Liposome preparation and preparation method thereof

Abstract

The invention provides a liposome preparation and a preparation method thereof, the liposome preparation comprises an ion pairing agent, phospholipid or a derivative thereof, sterol, a lipid molecule derivative and a drug compound, the ion pairing agent comprises at least one of a compound (I) and a compound (II), and the compound (II) comprises at least one of a compound (III) and a compound (III). Structures in the compound (I) and the compound (II) contain a plurality of functional groups (such as amide, carbonyl, thioether and the like), so that abundant binding sites are provided for drug molecules, and the compound (I) and the compound (II) can interact with different types of drug compounds (such as fat-soluble and water-soluble drugs); the liposome preparation is allowed to be combined with various drug molecules through hydrogen bonds, hydrophobic interaction or electrostatic interaction, and the liposome preparation adapts to physicochemical properties of different drugs. The compound (I) and the compound (II) further comprise different hydrophobic groups (such as alkyl chains) and hydrophilic groups (such as sulfonic acid, carboxylic acid and the like), so that the liposome preparation has the amphiphilic characteristic, not only can encapsulate hydrophobic drugs, but also can encapsulate hydrophilic drugs, and the variety and range of encapsulated drugs are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of drug synthesis, and in particular relates to a liposome preparation and a preparation method thereof. Background Art

[0002] Since Bangham first described liposomes in 1965 (J.Mol.Bio.13,238-252), there has been a sustained interest in and exploration of lipid nanocarrier systems for delivering active pharmaceutical ingredients (APIs). Throughout the history of liposome research and development, anticancer drugs have been selected as common encapsulation and delivery targets. Such APIs are usually rapidly metabolized, have toxic side effects, and have the risk of developing drug resistance. By encapsulating these APIs into liposomes, the APIs are endowed with the pharmacokinetic and pharmacodynamic characteristics of lipid-based nanocarriers, thereby reducing API-related toxicity and improving therapeutic effects. In addition, lipid-based nanocarriers can also achieve the following biological functions: overcome P-glycoprotein 1-mediated drug efflux, and aggregate drugs at tumor sites through enhanced permeability and retention effects (EPR), and can effectively escape after entering the cell endosomes to avoid being cleared.

[0003] One of the key requirements of liposomes in medical applications is the ability to load sufficient amounts of drugs to achieve therapeutic efficacy. Remote drug loading is a conventional liposome drug loading method that can achieve high drug encapsulation efficiency and high loading content (Chemistry and Chemistry of Lipids, 53 (1990) 37-46). This technology is to encapsulate drugs into pre-prepared liposomes. The "drug capture agent" is contained in the pre-prepared liposomes, and the former serves as a driving force to "pump" the drug from the extraliposomal medium into the liposome aqueous phase chamber and retain the drug there (Expert Opin. Drug Deliv., 8 (2011) 565-580). In order to achieve remote drug loading, after entering the liposome, the drug should be able to change from an uncharged substance that can diffuse through the lipid membrane to a charged substance that cannot diffuse through the lipid membrane. The degree of ionization of the drug compound depends on its pKa and pH value, and it needs to have a certain water solubility. In addition to pKa and solubility, other physicochemical properties of drugs also affect the results of remote drug delivery, such as molecular weight, polar and nonpolar surface area, solvent accessible surface area, logP and logD, and charge (Journal of Controlled Release, 139 (2009) 73-80). Considering all the above criteria, only amphiphilic weak bases or weak acids can meet all the requirements and are only applicable to a limited number of drug compounds. Summary of the invention

[0004] The technical purpose of the present invention is to provide a liposome preparation and a preparation method thereof, aiming to solve the problem that there are few types and a small range of drug compounds applicable to liposomes.

[0005] In order to solve the above technical problems, the present invention is implemented as follows: on the one hand, a liposome preparation is provided, the liposome preparation comprises an ion pairing agent, a phospholipid or a derivative thereof, a sterol, a lipid molecule derivative and a drug compound, the ion pairing agent comprises at least one of compound (I) and compound (II); The structural formula of the compound (I) is The structural formula of the compound (II) is , wherein Q includes at least one of O, NR, S, and -(CH2)n-, X includes at least one of C=O, C=NR, and C=S, Y includes at least one of alkylene, O, NR, and S, n is any one of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and m is any one of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, It includes at least one of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, cycloalkenyl, heterocyclic and hydrogen, and Z includes at least one of carboxylic acid, sulfuric acid, sulfonic acid, sulfinic acid, thiophenolic acid, phosphoric acid, monoester of phosphoric acid, diester of phosphoric acid, phosphonic acid, monoester of phosphonic acid, phosphinous acid, boric acid and monoester of boric acid.

[0006] On the other hand, a preparation method is provided for preparing a liposome preparation, the steps of the preparation method comprising: S1, dissolving the first drug compound, the ion pairing agent, the phospholipid or its derivative, the lipid molecule derivative and the sterol in an organic solvent to obtain a pretreatment solution, wherein the organic solvent comprises at least one of chloroform, methanol, ethanol and isopropanol; S2, mixing the pretreatment solution with a regulating solution to obtain a liposome preparation suspension, wherein the regulating solution comprises a buffer, an osmotic pressure regulator, a capture agent and a second drug compound; S3, heating the liposome preparation suspension, and obtaining a homogeneous solution by stirring or other homogenization operations; S4, performing dialysis, ultracentrifugation and / or size exclusion chromatography on the homogenous solution in sequence to remove excess drug compounds and obtain a pure homogenous solution; S5. Freeze-drying the pure homogeneous solution to obtain a dry liposome preparation.

[0007] Compared with the prior art, the liposome preparation and preparation method of the present invention have the following beneficial effects: The structures of compounds (I) and (II) contain multiple functional groups (such as amide, carbonyl, thioether, etc.), which provide abundant binding sites for drug molecules and can interact with different types of drug compounds (such as lipid-soluble and water-soluble drugs). The presence of these functional groups allows liposome preparations to bind to various drug molecules through hydrogen bonds, hydrophobic interactions or electrostatic interactions, thereby adapting to the physicochemical properties of different drugs. The structural design of compounds (I) and (II) contains different hydrophobic groups (such as alkyl chains) and hydrophilic groups (such as sulfonic acid, carboxylic acid, etc.). This structural design makes the liposome preparation have amphiphilic characteristics, that is, it can encapsulate both hydrophobic and hydrophilic drugs, thereby increasing the types and range of encapsulated drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 is a particle size distribution diagram of liposomes (PEM-L) loaded with pemetrexed in one embodiment of the present invention; Figure 2 is a particle size distribution diagram of venetoclax-loaded liposomes (VEN-L) in one embodiment of the present invention; Figure 3 This is a particle size distribution diagram of liposomes simultaneously loaded with venetoclax and cytarabine in one embodiment of the present invention; Figure 4 is a graph showing the relationship between the drug combination index and the degree of cell growth inhibition at different molar ratios of venetoclax and cytarabine in one embodiment of the present invention; Figure 5 In one embodiment of the present invention, the combination drug index graph of each drug molar ratio producing 75% effective cell growth inhibition and 90% effective cell growth inhibition at different molar ratios of venetoclax and cytarabine. DETAILED DESCRIPTION

[0009] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but are not to be construed as limiting the present invention. All other embodiments obtained by ordinary technicians in the field without creative work based on the embodiments of the present invention are within the scope of protection of the present invention.

[0010] In the description of the present invention, it is to be understood that the terms “center”, “longitudinal”, “lateral”, “length”, “width”, “thickness”, “up”, “down”, “front”, “back”, “left”, “right”, “vertical”, “horizontal”, “top”, “bottom”, “inside”, “outside”, “clockwise”, “counterclockwise”, “axial”, “circumferential”, “radial”, etc., indicating orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as limiting the present invention.

[0011] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0012] The present invention provides a liposome preparation, characterized in that the liposome preparation comprises an ion pairing agent, a phospholipid or a derivative thereof, and a sterol, wherein the ion pairing agent comprises at least one of compound (I) and compound (II); The structural formula of compound (I) is The structural formula of compound (II) is , wherein Q includes at least one of O, NR, S, and -(CH2)n-, X includes at least one of C=O, C=NR, and C=S, Y includes at least one of alkylene, O, NR, and S, n is any one of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and m is any one of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, It includes at least one of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, cycloalkenyl, heterocyclic and hydrogen, and Z includes at least one of carboxylic acid, sulfuric acid, sulfonic acid, sulfinic acid, thiophenolic acid, phosphoric acid, monoester of phosphoric acid, diester of phosphoric acid, phosphonic acid, monoester of phosphonic acid, phosphinous acid, boric acid and monoester of boric acid.

[0013] The structures of compound (I) and compound (II) contain multiple functional groups (such as amide, carbonyl, thioether, etc.), which provide abundant binding sites for drug molecules and can interact with different types of drug compounds (such as lipid-soluble and water-soluble drugs). The presence of these functional groups allows liposome preparations to bind to various drug molecules through hydrogen bonds, hydrophobic interactions or electrostatic interactions, thereby adapting to the physical and chemical properties of different drugs.

[0014] Moreover, these functional groups can form hydrogen bonds or electrostatic interactions with drug molecules or lipid molecules, thereby improving the encapsulation efficiency of liposome preparations for drugs and enhancing the stability of drugs in liposome preparations. This structural design helps to avoid drug leakage during preparation or storage, and ensures that the drug remains stable before being delivered to the target site.

[0015] The structural design of compounds (I) and (II) contains different hydrophobic groups (such as alkyl chains) and hydrophilic groups (such as sulfonic acid, carboxylic acid, etc.). This structural design makes the liposome preparation have amphiphilic characteristics, that is, it can encapsulate both hydrophobic drugs and hydrophilic drugs. The amphiphilic structure of the liposome preparation allows it to embed fat-soluble drugs into the lipid bilayer and encapsulate water-soluble drugs into the aqueous phase chamber, thereby accommodating a wider variety of drugs.

[0016] Moreover, this amphiphilic molecular structure helps it to be well distributed in the bilayer of liposome preparations, making the liposome preparations have good targeting and delivery effects in vivo. Specific functional groups (such as sulfonic acid groups and amine groups) can specifically bind to receptors on the cell membrane, thereby enhancing the targeting of liposome preparations, especially the targeted delivery effect on tumor cells or inflammatory tissues.

[0017] The ester bonds, amide bonds and other chemical bonds in the structure can be slowly degraded under specific enzymatic hydrolysis or pH conditions in the body, which means that compound (I) and compound (II) can be designed into a controlled release system to gradually release the drug over time or under specific conditions. This structural design is suitable for drug delivery systems that require sustained or controlled release, which can prolong the effective action time of the drug in the body, reduce the frequency of administration, and improve patient compliance.

[0018] The structures of compound (I) and compound (II) contain different hydrophobic chains (such as long-chain alkyl or unsaturated alkyl), which can adjust the fluidity of the bilayer of liposome preparations, making the membrane more flexible or rigid, thereby adjusting the drug release rate and stability. By selecting different hydrophobic chain lengths and unsaturation, the physical properties of liposome preparations can be adjusted to maintain appropriate fluidity and stability in different physiological environments.

[0019] The hydrophilic groups (such as sulfonic acid, carboxylic acid, amine, etc.) in the structures of compound (I) and compound (II) can increase the biocompatibility of liposome preparations in vivo, reduce the recognition and clearance of the immune system, and improve the long-circulation effect of liposome preparations. By rationally selecting these functional groups, the toxic and side effects of liposome preparations, especially the damage to normal cells, can be reduced, thereby improving the safety of drug delivery.

[0020] The structure of liposome preparations can effectively protect encapsulated biomacromolecules (such as enzymes or drugs) from degradation factors (such as enzymatic hydrolysis and oxidation) in the in vitro environment, thereby improving their stability in the body and prolonging their activity time.

[0021] Targeted drug delivery can be achieved by combining the liposome preparation structure with an ion pairing agent. This structure may have an affinity for specific cells or tissues, which helps to accurately deliver drugs to the target area and reduce damage to healthy cells. The ion pairing structure can also adjust the release rate to trigger the release of drugs in specific environments (such as pH, ion concentration, etc.), thereby improving the efficacy and safety of treatment. The hydrophilic and lipophilic parts of the liposome preparation can improve the solubility of the drug, especially for poorly soluble drugs. Liposome preparations can be used as carriers for lipophilic and hydrophilic drugs to enhance the absorption of drugs in the body and improve bioavailability. The encapsulation of liposome preparations prevents the drug from directly acting on non-targeted tissues, reducing toxic side effects. This can reduce the damage of drugs to normal cells and improve patient tolerance, especially for anticancer drugs with higher toxicity. The compounds in liposome preparations can slow down the release rate of drugs by forming a stable bilayer structure, giving them a longer half-life in the body, thereby reducing the frequency of administration and enhancing the efficacy.

