Lipid compound with efficient endosome escape and application thereof

Abstract

The invention discloses a lipid compound with a specific structure (formula (1)), vesicles or lipidosome prepared from the lipid compound have efficient endosome escape performance and have wide application prospects in the field of biological medicine. Specifically, the definition of each group has been stated in detail in the specification. The vesicles or lipidosome prepared from the lipid compound disclosed by the invention shows excellent cell compatibility in a physiological environment (pH 7.4). In an acidic environment of cell endosome, the vesicles or liposomes show excellent endosome escape efficiency. Due to the characteristic, the bioavailability of the medicine is remarkably improved, and the medicine is promoted to be more efficiently released into cytoplasm, so that the treatment effect is enhanced. In view of the excellent endosome escape characteristic, the lipid compound and the vesicles or lipidosome thereof have wide application potential in the field of biological medicine, and particularly have high industrialization value in the aspects of drug delivery, targeted therapy and the like. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a lipid compound with high-efficiency endosomal escape and application thereof. Background Art

[0002] The effectiveness of disease treatment usually depends on the bioavailability of the drug. Bioavailability refers to the proportion of a drug that can reach and effectively act on the target site after entering the systemic circulation, and usually involves the absorption, distribution, metabolism and excretion of the drug in the body. In the treatment of cancer, cardiovascular disease, genetic diseases, etc., drugs must be able to reach the target area quickly and efficiently to achieve the desired therapeutic effect. However, many drugs face the problem of low bioavailability in clinical applications. This problem not only affects the therapeutic effect, but may also have a negative impact on the safety of the drug.

[0003] The main reason for low bioavailability is usually closely related to the physicochemical properties of the drug. Many small molecule drugs, especially hydrophobic drugs, are often quickly cleared from the body due to their poor water solubility, making it difficult to reach effective therapeutic concentrations. In addition, the biological barrier system in the body (such as cell membranes, endosomes, lysosomes, etc.) also limits the absorption and distribution of drugs, further exacerbating this problem. Therefore, especially for those small molecule drugs with poor water solubility, how to improve their bioavailability has become one of the core challenges in drug delivery technology research.

[0004] In order to solve the problems of low drug bioavailability and poor therapeutic effect, researchers have developed various innovative drug delivery systems. As a classic class of drug delivery carriers, vesicles or liposomes have been widely used in the field of drug delivery due to their good biocompatibility, excellent drug encapsulation ability and controllable drug release characteristics. However, the operation of vesicles or liposomes in the body is not completely ideal, especially after the drug enters the target cell, it faces the huge challenge of endosomal escape.

[0005] When vesicles or liposomes enter cells, the drugs are first encapsulated in endosomes by endocytosis. Endosomes are intracellular membrane vesicles that are usually formed after endocytosis of the cell membrane. They are responsible for carrying drugs and transferring them to other parts through intracellular transport pathways. However, endosomes usually have a low pH environment and are rich in hydrolases, which provides favorable conditions for drug degradation. As a result, drugs tend to be rapidly degraded or lose their biological activity in endosomes and cannot be effectively released into the cytoplasm, thereby affecting the therapeutic effect of the drug. More seriously, if the drug cannot effectively escape the endosome, it cannot enter the nucleus or other target sites through the cytoplasm to exert its therapeutic effect, resulting in treatment failure.

[0006] Therefore, it is particularly important to develop vesicles or liposomes that can achieve efficient endosomal escape, thereby improving the bioavailability and therapeutic efficacy of drugs. Summary of the invention

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

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

[0009] A lipid compound with high-efficiency endosomal escape, characterized in that the compound has the following structure:

[0010]

[0011] Wherein Q1 and Q2 represent covalent bonds, and R1 and R2 represent long chains containing at least one hydrophobic group.

