothecins, vinblastines, doxorubicin, cyclosporins, flavonoids, dihydropyridines, retinoic acid, anthraquinones, volatile oils, podophyllotoxins, purine antagonists, pyrimidine antagonists, folic acid antagonists, gambogic acid, photosensitizers, proteins, and nucleic acids.

[0087] Protein-based drugs include antibodies or antibody-derived drugs (such as ADCs, antibody-nucleic acid conjugates, etc.), polypeptide hormones (such as insulin, growth hormone, follicle-stimulating hormone), cytokine drugs (such as interferon, granulocyte colony-stimulating factor, erythropoietin, thrombopoietin, interleukins), enzymes (such as human urokinase, human α-glucosidase), bone morphogenetic protein 2, hirudin, or other types of recombinant or natural proteins. Nucleic acid drugs include mRNA, shRNA, miRNA, siRNA, aptamer drugs, antisense oligonucleotides (ASOs), activating RNA (saRNA), or certain plasmids, or vectors carrying gene editing systems (such as the CRISPR-Cas9 system).

[0088] In some embodiments, the active drug carried is a vaccine active component.

[0089] In some embodiments, the liposomes are prepared in the form of a composition, and the preparation process may optionally include the steps of assembling the drug / adding pharmaceutically acceptable carriers, diluents, and adjuvants. Acceptable carriers, diluents, and adjuvants are nontoxic 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; low molecular weight polypeptides; 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, or sorbitol; starch, acacia, rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate.

[0090] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. For experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention, and may also be made to experimental manuals or conventional conditions in the art, other experimental methods known in the art, or conditions recommended by the manufacturer.

[0091] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0092] Example 1 Preparation of preformed lipids

[0093] 1. Prepare an organic solvent mixture according to the solvent ratios in Table 1. Add hydrogenated soybean phosphatidylcholine (HSPC), cholesterol, and polyethylene glycol 2000-distearylphosphatidylethanamine (PEG2000-DSPE) to the solvent mixture at a mass ratio of 3:1:1 to a concentration of 100 mg / ml.

[0094] 2. Place each lipid solution in a 40°C water bath, heat for 3-5 minutes, remove from the water, and observe for clarity. Then, place each lipid solution in a -40°C freezer and freeze overnight.

[0095] 3. Place the frozen sample in a freeze dryer, take it out after freeze drying for 12 hours, and store it in a sealed container at -20 degrees.

[0096] 4. Determination of the content of preformed lipids: Quantitatively weigh the preformed lipids prepared by freeze-drying, add anhydrous ethanol, and prepare a sample with a concentration of 1 mg / ml. The same method was used to prepare ethanol solutions of HSPC, cholesterol, and PEG2000-DSPE with gradient concentrations, as well as a mixed solution of the three lipids in a mass ratio of 3:1:1 as a reference. The concentrations of the three lipids in the preformed lipid ethanol solution and the reference solution were determined using a high-performance liquid chromatography (HPLC) electrospray ionization detector (CAD), and the mass ratio of the three was calculated. The results are shown in Table 2. Figure 1 HPLC-CAD spectra of three lipids.

[0097] Table 1. Organic solvent mixtures used for preformed lipid preparation (volume ratio)

[0098] Solvent No. acetone tert-Butanol Anhydrous ethanol water Dielectric constant of mixed solvent Is the lipid solution clear after heating? S1 5 90 5 0 12.07 no S2 10 85 5 0 12.56 no S3 20 75 5 0 13.54 no S4 15 75 10 0 13.73 no S5 20 70 10 0 14.22 yes S6 20 60 20 0 15.58 yes S7 20 55 15 10 21.82 no S8 20 50 15 15 25.28 no S9 20 45 15 20 28.74 no S10 25 45 15 15 25.77 no S11 25 50 15 10 22.31 no S12 25 55 15 5 18.85 no S13 30 50 10 10 22.12 no S14 30 45 10 15 25.58 no S15 30 40 10 20 29.04 no S16 20 55 20 5 19.04 no S17 20 60 15 5 18.36 yes S18 20 65 15 0 14.9 yes S19 20 55 25 0 16.26 yes S20 25 55 20 0 16.07 yes S21 25 60 15 0 15.39 yes