[0022] Phospholipids or their derivatives include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine, 1,2-heneicosanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, L-α-soy hydrogenated phosphatidylcholine, 1,2-dioleoyl-sn- At least one of glycerol-3-phosphoethanolamine, palmitoyloleoyl-phosphatidylethanolamine, 1,2-diphytyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-snglycero-3-phospho-rac-(1-glycerol) sodium salt, 1,2-distearoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-racemic glycerol) sodium salt, egg-derived glycerol phosphate, soybean-derived glycerol phosphate, and sphingomyelin.

[0023] Phospholipids are the main structural components of the bilayer of liposome preparations. Their functions include: phospholipid molecules have a hydrophilic head and a hydrophobic tail, spontaneously forming a bilayer in an aqueous solution, forming the basic structure of liposome preparations. The bilayer provides a hydrophilic and hydrophobic environment, which is convenient for encapsulating drugs with different solubility characteristics (such as water-soluble drugs and fat-soluble drugs). The hydrophobic tail region of the phospholipid bilayer can encapsulate fat-soluble drugs, while the aqueous phase cavity can accommodate hydrophilic drugs. By combining different phospholipids, the encapsulation rate of drugs can be adjusted and the stability of drug delivery can be improved.

[0024] Different types of phospholipids (such as saturated fatty acids and unsaturated fatty acids) affect the fluidity and flexibility of the membrane. Saturated phospholipid molecules (such as DPPC) can form a more rigid membrane, while unsaturated phospholipid molecules (such as DOPE) make the membrane more fluid and flexible, which can adapt to different drug delivery requirements.

[0025] Phospholipids usually have good biocompatibility and are similar in structure to cell membranes. They can effectively escape clearance by the immune system in the body and prolong the circulation time in the body. They have good biocompatibility and degradability and are suitable for in vivo drug delivery. The thickness and rigidity of the phospholipid bilayer will affect the release rate of the drug. By selecting different phospholipids, liposome preparations with different release rates can be designed to meet the needs of sustained or controlled release drug delivery.

[0026] The liposome preparation also includes lipid molecule derivatives, which include lipid molecules modified with polyethylene glycol. The lipid molecules modified with polyethylene glycol include at least one of phosphatidylethanolamine modified with polyethylene glycol, phosphatidyl modified with polyethylene glycol, ceramide modified with polyethylene glycol, dipropylamine modified with polyethylene glycol, diacylglycerol modified with polyethylene glycol, dialkylglycerol modified with polyethylene glycol, glyceride modified with polyethylene glycol, sterol modified with polyethylene glycol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] sodium salt.

[0027] The liposomal formulations also include drug compounds including actinomycin, alitretinoin, altretinoin, aminopterin, azacitidine, azathioprine, belotecan, bendamustine, bexarotene, bleomycin, camptothecin, capecitabine, carboplatin, carboquinone, carmustine, chlorhexidine, chlorouracil, cisplatin, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, decitabine, dicycloplatin, docetaxel, doxorubicin, entinostat, epirubicin, eribulin, execon, flufenamide, 5-fluorouracil, gemcitabine, gemeline, gemeline, idarubicin, amide , irinotecan, ixabepilone, lenalidomide, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, morphine, nedaplatin, nimustine, otracil, oxaliplatin, paclitaxel, pemetrexed, pirarubicin, prednisone, satraplatin, temozolomide, tesetaxel, thiotepa, tipiracil, topotecan, retinoic acid, triazinon, triethylene melamine, venetoclax, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, zabartinib, zolpidem, abemaciclib, acalabrutinib, afatinib, aflibercept, alectinib, avosidide, aximinib, afatinib Preventinib, axitinib, binimetinib, bortezomib, bosutinib, brigatinib cabozantinib, amitinib, carfilzomib, capivasertib, capmatinib, ceritinib, cobimetinib, crizotinib, dabrafenib, dasatinib, docotinib, duvelisib, encorafenib, entrectinib, erdafitinib, erlotinib, everolimus, fedratinib, fostamatinib, fruquintinib, fulvestrantinib, gefitinib, gilteritinib, ibrutinib, imatinib, infigratinib, ixazomib, lapatinib larotate, siniab, levatinib, lorlatinib, mistalin , mobotinib, melotinib, neratinib, nedasudil, nilotinib, nintedanib, osimertinib, pacritinib, palbociclib, pasidatinib, pazopanib, pemigatinib, peixitinib, pitobutinib, panatinib, pralsetinib, quizartinib, regorafenib, ripretinib, rituximab, ruxolitinib, simetinib, simetinib, sorafenib, sunitinib, temsirolimus, tipotinib, tivozanib, tofacitinib, trametinib, tracillin, tucatinib, upadacitinib, vandetanib, vemurafenib, vismodegib, and zabrutinib.

[0028] In one embodiment of the present invention, the drug compound includes a fat-soluble drug and a water-soluble drug, the liposome preparation includes a lipid bilayer and an aqueous phase cavity formed by the lipid bilayer, the lipid bilayer includes an ion pairing agent, a phospholipid or its derivative, a lipid molecule derivative and a sterol, the lipid bilayer is embedded with a fat-soluble drug, and the aqueous phase cavity is provided with a water-soluble drug.

[0029] That is, the liposome preparation can be a spherical liposome preparation or an elliptical liposome preparation. Among them, the spherical liposome preparation is closer to the cell membrane structure, has good biocompatibility, is not easy to cause immune response, and the drug release of the spherical liposome preparation is more uniform, which is suitable for long-acting sustained-release drugs. The spherical liposome preparation has good stability and is suitable for systemic distribution of drugs after intravenous injection. Due to the special shape of the elliptical liposome preparation, it is not easy to be recognized and cleared by phagocytes, and has a longer circulation time in the body. The rolling and retention effect of the elliptical liposome preparation in the blood vessel is stronger, and it is easy to target the lesion site in the blood flow, which is particularly suitable for tumor targeted therapy.

[0030] Examples of fat-soluble drugs include: paclitaxel (such as docetaxel, paclitaxel, etc.), vinca alkaloids (such as vinblastine, vincristine, vindesine, vinorelbine), acitretin, tretinoin, bexarotene, doxorubicin, epirubicin, daunorubicin, everolimus, lapatinib, dasatinib, erlotinib, afatinib, gefitinib, pemetrexed, flufenamide, temozolomide, tesetaxel, dabrafenib, sorafenib, sunitinib, trametinib, and vandetanib. Examples of water-soluble drugs include 5-fluorouracil, gemcitabine, cisplatin, carboplatin, cyclophosphamide, aminopterin, methotrexate, azathioprine, dacarbazine, cytarabine, bleomycin, azacitidine, decitabine, mitoxantrone, chlorouracil, bendamustine, carmustine, lomustine, daunorubicin, pemetrexed, idarubicin, tetrapene, and ibrutinib.

[0031] Lipid-soluble drugs are embedded in the lipid bilayer, making these hydrophobic drugs more stable in the body and avoiding rapid degradation or precipitation problems caused by poor water solubility. Water-soluble drugs are encapsulated in the aqueous phase chamber to prevent them from being degraded in the body environment, thereby extending their active time. Through this design, lipid-soluble and water-soluble drugs can be encapsulated at the same time, achieving the coordinated delivery of multiple drugs to meet the needs of combined treatment. For example, some anti-cancer therapies require the simultaneous delivery of lipid-soluble and water-soluble anti-cancer drugs to improve efficacy.

[0032] Lipid-soluble drugs are located in the lipid bilayer, and due to their strong hydrophobic interaction with the liposome preparation membrane, their release is usually delayed, thus achieving a sustained release effect. Water-soluble drugs are located in the aqueous phase chamber, and their release rate can be adjusted by controlling the permeability of the liposome preparation membrane or the degradation of the membrane. In this way, a rapid release or delayed release preparation can be designed according to the treatment needs.

[0033] By encapsulating drugs in liposome preparations, the toxic side effects of drugs on normal tissues can be effectively reduced. Liposome preparations have natural biocompatibility and can escape rapid clearance by the immune system, thereby achieving a long circulation time. Liposome preparations can also achieve targeting through surface modification, increase the aggregation of drugs at the target site, thereby enhancing the therapeutic effect of the drug while reducing damage to normal tissues.

[0034] For fat-soluble drugs, embedding in the lipid bilayer improves the dispersibility of the drug in water and increases in vivo absorption. Water-soluble drugs are encapsulated in the aqueous phase chamber to avoid degradation problems caused by direct exposure to the body, and improve solubility and stability.

[0035] In one embodiment of the present invention, when the phospholipid or its derivative includes at least one of sphingomyelin and double-tail lipid, the liposome preparation includes a plurality of cross-arranged nanotubular fibers and ion channel proteins arranged in the gaps of each nanotubular fiber, the nanotubular fibers include phospholipid or its derivative and sterol, the drug compound includes water-soluble drugs and sustained-release drugs, water-soluble drugs are arranged inside the nanotubular fibers, and the ion channel proteins form an ion channel system, which is used to pass the sustained-release drugs.

[0036] The tubular structure provides a larger lumen space, which can carry more drugs and is particularly suitable for the encapsulation of macromolecular drugs, such as proteins and peptides. Tubular liposome preparations can achieve slow release of drugs, which is conducive to long-term treatment. The tubular structure is relatively stable in the body and is particularly suitable for slow release and targeted delivery to specific parts, such as long-term treatment of local tumors.

[0037] The nanotubular fiber structure provides a protected inner cavity for water-soluble drugs, preventing the rapid degradation of drugs in the body environment, thereby improving the stability of the drugs. By adding ion channel proteins, liposome preparations can respond to specific ions or environmental stimuli, thereby achieving precise control of sustained-release drugs and improving the bioavailability of drugs.

[0038] The water-soluble drug is located in the lumen of the nanotubular fiber, and the sustained-release drug is embedded in the lipid bilayer, forming a dual drug delivery system. This design can deliver water-soluble and lipid-soluble drugs at the same time to meet the needs of combined treatment. This dual delivery system enables drugs to work synergistically and improve the therapeutic effect.

[0039] The embedding of ion channel proteins forms an ion channel system that can respond to external stimuli (such as pH, ion concentration, etc.) to control the release of sustained-release drugs. This design has advantages in specific environments such as the tumor microenvironment and can achieve targeted release of drugs in the lesion area. The responsiveness of the ion channel system enhances the controllability of drug release, allowing the drug to be released quickly under specific conditions, improving the accuracy and safety of treatment. Through the bilayer protection of liposome preparations, water-soluble drugs and sustained-release drugs can be released slowly, avoiding excessive distribution of drugs in the body and reducing toxic side effects on normal cells. The ion channel system enables drugs to be released in a concentrated manner at the lesion site, avoiding systemic drug exposure, thereby improving the safety of drugs, especially for highly toxic anticancer drugs.

[0040] Through the combined design of nanotubular fibers and ion channel systems, it can be applied to a variety of different drug types, especially the simultaneous delivery of highly hydrophilic biomacromolecule drugs and sustained-release small molecule drugs. This liposome preparation has strong adaptability and can be used in a variety of drug delivery scenarios such as anticancer drugs, antibiotics, and biomacromolecule drugs.

[0041] Sustained-release drugs are mainly drugs with strong fat solubility and slow metabolism in the body. Most of them are the same as fat-soluble drugs and will not be elaborated here.

[0042] Ion channel proteins include aquaporins, which control the passage of water molecules and regulate hydration status; sodium ion channel proteins, which trigger release in a high sodium environment; calcium ion channel proteins, which trigger release in response to biological signals; chloride ion channel proteins, which regulate acid-base balance and achieve pH-responsive release; potassium ion channel proteins, which are suitable for liposome formulation systems that respond to potential stimulation; and photosensitive ion channel proteins, which use light to control drug release and achieve site-specific release.

[0043] The particle size of liposome preparations is 50~500nm, for example, it can be 50nm, 150nm, 500nm. Liposome preparations with small particle size (50~100nm) can more easily pass through the vascular endothelium and tissue barriers, especially in tumor tissues, enhancing permeability and retention effect (EPR effect), and increasing aggregation at tumor sites. Liposome preparations with small particle size have a longer circulation time in the body and can more effectively evade recognition and clearance by the immune system, especially when the particle size is close to 50-100nm, the long circulation effect is more significant. Liposome preparations with small particle size are less likely to aggregate in the liver and spleen, reducing the clearance of non-targeted tissues, thereby improving targeting.

[0044] The inner cavity and bilayer structure of medium-sized liposome preparations (100-200nm) are more balanced, and they can carry both fat-soluble drugs and water-soluble drugs, with a higher drug loading capacity. Within the particle size range of 100-200nm, liposome preparations can achieve a good balance between circulation and targeting, and are suitable for drug delivery that requires long-term sustained release.

[0045] Liposome preparations with larger particle sizes (200-500nm) are usually suitable for local targeting, such as local injection or intratumoral injection. Large-particle liposome preparations are not easy to pass through cell barriers, but can stay at the injection site for a long time. Large-particle liposome preparations have a larger surface area, and drugs are more easily released from the membrane, which is suitable for drugs that need to be released quickly or local high-concentration treatment.

