Lipid compound with efficient endosome escape and application thereof

Abstract

The application discloses a lipid compound with a specific structure (formula (1)), and vesicles or liposomes prepared from the lipid compound have high endosome escape performance and wide application prospects in the biological medicine field. Specifically, the definitions of various groups are described in detail in the specification. The vesicles or liposomes prepared from the lipid compound of the application exhibit excellent cell compatibility in a physiological environment (pH 7.4). In an acidic environment of a cell endosome, the vesicles or liposomes exhibit excellent endosome escape efficiency. The characteristics significantly improve the bioavailability of drugs, promote the more efficient release of drugs into the cytoplasm, and thus enhance the treatment effect. In view of the excellent endosome escape characteristics, the lipid compound and the vesicles or liposomes thereof have wide application potential in the biological medicine field, especially in drug delivery and targeted treatment, and have high industrialization value.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a lipid compound with highly efficient endosomal escape and its applications. Background Technology

[0002] The effectiveness of disease treatment often depends on the bioavailability of drugs. Bioavailability refers to the proportion of a drug that, after entering systemic circulation, can reach and effectively act on its target site, typically involving processes such as drug absorption, distribution, metabolism, and excretion within the body. In the treatment of cancer, cardiovascular diseases, and genetic disorders, drugs must be able to reach the target area rapidly and efficiently to achieve the desired therapeutic effect. However, many drugs face the problem of low bioavailability in clinical applications, which not only affects treatment efficacy but may also negatively impact drug safety.

[0003] The main reason for low bioavailability is usually closely related to the physicochemical properties of drugs. Many small molecule drugs, especially hydrophobic drugs, are often rapidly eliminated from the body due to their poor water solubility, making it difficult to achieve effective therapeutic concentrations. Furthermore, the body's biological barrier systems (such as cell membranes, endosomes, and lysosomes) also limit drug absorption and distribution, further exacerbating this problem. Therefore, improving the bioavailability of small molecule drugs, especially those with poor water solubility, has become one of the core challenges in drug delivery technology research.

[0004] To address the issues of low drug bioavailability and poor therapeutic efficacy, researchers have developed various innovative drug delivery systems. Vesicles or liposomes, as classic drug delivery carriers, have been widely used in the field of drug delivery due to their good biocompatibility, excellent drug encapsulation capabilities, and controllable drug release characteristics. However, the operation of vesicles or liposomes in vivo is not entirely ideal, especially after the drug enters the target cells, where they face the significant challenge of endosome escape.

[0005] Once vesicles or liposomes enter a cell, the drug is first encapsulated within an endosome through endocytosis. An endosome is a membrane-bound vesicle within the cell membrane, typically formed after endocytosis, responsible for carrying the drug and transporting it to other sites via intracellular transport pathways. However, endosomes usually have a low pH environment and are rich in hydrolytic enzymes, providing favorable conditions for drug degradation. As a result, drugs often rapidly degrade or lose their biological activity within the endosome, failing to be effectively released into the cytoplasm, thus affecting therapeutic efficacy. More seriously, if the drug cannot effectively escape the endosome, it cannot cross the cytoplasm to reach the nucleus or other target sites to exert its therapeutic effect, leading to treatment failure.

[0006] Therefore, it is particularly important to develop vesicles or liposomes that can achieve efficient endosome escape, thereby improving drug bioavailability. Summary of the Invention

[0007] The purpose of this invention is to provide a lipid compound with efficient endosome escape and its application. The vesicles or liposomes prepared by the lipid compound with the novel structure of this invention have excellent endosome escape ability.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A lipid compound with highly efficient endosomal escape, characterized in that the compound has the following structure:

[0010] Where Q1 and Q2 represent covalent bonds, and R1 and R2 represent long chains containing at least one hydrophobic group.

[0011] The lipid compound with highly efficient endosomal escape is characterized in that:

[0012] S1: The lipid compound contains an indole residue, to which a hydrophobic long chain R1 is attached at the ortho position of the NH group, and / or a hydrophobic long chain R2 is attached at the meta position, wherein the hydrophobic long chain R1 and the hydrophobic long chain R2 may be structurally identical.

[0013] S2: Indole residues are connected to a hydrophobic long chain via covalent bonds Q1 and / or Q2. The covalent bonds Q1 and / or Q2 include amide bonds, ester bonds, ether bonds, thioether bonds, amine bonds, nitro bonds, fluoroolefin bonds, disulfide bonds, olefin bonds, or azide bonds. Covalent bond Q1 can be structurally equivalent to covalent bond Q2.