[0099] Table 2. Concentrations and ratios of the three lipids in preformed lipids prepared from different organic solvent mixtures

[0100]

[0101]

[0102] According to the mass ratio requirements of the three lipids HSPC, cholesterol, and DSPE-PEG2000 in the liposome formulation (58-62%, 19-21%, and 19-21%, respectively), the proportions of the preformed lipids numbered 5, 6, 8, and 9 in Table 2 were unqualified. The rest met the predetermined requirements and were consistent with the control.

[0103] Example 2 Characterization of the physical state and intermolecular interactions of preformed lipids

[0104] A small amount of the 9 groups of pre-made lipid powders and reference substances in Table 2 and a simple mixture of HSPC, cholesterol and DSPE-PEG2000 prepared at a mass ratio of 3:1:1 were taken and their infrared spectra were measured using a Nicolet iS20 Fourier infrared spectrometer (Thermo Corporation, USA).

[0105] The infrared spectrum results are as follows Figure 2 As shown, 3400cm -1 The main difference between the infrared spectra of preformed lipids and simple mixtures is the peak at 1700 cm -1 ~600cm -1 The differences in the fingerprint regions (marked by light and dark circles, respectively). We believe that there are no new chemical species in the preformed lipids, but that the forces between the molecules are different compared to simple mixtures, making them easier to dissolve in organic solvents such as ethanol.

[0106] Figure 3 These are infrared spectra of preformed lipids prepared using two different solvent formulations. While the concentrations and ratios of the three lipids in preformed lipid S17 met expectations, the lipid ratio in preformed lipid S12 did not meet the 3:1:1 requirement, and the DSPE-PEG2000 content was low. Comparing the infrared spectra of S17 and S12 reveals virtually no difference, indicating that the unsatisfactory lipid ratio in S12 is not due to chemical bonds formed during the preparation process or changes in the lipid's molecular structure.

[0107] HSPC, cholesterol, DSPE-PEG2000, a simple mixture of the three (HSPC / cholesterol / DSPE-PEG2000=3:1:1, w / w), the preformed lipid numbered 9 in Table 2, and the control were taken, and the diffraction behavior of each sample was measured in the range of 5-70 degrees using a D8 Advance X-ray diffractometer (Bruker, UK).

[0108] like Figure 4As shown, cholesterol is a typical crystal with high-intensity crystal diffraction peaks. DSPE-PEG2000 also has a crystalline structure, while HSPC has a weaker crystal structure. After mixing the three and grinding them into a fine powder, the crystal diffraction peaks of cholesterol and DSPE-PEG2000 in the mixture weakened, but still existed. Compared with the preformed lipid (S17), the diffraction peaks of cholesterol and DSPE-PEG2000 were found to have almost disappeared. Figure 5 It is more clearly shown that the simple mixture still has relatively obvious crystal diffraction peaks (including cholesterol and DSPE-PEG2000), while the preformed lipid (S17) is very weak. Figure 6 Looking at the XRD patterns of the two preformed lipids, we can see that S12 still has some crystalline diffraction peaks of cholesterol and DSPE-PEG2000, while S17 is almost amorphous. We believe this is the main reason why the ratio of the three lipids in the preformed lipid S12 does not meet expectations.

[0109] In summary, the preparation method of preformed lipids represented by S17 mainly changes the intermolecular forces in the preformed lipid composition through a mixed solvent with a specific dielectric constant, thereby changing the physical state of the lipid (mainly cholesterol), transforming its crystals into amorphous, so that it can dissolve in organic solvents such as ethanol.

[0110] Because solids or powders prepared by freeze-drying often have a loose structure, and tert-butanol, in particular, has a very low freezing point, it forms numerous needle-like channels during the freeze-drying process. These channels further increase the surface area of ​​the preformed lipids, allowing them to be added directly to water for rapid and sufficient hydration. Their solubility is also greatly increased in ethanol, so if the tert-butanol content is too low, it will be impossible to maintain a good ratio of the preformed lipids.