[0046] In one embodiment of the present invention, the liposome preparation can be a sheet formed by the cross-section of an overall sheet or nano-tubular liposome preparation. The sheet-like liposome preparation can release the drug faster due to its large surface area, and is suitable for drugs that need to take effect in a short time. The sheet-like structure is easily adsorbed on the surface of local tissues and is suitable for local administration, such as drug delivery on the mucosal surface or the skin surface. The sheet-like structure is not easy to penetrate the tissue barrier in the body, and is suitable for local treatment to avoid adverse reactions caused by systemic distribution.

[0047] Embodiment 1: Embodiment 1 of the present invention provides a preparation method for preparing a liposome preparation, and the steps of the preparation method include: S1. Dissolving the first drug compound, the ion pairing agent, the phospholipid or its derivative, and the sterol in an organic solvent to obtain a pretreatment solution, wherein the organic solvent comprises at least one of chloroform, methanol, ethanol, and isopropanol.

[0048] Sterols include cholesterol, β-sitosterol, stigmasterol, campesterol, lanosterol, dehydrocholesterol, ergosterol, and 7-dehydrocholesterol. Sterol molecules can be embedded in the phospholipid bilayer, and by interacting with the hydrophobic tails of the phospholipid molecules, they reduce the gaps in the bilayer and increase the rigidity of the membrane. Liposome preparations are more stable in vivo or in vitro environments, are not easily deformed or disintegrated, and are suitable for preparations for long-term delivery.

[0049] Sterols can moderately regulate the fluidity of the bilayer, preventing the membrane from being too soft and preventing the membrane from being too rigid and resulting in fragility. This fluidity regulation makes liposome preparations more suitable for circulation in complex biological environments. Sterols can reduce the permeability of the bilayer, especially to small molecules and water molecules. This can prevent the drug in the liposome preparation from leaking out prematurely. This property is particularly important in preparations that require long-term sustained release, helping to achieve more stable drug delivery.

[0050] The rigid structure of sterols helps liposome preparations resist external mechanical stress, temperature changes, pH fluctuations, etc., so that they can maintain structural integrity in different environments. Especially in the blood circulation in the body or under external storage conditions, sterols can protect liposome preparations from disintegration and improve their stability.

[0051] Sterols can help liposome preparations form special structures such as tubular and sheet-like structures at a certain ratio, which helps to increase drug loading and adjust release patterns. In nanotubular liposome preparations, sterols can help form and stabilize tubular structures by increasing membrane rigidity.

[0052] By dissolving the drug, lipids and sterols in an organic solvent, ensure that the ingredients are fully mixed. Sterols (such as cholesterol) can enhance the rigidity of the lipid bilayer, provide stability, and help form the membrane structure of the liposome formulation. The addition of ion pairing agents helps to enhance the interaction between the drug and lipids, allowing the drug to be more effectively embedded in the bilayer or enter the aqueous phase cavity. Selecting an appropriate organic solvent ensures the solubility of the drug, phospholipids and sterols, which helps the formation and uniform distribution of the liposome formulation in subsequent steps.

[0053] For the step of preparing the nanotubular liposome preparation, the ratio of sterol can be appropriately increased so that the ratio of sterol to phospholipid is within the range of 40:60 or higher. The rigid structure of sterol helps the bilayer to extend along the axial direction and promotes the formation of nanotubular structure. Sphingomyelin or specific double-tail lipids (such as double-tailed phosphatidylethanolamine) are used. Such lipid molecules can form higher rigidity and stability in the bilayer, which helps the spontaneous formation of tubular structure.

[0054] S2. Mixing the pretreatment solution with the regulating solution to obtain a liposome preparation suspension, wherein the regulating solution includes a buffer, an osmotic pressure regulator, a capture agent and a second drug compound.

[0055] The buffer includes at least one of phosphate buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, citric acid buffer, and tris(hydroxymethylaminomethane) buffer; the osmotic pressure regulator includes at least one of sodium chloride, glucose, mannitol, and sucrose; and the capture agent includes at least one of cyclodextrin and its derivatives, ammonium sulfate, diammonium hydrogen phosphate, and trisodium citrate.

[0056] Adjusting the buffer and trapping agent in the solution helps optimize the internal environment of the liposome preparation, making it easier for drug molecules to be encapsulated. For example, using an appropriate buffer can make the pH value of the internal environment more suitable for drug encapsulation and improve the adsorption effect of liposome preparations on water-soluble drugs or ionic drugs. Capture agents (such as pH gradient capture agents) can enhance the encapsulation efficiency of water-soluble or polar drugs, and are particularly suitable for molecules with higher polarity or charged drugs. Osmotic pressure regulators can adjust the osmotic pressure of the liposome preparation suspension to make it close to the osmotic pressure of body fluids, prevent the liposome preparation from swelling or shrinking in the in vivo and in vitro environment, and maintain structural stability.

[0057] During homogenization and storage, the osmotic pressure regulator can reduce the tension of the liposome preparation membrane, avoid membrane rupture or structural deformation, and improve the stability of the liposome preparation in vivo and in vitro. By adjusting the pH value and ionic strength of the solution, the buffer can affect the fluidity and permeability of the liposome preparation membrane, thereby adjusting the release rate of the drug. For example, by selecting the appropriate buffer type and concentration, the diffusion and release of water-soluble drugs can be controlled. The addition of a capture agent can also form a drug precipitate inside the liposome preparation, so that the drug remains inside the liposome preparation, reducing leakage, thereby achieving a sustained release effect. Since the pretreatment solution contains a first class of drug compounds and the adjustment solution contains a second class of drug compounds, a dual drug system containing different drugs can be formed by mixing the two solutions. The first class of drugs can be fat-soluble drugs embedded in the bilayer of the liposome preparation, while the second class of drugs can be water-soluble drugs encapsulated in the aqueous phase chamber. In this way, dual drug delivery can be achieved to meet the needs of combined therapy.

[0058] In steps S1 and S2, it also includes: Select appropriate fluorescent dyes for the first type of drugs (lipid-soluble drugs) and the second type of drugs (water-soluble drugs). Fluorescent dye selection: For lipid-soluble drugs, hydrophobic fluorescent dyes such as NileRed, BODIPY, and DiI are recommended, and for water-soluble drugs, hydrophilic fluorescent dyes such as FITC, RhodamineB, and AlexaFluor are recommended.

[0059] The ideal fluorescent dye should be soluble in the phase where the drug is located (lipid soluble or water soluble) and have different emission wavelengths so that they can be distinguished during detection. The selected fluorescent dye is labeled with the drug, which can be achieved by covalent binding or physical encapsulation to ensure that the fluorescent signal can accurately reflect the distribution of the drug. Liposomes are prepared using a solution containing the fluorescently labeled first and second drugs, and the fluorescently labeled drugs are evenly distributed in the liposome preparation.

[0060] A fluorescently labeled first type of drug is added to the pretreatment solution, and a fluorescently labeled second type of drug is added to the adjustment solution. The prepared liposome preparation suspension is imaged using a fluorescence microscope to observe the distribution of the fluorescent dye to ensure that the two types of drugs are accurately positioned in the liposomes.

[0061] By selecting appropriate excitation and emission filters, the fluorescence signals of fat-soluble drugs in the liposome bilayer and water-soluble drugs in the aqueous phase chamber can be distinguished. By observing the fluorescence intensity at different positions, the distribution of drugs in the liposome preparation can be preliminarily judged to confirm that fat-soluble drugs are embedded in the bilayer and water-soluble drugs are encapsulated in the aqueous phase chamber.

[0062] The liposome suspension was analyzed by fluorescence spectrophotometer to obtain the fluorescence intensity at different wavelengths, and the standard curve was used for quantification, and the fluorescence intensity was converted into the concentration of the drug to calculate the encapsulation rate of fat-soluble drugs and water-soluble drugs.

[0063] Each fluorescent dye has different excitation and emission wavelengths, which can accurately distinguish and quantify the fluorescence signals of fat-soluble and water-soluble drugs. The specific structure of the fluorescence spectrophotometer is known to those skilled in the art and will not be described in detail here.

[0064] The accuracy of the data can be verified by three replicate experiments to ensure that the distribution of the drug within the liposome meets the design requirements.

[0065] The specific steps are as follows: prepare a series of drug solutions with known concentrations (usually fluorescently labeled drug solutions) that cover the expected measurement range. Three or more replicate samples are usually prepared for each concentration to ensure the accuracy and reliability of the data.

[0066] Measure the fluorescence intensity of each solution of known concentration using a fluorescence spectrophotometer or fluorescence microscope. Each sample is measured at the same excitation and emission wavelengths to ensure comparability of the results.

[0067] Record the fluorescence intensity corresponding to each concentration. Usually, the average fluorescence intensity is used as the representative data of the concentration. With the known drug concentration as the horizontal coordinate (X-axis) and the corresponding fluorescence intensity as the vertical coordinate (Y-axis), a scatter plot is drawn on the coordinate system. A curve is fitted through these points, which is the standard curve. If a straight line is obtained, this indicates a linear relationship between concentration and fluorescence intensity. If the fluorescence intensity and concentration show nonlinearity in a higher range, it can be processed by curve fitting. After the standard curve is drawn, a linear regression equation is obtained, such as , where Y is the fluorescence intensity and X is the known drug concentration, so k and b can be calculated. Substituting the new fluorescence intensity into the regression equation, the concentrations of fat-soluble drugs and water-soluble drugs can be known, and the encapsulation rate = drug concentration / total concentration.

[0068] If the encapsulation rate or distribution does not meet the requirements, the preparation process can be optimized, such as adjusting the lipid ratio, solution pH or stirring speed, to improve the encapsulation and distribution of the drug. For therapeutic drugs that require precise delivery, such as anticancer drugs and antibiotics, the encapsulation rate is usually required to be higher to ensure that sufficient drug doses are effectively delivered to the target tissue. An encapsulation rate of 70-98% is considered to be an ideal range.

[0069] For the step of preparing the nanotubular liposome preparation, the pretreatment solution and the adjustment solution are mixed in a microchannel using microfluidics technology, and the microfluidic environment can better control the direction and morphology of lipid self-assembly, which is conducive to the formation of tubular structures. Alternatively, a slow evaporation method can be used to gradually remove the organic solvent and control the arrangement direction of lipid molecules, thereby promoting the formation of tubular structures.

[0070] S3. Heat the liposome preparation suspension and obtain a homogenous solution by stirring or other homogenization operations.

[0071] For the steps of preparing nano-tubular liposome preparations, a relatively low temperature (e.g., about 50°C) is used for molding in the heating step, and the mixture is gradually cooled after heating so that the lipid bilayer is arranged axially to form nano-tubular fibers. Further thermal cycle treatment (heating-cooling cycle) can improve the crystallinity of the bilayer and the stability of the tubular structure.

[0072] Then, the ion channel protein (such as aquaporin or sodium ion channel protein) is dissolved in a low temperature buffer to ensure that the structure and activity of the protein are not damaged. The ion channel protein solution is slowly added to the homogenized solution using a gradual dropwise addition method to ensure uniform distribution and gradual fusion.

[0073] Perform slow stirring at low temperature (such as 4°C) to prevent channel protein denaturation and promote the embedding of ion channel proteins into the gaps of the bilayer of liposome preparations. Slow stirring helps the correct positioning and embedding of ion channel proteins, avoids damage to the protein structure caused by high temperature or violent stirring, and thus maintains the activity and stability of the protein.

[0074] To further enhance the binding of ion channel proteins to the liposome membrane, a slow heat treatment and cooling cycle can be performed. This operation can help the liposome membrane rearrange and help the ion channel protein to effectively fuse with the bilayer. The heat treatment temperature and time should be carefully controlled (generally below 50°C and no more than a few minutes) to ensure that the activity of the protein is not affected.

[0075] After the protein embedding step is completed, the structure of the liposome preparation is observed using a transmission electron microscope (TEM) or a fluorescence microscope to ensure that the ion channel protein is successfully embedded in the liposome preparation membrane. In addition, dynamic light scattering (DLS) or other particle size measurement techniques can be used to detect whether the liposome preparation still maintains a uniform particle size range and verify the structural integrity. The specific structures of the above-mentioned experimental equipment and observation equipment are known to those skilled in the art and will not be repeated here.

[0076] S4. The homogenous solution is subjected to dialysis, ultracentrifugation and / or size exclusion chromatography in sequence to remove excess drug compounds and obtain a pure homogenous solution.

[0077] Dialysis, ultracentrifugation, and size exclusion chromatography can effectively remove unencapsulated free drug compounds in the liposome suspension, ensuring that the liposome contains only encapsulated drugs. Removing free drugs can reduce the side effects of drugs, because unencapsulated drugs will diffuse rapidly and may have adverse effects on normal tissues. Purified liposomes can better achieve targeted delivery and sustained release, improving the safety and effectiveness of treatment.