[0014] S3: When only the ortho-position R1 hydrophobic long chain or the meta-position R2 hydrophobic long chain exists, the groups on the indole residues corresponding to covalent bonds Q1 and Q2 can exist independently.

[0015] Lipid compounds possessing highly efficient endosomal escape capabilities are characterized in that the ortho-R1 hydrophobic long chain and / or meta-R2 hydrophobic long chain are single C1464 ... 12 -C 20 Straight-chain alkyl, and / or a single C 12 -C 20 Straight-chain alkyl groups containing olefin structures.

[0016] Lipid compounds exhibiting efficient endosomal escape, characterized in that the ortho-R1 hydrophobic long chain and / or meta-R2 hydrophobic long chain are both C1464-C ... 10 -C 20 Alkyl groups are covalently linked to indole residues via Q1 and / or Q2.

[0017] Lipid compounds exhibiting efficient endosomal escape, characterized in that the ortho-R1 hydrophobic long chain and / or meta-R2 hydrophobic long chain are single C46 ... 12 -C 20 Straight-chain alkyl, and / or a single C 12 -C 20 Straight-chain alkyl groups containing an olefin structure, and / or two C2Cs 10 -C 20 The alkyl group is linked to the indole residue via covalent bonds Q1 and / or Q2.

[0018] A lipid compound with efficient endosomal escape, characterized in that the ortho-R1 hydrophobic long chain and / or meta-R2 hydrophobic long chain both contain thioether bond structures.

[0019] A lipid compound with efficient endosomal escape, characterized in that the ortho-R1 hydrophobic long chain and / or meta-R2 hydrophobic long chain are long chains containing aromatic groups, wherein the aromatic groups are selected from phenyl or naphthyl and contain C1-C6 alkyl or halogen substitutions.

[0020] The lipid compound of the present invention possesses highly efficient endosome escape capability, with indole residues as terminal groups and covalently linked to a hydrophobic chain. Vesicles or liposomes prepared using this lipid compound exhibit highly efficient endosome escape capability. Furthermore, any modifications based on this structure are within the scope of protection of the present invention and are inspired by the present invention.

[0021] As a preferred embodiment, the aforementioned lipid compound with highly efficient endosomal escape is preferably derived from the following structures:

[0022]

[0023]

[0024] This invention relates to a lipid compound with efficient endosomal escape function, its stereoisomers and tautomers, and its application in the preparation of vesicles or liposomes containing the lipid compound, with the aim of improving the efficiency of disease treatment.

[0025] The aforementioned drug carrier with highly efficient endosomal escape includes vesicles or liposomes composed of a lipid compound and comprising the following components: a lipid compound, a loaded drug reagent, a drug adjuvant, and lecithin, cholesterol, or a complex of both.

[0026] The aforementioned drug-loaded vesicles or liposomes with efficient inentome escape and containing the lipid compound should be noted as follows: the composition of the vesicles or liposomes is not limited and can be a combination of known substances or an unknown substance. As long as the vesicles or liposomes with efficient inentome escape adopt the structure of the present invention, they are all within the protection scope of the present invention and are all inspired by the present invention.

[0027] In one embodiment, the drug-loaded vesicle or liposome containing the lipid compound and possessing efficient endosomal escape has a lipid compound content of 1-30%.

[0028] The aforementioned drug-loaded vesicles or liposomes with efficient inentome escape and containing the lipid compound include drug reagents such as nucleic acid molecules, small molecule compounds, polypeptides, proteins, or combinations thereof. The selection and combination of drug reagents are not limited. Any vesicles or liposomes with efficient inentome escape that adopt the structure of the present invention are within the protection scope of the present invention and are inspired by the present invention.

[0029] The advantages of this invention are:

[0030] Vesicles or liposomes constructed using the lipid compounds of this invention with high endosome escape efficiency exhibit excellent endosome escape efficiency. This high efficiency is achieved through their hydrophobic and aromatic structures, enabling them to interact with the lipid bilayer of the endosome membrane. The hydrophobicity of indole residues facilitates their insertion into the endosome membrane, interfering with membrane stability and integrity. Furthermore, the aromatic ring of indole can interact with intramembrane lipids or membrane proteins through π-π stacking, altering the membrane's physical properties and leading to increased membrane fluidity or rupture. These combined effects allow the indole group to effectively disrupt the endosome membrane structure, thereby promoting the release of drugs or other active molecules and significantly enhancing the endosome escape efficiency of the drug delivery system. Vesicles or liposomes prepared using the structure of this invention exhibit excellent endosome escape efficiency and are synthesized using a simple process. Attached Figure Description

[0031] Figure 1 This is the 1H NMR spectrum of indole-2-carboxylic acid-oleylamine prepared in this invention.