[0111] Example 3 Preparation of blank liposomes

[0112] 1. Use direct hydration method to prepare blank liposomes (without drug) with preformed lipids and compare with simple lipid mixtures

[0113] Weigh 150 mg of preformed lipids (HSPC / cholesterol / DSPE-PEG2000=3:1:1, w / w); weigh 90 mg of HSPC, 30 mg of cholesterol and 30 mg of DSPE-PEG2000 (simple lipid mixture); add to 3 ml of ammonium sulfate solution (250 mM, pH 5.5), vortex and shake for 1 min. Observe and see that the preformed lipid group has fewer bubbles, while the simple mixture group has significantly more bubbles ( Figure 7 ).

[0114] The two lipid suspensions were placed in a 60°C water bath and magnetically stirred for 20 minutes. They were then extruded through polycarbonate membranes of 200 nm, 100 nm, 80 nm, and 50 nm using an extruder (Lipexextruder, Evonik). After the blank liposomes prepared from the simple mixture group passed through the membrane, white granular residues were clearly visible on the 200 nm extruded membrane. Upon closer inspection, crystals ( Figure 8 Since both HSPC and DSPE-PEG2000 are amorphous, the precipitate on the extrusion membrane is primarily cholesterol. Because the precipitate clogs the extrusion membrane, requiring replacement for continued extrusion, preparing blank liposomes with a simple lipid mixture requires more time during the extrusion process (Table 3).

[0115] Table 3. Extrusion time of blank liposomes prepared by direct hydration of preformed lipids and simple lipid mixtures

[0116]

[0117] The two groups of blank liposomes were diluted with physiological saline and the particle size and particle size distribution were measured by dynamic light scattering (Zetasizer NanoZS90, Malvern, UK). Figure 9 ). It can be seen that the average particle size of blank liposomes prepared with preformed lipids is smaller and significantly more uniform.

[0118] 2. Prepare blank liposomes (without drug) using preformed lipids using the ethanol injection method and compare with a simple lipid mixture

[0119] Weigh 150mg of pre-made lipids (HSPC / cholesterol / DSPE-PEG2000=3:1:1, w / w), add all of them to a small amount of anhydrous ethanol, and heat in a 40℃ water bath to dissolve; weigh 90mgHSPC, 30mg cholesterol and 30mgDSPE-PEG2000 (simple lipid mixture); first add cholesterol to anhydrous ethanol, heat to dissolve, then cool, and add the remaining two lipids to dissolve. Then add 3ml of ammonium sulfate solution (250mM, pH 5.5), stir magnetically in a 60-degree Celsius water bath for 20min, hydrate, and prepare liposomes. Use the same method as in 1 to extrude through the membrane; measure the particle size of the two groups of blank liposomes after extrusion, and the results are as follows Figure 10 shown.

[0120] Table 4. Lipid dissolution and extrusion steps for blank liposomes prepared using preformed lipids and simple mixtures in the ethanol injection method

[0121]

[0122] 3. Characterization of lipid concentration, ratio, and thermodynamics of blank liposomes prepared with preformed lipids and comparison with blank liposomes prepared with simple lipid mixtures

[0123] Blank liposomes prepared using the two methods in steps 2 and 3, using preformed lipids and simple lipid mixtures as excipients, were dialyzed overnight against 10% sucrose solution to remove ammonium sulfate from the external aqueous phase. The particle size of the four groups of dialyzed blank liposomes was determined by dynamic light scattering. The lipid concentrations in the four groups of blank liposomes were determined using HPLC-CAD, and the lipid ratios were calculated. The thermodynamic behavior of the four groups of blank liposomes was determined using differential scanning calorimetry (MicroCal capillary DSC, Malvern, UK) (heating scan rate, 1°C / min; 10% sucrose solution was used as the reference solution).

