A lipid compound and its application

Abstract

This invention provides a lipid compound and its applications, the results of which are shown in Formula I. The lipid compound provided by this invention can be used for targeted delivery of bioactive molecules to splenic T cells, particularly suitable for transporting negatively charged nucleic acid molecules, such as mRNA, providing a potential delivery strategy for in vivo specific gene editing of T cells. This invention also provides a bioactive substance delivery system comprising the lipid compound, which is loaded with bioactive substances. The bioactive substance delivery system of this invention, after intravenous administration, can efficiently target spleen tissue and selectively deliver active substances to T cells within the spleen, achieving efficient T cell transfection and immune activation. The delivery system of this invention has a simple synthesis process and strong structural tunability, and has broad application prospects in the fields of T cell immunotherapy and gene therapy.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical compound technology, specifically relating to a lipid compound and its applications. Background Technology

[0002] T cells are key effector cells in adaptive immunity, playing a central role in anti-infection and anti-tumor immunity. Gene editing of T cells can help regulate the effectiveness of adaptive immune responses. Furthermore, targeted delivery of chemotherapeutic drugs to T-cell lymphoma cells holds great clinical promise for the treatment of T-cell lymphoma. However, while existing delivery systems (such as lipid molecules and polymers) can achieve organ-level targeting, their in vivo T-cell specific transfection efficiency remains unsatisfactory, becoming a major bottleneck for in vivo T-cell gene editing. Although CD3 antibody or peptide modification can enhance the targeting specificity of delivery systems to T cells, excessive binding of CD3 antibodies may trigger abnormal T-cell activation and related toxicity, posing risks.

[0003] CN119074664A discloses a nanodelivery system for targeting and regulating CAR-T cells and its application, belonging to the field of nanomedicine and tumor therapy technology. The nanodelivery system comprises drug-loaded DLNPs and NT cells. This invention achieves precise targeting of CAR-T cells to tumor sites using nanotechnology, and regulates the activity of CAR-T cells or modulates the tumor microenvironment of CAR-T cells through drug-loaded DLNPs, solving problems such as CAR-T cell depletion, insufficient killing activity, or cytokine storm caused by CAR-T cell overdose.

[0004] CN107557393A discloses a magnetic nanomaterial-mediated CRISPR / Cas9 T-cell intracellular delivery system, its preparation method, and its applications. The preparation method includes the following steps: 1) modifying a cluster of magnetic nanoparticles of a certain size with a cationic polymer to obtain a nanocarrier; 2) providing a CRISPR / Cas9 system expression plasmid targeting a specific gene; 3) co-incubating the nanocarrier obtained in step 1) with the CRISPR / Cas9 system expression plasmid targeting the specific gene to obtain a nanocomposite; and 4) co-incubating the nanocomposite obtained in step 3) with T cells under a magnetic field to obtain a magnetic nanomaterial-mediated CRISPR / Cas9 T-cell intracellular delivery system. This invention provides a simple, safe, and efficient method for editing target genes within T cells, showing promising application prospects in tumor immunotherapy.

[0005] However, the methods described above have not completely solved the problem of low specific transfection efficiency in existing technologies. Therefore, how to provide a novel T-cell targeted delivery system has become an urgent problem to be solved. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a lipid compound and its applications. The lipid compound provided by the present invention can be used for targeted delivery of bioactive molecules (such as DNA, mRNA, proteins, peptides, chemotherapeutic drugs, etc.) to splenic T cells, and is particularly suitable for transporting negatively charged nucleic acid molecules, such as mRNA, providing a potential delivery strategy for in vivo specific gene editing of T cells.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a lipid compound, the result of which is shown in Formula I: Formula I R1, R2, R3, and R4 are independently selected from hydrogen, substituted or unsubstituted straight-chain or branched C1-30 alkyl groups, substituted or unsubstituted straight-chain or branched C2-30 alkenyl groups, substituted or unsubstituted straight-chain or branched C2-30 alkynyl groups, -CH2CH(OH)R5, and -CH2COOR5. , or Any one of R1, R2, R3, and R4, and any one of them is At least one of them is or In which the carbon atoms on the alkyl, alkenyl, or alkynyl groups are not substituted or at least one carbon atom is independently substituted by an oxygen atom, a sulfur atom, or a nitrogen atom.