[0078] Dialysis, ultracentrifugation and size exclusion chromatography can remove impurities and unformed liposome preparations introduced during the preparation process. High-purity and uniform liposome preparations have better biocompatibility and stability in the body, which can improve the delivery efficiency of drugs and ensure that the preparations have consistent quality and expected effects.

[0079] Ultracentrifugation and size exclusion chromatography can separate and select based on particle size, making the particle size of liposome preparations more uniform. Uniform particle size distribution ensures that liposome preparations have consistent distribution and release characteristics in the body, helps to achieve uniform drug release in specific tissues or cells, reduces the uncontrollability of in vivo distribution, and is suitable for long-acting and targeted drug delivery.

[0080] The removal of unbound small molecules and unstable components during dialysis and ultracentrifugation helps stabilize the structure of liposome preparations. Liposome preparations with stable structures can maintain integrity during storage and in vivo use, reduce spontaneous release and decomposition, extend the shelf life and storage stability of liposome preparations, and are particularly suitable for long-term delivery systems.

[0081] The purified liposome preparation contains only encapsulated drugs, removing free drugs and impurities that may cause adverse reactions to normal cells. Reducing the irritation and toxicity of free drugs and impurities to normal tissues improves the biosafety of liposome preparations, ensures that the drugs act more concentratedly on the lesion site, and improves the accuracy of treatment and patient tolerance.

[0082] The specific effects of each method: Dialysis: Suitable for removing small molecule impurities and unencapsulated small molecule drugs, especially suitable for liposome preparations that need to remove unencapsulated water-soluble drugs. Ultracentrifugation: It can separate liposome preparations of different particle sizes and remove particles that do not meet the particle size requirements. It is suitable for small-scale preparations and preparations that require high purity. Size exclusion chromatography: It can accurately separate liposome preparations of different sizes, suitable for purification during large-scale production, and can effectively control the particle size distribution of liposome preparations. The equipment used in the above method is known to those skilled in the art and will not be described here.

[0083] S5. Freeze-drying the purified homogenous solution to obtain a dry liposome preparation.

[0084] Freeze drying (lyophilization) can remove water from liposome preparations and convert them into dry powder. This dry form is more stable than the liquid state and can significantly extend the storage time of liposome preparations. Dried liposome preparations can be stored for a longer time at low temperatures, reducing the risk of degradation and denaturation, and are suitable for long-term storage and transportation, especially for drug production and clinical applications.

[0085] After removing the water, the chemical components in the liposome preparation are less likely to undergo hydrolysis or oxidation reactions, reducing the risk of degradation of the drug components. In a dry state, the physical structure of the liposome preparation is more stable, reducing the possibility of aggregation, precipitation or deformation of the liposome preparation, thereby maintaining the structural integrity of the preparation and the effectiveness of the drug.

[0086] Freeze-dried liposome preparations can be reconstituted by adding appropriate solvents (such as saline or buffer) before use to restore them to liquid form while maintaining the original particle size and drug distribution characteristics. The freeze-dried liposome preparations can quickly restore their original dispersion state after reconstitution, which is convenient for clinical use while ensuring that the bioavailability and efficacy of the drug are not affected.

[0087] In a dry state, the sensitivity of liposome preparations to temperature is greatly reduced, and cold chain transportation is not required, which simplifies storage and transportation conditions. Dried liposome preparations can be stored at room temperature, reducing the cost and difficulty of cold chain transportation, and are particularly suitable for large-scale production and distribution. In a dry state, liposome preparations do not contain moisture, making it difficult for microorganisms to grow in them, and can effectively prevent microbial contamination. The microbial safety of the preparation is increased, making it suitable for long-term storage, and reducing the risk of microbial degradation of drug ingredients.

[0088] The specific structure of the freeze-drying equipment is known to those skilled in the art and will not be described in detail here.

[0089] All commercially available chemical reagents and solvents used in all examples of the present invention were purchased from SigmaAldrich (St. Louis, Missouri), FisherScientific (Hampton, New Hampshire), Ambeed (Arlington Heights, Illinois), OakwoodChemical (Estill, South Carolina) and TCIAmerica (Portland, Oregon) and used without further purification. Proton NMR spectra were recorded at room temperature by Agilent 500 MHz spectrometer. Chemical shifts (δ) are expressed in parts per million, referenced to residual solvent signals; all coupling constants (J) are reported in Hertz.

[0090] Embodiment 2: Compound 001: Synthesis of (3β)-cholest-5-en-3-yl glycine ester.

[0091] Place N-(tert-Butyloxycarbonyl)glycine (2.24 g, 11.8 mmol) and a magnetic stirrer in a round-bottom flask, add anhydrous dichloromethane (50 ml) at room temperature, stir at room temperature, then add 4-dimethylaminopyridine (93 mg, 0.761 mmol), N,N-diisopropylethylamine (2.0 ml, 11.4 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.34 g, 12.2 mmol) in sequence, stir at room temperature for 15 minutes, then add cholesterol (3.03 g, 7.76 mmol) all at once, stir overnight, add another portion of N-(tert-Butyloxycarbonyl)glycine (0.68 g, 3.88 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.0 g, 5.22 mmol), and stir again overnight. The reaction mixture was quenched with saturated NaHCO3 solution (50ml), then diluted with dichloromethane (50ml) and extracted. The aqueous phase was further extracted with dichloromethane (3x50ml), then all organic phases were combined and washed with 0.5N HCl (2x50ml) and brine (50ml). The organic phase was dried over Na2SO4 and concentrated to obtain a white solid. This solid was dissolved in ethyl acetate (150ml), washed with 0.5N HCl (100ml), then washed with brine (100ml), then dried over Na2SO4 and concentrated to obtain the tert-butyloxycarbonyl protected intermediate. The intermediate was added to a round bottom flask equipped with a magnetic rotor and dissolved in ether (50ml). At room temperature, a solution of dioxane hydrochloride (4.0M, 75ml, 300mmol) was added and the mixture was stirred for 1 hour. Another portion of a solution of dioxane hydrochloride (4.0M, 50ml, 200mmol) was added and the mixture was stirred for 1 hour. The reaction was then poured into ether (200 ml), and the resulting precipitate was isolated by vacuum filtration, washed with ether (5 x 50 ml) and dried to give the hydrochloride of the title compound (2.64 g, 69.8% yield in 2 steps) as a fluffy white solid. Saturated NaHCO3 solution (100 ml) was added to 1.7 g of the hydrochloride, and the hydrochloride was converted into a free base by extraction with dichloromethane (3 x 75 ml). The combined organic phases were dried over Na2SO4, concentrated and dried again to give the title compound as a powdery white solid.

[0092] 1HNMR proton nuclear magnetic resonance spectrum (CDCl3, 500 MHz) δ: 5.38 (s, 1H), 4.67 (s, 1H), 3.39 (s, 2H), 2.32 (d, J = 7.1 Hz, 2H), 2.03-1.92 (m, 2H), 1.89-1.78 (m, 3H), 1.63-1.41 (m, 10H), 1.41-1.30 (m, 5H), 1.19-1.05 (m, 6H), 1.01 (s, 5H), 0.91 (d, J = 6.6 Hz, 3H), 0.86 (dd, J = 6.6, 2.2 Hz, 6H), 0.67 (s, 3H).

[0093] Embodiment 3: Compound 003: Synthesis of (3β)-cholest-5-en-3-yl (N,N-dimethyl)glycine ester.

[0094] In a dry round-bottom flask equipped with a magnetic rotor, N,N-dimethylglycine (1.07 g, 10.4 mmol) and anhydrous dichloromethane (40 ml) were added. The solution was stirred at room temperature and the following reagents were added in the following order: 4-dimethylaminopyridine (67 mg, 0.548 mmol), N,N-diisopropylethylamine (1.4 ml, 1.04 g, 8.05 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (2.20 g, 11.4 mmol). The solution was stirred at room temperature for 15 minutes, and then cholesterol (2.0 g, 5.17 mmol) was added in one portion. Dimethylformamide (10 ml) was added to promote dissolution. The mixture was stirred at room temperature overnight, quenched with saturated NaHCO3 solution (50 ml), and extracted with dichloromethane (2 x 100 ml). The organic phases were combined, washed with brine (50 ml), and the organic phases were dried over Na2SO4 and concentrated. The residue was purified by flash column chromatography to give the product as a white solid (1.82 g, 74.8%).

[0095] 1HNMR proton nuclear magnetic resonance spectrum (CDCl3, 500 MHz) δ: 5.38 (s, 1H), 4.69 (s, 1H), 3.13 (s, 2H), 2.39-2.31 (m, 1H, overlaps with 2.34), 2.34 (s, 6H, overlaps with 2.39-2.31), 2.07-1.92 (m, 2H), 1.92-1.76 (s, 3H), 1.70–1.24 (m, 11H), 1.25–1.03 (m, 8H), 1.03–0.96 (m, 3H, overlapping with 1.01), 1.01 (s, 3H, overlapping with 1.03–0.96) 0.91 (d, J = 6.5 Hz, 3H), 0.86 (dd, J = 6.6, 2.3 Hz, 6H), 0.67 (s, 3H).

[0096] Embodiment 4: Compound 004: Synthesis of (3β)-cholest-5-en-3-yl lysine ester.

[0097] In a dry round-bottom flask equipped with a magnetic rotor, cholesterol (1.0 g, 2.59 mmol) was added and dissolved in anhydrous dichloromethane (20 mL). (2S)-2,6-Bis[(tert-butyloxycarbonyl)amino]hexanoic acid (1.04 g, 3.00 mmol) and 4-dimethylaminopyridine (40 mg, 0.327 mmol) were added and the mixture was cooled to 0°C. A solution of N,N'-dicyclohexylcarbodiimide (690 mg, 3.34 mmol) in anhydrous dichloromethane (10 mL) was added via syringe over 1 min and the mixture was warmed to room temperature overnight. The reaction was quenched with saturated NaHCO3 solution (50 mL), filtered, and the filter cake was washed with dichloromethane (2 x 50 mL). The filtrate was transferred to a separatory funnel and deionized water (50 mL) was added to extract the mixture. The aqueous phase was extracted again with dichloromethane (100 mL), and the combined organic phases were washed with brine (100 mL). The organic phase was dried over Na2SO4, concentrated, and the residue was resuspended in ether (10 mL). The solid was filtered, washed with ether (2x10 mL), and the filtrate was concentrated to give the tert-butyloxycarbonyl protected intermediate, which was used without further purification. The 4.0 M intermediate was added to a round-bottom flask equipped with a magnetic rotor and dissolved in ether (25 mL). At room temperature, a solution of dioxane hydrochloride (4.0 M, 60 mL, 240 mmol) was added in one portion, and the mixture was stirred overnight. The reactants were diluted with ether (100 mL) and cooled to -20 ° C. The resulting solid was isolated by vacuum filtration, washed with ether (3x50 mL) and dried to give the dihydrochloride salt of the title compound (1.02 g) as a powdery white solid. A portion of this dihydrochloride salt was added to saturated NaHCO3 solution (100 mL) and extracted with dichloromethane (3 x 75 mL) to convert to the free base. The combined organic phases were dried over Na2SO4, concentrated and dried again to give the title compound (800 mg, 51.9%) as a sticky solid.

[0098] Embodiment 5: Compound 005: Synthesis of (3β)-cholest-5-en-3-yl arginine ester.

[0099] In a dry round-bottom flask equipped with a magnetic rotor, N2-(tert-butyloxycarbonyl)-N5-{N-[(2,2,4,6,7-pentamethyl-2,3-dihydro-1-benzofuran-5-yl)sulfonyl]carbamimidoyl}-L-ornithine (2.0 g, 3.80 mmol) and anhydrous dichloromethane (40 ml) were added. 4-Dimethylaminopyridine (47 mg, 0.385 mmol) and cholesterol (1.92 g, 4.96 mmol) were added and the mixture was cooled to 0°C. A solution of N,N'-dicyclohexylcarbodiimide (1.03 g, 4.99 mmol) in anhydrous dichloromethane (10 mL) was added in one portion via syringe. The mixture was stirred at 0°C for 10 minutes and then warmed to room temperature overnight. The reaction was quenched with deionized water (75 mL) and the mixture was transferred to a separatory funnel. The aqueous phase was extracted with dichloromethane (3x50mL) and the combined organic phases were washed with brine (100mL). The organic phase was dried over Na2SO4 and concentrated to a sticky solid, which was purified by flash column chromatography to give the tert-butyloxycarbonyl protected intermediate (1.65g, 48.5%) as a white solid. This intermediate (1.65g, 1.84mmol) was dissolved in anhydrous dichloromethane (25mL) in a round bottom flask equipped with a magnetic rotor and trifluoroacetic acid (14mL, 20.8g, 182mmol) was added via syringe over 1 minute. The mixture was stirred at room temperature overnight and then carefully quenched with saturated NaHCO3 solution (150mL). The pH was further adjusted with NaOH pellets until pH>9. The mixture was extracted with ethyl acetate (2x200mL) and the combined organic phases were washed with brine (100mL). The organic phase was dried over Na2SO4 and concentrated to give the crude product as an oily solid. The crude product was suspended in diethyl ether (about 10 mL), the solid was filtered off and washed with diethyl ether (about 10 mL). The isolated solid was dried to give the title compound as a yellow lumpy solid (650 mg, 65%).