[0124] The particle sizes of the four groups of blank liposomes after dialysis were all around 100-110 nm, with a uniform particle size distribution. Among them, the average particle size of the liposomes prepared by the ethanol injection method was the smallest (Table 5).

[0125] Table 5. Particle size and PDI of four groups of blank liposomes after dialysis (mean ± SD, n = 3)

[0126]

[0127] In terms of lipid concentration and ratio, the cholesterol concentration in the liposomes prepared by direct hydration of a simple lipid mixture was significantly lower, and the DSPE-PEG2000 concentration was also lower, resulting in the mass ratio of the three lipids in the liposomes significantly deviating from the input ratio of 3:1:1 (Table 6). Combined with the solid precipitation observed on the extrusion membrane during the extrusion process, this indicates that when a simple lipid mixture is used as a raw material and liposomes are prepared by direct hydration, the lipids cannot be fully hydrated, especially cholesterol precipitation, thus affecting the lipid concentration and ratio of the prepared liposomes. The concentrations and ratios of the three lipids in the blank liposomes obtained by the two preparation methods of prefabricated lipids are consistent with the ratios in the prefabricated lipids, indicating that the prefabricated lipids ensure the full hydration and uniform dispersion of each lipid component.

[0128] Table 6. Lipid content and ratio of blank liposomes prepared with different raw materials and preparation methods

[0129]

[0130] The phase transition behavior and thermodynamic parameters of lipid membranes are closely related to the lipid concentration and the ratio of the three lipids. Therefore, based on the quantitative determination of the concentration of each lipid in the lipid membrane, we also characterized the phase transition behavior of each group of blank liposomes. Figure 11As shown, the phase transition temperatures (Tm) of the four groups of liposomes are all around 52 degrees, which is the phase transition temperature of HSPC. Except for the liposomes prepared by direct hydration of a simple mixture, the DSC curves of the other three blank liposome samples completely overlap. Among them, the phase transition peak of the blank liposomes prepared by direct hydration of a simple lipid mixture is significantly higher and narrower, while the phase transition peaks of the other three groups of blank liposomes are all flat. This is mainly because the proportion of cholesterol in the liposomes prepared by direct hydration of a simple mixture is significantly lower, so the phase transition of the lipid membrane from the colloidal phase (solid ordered phase) to the liquid crystal phase (liquid ordered phase) is more significant, showing a high and narrow phase transition peak; while the other three groups of liposomes, due to containing more cholesterol (molar ratio of about 40%), are in the liquid disordered phase, and the transition to the liquid crystal phase is less significant.

[0131] The thermodynamic parameter values ​​obtained after quantitative fitting of the DSC curves of the four groups of blank liposomes (Table 7) also show that the liposomes prepared by the simple mixture using the direct hydration method have a different lipid membrane structure from the other three groups and have a higher phase transition enthalpy and consistency (ΔT 1 / 2 Thermodynamic parameters of the remaining three groups of liposomes were similar to those reported in the literature. This indicates that the liposomes prepared using preformed lipids and the liposomes prepared using the ethanol infusion method maintained a 3:1:1 ratio of the three lipids in their lipid membranes, and their lipid membrane structures were identical and consistent with those of blank liposomes reported in the literature.

[0132] Table 7. Thermodynamic parameters of blank liposomes in each group

[0133]

[0134] Summarize:

[0135] (1) Using preformed lipids as raw materials, blank liposomes can be prepared by direct hydration and ethanol injection methods. The concentrations and ratios of the lipid components and the lipid membrane structure of the blank liposomes obtained are consistent with expectations.

[0136] (2) Compared with simple lipid mixtures, the use of pre-made lipids in the direct hydration method for preparing liposomes can reduce the generation of foaming during stirring, ensuring the correct concentration and ratio of the three lipids in the formulation. It can also completely eliminate the possible precipitation of lipids (especially cholesterol) during future large-scale extrusion production and the resulting solvent residue problem.