[0008] R5 is selected from hydrogen, substituted or unsubstituted straight-chain or branched C1-30 alkyl, substituted or unsubstituted straight-chain or branched C2-30 alkenyl, substituted or unsubstituted straight-chain or branched C2-30 alkynyl, wherein the carbon atom on the alkyl, alkenyl, or alkynyl group is not substituted or at least one carbon atom is independently substituted by an oxygen atom, a sulfur atom, or a nitrogen atom.

[0009] R6, R7, and R8 are independently selected from hydrogen, C1-3 alkyl, or C1-3 alkoxy.

[0010] R9 is selected from hydrogen, C1-3 alkyl, C1-3 alkoxy, or hydroxyl.

[0011] n is an integer independently selected from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7 or 8), m is an integer independently selected from 0 to 8 (e.g., 0, 1, 2, 3, 4, 5, 6, 7 or 8), and m being 0 indicates that the group does not exist here.

[0012] The lipid compounds with the aforementioned specific structures can be used to target and deliver bioactive molecules (such as DNA, mRNA, proteins, peptides, chemotherapeutic drugs, etc.) to spleen T cells, and are particularly suitable for transporting negatively charged nucleic acid molecules, such as mRNA, providing a potential delivery strategy for in vivo gene editing of T cells.

[0013] Preferably, the substituents are independently selected from any one of halogens, hydroxyl groups, straight-chain or branched C1-10 alkyl groups, and straight-chain or branched C1-10 alkoxy groups.

[0014] Preferably, any one of R1, R2, R3, and R4 is And at least one of them is , where R6, R7, R8, n, and m have the same defined range as described above.

[0015] The structure contains two adjacent cis double bonds, which enables it to exhibit high encapsulation efficiency and good cell transfection rate when subsequently applied to delivery systems to encapsulate active substances (such as nucleic acids, like mRNA). Furthermore, it results in more uniform lipid nanoparticle sizes during preparation. The lipid compounds of this invention are particularly suitable for preparing solid-structured nanoparticles.

[0016] Preferably, R6, R7, and R8 are selected from hydrogen; Preferably, R9 is selected from hydrogen.

[0017] Preferably, n is independently selected from an integer between 4 and 8; Preferably, m is independently selected from an integer between 4 and 8.

[0018] Preferably, the lipid compound is selected from any one of the following structural compounds: .

[0019] The lipid compounds with the above-described structures can be synthesized using methods known in the prior art, and this invention does not impose any limitations on them. For example, they can be prepared using the Ugi reaction: Secondly, the present invention provides the application of the lipid compounds described above in the preparation of bioactive substance delivery systems.

[0020] The lipid compound possesses several properties suitable for preparing drug delivery systems: 1) the ability to lipid complex and “protect” unstable agents; 2) the ability to buffer pH in vivo; 3) the ability to act as a “proton sponge” and induce in vivo dissolution; and 4) the ability to neutralize the charge on negatively charged active substances.

[0021] The delivery system formed from the lipid compounds of the present invention can also be modified with targeting molecules to make it a targeting agent capable of targeting specific cells, tissues, or organs. The targeting molecule may be present throughout the delivery system or may be located only on its surface. The targeting molecule may be a protein, peptide, glycoprotein, lipid, small molecule, nucleic acid, etc., examples of which include (but are not limited to) antibodies, antibody fragments, low-density lipoprotein (LDL), transferrin, asialycoprotein, receptor ligands, sialic acid, aptamers, etc.

[0022] The delivery system formed from the lipid compounds of the present invention can be combined with one or more pharmaceutical excipients to form pharmaceutical compositions suitable for administration to animals (including humans). The term "pharmaceutical excipient" means any type of non-toxic, inert solid, semi-solid, or liquid filler, diluent, etc., including but not limited to sugars such as lactose, trehalose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; gelatin; talc; oils such as peanut oil, cottonseed oil, safflower oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; surfactants such as Tween 80; buffers such as phosphate buffers, acetate buffers, and citrate buffers; colorants, sweeteners, flavorings and aromas, preservatives, and antioxidants, etc.

[0023] The pharmaceutical compositions of the present invention can be administered to humans and / or animals orally, rectally, intravenously, intramuscularly, vaginally, intranasally, intraperitoneally, buccally, or orally or via nasal spray.