[0012] The lipid compound having high efficiency of endosome escape is characterized by:

[0013] S1: The lipid compound contains an indole residue, on which a hydrophobic long chain R1 is connected at the ortho position of the NH group, and / or a hydrophobic long chain R2 is connected at the meta position, wherein the R1 hydrophobic long chain and the R2 hydrophobic long chain may be identical in structure.

[0014] S2: The indole residue is connected to the hydrophobic long chain via a covalent bond Q1 and / or Q2. The covalent bond Q1 and / or Q2 comprises an amide bond, an ester bond, an ether bond, a thioether bond, an amine bond, a nitro bond, a fluoroolefin bond, a disulfide bond, an olefin bond or an azide bond, and the covalent bond Q1 may be structurally equivalent to the covalent bond Q2.

[0015] S3: When only the ortho-position R1 hydrophobic long chain or the meta-position R2 hydrophobic long chain exists, the groups on the indole residue corresponding to the covalent bonds Q1 and Q2 can exist independently.

[0016] The lipid compound with high-efficiency endosomal escape ability is characterized in that the ortho-position R1 hydrophobic long chain and / or the meta-position R2 hydrophobic long chain are single C 12 -C 20 Straight chain alkyl, and / or a single C 12 -C 20 Straight chain alkyl and contains olefin structure.

[0017] The lipid compound with high efficiency of endosome escape is characterized in that the ortho-position R1 hydrophobic long chain and / or the meta-position R2 hydrophobic long chain are C 10 -C 20alkyl group and is linked to the indole residue via a covalent bond Q1 and / or Q2.

[0018] The lipid compound with high efficiency of endosome escape is characterized in that the ortho-position R1 hydrophobic long chain and / or the meta-position R2 hydrophobic long chain are single C 12 -C 20 Straight chain alkyl, and / or a single C 12 -C 20 Straight chain alkyl containing olefin structure and / or two C 10 -C 20 The alkyl group is linked to the indole residue via a covalent bond Q1 and / or Q2.

[0019] A lipid compound with high-efficiency endosomal escape, characterized in that the ortho-position R1 hydrophobic long chain and / or the meta-position R2 hydrophobic long chain both contain a thioether bond structure.

[0020] A lipid compound with efficient endosomal escape, characterized in that the ortho-position R1 hydrophobic long chain and / or the meta-position R2 hydrophobic long chain are long chains containing aromatic groups, the aromatic groups are selected from phenyl or naphthyl, and contain C1-C6 alkyl or halogen substitutions.

[0021] The lipid compound with high efficiency of endosome escape of the present invention has an indole residue as the terminal group and is connected to a hydrophobic chain through a covalent bond, and the vesicle or liposome prepared by using the lipid compound has the ability of high efficiency of endosome escape. In addition, any improvement based on the structure is within the protection scope of the present invention and is inspired by the present invention.

[0022] The aforementioned lipid compound with high efficiency of endosome escape is, as a preferred embodiment, preferably selected from the following structures:

[0023]

[0024] The present invention relates to a lipid compound with high-efficiency endosomal escape function and its stereoisomers and tautomers, and uses the lipid compound in preparing vesicles or liposomes containing the lipid compound, aiming to improve the efficiency of disease treatment.

[0025] The aforementioned drug carrier with high-efficiency endosomal escape includes vesicles or liposomes, which are composed of the lipid compound and contain the following components: lipid compound, loaded drug agent, drug adjuvant, and lecithin, cholesterol or a complex of the two.

[0026] The aforementioned drug-loaded vesicle or liposome with efficient endosomal escape and containing the lipid compound should be noted that the components of the vesicle or liposome are not limited and can be a composition of existing known substances or a composition of unknown substances. As long as the vesicle or liposome with efficient endosomal escape adopts the structure of the present invention, it is within the protection scope of the present invention and is inspired by the present invention.

[0027] The aforementioned drug-loaded vesicle or liposome having high-efficiency endosomal escape and comprising the lipid compound, as an embodiment, has a content of the lipid compound of 1-30%.