[0137] (3) When preparing blank liposomes using the ethanol injection method, all preformed lipids can be added to ethanol and dissolved at a lower temperature. This method is easier to operate than a simple mixture, simplifies the operation process, and improves the chemical stability of the lipids. Compared with the direct hydration method, it can be an option for lipid-soluble drugs.

[0138] Example 4: Preparation of Doxorubicin Liposomes Using Preformed Lipids

[0139] Referring to commercially available doxorubicin liposomes (Doxil, Janssen Pharmaceuticals, USA), a doxorubicin solution was added to each blank liposome provided in Example 3. The drug was loaded by magnetic stirring at 60°C for approximately 20 minutes to prepare drug-loaded liposomes with a target drug concentration of 2 mg / ml and a total lipid concentration of approximately 16 mg / ml. The particle size and size distribution of the drug-loaded liposomes were determined by dynamic light scattering, and the entrapment efficiency of the liposomes was determined by resin adsorption.

[0140] As shown in Table 8, the blank liposomes prepared by direct hydration of a simple mixture had a very low encapsulation efficiency for doxorubicin (this is most likely related to the precipitation of cholesterol during hydration and extrusion. After cholesterol precipitation, the lipid formulation changed, the permeability increased, and the drug loading gradient decreased); the encapsulation efficiency of the other three blank liposomes for doxorubicin was about 99%.

[0141] Table 8. Particle size, particle size distribution (mean ± SD, n = 3) and encapsulation efficiency of four groups of drug-loaded liposomes

[0142]

[0143] For the three groups of drug-loaded liposomes with an encapsulation efficiency of 99%, the morphology of the liposomes was further observed using cryo-transmission electron microscopy ( Figure 12 The three groups of liposomes had similar morphologies, all in the shape of coffee beans, and contained rod-shaped doxorubicin-ammonium sulfate nanocrystals.

[0144] The concentrations of the three lipids in the three groups of drug-loaded liposomes with an encapsulation efficiency of 99% were determined using HPLC-CAD. The concentrations of the individual lipid components and the total lipid concentration in the three groups of drug-loaded liposomes were consistent with expectations and were consistent with the commercially available product Doxil (Table 9).

[0145] Table 9. Concentrations of three lipids and total lipid concentration in drug-loaded liposomes

[0146]

[0147]

[0148] The thermodynamic behavior of the three groups of drug-loaded liposomes with high encapsulation efficiency was characterized by DSC. The test conditions were the same as those of blank liposomes. Figure 13 As shown, two endothermic phase transition peaks were observed in the DSC of all three groups of drug-loaded liposomes: the flat phase transition peak with a Tm of approximately 52°C represents the phase transition of the lipid membrane, while the sharp, narrow peak with a Tm of approximately 70°C represents the melting peak of doxorubicin nanocrystals in the internal aqueous phase. This phase transition behavior is consistent with that of Doxil reported in the literature, indicating that the drug-loaded liposomes formed from the two groups of blank liposomes prepared using preformed lipids, after loading doxorubicin, have very similar structures (including lipid membrane structure and nanocrystal structure) to Doxil, and therefore have similar thermodynamic parameters (Table 10).

[0149] Table 10. Thermodynamic parameters of the prepared doxorubicin liposomes and the thermodynamic parameter values ​​of Doxil and Lipodox reported in the literature (Lipodox is a generic doxorubicin liposome from Sun Pharma, India)

[0150]

[0151] In vitro release rate study of three groups of drug-loaded liposomes:

[0152] Three groups of prepared doxorubicin liposomes were added to the release medium at a volume ratio of 1:50 using normal saline (pH 7.4) (ammonium ions). Dowex resin was then added to adsorb the released drug, creating a sink condition. Samples were collected at different time points to determine the liposome-encapsulated drug concentration. The cumulative release percentage was calculated and compared with the release behavior of Doxil.

[0153] like Figure 14 As shown in the figure, in normal saline without ammonium ions, the drug release rates of the three groups of drug-loaded liposomes were very slow, with the cumulative release percentages after 32 hours being less than 10%, indicating that the three groups of preparations maintained stable drug loading performance; under the release-promoting effect of ammonium ions, the release of the three groups of preparations was significantly accelerated, and the trends and cumulative release percentages at each time point were very close, and very close to the release rate of Doxil.