[0032] Figure 2 This is a particle size distribution diagram of the liposomes containing 5% indole-2-carboxylic acid-oleylamine prepared according to the present invention.

[0033] Figure 3 This is an intracytoplasmic escape fluorescence image of liposomes containing different amounts of indole-2-carboxylic acid-oleylamine prepared according to the present invention.

[0034] Figure 4This is an intima-body escape fluorescence colocalization map of liposomes containing different amounts of indole-2-carboxylic acid-oleylamine prepared according to the present invention. Detailed Implementation

[0035] To better understand the technical content of the present invention, specific embodiments are described below in conjunction with the accompanying drawings.

[0036] Various aspects of the invention are described in this disclosure with reference to the accompanying drawings, which illustrate numerous illustrative embodiments. The embodiments of this disclosure are not necessarily intended to encompass all aspects of the invention. It should be understood that the various concepts and embodiments described above, as well as those described in more detail below, can be implemented in any of many ways, because the concepts and embodiments disclosed herein are not limited to any particular implementation. Furthermore, some aspects of the invention disclosed may be used alone or in any suitable combination with other aspects of the invention disclosed.

[0037] To facilitate better understanding, the present invention will be further illustrated below with several specific examples. It should be noted that the following embodiments are examples of the present invention and are used only to illustrate the invention, not to limit it. Other combinations and various modifications within the scope of the present invention can be made without departing from its spirit or scope.

[0038] [Example 1] Preparation of indole lipid compounds

[0039] Hydrophobic long chains, such as oleylamine, and covalent bonds, such as amide bonds, are examples.

[0040] Weigh out oleylamine (OAM) (500 mg, 3.10 mmol), indole-2-carboxylic acid (ICA) (554 mg, 2.06 mmol), HOBt (470 mg, 2.06 mmol), and EDC·HCl (590 mg, 2.06 mmol) and place them in a 50 mL reaction flask. The flask was repeatedly evacuated and purged with nitrogen three times. Then, 20.7 mL of dichloromethane (DCM) was injected using a syringe to dissolve the product. After reacting in an ice bath for 30 minutes, while maintaining the ice bath with stirring, DIPEA (4.01 g, 31 mmol) was slowly injected into the reaction flask using a syringe. The reaction was stirred at room temperature for 48 hours. After the reaction was complete, DCM was removed by rotary evaporation, and the product was dissolved in 200 mL of chloroform, followed by washing with 5% NaHCO3 and saturated NaCl water. The product was then dried overnight with anhydrous Na2SO4, filtered, and concentrated. Finally, the compound was purified by column chromatography (mobile phase: n-hexane and ethyl acetate, volume ratio 4:1) to obtain a white powdery lipid compound, indole-2-carboxylic acid-oleylamine (OAM-ICA), in 89% yield. The analysis was performed using nuclear magnetic resonance (NMR). 1- H NMR spectrum ( Figure 1Analysis revealed a characteristic peak of oleylamine at 1.25 ppm and a characteristic peak of indole at 7.0-7.75 ppm, confirming the successful synthesis of indole-2-carboxylic acid-oleylamine.

[0041] [Example 2] Preparation of blank liposomes modified with indole lipid compounds

[0042] Taking blank liposomes prepared with a content of 15% lipid compound (indole-2-carboxylic acid-oleylamine) as an example

[0043] First, 11 mg of indole-2-carboxylic acid-oleylamine, 50 mg of phospholipid (such as lecithin), and 10 mg of cholesterol were dissolved in an appropriate amount of organic solvent (such as chloroform) to form a homogeneous solution. Then, the solvent was removed under a nitrogen stream or by rotary evaporation to obtain a homogeneous film. Next, the film was hydrated with deionized water (approximately 5 mL) and subjected to ultrasonic treatment or warm water bath agitation until a homogeneous liposome suspension was formed. To adjust the liposome particle size, a high-pressure homogenizer was used for homogenization, controlling the particle size within the range of 100-200 nm. Finally, unencapsulated indole-2-carboxylic acid-oleylamine and solvent were removed by dialysis or gel filtration to obtain purified blank liposomes, which were stored at 4°C for later use. Their size and distribution were measured using a nanoparticle size analyzer. Figure 2 As can be seen, the liposomes are uniform in size, about 150 nm, and have a narrow size distribution.