[0024] Thirdly, the present invention provides a bioactive substance delivery system, the bioactive substance delivery system comprising the lipid compounds described above.

[0025] Preferably, the bioactive substance delivery system comprises either nanoparticles or microbubbles.

[0026] The aforementioned bioactive substance delivery system can efficiently target spleen tissue after intravenous administration and selectively deliver active substances to T cells within the spleen, achieving efficient transfection and immune activation of T cells. It has the advantages of simple synthesis process and strong structural tunability, and has broad application prospects in the fields of T cell immunotherapy and gene therapy.

[0027] Fourthly, the present invention also provides a pharmaceutical composition comprising the bioactive substance delivery system described above. The pharmaceutical composition may be administered to humans and / or animals orally, rectally, intravenously, intramuscularly, vaginally, intranasally, intraperitoneally, buccally, or via oral or nasal spray.

[0028] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a lipid compound with a specific structure that can be used to target and deliver bioactive molecules (such as DNA, mRNA, proteins, peptides, chemotherapeutic drugs, etc.) to spleen T cells. It is particularly suitable for transporting negatively charged nucleic acid molecules, such as mRNA, providing a potential delivery strategy for in vivo gene editing of T cells. Attached Figure Description

[0029] Figure 1 This is a graph showing the in vitro cell transfection efficiency results of LucRNA VI@LNP; Figure 2 This is an in vitro cell-killing effect diagram of PTX VI@LNP; Figure 3 This is a diagram showing the distribution of LucRNA VI@LNP in internal organs; Figure 4 This is a diagram showing the transfection of GFP mRNA VI@LNP into spleen immune cells; Figure 5 This is a diagram showing the results of specific transfection of mouse spleen T cells with GFP mRNA VI@LNP. Detailed Implementation

[0030] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0031] As used herein, the term "alkyl" refers to a saturated hydrocarbon group obtained by removing a single hydrogen atom from a hydrocarbon moiety containing 1 to 30 carbon atoms. Examples of alkyl groups include (but are not limited to) methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, and n-dodecyl.

[0032] The term "alkenyl" refers to a monovalent group obtained from a hydrocarbon moiety having at least one carbon-carbon double bond by removing a single hydrogen atom. Alkenyl groups include vinyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, etc.

[0033] The term "alkynyl" refers to a monovalent group obtained from a hydrocarbon having at least one carbon-carbon triple bond by removing a single hydrogen atom. Representative alkynyl groups include ethynyl, 2-propynyl (propynyl), and 1-propynyl.

[0034] The term "alkoxy" refers to an alkyl group, as defined above, that is attached to a parent molecule by an oxygen atom. Examples of alkoxy groups include (but are not limited to) methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy, and n-hexoxy.

[0035] The term "halogen" refers to an atom selected from fluorine, chlorine, bromine, and iodine.

[0036] The terms “substitution” (with or without the preceding term “optionally”) and “substituent” refer to the ability to transform one functional group into another, provided that the valences of all atoms are maintained. When more than one position in any particular structure can be substituted by more than one substituent selected from a specified group, the substituents may be the same or different at each position.

[0037] Example: Synthesis of lipid compounds Synthesis of 2-octyl-1-dodecanoic acid. 10 g of 2-octyl-1-dodecanoic acid was added to 100 mL of dichloromethane. 19.1 g of Dess-Martin reagent was added while stirring in an ice bath. The mixture was then stirred at 20 °C for 3 h. Thin-layer chromatography (TLC) showed the reaction was complete. The product was purified by silica gel column chromatography to obtain the target product, 2-octyl-1-dodecanoic acid.

[0038] Synthesis of Product I: 355.8 mg of 2-octyl-1-dodecanoic acid was dissolved in 2 mL of anhydrous methanol, and 321 mg of oleylamine was added. After mixing thoroughly, the mixture was reacted at 40 °C for 1 h, and the reaction progress was monitored by TLC. After the aldehyde and amine reacted completely, 168.2 mg of 3-(imidazol-4-yl)propionic acid was added to the reaction solution, and the mixture was reacted at 40 °C for 30 min. Finally, 118.9 mg of isonitrile methyl acetate was added to the reaction solution, and the mixture was reacted overnight at 40 °C, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was evaporated to dryness and dissolved in 20 mL of a DCM:MeOH mixture (v / v):10:1). The solution was washed with saturated sodium bicarbonate and saturated brine, respectively, and the organic phase was enriched. The target product I was collected by column purification, yielding 100 mg of product.