[0154] The results of in vitro release experiments showed that the release behaviors of the three groups of doxorubicin liposomes prepared were consistent and similar to that of Doxil.

[0155] Summarize:

[0156] Compared to the currently common process of preparing blank liposomes using ethanol injection, which uses simple lipid mixtures as raw materials, blank liposomes prepared using preformed lipids (including direct hydration and ethanol injection methods) achieve stable and effective drug delivery, ensuring a high process success rate. The resulting doxorubicin liposomes closely match the original research in terms of particle size, morphology, thermodynamic parameters, and in vitro release behavior.

[0157] Example 5: Preparation of Irinotecan Liposomes Using Preformed Lipids

[0158] 1. Preparation of Blank Liposomes

[0159] 90 mg of preformed lipids (DSPC / cholesterol / DSPE-PEG2000 = 61.6:38.1:0.3, molar ratio) were weighed and added to a small amount of anhydrous ethanol and dissolved in a 40°C water bath. 67 mg of DSPC, 21.8 mg of cholesterol, and 1.2 mg of DSPE-PEG2000 (a simple lipid mixture) were weighed. Cholesterol was first added to the anhydrous ethanol and heated to dissolve, then cooled, and the remaining two lipids were added to dissolve. Then, 3 ml of sucrose octasulfate triethylamine solution (81.25 mM, pH 5.5) was added, and the mixture was magnetically stirred in a 60°C water bath for 20 minutes to allow hydration and prepare liposomes. Liposomes were extruded through a membrane using a liposome extruder. The particle size of two sets of blank liposomes after extrusion was measured.

[0160] 2. Determination of lipid concentration and ratio of blank liposomes prepared from pre-made lipids and comparison with blank liposomes prepared from simple lipid mixtures

[0161] Blank liposomes prepared using the preformed lipids and simple lipid mixtures described in Example 1 were dialyzed overnight against normal saline to remove sucrose octasulfate triethylamine from the external aqueous phase. The particle size of the blank liposomes before and after dialysis was determined using dynamic light scattering. The lipid concentration in the blank liposomes was determined using the HPLC-CAD quantification method described in the first set of examples, and the lipid ratio was calculated.

[0162] The particle sizes of blank liposomes prepared with preformed lipids and simple lipid mixtures were approximately 100-110 nm before and after dialysis, with a uniform particle size distribution (Table 11).

[0163] Table 11. Particle size and PDI of blank liposomes before and after dialysis (mean ± SD, n = 3)

[0164]

[0165] In terms of lipid concentration and ratio, the blank liposomes prepared from a simple lipid mixture had a low input of DSPE-PEG2000 (preparing 3 ml of blank liposomes with only 1 mg added) and a loss during the preparation process, so the measured concentration of DSPE-PEG2000 in the blank liposomes was lower than the theoretical value, resulting in a change in the ratio of the three lipid components. In the blank liposomes prepared from prefabricated lipids, the concentrations and ratios of the three lipids were as expected (Table 12). This result illustrates that using prefabricated lipids as adjuvants can avoid the loss of low-content lipids during the preparation process, thereby ensuring the ratio and concentration of each lipid component in the blank liposomes.

[0166] Table 12. Lipid content and ratio of blank liposomes prepared with different raw materials and preparation methods

[0167]

[0168] 3. Irinotecan loading capacity of blank liposomes prepared from preformed lipids and compared with blank liposomes prepared from simple lipid mixtures

[0169] Referring to the commercially available irinotecan liposomes (Onivyde), irinotecan hydrochloride solution was added to each blank liposome and magnetically stirred at 55°C for approximately 20 minutes for drug loading. This resulted in drug-loaded liposomes with a target drug concentration of 4.3 mg / ml and a total lipid concentration of approximately 9.2 mg / ml. The particle size and size distribution of the drug-loaded liposomes were determined by dynamic light scattering; the zeta potential of the liposomes was measured by electrophoresis (Zetasizer NanoZS90, Malvern, UK); and the entrapment efficiency of the liposomes was determined by resin adsorption. The average particle size and entrapment efficiency of the two liposome groups were identical (Table 13).