[0028] The aforementioned drug-loaded vesicle or liposome with efficient endosomal escape and containing the lipid compound, the drug agent carried includes: nucleic acid molecules, small molecule compounds, polypeptides, proteins, or combinations thereof; the selection and combination formula of the drug agent carried are not limited, as long as the vesicle or liposome with efficient endosomal escape adopts the structure of the present invention, it is within the protection scope of the present invention and is inspired by the present invention.

[0029] The present invention is beneficial in that:

[0030] The vesicle or liposome constructed by the lipid compound with efficient endosomal escape of the present invention has excellent endosomal escape efficiency. The reason why it can achieve efficient endosomal escape is that its hydrophobic and aromatic structure enables it to interact with the lipid bilayer of the endosomal membrane. The hydrophobicity of the indole residue makes it easy to insert into the endosomal membrane, interfering with the stability and integrity of the membrane. In addition, the aromatic ring of indole can interact with the lipids or membrane proteins in the membrane through π-π stacking, changing the physical properties of the membrane, resulting in increased fluidity or rupture of the membrane. These combined effects enable the indole group to effectively destroy the endosomal membrane structure, thereby promoting the release of drugs or other active molecules, thereby significantly enhancing the endosomal escape efficiency of the drug delivery system. The vesicle or liposome prepared by the structure of the present invention not only has excellent endosomal escape efficiency, but also has good safety, high biocompatibility, and a simple synthesis process, which is suitable for industrial application in the field of biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the nuclear magnetic hydrogen spectrum of indole-2-carboxylic acid-oleylamine prepared by the present invention.

[0032] Figure 2 The figure is a particle size distribution diagram of the liposome containing 5% indole-2-carboxylic acid-oleylamine prepared by the present invention.

[0033] Figure 3 It is the fluorescence graph of endosomal escape of liposomes containing different contents of indole-2-carboxylic acid-oleylamine prepared by the present invention.

[0034] Figure 4 It is a fluorescence co-localization diagram of endosomal escape of liposomes containing different contents of indole-2-carboxylic acid-oleylamine prepared by the present invention. DETAILED DESCRIPTION

[0035] In order to better understand the technical content of the present invention, specific embodiments are given and described as follows in conjunction with the accompanying drawings.

[0036] Various aspects of the present invention are described in this disclosure with reference to the accompanying drawings, in which many illustrative embodiments are shown. The embodiments of the present disclosure are not necessarily intended to include all aspects of the present invention. It should be understood that the various concepts and embodiments introduced above, as well as those described in more detail below, can be implemented in any of many ways, because the concepts and embodiments disclosed by the present invention are not limited to any implementation. In addition, some aspects disclosed by the present invention can be used alone or in any appropriate combination with other aspects disclosed by the present invention.

[0037] In order to facilitate better understanding, the present invention is further described below in conjunction with several specific examples. It should be noted that the following embodiments are examples of the present invention and are only used to illustrate the present invention, but not to limit the present invention. Other combinations and various improvements within the concept of the present invention may be carried out without departing from the gist or scope of the present invention.

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

[0039] The hydrophobic long chain is oleylamine, and the covalent bond is amide bond.

[0040] Weigh 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), place them in a 50 mL reaction bottle, and repeatedly evacuate and pass nitrogen for 3 times. Subsequently, inject 20.7 mL of dichloromethane (DCM) using a syringe to dissolve it. After reacting for 30 minutes under ice bath conditions, keep the ice bath and stir, slowly inject DIPEA (4.01 g, 31 mmol) into the reaction bottle with a syringe, and stir the reaction at room temperature for 48 hours. After the reaction is completed, remove DCM by rotary evaporation, dissolve the product with 200 mL of chloroform, and then wash with 5% NaHCO3 and saturated NaCl water in turn. Then, dry it with anhydrous Na2SO4 overnight, filter and concentrate. Finally, the lipid compound indole-2-carboxylic acid-oleylamine (OAM-ICA) was purified by column chromatography (mobile phase: n-hexane and ethyl acetate, volume ratio: 4:1) with a yield of 89%. 1- H NMR spectrum ( Figure 1 ) analysis, the characteristic peak of oleylamine can be observed at 1.25ppm, and the characteristic peak of indole can be observed at 7.0-7.75ppm, which proves the successful synthesis of indole-2-carboxylic acid-oleylamine.