[0100] Embodiment 6: Compound 006: Synthesis of (3β)-cholest-5-en-3-yldimethylamino-β-alanine ester.

[0101] Cholesterol (2.01 g, 5.20 mmol) and anhydrous dichloromethane (25 ml) were added to a dry round bottom flask equipped with a magnetic rotor. The solution was cooled to 0°C and trifluoroacetic acid (1.25 ml, 908 mg, 8.97 mmol) was added. Then, 3-bromopropionyl chloride (780 µL, 1.33 g, 7.75 mmol) was added via syringe over 1 min and the solution was stirred at 0°C for 1 h. The reaction was carefully quenched with saturated NaHCO3 solution (100 mL) and diluted with ethyl acetate (75 ml). The mixture was transferred to a separatory funnel, extracted, and the aqueous layer was further extracted with ethyl acetate (3 x 75 ml). The combined organics were washed with brine (100 ml), dried over Na2SO4, and concentrated to give the intermediate bromide (2.52 g, quantitative) which was used without further purification. A magnetic rotor and acetone: dimethylformamide (4:1, 40 mL) were added to a round-bottom flask containing the bromide intermediate (2.52 g, 5.20 mmol). Then, dimethylamine hydrochloride (848 mg, 10.4 mmol) and K2CO3 (2.15 g, 15.6 mmol) were added and the mixture was stirred vigorously at room temperature overnight. The reaction was filtered through a Buchner funnel and the filtrate was concentrated to give the title compound (910 mg, 36% yield over 2 steps) as an off-white solid.

[0102] 1HNMR proton nuclear magnetic resonance spectrum (CDCl3, 500 MHz) δ: 5.37 (s, 1H), 4.63 (s, 1H), 2.60 (t, J = 6.0 Hz, 2H), 2.45 (t, J = 6.0 Hz, 2H), 2.35-2.28 (m, 2H), 2.24 (s, 6H), 2.06-1.92 (m, 2H), 1.92-1.77 (m, 3H), 1.66-1.27 (m, 11H), 1.20-1.06 (m, 6H), 1.04-0.97 (m, 6H), 0.93-0.81 (m, 10H), 0.67 (s, 3H).

[0103] Embodiment 7: Compound 007: Synthesis of (3β)-cholest-5-en-3-yl[2-(diethylamino)ethyl]carbamate.

[0104] In a dry round-bottom flask equipped with a magnetic rotor, anhydrous dichloromethane (150 mL) and cholesterol chloroformate (10.0 g, 22.3 mmol) were added. The reaction was cooled to 0°C and triethylamine (4.8 mL, 3.48 g, 34.4 mmol) was added. Diethylethylenediamine (4.8 mL, 3.93 g, 33.8 mmol) was added via syringe over 1 min and the reaction was stirred at 0°C for 40 min. The reaction was quenched with saturated NaHCO3 (150 mL) and transferred to a separatory funnel. The aqueous layer was extracted with dichloromethane (2x150 mL) and the combined organics were washed with brine (150 mL). The organic phase was dried over Na2SO4 and concentrated, and the residue was suspended in methanol (200 mL). The mixture was cooled to -20°C in a refrigerator and the resulting solid was collected by vacuum filtration. The solid was washed with cold methanol (3 x 75 mL) and dried to give the title compound (10.5 g, 89.0%) as a white powdery solid.

[0105] 1HNMR proton nuclear magnetic resonance spectrum (CDCl3, 500 MHz) δ: 5.37 (s, 1H), 5.12 (s, 1H), 4.49 (s, 1H), 3.20 (s, 2H), 2.50 (q, J = 5.4 Hz, Hz, 6H), 2.40-2.23 (m, 2H), 2.06-1.91 (m, 2H), 1.91-1.75 (s, 3H), 1.64-1.39 (m, 7H), 1.40-1.28 (m, 3H), 1.22-0.81 (m, 29H), 0.67 (s, 3H).

[0106] Embodiment 8: Compound 008: Synthesis of (3β)-cholest-5-en-3-yl[3-(diethylamino)propyl]carbamate.

[0107] In a dry round bottom flask equipped with a magnetic rotor, add anhydrous dichloromethane (25 mL) and cholesterol chloroformate (1.51 g, 3.36 mmol). Cool the reaction to 0°C and add triethylamine (700 µL, 508 mg, 5.02 mmol). Add diethylpropylenediamine (800 µL, 660 mg, 5.06 mmol) via syringe over 1 min and stir the reaction at 0°C for 1 h. Quench the reaction mixture with saturated NaHCO3 solution (100 mL) and transfer to a separatory funnel. Extract the aqueous phase with dichloromethane (2 x 100 mL) and wash the combined organic phases with brine (100 mL). Dry the organic phase over Na2SO4 and concentrate to give the title compound (1.25 g, 68.6%) as a white solid.

[0108] 1HNMR proton nuclear magnetic resonance spectrum (CDCl3, 500 MHz) δ: 5.79 (s, 1H), 5.36 (s, 1H), 4.53-4.41 (m, 1H), 3.22 (s, 2H), 2.53-2.42 (m, 6H), 2.40-2.20 (m, 2H), 2.05-1.91 (s, 2H), 1.91-1.78 (s, 3H), 1.67-1.21 (m, 13H), 1.20-0.79 (m, 28H), 0.67 (s, 3H).

[0109] Embodiment 9: Compound 009: Synthesis of (3β)-cholest-5-en-3-yl[4-(dimethylamino)]butyrate.

[0110] (3β)-cholest-5-en-3-yl-4-bromobutyrate (2.55 g, 4.76 mmol) was added to a round-bottom flask equipped with a magnetic rotor and tetrahydrofuran:dimethylformamide (1:1, 20 mL) was added. K2CO3 (3.0 g, 21.7 mmol) and dimethylamine hydrochloride (1.50 g, 18.4 mmol) were added and the mixture was stirred at room temperature for 24 h. Upon completion, deionized water (100 mL) and dichloromethane (150 mL) were added and the mixture was transferred to a separatory funnel. The aqueous phase was extracted again with dichloromethane (100 mL) and the combined organic phases were washed with brine (100 mL). The organic phase was dried over Na2SO4 and concentrated, and the residue was purified by flash column chromatography to give the product (1.65 g, 69.6%) as a white solid.

[0111] Embodiment 10: Compound 010: Synthesis of (3β)-cholest-5-en-3-yl[4-(diethylamino)]butyrate.

[0112] (3β)-cholest-5-en-3-yl-4-bromobutyrate (2.17 g, 4.05 mmol) was added to a round bottom flask with a magnetic rotor and tetrahydrofuran:dimethylformamide (1:1, 20 mL) was added. K2CO3 (2.01 g, 14.5 mmol) and diethylamine (1.2 mL, 848 mg, 11.6 mmol) were added and the mixture was stirred at room temperature overnight. Since there was starting material remaining, the reaction was heated to 50°C and allowed to react for an additional 8 hours. After the reaction was complete, the reaction was diluted with dichloromethane (100 mL), vacuum filtered, and the filter cake was washed with dichloromethane (2 x 50 mL). The filtrate was concentrated and the residue was purified by flash column chromatography to give the title compound (1.27 g, 59.6%) as a white solid.

[0113] 1HNMR proton nuclear magnetic resonance spectrum (CDCl3, 500 MHz) δ: 5.37 (s, 1H), 4.60 (s, 1H), 2.60-2.48 (m, 4H), 2.48-2.37 (m, 2H), 2.33-2.26 (m, 4H), 2.05-1.91 (m, 2H), 1.89-1.69 (m, 5H), 1.64-1.20 (m, 12H), 1.19-0.78 (m, 27H), 0.67 (s, 3H).

[0114] Embodiment 11: Compound 011: Synthesis of (3β)-cholest-5-en-3-yl-2-diethylglycinate.

[0115] (3β)-cholest-5-en-3-yl-2-chloroacetate (1.20 g, 2.59 mmol) was added to a round-bottom flask equipped with a magnetic rotor, and tetrahydrofuran (10 mL) and dimethylformamide (8 mL) were added. K2CO3 (1.92 g, 13.4 mmol) and diethylamine (1.0 mL, 707 mg, 9.66 mmol) were added, and the mixture was stirred at room temperature for 24 hours. Upon completion, the reaction was diluted with dichloromethane (100 mL), vacuum filtered, the filter cake was washed with dichloromethane (100 mL), and the filtrate was dried and concentrated to give the title compound (1.22 g, 94.5%) as an off-white solid.

[0116] 1HNMR proton nuclear magnetic resonance spectrum (CDCl3, 500 MHz) δ: 5.36 (s, 1H), 4.65 (s, 1H), 3.29 (s, 2H), 2.65 (q, J = 7.2 Hz, 4H), 2.32 (d, J = 7.2 Hz, 2H), 2.07–1.74 (m, 5H), 1.47 (dd, J = 64.8, 51.6 Hz, 12H), 1.18–0.81 (m, 27H), 0.67 (s, 3H).

[0117] Embodiment 12: Compound 012: Synthesis of (3β)-cholest-5-en-3-yl-4-pyrrolidinylbutyrate.

[0118] (3β)-cholest-5-en-3-yl-4-bromobutyrate (630 mg, 1.17 mmol) was added to a round-bottom flask with a magnetic stirrer, followed by tetrahydrofuran:dimethylformamide (1:1, 10 mL). K2CO3 (752 mg, 5.44 mmol) and pyrrolidine (300 µL, 255 mg, 3.58 mmol) were added, and a reflux condenser was installed on the flask. The mixture was heated to 60°C for 2 hours. After the reaction was completed, the reaction was diluted with deionized water (100 mL), transferred to a separatory funnel, and extracted with dichloromethane (2x100 mL). The combined organic phases were washed with brine (100 mL), dried over Na2SO4, and concentrated to an oily solid crude product. The crude product was suspended in methanol (75 mL) and placed in a -20°C refrigerator overnight. The resulting solid was filtered, washed with cold methanol (3 x 15 mL) and dried to give the title compound (350 mg, 56.9%) as a shiny white solid.

[0119] 1H NMR proton nuclear magnetic resonance (CDCl3, 500 MHz) δ: 5.36 (s, 1H), 4.60 (s, 1H), 2.53-2.40 (m, 6H), 2.37-2.26 (m, 4H), 2.05-1.91 (m, 2H), 1.90-1.70 (m, 9H), 1.62-1.38 (m, 8H), 1.38-1.03 (m, 11H), 1.04-0.93 (m, 5H), 0.88 (dd, J = 24.6, 6.6 Hz, 9H), 0.67 (s, 3H).

[0120] Embodiment 13: Compound 013: Synthesis of (3β)-cholest-5-en-3-yl (2-pyrrolidinyl)ethylcarbamate.

[0121] In a dry round-bottom flask equipped with a magnetic rotor, add anhydrous dichloromethane (20 mL) and cholesterol chloroformate (1.03 g, 2.29 mmol). Cool the reaction to 0°C and add triethylamine (480 µL, 348 mg, 3.44 mmol). Add 1-(2-aminoethyl)pyrrolidine (420 µL, 378 mg, 3.31 mmol) via syringe over 1 min and stir the reaction at 0°C for 2 h. Quench the reaction with saturated NaHCO3 solution (100 mL) and transfer to a separatory funnel. Extract the aqueous phase with dichloromethane (2 x 100 mL) and wash the combined organic phases with brine (100 mL). Dry the organic phase over Na2SO4, concentrate, and suspend the residue in methanol (75 mL). Place the mixture in a -20°C freezer and collect the resulting solid by vacuum filtration. The solid was washed with cold methanol (2 x 50 mL) and dried to give the title compound (1.04 g, 85.9%) as a white solid.

[0122] 1HNMR proton nuclear magnetic resonance spectrum (CDCl3, 500 MHz) δ: 5.36 (s, 1H), 5.16 (s, 1H), 4.48 (s, 1H), 3.27 (s, 2H), 2.61-2.53 (m, 2H), 2.53-2.45 (m, 3H), 2.40-2.16 (m, 2H), 2.08-1.69 (m, 10H), 1.63-1.30 (m, 10H), 1.21-0.83 (m, 23H), 0.67 (s, 3H).

[0123] Embodiment 14: Compound 014: Synthesis of trans-3,7-dimethylocta-2,6-dien-1-yl-4-(diethylamino)butyrate.