[0170] Table 13. Particle size, particle size distribution, zeta potential (mean ± SD, n = 3) and encapsulation efficiency of irinotecan liposomes

[0171] Lipid raw materials 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

[0172] The morphology of the two groups of irinotecan liposomes was basically the same ( Figure 15 ). Components with higher electron density are visible inside, but there is no clear fine structure.

[0173] The concentrations of the individual lipid components in the two irinotecan-loaded liposomes were determined using HPLC-CAD. The total lipid concentration of both liposomes was close to that of the commercial product (9.15 mg / ml). However, similar to the concentrations and ratios of the individual lipid components in the blank liposomes, the concentrations and ratios of the three lipids in the irinotecan liposomes prepared using preformed lipids were consistent with expectations. In particular, 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). In the liposomes prepared using a simple lipid mixture as an excipient, the concentration and ratio of DSPE-PEG2000 were both lower than those of the commercial product (Table 14).

[0174] Table 14. Concentrations of three lipids and total lipid concentration in drug-loaded liposomes

[0175]

[0176] The difference in DSPE-PEG2000 concentration between the two groups of irinotecan-loaded liposomes in Table 14 is also reflected in the difference in zeta potential between the two liposomes (Table 13). In the drug-loaded liposomes prepared from the simple mixture, the DSPE-PEG2000 concentration is low, resulting in greater exposure of the negative charge of the phosphate group on the DSPC head in the lipid membrane, leading to a higher absolute zeta potential.

[0177] Study on the in vitro release rate of two groups of irinotecan-loaded liposomes:

[0178] Two sets of irinotecan liposomes were added to the release medium at a volume ratio of 1:50 using normal saline (ammonium ions) at pH 7.4. Dowex resin was then added to adsorb the released drug, creating a sink condition. Samples were taken at different time points to determine the drug concentration encapsulated in the liposomes. The cumulative release percentage was calculated and compared with the release behavior of Onivyde.

[0179] like Figure 16 As shown in the data, in normal saline without ammonium ions, the drug release rates of the two groups of drug-loaded liposomes were very slow, with the cumulative release percentages over 24 hours being less than 10%, indicating that the preparations maintained stable drug loading performance. Under the release-promoting effect of ammonium ions, the release of the two groups of preparations was significantly accelerated, and the trends and cumulative release percentages at each time point were very close, and very close to the release rate of Onivyde.

[0180] Summarize:

[0181] (1) Blank liposomes prepared by ethanol injection using preformed lipids with a molar ratio of DSPC / cholesterol / DSPE-PEG2000 = 61.6:38.1:0.3 as raw materials had similar particle size and distribution to blank liposomes prepared from a simple mixture. However, the preformed lipid group can better maintain the concentration of each lipid component, especially the low-content lipid component (DSPE-PEG2000), which is beneficial for maintaining the ratio of lipid components and conforming to the input ratio.

[0182] (2) Compared with the currently common process of preparing blank liposomes using ethanol injection with a simple lipid mixture as raw material, blank liposomes prepared with preformed lipids can stably and efficiently load irinotecan. The particle size and morphology of the resulting irinotecan liposomes are similar to those prepared with a simple mixture. The lipid concentration, ratio, and in vitro release rate of the irinotecan liposomes prepared with preformed lipids are consistent with those of commercially available products.

[0183] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the concept of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims, and the description and drawings may be used to interpret the content of the claims.