[0044] Table 1. Preparation of liposomes modified with different contents of indole-2-carboxylic acid-oleylamine

[0045] Indole-2-carboxylic acid-oleylamine (mg) Phospholipids (mg) Cholesterol (mg) content(%) 1 26 50 10 30 2 13 50 10 17 3 6.5 50 10 9 4 3 50 10 5 5 1.5 50 10 2 6 0.7 50 10 1

[0046] Table 2. Preparation of liposomes containing different types of lipid compounds

[0047]

[0048] [Example 3] Preparation of liposomes modified with indole lipid compounds loaded with doxorubicin (DOX)

[0049] Taking doxorubicin (DOX), a hydrophobic drug with a drug loading of 15%, and indole-2-carboxylic acid-oleylamine liposomes with a content of 15% as an example...

[0050] First, weigh out appropriate proportions of phospholipids (e.g., lecithin, 50 mg), cholesterol (10 mg), and indole-2-carboxylic acid-oleylamine (OAM, 10 mg) and dissolve them in a suitable amount of organic solvent (e.g., chloroform, 10 mL). Then, add hydrophobic doxorubicin (DOX, 15 mg) to the above solution, ensuring complete dissolution and uniform dispersion of the drug with the lipids. Remove the organic solvent from the solution using a rotary evaporator to form a homogeneous lipid film. Next, add an appropriate amount of deionized water (e.g., 5 mL) to the resulting lipid film and hydrate it under ultrasonic vibration or a warm water bath until a homogeneous liposome suspension is formed. To ensure the homogeneity of the liposomes and an appropriate particle size (generally controlled within the range of 100-200 nm), homogenize the suspension using a high-pressure homogenizer. Finally, remove unencapsulated DOX and other solvents by dialysis or gel filtration to purify the liposomes. Finally, indole-2-carboxylic acid-oleylamine drug-loaded liposomes containing hydrophobic doxorubicin were obtained and stored in a 4°C refrigerator for later use, avoiding high temperature and direct sunlight.

[0051] Table 3. Preparation of doxorubicin liposomes with different drug loading capacities

[0052] Phospholipids (mg) Cholesterol (mg) Indole-2-carboxylic acid-oleylamine (mg) Doxorubicin (mg) Drug loading (%) 1 50 10 10 30 30 2 50 10 10 15 15 3 50 10 10 8 8 4 50 10 10 4 4 5 50 10 10 2 2

[0053] [Example 4] Intracytoplasmic escape experiment of liposomes modified with indole lipid compounds loaded with doxorubicin (DOX).

[0054] First, GL261 mouse glioma cells were seeded in culture plates and cultured until 80% confluence. Liposomes modified with a lipid compound encapsulating DOX were then added, and incubation continued for 2 and 4 hours. During incubation, endosomes were stained with Lysotracker Green to observe the interaction between liposomes and endosomes. After incubation, fluorescence images of the cells were captured using a laser confocal microscope to observe the distribution of red DOX fluorescence and whether it escaped from the endosomes into the cytoplasm. The endosome escape ability of liposomes in GL261 cells was assessed by analyzing the fluorescence distribution and intensity at different time points. Figure 3 Endosome escape fluorescence and Figure 4 Quantitative fluorescence analysis of endosome escape revealed that the higher the amount of indole-2-carboxylic acid-oleylamine, the weaker the green fluorescence, indicating a stronger endosome escape capability of the liposome. This suggests that indole-2-carboxylic acid-oleylamine facilitates efficient endosome escape from liposomes.

[0055] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A lipid compound with highly efficient endosomal escape capability, characterized in that, The lipid compound has a structure as shown in formula (I): Formula (I) The lipid compound uses indole residues as end groups, wherein the indole nitrogen atom (NH) is connected to the hydrophobic chain of oleylamine via an amide bond at the ortho position to form an amphiphilic molecule.

2. The lipid compound according to claim 1, characterized in that: The lipid compound is used to construct at least one selected from vesicles and liposomes, and the lipid compound is present in the vesicles or liposomes at a concentration of 1%–30%.

3. The use of the lipid compound of claim 1 in the preparation of vesicles and liposomes that promote drug endosome escape.

4. The application according to claim 3, characterized in that: The drugs encapsulated in the vesicles and liposomes are selected from small molecule drugs, nucleic acid drugs, or protein drugs.

5. The application according to claim 3, characterized in that: The drug loading capacity of the vesicles and liposomes is 2%–30%.

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