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

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

[0043] First, 11 mg of indole-2-carboxylic acid-oleylamine, 50 mg of phospholipids (such as lecithin) and 10 mg of cholesterol are dissolved in an appropriate amount of an organic solvent (such as chloroform) to form a uniform solution. Then, the solvent is removed under a nitrogen flow or by rotary evaporation to obtain a uniform film. Next, deionized water (about 5 mL) is used to hydrate the film, and ultrasonic treatment or warm water bath shaking is performed until a uniform liposome suspension is formed. In order to adjust the particle size of the liposomes, a high-pressure homogenizer is used for homogenization so that the particle size is controlled within the range of 100-200 nm. Finally, the unencapsulated indole-2-carboxylic acid-oleylamine and the solvent are removed by dialysis or gel filtration to obtain purified blank liposomes, which are stored in a 4°C refrigerator for use. The size and distribution of the liposomes are tested by a nanoparticle size analyzer ( Figure 2 ), it can be seen that the liposomes have a uniform size of about 150 nm and a narrow size distribution.

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

[0045]

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

[0047]

[0048]

[0049] [Example 3] Preparation of liposomes modified with indole lipid compounds encapsulating doxorubicin (DOX)

[0050] Take the hydrophobic drug doxorubicin (DOX) with a drug loading of 15% and the indole-2-carboxylic acid-oleylamine liposome with a drug loading of 15% as an example.

[0051] First, weigh appropriate proportions of phospholipids (such as lecithin, 50 mg), cholesterol (10 mg) and indole-2-carboxylic acid-oleylamine (OAM, 10 mg) and dissolve them in an appropriate amount of organic solvent (such as chloroform, 10 mL). Then, add hydrophobic doxorubicin (DOX, 15 mg) to the above solution to ensure that the drug and lipids are completely dissolved and evenly dispersed. The solution is removed from the organic solvent by a rotary evaporator to form a uniform lipid film. Next, add an appropriate amount of deionized water (such as 5 mL) to the resulting lipid film and hydrate it under ultrasonic vibration or warm water bath conditions until a uniform liposome suspension is formed. In order to ensure the uniformity and appropriate particle size of the liposomes (generally controlled in the range of 100-200 nm), the suspension is homogenized by a high-pressure homogenizer. Finally, dialysis or gel filtration is used to remove unencapsulated DOX and other solvents to purify the liposomes. Finally, the indole-2-carboxylic acid-oleylamine drug-loaded liposomes encapsulating the hydrophobic doxorubicin drug were obtained and stored in a refrigerator at 4° C. for use, avoiding high temperature and direct sunlight.

[0052] Table 3 Preparation of doxorubicin liposomes with different drug loadings

[0053]

[0054] [Example 4] Endosomal escape experiment of liposomes modified with indole lipid compounds encapsulating doxorubicin (DOX)

[0055] First, GL261 mouse glioma cells were inoculated in a culture plate and cultured to 80% confluence. Liposomes modified with lipid compounds encapsulating DOX were then added and incubated for 2 hours and 4 hours. During the incubation period, LysotrackerGreen was used to stain the endosomes of the cells in order to observe the interaction between liposomes and endosomes. After the incubation period, a laser confocal microscope was used to capture fluorescent images of the cells to observe the red fluorescence distribution of DOX and whether it escaped from the endosomes into the cytoplasm. The endosomal escape ability of liposomes in GL261 cells was evaluated by analyzing the fluorescence distribution and intensity at different time points. Figure 3 Endosomal escape fluorescence and Figure 4 Endosomal escape fluorescence quantitative analysis shows that the more indole-2-carboxylic acid-oleylamine there is, the weaker the green fluorescence is, indicating that the liposome endosomal capacity is stronger, indicating that indole-2-carboxylic acid-oleylamine helps liposomes achieve efficient endosomal escape.