[0124] Trans-3,7-dimethylocta-2,6-dien-1-yl-4-bromobutyrate (1.0 g, 3.29 mmol) was added to a round-bottom flask with a magnetic rotor, and tetrahydrofuran: dimethylformamide (1:1, 20 mL) was added. K2CO3 (1.85 g, 13.4 mmol) and diethylamine (1.1 mL, 777 mg, 10.6 mmol) were added, and the mixture was stirred at room temperature for 24 hours. After the reaction was completed, the reactant was concentrated and the residue was purified by flash column chromatography to obtain the title compound (520 mg, 53.4%) as a transparent light yellow oil.

[0125] Embodiment 15: Compound 015: Synthesis of (3β)-cholest-5-en-3-yl[(3-aminopropyl-methyl)amino]propylcarbamate.

[0126] In a dry round-bottom flask equipped with a magnetic rotor, add anhydrous dichloromethane (30 mL) and cholesterol chloroformate (1.22 g, 2.71 mmol). The reaction was cooled to 0°C and triethylamine (1.2 mL, 871 mg, 7.29 mmol) was added. A solution of 3,3'-diamino-N-methyldipropylamine (1.06 g, 7.29 mmol) in anhydrous dichloromethane (10 mL) was added via syringe over 3 minutes and stirred at 0°C for 1 hour. The reaction was quenched with saturated NaHCO3 solution (100 mL) and the mixture was transferred to a separatory funnel. The aqueous phase was extracted with dichloromethane (2x100 mL) and the combined organic phases were washed with brine (100 mL). The organic phase was dried over Na2SO4 and concentrated, and the residue was suspended in methanol (100 mL). The mixture was placed in a freezer at -20°C and the resulting solid was collected by vacuum filtration, washed with cold methanol (3 x 50 mL) and dried to afford the title compound (1.12 g, 74.2%) as a lumpy white solid.

[0127] 1H NMR proton nuclear magnetic resonance (CDCl3, 500 MHz) δ: 5.49 (s, 1H), 5.35 (s, 1H), 4.47 (s, 1H), 3.22 (s, 2H), 2.43-2.07 (m, 6H), 2.04-1.71 (m, 7H), 1.71-1.25 (m, 17H), 1.25-0.94 (m, 21H), 0.94-0.80 (m, 14H), 0.66 (s, 3H).

[0128] Embodiment 16: Compound 016: Synthesis of (3β)-cholest-5-en-3-yl bis-(2-diethylamino)ethylcarbamate.

[0129] In a dry round bottom flask equipped with a magnetic rotor was added anhydrous dichloromethane (25 mL) and cholesterol chloroformate (2.0 g, 4.45 mmol). The reaction was cooled to 0°C and triethylamine (1.3 mL, 944 mg, 9.33 mmol) was added. N,N,N',N'-tetraethyldiethylenetriamine (1.7 mL, 1.42 g, 6.59 mmol) was added via syringe over 1 min and allowed to warm to room temperature overnight. The reaction was quenched with saturated NaHCO3 (100 mL) and the mixture was transferred to a separatory funnel. The aqueous phase was extracted with dichloromethane (2 x 100 mL) and the combined organic phases were washed with brine (100 mL). The organic phase was dried over Na2SO4, concentrated and purified by flash column chromatography to give the title compound (880 mg, 31.5%) as a viscous yellow oil.

[0130] 1H NMR proton nuclear magnetic resonance (CDCl3, 500 MHz) δ: 5.36 (s, 1H), 5.31-5.26 (m, 1H), 4.49 (s, 1H), 4.11 (s, 1H), 3.44-3.20 (m, 4H), 2.72-2.46 (m, 9H), 2.43-2.19 (m, 2H), 2.09-1.75 (m, 5H), 1.64-1.19 (m, 11H), 1.19-0.80 (m, 25H), 0.66 (s, 3H).

[0131] Embodiment 17: Compound 017: Synthesis of 2-(pyrrolidin-1-yl)ethyl deoxycholate.

[0132] Deoxycholic acid (1.02 g, 2.60 mmol) and dimethylformamide:tetrahydrofuran (1:1, 10 mL) were added to a dry round-bottom flask equipped with a magnetic rotor. K2CO3 (1.07 g, 7.74 mmol) and 1-(2-chloroethyl)pyrrolidine hydrochloride (652 mg, 3.83 mmol) were added at room temperature and stirred for 16 hours. The reaction was then heated to 50°C in an oil bath and stirred for 5 hours. The reaction mixture was then cooled to room temperature, quenched with saturated NaHCO3 solution, and transferred to a separatory funnel. The mixture was extracted with dichloromethane (2x100 mL), and the combined organic phases were washed with brine, dried over Na2SO4, and concentrated. The residue was purified by flash column chromatography (SiO2, 9:1 dichloromethane:methanol with 0.1% NH4OH) to give the title compound (620 mg, 48.8%) as a viscous white solid.

[0133] 1H NMR proton nuclear magnetic resonance (CDCl3, 500 MHz) δ: 4.17 (t, J = 4.8 Hz, 2H), 3.94 (s, 1H), 3.61-3.51 (m, 1H), 2.71 (t, J = 4.6 Hz, 2H), 2.58-2.50 (m, 4H), 2.40-2.30 (m, 1H), 2.28-2.17 (m, 1H), 1.83-1.44 (m, 18H), 1.43-1.28 (m, 6H), 1.28-1.18 (m, 3H), 1.16-1.00 (m, 2H), 0.98-0.90 (m, 4H), 0.88 (s, 3H), 0.65 (s, 3H).

[0134] Embodiment 18: Compound 018: Synthesis of 2-hexyldecyl (4-pyrrolidin-1-yl) butyrate.

[0135] 2-Hexyldecyl-4-bromobutyrate (1.52 g, 3.88 mmol) was added to a round bottom flask with a magnetic rotor and tetrahydrofuran:dimethylformamide (1:1, 20 mL) was added. K2CO3 (2.58 g, 18.7 mmol) and pyrrolidine (1.0 mL, 852 mg, 12.0 mmol) were added and the mixture was heated to 50°C and stirred for 4 hours. Upon completion, the reaction was diluted with dichloromethane (50 mL), filtered, and the residue was washed with dichloromethane (3 x 50 mL). The filtrate was concentrated and the resulting oil was dissolved in ethyl acetate (100 mL). The organic phase was washed with deionized water (2 x 75 mL), then with brine (75 mL), dried over Na2SO4 and concentrated to give the title compound (1.19 g, 80.4%) as a clear, bright yellow oil.

[0136] Embodiment 19: Compound 019: Synthesis of bis[(3β)-cholest-5-en-3-yl]-3,3'-[(2-(pyrrolidin-1-yl)ethyl)azanediyl]dipropionate.

[0137] In a dry scintillation vial equipped with a magnetic rotor, isopropanol:hexafluoroisopropanol (3:1, 10 mL) was added to (3β)-cholest-5-ene-3-acrylate (1.08 g, 2.45 mmol) and stirred to form a slurry. A solution of 1-(2-aminoethyl)pyrrolidine (133 mg, 1.16 mmol) in isopropanol:hexafluoroisopropanol (3:1, 5 mL) was added and the mixture was stirred at 80°C for 8 hours. After cooling, a solid precipitated from the solution. The solid was filtered and washed with methanol (2x20 mL) and dried. The title compound (260 mg, 22.6%) was obtained as an off-white solid.

[0138] Embodiment 20: Compound 020: Synthesis of (3β)-cholest-5-en-3-yl-6-(pyrrolidin-1-yl)hexanoate.

[0139] (3β)-Cholest-5-en-3-yl-6-bromohexanoate (1.09 g, 1.93 mmol) was added to a round-bottom flask equipped with a magnetic rotor and tetrahydrofuran (15 mL) was added. K2CO3 (1.35 g, 9.76 mmol) and pyrrolidine (460 µL, 392 mg, 5.51 mmol) were added and the mixture was heated to 60°C and stirred for 5.5 hours. Upon completion, the reaction mixture was diluted with ethyl acetate (100 mL) and the organic phase was washed with deionized water (100 mL) and brine (100 mL). The organic phase was dried over Na2SO4, concentrated, and the resulting oil was suspended in methanol (50 mL). The resulting solid was collected by filtration, washed with methanol (2 x 50 mL), and dried to give the title compound (610 mg, 57.0%) as a white solid.

[0140] Embodiment 21: Compound 021: Synthesis of (3β)-cholest-5-en-3-yl-6-(diethylamino)hexanoate.

[0141] (3β)-Cholest-5-en-3-yl-6-bromohexanoate (570 mg, 1.01 mmol) was added to a round bottom flask with a magnetic stirrer and tetrahydrofuran (15 mL) was added. K2CO3 (708 mg, 5.12 mmol) and diethylamine (400 µL, 283 mg, 3.87 mmol) were added and the mixture was heated to 60°C and stirred for 3 hours. Another portion of diethylamine (500 µL, 354 mg, 4.84 mmol) was added and the mixture was reheated to 60°C for 6 hours. Upon completion, the reaction mixture was diluted with deionized water (100 mL) and extracted with ethyl acetate (2 x 100 mL). The combined organic phases were washed with brine (100 mL), dried over Na2SO4, concentrated, and the residue was suspended in methanol (30 mL). The resulting solid was collected by filtration, washed with methanol (2 x 25 ml) and dried to give the title compound (330 mg, 58.8%) as a white solid.

[0142] Embodiment 22 Compound 022: Synthesis of (3β)-cholest-5-en-3-yl[3-(4-methyl-1-piperazine)propyl]carbamate.

[0143] In a dry round bottom flask equipped with a magnetic rotor was added anhydrous dichloromethane (15 mL) and cholesterol chloroformate (1.19 g, 2.65 mmol). The reaction was cooled to 0°C and triethylamine (500 µL, 363 mg, 3.59 mmol) was added. 1-(3-aminopropyl)-4-methylpiperazine (632 mg, 4.02 mmol) was added in one portion at 0°C and stirred for 1 hour. The mixture was quenched with saturated NaHCO3 solution (100 mL) and the reaction mixture was transferred to a separatory funnel. The aqueous phase was extracted with dichloromethane (2 x 100 mL) and the combined organic phases were washed with brine (100 mL). The organic phase was dried over Na2SO4, concentrated and purified by flash column chromatography to give the title compound (900 mg, 59.6%) as a low melting white solid.

[0144] Embodiment 23: Compound 023: Synthesis of (3β)-cholest-5-en-3-yl-L-proline ester.

[0145] In a dry round-bottom flask equipped with a magnetic rotor, N-tert-butyloxycarbonyl-L-proline (2.23 g, 10.3 mmol) and anhydrous dichloromethane (20 mL) were added. The solution was stirred at room temperature and the following reagents were added in the following order: 4-dimethylaminopyridine (72.4 mg, 0.592 mmol), N,N-diisopropylethylamine (1.4 mL, 1.04 g, 8.05 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (2.0 g, 10.4 mmol). The solution was stirred at room temperature for 15 minutes, and then cholesterol (2.02 g, 5.22 mmol) was added in one portion. The mixture was stirred at room temperature overnight, quenched with saturated NaHCO3 (100 ml), and extracted with dichloromethane (2x100 ml). The combined organic phases were washed with brine (100 ml), dried over Na2SO4 and concentrated to give the tert-butyloxycarbonyl protected intermediate which was used without further purification. The intermediate was added to a round bottom flask equipped with a magnetic rotor and dissolved in ether (75 ml). A hydrochloric acid dioxane solution (4.0 M, 75 ml, 300 mmol) was added at once at room temperature and the mixture was stirred for 2.5 hours. The mixture was basified with saturated NaHCO3 until pH>9 and the mixture was extracted with ethyl acetate (2x100 ml). The organic phase was concentrated and the residue was resuspended in acetone (~10 ml). The solid was collected by filtration and dried to give the title compound (450 mg, 17.8%) as an off-white solid.

[0146] Embodiment 24: Compound 024: Synthesis of N-[2-(diethylamino)ethyl]oleamide.

[0147] Anhydrous dichloromethane (15 mL) and oleyl chloride (1.6 mL, 1.46 g, 4.85 mmol) were added to a dry round-bottom flask equipped with a magnetic rotor. The reaction was cooled to 0°C and triethylamine (1.0 mL, 726 mg, 7.17 mmol) was added. Diethylethylenediamine (1.0 mL, 827 mg, 7.11 mmol) was added once via syringe at 0°C and stirred for 1.5 hours. The reaction was quenched with saturated NaHCO3 solution (100 mL) and the mixture was transferred to a separatory funnel. The aqueous layer was extracted with dichloromethane (2x100 mL) and the combined organic phases were washed with brine (100 mL). The organics were dried over Na2SO4, concentrated and purified by flash column chromatography to give the title compound (390 mg, 21.0%) as a viscous yellow oil.