Claims

1. A lipid composition comprising a lyophilized powder of phospholipids and cholesterol in a ratio suitable for preparing liposomes, wherein at least 90% of the cholesterol is in an amorphous form; the phospholipids include phosphatidylcholine and PEGylated phospholipids; The phosphatidylcholine includes one or more of dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, hydrogenated soybean lecithin, dimyristoylphosphatidylcholine, dilauroylphosphatidylcholine, 1-stearoyl-2-palmitoylphosphatidylcholine, 1-myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl-2-myristoylphosphatidylcholine, 1-stearoyl-2-myristoylphosphatidylcholine and 1-palmitoyl-2-stearoylphosphatidylcholine; The preparation method of the lipid composition comprises: 1) fully dissolving the phospholipid and the cholesterol in a solvent; 2) freeze-drying to remove the solvent; The solvent is a mixed system of acetone, tert-butanol, anhydrous ethanol and water; the volume percentages of acetone, tert-butanol, anhydrous ethanol and water are 20%: (60%-65%): 15%: (0-5%) respectively.

2. The lipid composition of claim 1, wherein the cholesterol accounts for 38 mol % to 42 mol % of the lipid composition.

3. The lipid composition of claim 2, wherein the cholesterol accounts for 38 mol % to 40 mol % of the lipid composition.

4. The lipid composition according to any one of claims 1 to 3, wherein the content of the PEGylated phospholipid is less than 8 mol%.

5. The lipid composition according to any one of claims 1 to 3, wherein the PEGylated phospholipid is polyethylene glycol-distearoylphosphatidylethanolamine.

6. The lipid composition according to claim 5, wherein the PEGylated phospholipid is polyethylene glycol-distearoylphosphatidylethanolamine 2000.

7. A method for preparing the lipid composition according to any one of claims 1 to 6, comprising: 1) fully dissolving the phospholipid and the cholesterol in a solvent; 2) freeze-drying to remove the solvent; The solvent is a mixed system of acetone, tert-butanol, anhydrous ethanol and water; the volume percentages of acetone, tert-butanol, anhydrous ethanol and water are 20%: (60%-65%): 15%: (0-5%) respectively. The method according to claim 7 , wherein the freeze-drying temperature is -30° C. to -50° C.

9. method according to claim 8, the time of described freeze drying is 8h~48h.

10. The method according to any one of claims 7 to 9, wherein the freeze-dried lipid composition is pulverized and / or packaged.

11. A method for preparing liposomes, comprising: Dispersing the lipid composition according to any one of claims 1 to 6 into a hydration medium and optionally loading it with an active drug; The hydration medium is water or an aqueous solution without an organic solvent.

12. The preparation method according to claim 11, wherein the lipid composition is directly dispersed into the hydration medium.

13. The preparation method according to claim 11, wherein the lipid composition is first injected into a water-miscible organic dispersion to dissolve the lipid composition, and then the resulting solution is dispersed into a hydration medium. The preparation method according to claim 13 , wherein the water-miscible organic dispersion comprises ethanol. The preparation method according to claim 14 , wherein the temperature t of the mixed system of the lipid composition and ethanol is 40° C. ≤ t ≤ 70° C. The preparation method according to claim 15 , wherein the temperature t of the mixed system of the lipid composition and ethanol is 40° C. ≤ t ≤ 60° C. The preparation method according to claim 16 , wherein the temperature t of the mixed system of the lipid composition and ethanol is 50° C.≤t≤60° C. The preparation method according to claim 15 , wherein the temperature t of the mixed system of the lipid composition and ethanol is 50° C.≤t≤70° C.

19. The preparation method according to any one of claims 11 to 18, further comprising regulating the average particle size of the liposomes by mechanical means.

20. The preparation method according to claim 19, wherein the mechanical method comprises one or more of ball milling, air flow milling, high-speed shearing, high-pressure homogenization, and high-pressure extrusion.

21. The preparation method according to any one of claims 11 to 18 and 20, wherein the active drug comprises any one or more of taxanes, camptothecins, vinblastines, doxorubicin, cyclosporins, flavonoids, dihydropyridines, retinoids, anthraquinones, volatile oils, podophyllotoxins, purine antagonists, pyrimidine antagonists, folic acid antagonists, gambogic acids, photosensitizers, proteins, and nucleic acids.

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