[0056] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A lipid compound with high-efficiency endosomal escape ability, characterized in that: The lipid compound is an amphiphilic molecule with an indole residue as the terminal group and connected to the hydrophobic chain through a covalent bond. The structural formula of the lipid compound is as follows, wherein Q1 and Q2 represent covalent bonds, and R1 and R2 represent long chains containing at least one hydrophobic group.

2. A lipid compound with high-efficiency endosomal escape ability according to claim 1, characterized in that: S1: The lipid compound contains an indole residue, on which a hydrophobic long chain R1 is connected at the ortho position of the NH group, and / or a hydrophobic long chain R2 is connected at the meta position, wherein the R1 hydrophobic long chain and the R2 hydrophobic long chain may be identical in structure. S2: The indole residue is connected to the hydrophobic long chain via a covalent bond Q1 and / or Q2. The covalent bond Q1 and / or Q2 comprises an amide bond, an ester bond, an ether bond, a thioether bond, an amine bond, a nitro bond, a fluoroolefin bond, a disulfide bond, an olefin bond or an azide bond, and the covalent bond Q1 may be structurally equivalent to the covalent bond Q2. S3: When only the ortho-position R1 hydrophobic long chain or the meta-position R2 hydrophobic long chain exists, the groups on the indole residue corresponding to the covalent bonds Q1 and Q2 can exist independently.

3. The lipid compound with high-efficiency endosomal escape ability according to claims 1 and 2, characterized in that: The ortho-position R1 hydrophobic long chain and / or the meta-position R2 hydrophobic long chain are single C12-C20 straight-chain alkyl groups, and / or are single C12-C20 straight-chain alkyl groups and contain olefin structures.

4. The lipid compound with high-efficiency endosomal escape according to claims 1 and 2, characterized in that: The ortho-position R1 hydrophobic long chain and / or meta-position R2 hydrophobic long chain are both C10-C20 alkyl groups, and are connected to the indole residue via covalent bonds Q1 and / or Q2.

5. The lipid compound with high-efficiency endosomal escape according to claims 1 and 2, characterized in that: The ortho-position R1 hydrophobic long chain and / or meta-position R2 hydrophobic long chain is a single C12-C20 straight-chain alkyl group, and / or a single C12-C20 straight-chain alkyl group containing an olefin structure, and / or two C10-C20 alkyl groups are connected to the indole residue via covalent bonds Q1 and / or Q2.

6. The lipid compound with high-efficiency endosomal escape according to claims 1 and 2, characterized in that: The ortho-position R1 hydrophobic long chain and / or the meta-position R2 hydrophobic long chain both contain a thioether bond structure.

7. The lipid compound with high-efficiency endosomal escape according to claims 1 and 2, characterized in that: The ortho-position R1 hydrophobic long chain and / or meta-position 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.

8. The lipid compound with high-efficiency endosomal escape according to claim 1, characterized in that: The lipid compound can be used to prepare vesicles or liposomes and significantly improve the endosome escape efficiency. The content of the lipid compound in the vesicles or liposomes is 1-30%.

9. According to claim 8, the present invention relates to a highly efficient endosomal escape vesicle or liposome constructed using lipid compounds, characterized in that: Vesicles or liposomes can encapsulate small molecule drugs, nucleic acid drugs or protein drugs to achieve rapid escape from endosomes, thereby significantly improving the bioavailability of drugs and are suitable for the clinical treatment of diseases.

10. According to claims 1 to 9, the drug-loaded vesicle or liposome has high-efficiency endosomal escape performance, characterized in that: The drug loading of vesicles or liposomes is 2-30%.

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