[0148] Embodiment 25: Compound 025: Synthesis of (3β)-cholest-5-en-3-yl 3-(4-methyl-1-piperazine)propanoate.

[0149] In a dry round-bottom flask equipped with a magnetic rotor, intermediate II (644 mg, 1.46 mmol) was added, followed by isopropanol:hexafluoroisopropanol (3:1, 12 mL) to stir to form a slurry. N-methylpiperazine (260 µL, 234 mg, 2.34 mmol) was added via syringe, and the mixture was stirred at 80 °C for 50 minutes. After cooling, the reaction mixture was concentrated and the residue was suspended in deionized water (75 mL). The aqueous phase was extracted with ethyl acetate (2 x 100 mL), and the combined organic phases were washed with deionized water (75 mL) and brine (75 mL). The organic phase was dried over Na2SO4, concentrated, and the residue was suspended in methanol (50 mL). The resulting solid was filtered, washed with methanol (2 x 25 mL), and dried to give the title compound (500 mg, 22.6%) as an off-white solid.

[0150] Embodiment 26: Compound 026: Synthesis of (3β)-cholest-5-en-3-yl-2-(4-methyl-1-piperazine)acetate.

[0151] (3β)-cholest-5-en-3-yl 2-chloroacetate (1.01 g, 2.18 mmol) was added to a round-bottom flask equipped with a magnetic rotor and tetrahydrofuran (15 mL) was added. K2CO3 (1.70 g, 12.3 mmol) and N-methylpiperazine (720 µL, 650 mg, 6.50 mmol) were added and the mixture was stirred at 60°C for 6 hours. After the reaction was complete, the reaction mixture was diluted with deionized water (100 mL) and the aqueous phase was extracted with ethyl acetate (2 x 100 mL). The combined organic phases were washed with deionized water (100 mL) and then with brine (100 mL), dried over Na2SO4, and concentrated. The resulting solid was suspended in methanol (50 mL) and the resulting solid was collected by filtration. The solid was washed with methanol (2 x 25 mL) and dried to give the title compound (750 mg, 65.8%) as an off-white solid.

[0152] Embodiment 27: Compound 027: Synthesis of (3β)-cholest-5-en-3-yl-glycine methyl ester.

[0153] In a dry round-bottom flask equipped with a magnetic rotor, N-tert-butyloxycarbonyl-sarcosine (1.97 g, 10.4 mmol) and anhydrous dichloromethane (25 mL) were added. The solution was stirred at room temperature and the following reagents were added in the following order: 4-dimethylaminopyridine (72 mg, 0.589 mmol), N,N-diisopropylethylamine (1.4 mL, 1.04 g, 8.05 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (2.0 g, 10.4 mmol). The solution was stirred at room temperature for 15 minutes, and then cholesterol (2.0 g, 5.17 mmol) was added in one portion. The mixture was stirred at room temperature overnight, quenched with saturated NaHCO3 solution (100 mL), and extracted with dichloromethane (2x100 mL). The combined organic phases were washed with brine (100 mL), dried over Na2SO4, and concentrated to give the tert-butyloxycarbonyl protected intermediate, which was used without further purification. The intermediate mixture was added to a round-bottom flask equipped with a magnetic rotor and dissolved in anhydrous dichloromethane (75 mL). Trifluoroacetic acid (10 mL, 14.9 g, 130 mmol) was slowly added over 1 minute at room temperature and the mixture was stirred for 30 minutes. At this time, deionized water (100 mL) was added and the mixture was basified with saturated NaHCO3 solution until pH>9. The mixture was transferred to a separatory funnel and extracted with dichloromethane (2x100 mL). The combined organic phases were washed with brine (100 mL), dried over Na2SO4 and concentrated to a yellow oily crude product. The crude oil was dissolved in ether (20 mL) and ethereal hydrochloric acid (2.0 M, 30 mL) was added to prepare the hydrochloride salt. The solid was collected by filtration, washed with ether (2x50 mL) and dried to obtain a sticky solid. The solid was basified with saturated NaHCO3 solution (100 mL) and the aqueous phase was extracted with dichloromethane (2x100 mL). The combined organic phases were washed with brine (100 mL), dried over Na2SO4 and concentrated to give a solid. The solid was suspended in acetone (25 mL), filtered, washed with acetone (2 x 50 mL) and dried to give the title compound (350 mg, 13.8%) as a lumpy white solid.

[0154] Embodiment 28: Compound 028: Synthesis of (3β)-cholest-5-en-3-yl-N-2-(dimethylaminoethyl)-N-methylglycinate.

[0155] (3β)-Cholest-5-en-3-yl-2-chloroacetate (1.01 g, 2.18 mmol) was added to a round-bottom flask equipped with a magnetic rotor and tetrahydrofuran (10 mL) was added. K2CO3 (917 mg, 6.63 mmol) and N,N,N'-trimethylethane-1,2-diamine (560 µL, 440 mg, 4.30 mmol) were added and the mixture was stirred at 60°C for 4 hours. After the reaction was completed, the reaction was diluted with ethyl acetate (100 mL) and the organic phase was washed with deionized water (2 x 100 mL) and brine (100 mL). The organic phase was dried over Na2SO4, concentrated, and the residue was purified by flash column chromatography to give the title compound (510 mg, 41.8%) as a yellow solid.

[0156] Embodiment 29: Compound 029: Synthesis of (3β)-cholest-5-en-3-yl-[2-(4-methyl-1-piperazine)ethyl]glycine ester.

[0157] (3β)-cholest-5-en-3-yl-2-chloroacetate (1.0 g, 2.16 mmol) was added to a round-bottom flask equipped with a magnetic rotor and tetrahydrofuran (15 mL) was added. K2CO3 (1.80 g, 13.0 mmol) and 2-(4-methyl-1-piperazine)ethylamine (1.54 g, 10.8 mmol) were added and the mixture was stirred at 60°C for 5 hours. After the reaction was completed, the reaction mixture was diluted with deionized water (100 mL) and the aqueous phase was extracted with ethyl acetate (3x100 mL). The organic phase was washed with deionized water (100 mL) and then with brine (100 mL), dried over Na2SO4 and concentrated to give the title compound (380 mg, 30.9%) as a viscous yellow oil.

[0158] Embodiment 30: Compound 030: Synthesis of (3β)-cholest-5-en-3-ylbis[3-(dimethylamino)propyl]glycine ester.

[0159] (3β)-cholest-5-en-3-yl-2-chloroacetate (1.0 g, 2.16 mmol) was added to a round-bottom flask equipped with a magnetic rotor and tetrahydrofuran (15 mL) was added. K2CO3 (1.50 g, 10.9 mmol) and 3,3'-iminobis(N,N-dimethylpropylamine) (1.4 mL, 1.18 g, 6.30 mmol) were added and the mixture was stirred at 60°C for 2 hours. After the reaction was complete, the reaction mixture was diluted with deionized water (100 mL) and the aqueous phase was extracted with ethyl acetate (3 x 100 mL). The organic phase was washed with deionized water (100 mL) and then with brine (100 mL), dried over Na2SO4 and concentrated to give the title compound (220 mg, 16.5%) as a waxy amber solid.

[0160] Embodiment 31: Compound 031: Synthesis of 3-(pyrrolidin-1-yl)propane-1,2-distearate.

[0161] 3-Chloropropane-1,2-distearate (1.03 g, 1.60 mmol) was added to a round bottom flask with a magnetic stir bar and tetrahydrofuran (15 mL) was added. K2CO3 (1.03 g, 7.45 mmol) and pyrrolidine (400 µL, 341 mg, 4.79 mmol) were added and the mixture was stirred at room temperature overnight. The next morning, the reaction was heated to 60°C for 5 hours and then quenched with saturated NaHCO3 solution (100 mL). The aqueous phase was extracted with ethyl acetate (2 x 100 mL) and the combined organic phases were washed with brine (100 mL). The organic phase was dried over Na2SO4, concentrated and the residue was suspended in methanol (50 mL). The resulting solid was collected by filtration and washed with methanol (2 x 25 mL) to obtain the first batch of solids. The filtrate was cooled to -20°C in a refrigerator, the resulting solid was filtered and washed with ice-cold methanol (3 x 20 mL) to obtain the second batch of solids. The combined solids were dried to give the title compound (610 mg, 56.5%) as a white solid.

[0162] Embodiment 32: Compound 032: Synthesis of 3-[2-(dimethylamino)ethyl]-methylaminopropane-1,2-distearate.

[0163] 3-Chloropropane-1,2-distearate (1.06 g, 1.64 mmol) was added to a round bottom flask with a magnetic stir bar and tetrahydrofuran (15 mL) was added. K2CO3 (650 mg, 4.70 mmol) and N,N,N'-trimethylethane-1,2-diamine (420 µL, 330 mg, 3.23 mmol) were added and the mixture was heated to 60°C for 3 hours. Upon completion, the reaction was quenched with saturated NaHCO3 solution (100 mL) and the aqueous phase was extracted with ethyl acetate (2 x 100 mL). The combined organic phases were washed with brine (100 mL), dried over Na2SO4 and concentrated to give the title compound (450 mg, 38.8%) as a blocky off-white solid.

[0164] Embodiment 33: Compound 033: Synthesis of (3β)-cholest-5-en-3-yl-carbamoylglycine.

[0165] To a dry round-bottom flask equipped with a magnetic rotor was added the hydrochloride salt of compound 001 (690 mg, 1.44 mmol) and anhydrous dimethylformamide (10 mL). N,N-diisopropylethylamine (750 µL, 556 mg, 4.30 mmol) and 1H-pyrazole-1-carboximide hydrochloride (365 mg, 2.50 mmol) were added to the mixture at room temperature. The reaction was heated to 50 °C for 3 hours and then quenched with saturated NaHCO3 solution (100 mL). When an attempt was made to extract the aqueous phase with dichloromethane, a solid formed at the interface of the organic and aqueous phases. The mixture was filtered and the resulting solid was suspended in methanol (20 mL). The solid was collected by filtration, washed with methanol (2x10 mL), and dried to give the title compound (180 mg, 25.8%) as a white solid.

[0166] Embodiment 34: Compound A: Preparation of crude liposome formulations containing pemetrexed (PEM).

[0167] mPEG2000-DSPE, DSPC, cholesterol, an ion pairing agent in Formula I and pemetrexed (PEM) were dissolved in ethanol. The organic phase was slowly injected into an aqueous buffer at about 45-75°C while rapidly stirring for about 30-45 minutes for emulsification. The emulsion was cooled in an ice bath to obtain a crude liposome preparation suspension (PEM-L) encapsulating pemetrexed. The drug encapsulation efficiency of crude PEM-L is shown in the table below (Table 1). The data show that the encapsulation efficiency of some ion pairing agents is less than 70%, which is not suitable for encapsulation of pemetrexed.

[0168] Table 1: Effects of different ion pairing agents on the encapsulation efficiency of pemetrexed.

[0169] Example 35: Preparation of refined liposome preparations encapsulating pemetrexed (PEM).

[0170] mPEG2000-DSPE, DSPC, cholesterol, the ion pairing agent in Formula I and pemetrexed (PEM) are dissolved in ethanol. The organic phase is slowly injected into the aqueous buffer phase at about 45-75°C while rapidly stirring for about 30-45 minutes to allow the crude liposome preparation to form. The crude liposome preparation is then extruded through a filter membrane with different pore sizes of 50-400 nm at about 45-75°C multiple times to obtain the desired liposome preparation particle size and particle size distribution. The extruded liposome preparation is cooled in an ice bath and then purified by dialysis to produce a liposome preparation (PEM-L) containing PEM. The final product of the drug-loaded liposome preparation usually has an average particle size between 50 and 150 nm.

[0171] Example 36: Characterization of the physicochemical properties of liposome formulations encapsulating pemetrexed (PEM).

[0172] The liposome preparation (PEM-L) containing pemetrexed formed by the ion pairing agent in Formula I was physicochemically characterized. The drug-loaded liposome preparation was prepared according to the preparation method of Example 35. The average particle size, particle size distribution (PDI) and PEM encapsulation efficiency of the liposome preparation suspension are listed in Table 2. Due to the leakage characteristics of the tumor vasculature, nanoparticles with a particle size of <200 nm will show preferential accumulation at the tumor site, which is a key feature of the enhanced permeability and retention (EPR) effect of the liposome preparation.

[0173] Table 2 shows the average particle size (nm) and particle size distribution (PDI) of liposomes with different drug pairing agents. The average particle size of all formulations is around 100nm (specific values ​​range from 101.2nm to 103.5nm), and the particle size distribution (PDI) is relatively low (0.087 to 0.133). This shows that the prepared liposome particle size is around 100nm and is evenly distributed, meeting the requirements of small particle size. Liposomes with a particle size below 200nm are conducive to entering tumor tissues through enhanced permeation and retention effect (EPR effect) in the body circulation, thereby improving the targeted delivery effect of drugs.

[0174] Table 2 shows the encapsulation efficiency (%) of liposome preparations with different drug pairing agents, ranging from 78.9% to 98.8%. This shows that there are certain differences in the encapsulation efficiency of drugs under different formulations. The formula encapsulation efficiency of compound 007 is the highest, at 98.8%, while the encapsulation efficiency of compound 010 is relatively low, at 78.9%. This shows that the selection of different pairing agents has a significant effect on the encapsulation efficiency of drugs.

[0175] Table 2: Effects of different ion pairing agents on the particle size, particle size distribution and pemetrexed encapsulation efficiency of liposome preparations.

[0176] Example 37: Preparation of liposome formulations encapsulating Venetoclax (VEN).

[0177] DSPG, DSPC, cholesterol and Venetoclax (VEN) were dissolved in a mixture of chloroform and methanol at about 50-80°C. The solution was acidified and then the solvent was removed by spray drying. The resulting yellow powder was rehydrated in an aqueous phase consisting of about 5-50mM HEPES, pH 4-7 buffer at a temperature of 45-75°C while rapidly stirring for about 30-60 minutes to achieve emulsification. The emulsion was extruded at about 45-75°C using multiple 50-400nm filters to obtain the desired liposome preparation particle size and particle size distribution. The extruded emulsion was finally cooled in an ice bath to produce a lipid suspension (VEN-L) encapsulated by VEN.

[0178] Example 38: Preparation of complex liposome formulations encapsulating Venetoclax (VEN) and Cytarabine (CYT).

[0179] DSPG, DSPC, cholesterol and venetoclax VEN were dissolved in a mixture of chloroform and methanol at about 50-80°C. The solution was acidified and then the solvent was removed by spray drying. The resulting yellow powder was dispersed in a buffer containing about 5-50 mM HEPES, pH 4-7, at 45-75°C for rehydration, in which cytarabine CYT was also dissolved. Rapid stirring was performed for about 30-60 minutes to achieve emulsification. The emulsion was extruded at about 45-75°C using multiple 50-400 nm filters to obtain the desired liposome preparation particle size and particle size distribution. The extruded emulsion was cooled in an ice bath to produce a drug-loaded liposome preparation suspension. The crude liposome preparation suspension was then purified by dialysis to produce the final product of VEN / CYT liposome preparation (VEN / CYT-L). The physicochemical properties of VEN-L and VEN / CYT-L are shown in Table 3.

[0180] The data in Table 3 illustrate the following points: The particle size of liposomes encapsulating only a single drug (VEN-L) was small, while the particle size of liposomes encapsulating two drugs (VEN / CYT-L) was significantly larger. Both formulations had high drug encapsulation efficiencies, especially the composite liposomes (VEN / CYT-L), which had encapsulation efficiencies of 96.2% and 99.4% for the two drugs, respectively, which was conducive to efficient delivery. The high encapsulation efficiency and moderate particle size make these liposomes suitable for combined drug therapy, which helps to achieve synergistic effects and improve therapeutic effects.

[0181] Table 3: Physicochemical characterization of VEN-L and VEN / CYT-L.

[0182] Example 39: Evaluation of the efficacy of venetoclax and cytarabine in combination.

[0183] For a combination drug regimen, two or more drugs may exhibit synergistic, additive or antagonistic interactions, and the above drug effects depend specifically on the ratio between the combined drugs. In order to determine the combination ratio (VEN / CYT) of Venetoclax and Cytarabine that has a synergistic effect, this experiment studied a wide range of VEN:CYT drug molar ratios using relevant cell lines. The synergistic, additive or antagonistic effects between Venetoclax and Cytarabine were determined using 400:1 to 1:1, preferably 1:1, 2:1, 4:1, 100:1, 200:1 and 400:1 to determine the drug effects produced for different cancer cells (e.g., MV4-11 leukemia cells) at different molar ratios of CYT:VEN. Figure 4Shown are the changes in CI values ​​as a function of cell growth inhibition expressed as the fractional effect (Fa) on MV4-11 cells at various CYT to VEN molar ratios. Synergism was observed at CYT:VEN ratios of 1:1, 2:1, 4:1, 100:1, 200:1, and 400:1, as CI was well below 0.9 over the Fa range. These results suggest that 1:1, 2:1, 4:1, 100:1, 200:1, and 400:1 CYT:VEN molar ratios are synergistic at concentrations that result in significant tumor cell death. In contrast, antagonism (i.e., CI>1.1) was observed when the CYT:VEN molar ratio was 50:1 and Fa was above 0.5, and when Fa was above 0.65 at a CYT:VEN molar ratio of 25:1. The dependence of CI on the CYT:VEN ratio was also observed. Figure 5 The table shows that the CI values ​​at drug concentrations sufficient to cause 75% (ED75) and 90% (ED90) tumor cell growth inhibition at different CYT:VEN molar ratios are compared. The strongest synergistic effect was observed when the CYT:VEN ratio was between 1:1 and 4:1. Based on the above results, a CYT:VEN molar ratio of 2:1 was selected as a fixed drug ratio to prepare a liposome formulation drug product for the treatment of leukemia.

[0184] Figure 1 Particle size distribution of pemetrexed-loaded liposomes (PEM-L) is shown.

[0185] Figure 2 Particle size distribution of venetoclax-loaded liposomes (VEN-L) is shown.

[0186] Figure 3 The particle size distribution of liposomes loaded with venetoclax and cytarabine is shown. The molar ratio of venetoclax to cytarabine in this liposome is 1:8.

[0187] Figure 4 Results of the study of the pharmacodynamic synergy between venetoclax and cytarabine in leukemia cells (MV4-11) in vitro are presented. The relationship between the Combination Index (CI) and the degree of cell growth inhibition (Fraction affected, Fa) at different molar ratios of venetoclax and cytarabine is shown in the figure. CI values ​​>1.1, 0.9-1.1, and <0.9 indicate drug antagonism, additive, and synergistic effects, respectively.

[0188] Figure 5The combination index (CI) of each drug molar ratio producing 75% effective cell growth inhibition (ED75) and 90% effective cell growth inhibition (ED90) at different molar ratios of venetoclax and cytarabine is shown.

[0189] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A liposome preparation, characterized in that: The liposome preparation comprises an ion pairing agent, a phospholipid or a derivative thereof, a sterol, a lipid molecule derivative and a drug compound, wherein the ion pairing agent comprises at least one of compound (I) and compound (II); The structural formula of the compound (I) is The structural formula of the compound (II) is , wherein Q includes at least one of O, NR, S, and -(CH2)n-, X includes at least one of C=O, C=NR, and C=S, Y includes at least one of alkylene, O, NR, and S, n is any one of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and m is any one of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, It includes at least one of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, cycloalkenyl, heterocyclic and hydrogen, and Z includes at least one of carboxylic acid, sulfuric acid, sulfonic acid, sulfinic acid, thiophenolic acid, phosphoric acid, monoester of phosphoric acid, diester of phosphoric acid, phosphonic acid, monoester of phosphonic acid, phosphinous acid, boric acid and monoester of boric acid.

2. The liposome preparation according to claim 1, characterized in that Some structural formulas of the ion pairing agent are: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 3. The liposome preparation according to claim 1, characterized in that The phospholipids or their derivatives include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine, 1,2-heneicosanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, L-α-soybean hydrogenated phosphatidylcholine, 1,2-dioleoyl-sn -glycerol-3-phosphoethanolamine, palmitoyloleoyl-phosphatidylethanolamine, 1,2-diphytyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-snglycero-3-phospho-rac-(1-glycerol) sodium salt, 1,2-distearoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-racemic glycerol) sodium salt, egg-derived glycerol phosphate, soybean-derived glycerol phosphate, and at least one of sphingomyelin.

4. The liposome preparation according to claim 1, characterized in that The lipid molecule derivatives include polyethylene glycol-modified lipid molecules, and the polyethylene glycol-modified lipid molecules include at least one of polyethylene glycol-modified phosphatidylethanolamine, polyethylene glycol-modified phosphatidyl, polyethylene glycol-modified ceramide, polyethylene glycol-modified dipropylamine, polyethylene glycol-modified diacylglycerol, polyethylene glycol-modified dialkylglycerol, polyethylene glycol-modified glyceride, polyethylene glycol-modified sterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] sodium salt.

5. The liposome preparation according to claim 1, characterized in that The drug compounds include actinomycin, alitretinoin, altretinoin, aminopterin, azacitidine, azathioprine, belotecan, bendamustine, bexarotene, bleomycin, camptothecin, capecitabine, carboplatin, carboquinone, carmustine, chlorhexidine, chlorouracil, cisplatin, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, decitabine, dicycloplatin, docetaxel, doxorubicin, entinostat, epirubicin, eribulin, execon, flufenamide, 5-fluorouracil, gemcitabine, gemeline, gemiracil, idarubicin, irinotecan, ixapilinamide, irinotecan, and saxapyramide. lon, lenalidomide, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, morphine, nedaplatin, nimustine, otracil, oxaliplatin, paclitaxel, pemetrexed, pirarubicin, prednisone, satraplatin, temozolomide, tesetaxel, thiotepa, tipiracil, topotecan, tretinoin, triazinequinone, triethylene melamine, venetoclax, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, zabartinib, zolpidem, abemaciclib, acalabrutinib, afatinib, aflibercept, alectinib, avosidide, aximinib, apretinib, acimetidine tinib, binimetinib, bortezomib, bosutinib, brigatinib cabozantinib, amitinib, carfilzomib, capivasertib, capmatinib, ceritinib, cobimetinib, crizotinib, dabrafenib, dasatinib, docotinib, duvelisib, encorafenib, entrectinib, erdafitinib, erlotinib, everolimus, fedratinib, fostamatinib, fruquintinib, fulvestrantinib, gefitinib, gilteritinib, ibrutinib, imatinib, infigratinib, ixazomib, lapatinib, cilathione, siniab, levatinib, lorlatinib, mistalin, mobo At least one of: tinib, melotinib, neratinib, nedasudil, nilotinib, nintedanib, osimertinib, pacritinib, palbociclib, pasidatinib, pazopanib, pemigatinib, peixitinib, pitobutinib, panatinib, pragatinib, quizartinib, regorafenib, ripretinib, rituximab, ruxolitinib, simetinib, simetinib, sorafenib, sunitinib, temsirolimus, tipotinib, tivozanib, tofacitinib, trametinib, tracillin, tucatinib, upadacitinib, vandetanib, vemurafenib, vismodegib, and zabrutinib.

6. The liposome preparation according to claim 5, characterized in that The drug compound includes a fat-soluble drug and a water-soluble drug, the liposome preparation includes a lipid bilayer and an aqueous phase cavity formed by the lipid bilayer, the lipid bilayer includes the ion pairing agent, the phospholipid or its derivative, the lipid molecule derivative and the sterol, the fat-soluble drug is embedded in the lipid bilayer, and the water-soluble drug is arranged in the aqueous phase cavity.

7. The liposome preparation according to claim 5, characterized in that When the phospholipid or its derivative includes at least one of sphingomyelin and double-tail lipid, the liposome preparation includes a plurality of cross-arranged nanotubular fibers and ion channel proteins arranged in the gaps of each of the nanotubular fibers, the nanotubular fibers include the phospholipid or its derivative and the sterol, the drug compound includes a water-soluble drug and a sustained-release drug, the water-soluble drug is arranged inside the nanotubular fibers, the ion channel proteins form an ion channel system, and the ion channel system is used to pass the sustained-release drug.

8. The liposome preparation according to claim 5 or 6, characterized in that The particle size of the liposome preparation is 50-500 nm.

9. A preparation method, characterized in that: For preparing the liposome preparation according to any one of claims 1 to 8, the preparation method comprises the steps of: S1, dissolving the first drug compound, the ion pairing agent, the phospholipid or its derivative, the lipid molecule derivative and the sterol in an organic solvent to obtain a pretreatment solution, wherein the organic solvent comprises at least one of chloroform, methanol, ethanol and isopropanol; S2, mixing the pretreatment solution with a regulating solution to obtain a liposome preparation suspension, wherein the regulating solution comprises a buffer, an osmotic pressure regulator, a capture agent and a second drug compound; S3, heating the liposome preparation suspension, and obtaining a homogeneous solution by stirring or other homogenization operations; S4, performing dialysis, ultracentrifugation and / or size exclusion chromatography on the homogenous solution in sequence to remove excess drug compounds and obtain a pure homogenous solution; S5. Freeze-drying the pure homogeneous solution to obtain a dry liposome preparation.

10. The preparation method according to claim 9, characterized in that: The buffer includes at least one of phosphate buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, citric acid buffer, and tris(hydroxymethylaminomethane) buffer; the osmotic pressure regulator includes at least one of sodium chloride, glucose, mannitol, and sucrose; and the capture agent includes at least one of cyclodextrin and its derivatives, ammonium sulfate, diammonium hydrogen phosphate, and trisodium citrate.

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