[0039] 1HNMR(400 MHz, CDCl3): δ0.880.92(m, 9H), 2.02-2.03(m, 4H), 2.71-2.74(m, 2H), 2.99-3.02(m, 2H), 3.78(s, 3H), 5.36-5.37(m, 2H), 6.86(s, 1H), 7.62(s, 1H).

[0040] Synthesis of II: 355.8 mg of 2-octyl-1-dodecanoic acid was dissolved in 2 mL of anhydrous methanol, and 321 mg of oleylamine was added. After mixing thoroughly, the mixture was reacted at 40 °C for 1 h, and the reaction progress was monitored by TLC. After the aldehyde and amine reacted completely, 168.2 mg of 3-(1-imidazolyl)propionic acid was added to the reaction solution, and the mixture was reacted at 40 °C for 30 min. Finally, 118.9 mg of isonitrile methyl acetate was added to the reaction solution, and the mixture was reacted overnight at 40 °C, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was evaporated to dryness and dissolved in 20 mL of a DCM:MeOH mixture (v / v):10:1). The solution was washed with saturated sodium bicarbonate and saturated brine, respectively, and the organic phase was enriched. The target product II was collected by column purification, yielding 201 mg of product.

[0041] 1 HNMR(400 MHz, CDCl3): δ0.860.90(m, 9H), 1.98-2.01(m, 4H), 2.78-2.81(m, 2H), 3.14-3.16(m, 2H), 3.73(s, 3H), 5.30-5.38(m, 2H), 6.95(s, 1H), 7.03(s, 1H), 7.55(s, 1H).

[0042] Synthesis of III: 355.8 mg of 2-octyl-1-dodecanoic acid was dissolved in 2 mL of anhydrous methanol, and 321 mg of oleylamine was added. After mixing thoroughly, the mixture was reacted at 40 °C for 1 h, and the reaction progress was monitored by TLC. After the aldehyde and amine reacted completely, 168.2 mg of 3-(1-pyrazolyl)propionic acid was added to the reaction solution, and the mixture was reacted at 40 °C for 30 min. Finally, 118.9 mg of isonitrile methyl acetate was added to the reaction solution, and the mixture was reacted overnight at 40 °C, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was evaporated to dryness and dissolved in 20 mL of a DCM:MeOH mixture (v / v):10:1). The solution was washed with saturated sodium bicarbonate and saturated brine, respectively, and the organic phase was enriched. The target product III was collected by column purification, yielding 518 mg of product.

[0043] 1HNMR(400 MHz, CDCl3): δ0.870.91(m, 9H), 2.01-2.04(m, 4H), 2.95-2.99(m, 2H), 3.16-3.19(m, 2H), 3.74(s, 3H), 5.36-5.37(m, 2H), 6.19(s, 1H), 7.28(s, 1H), 7.47(s, 1H), 7.51(s, 1H).

[0044] Synthesis of IV: 355.8 mg of 2-octyl-1-dodecanoic acid was dissolved in 2 mL of anhydrous methanol, and 321 mg of oleylamine was added. After mixing thoroughly, the mixture was reacted at 40 °C for 1 h, and the reaction progress was monitored by TLC. After the aldehyde and amine reacted completely, 262.4 mg of 3-(2-isopropyl-1H-imidazol-1-yl)propionate was added to the reaction solution, and the mixture was reacted at 40 °C for 30 min. Finally, 118.9 mg of isonitrile methyl acetate was added to the reaction solution, and the mixture was reacted overnight at 40 °C, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was evaporated to dryness and dissolved in 20 mL of a DCM:MeOH mixture (v / v):10:1). The solution was washed with saturated sodium bicarbonate and saturated brine, respectively, and the organic phase was enriched. The target product IV was collected by column purification, yielding 170 mg of product.

[0045] 1 HNMR(400 MHz, CDCl3): δ0.880.92(m, 9H), 2.00-2.03(m, 4H), 2.77-2.81(m, 2H), 3.18-3.22(m, 3H), 3.75(s, 3H), 5.32-5.38(m, 2H), 6.86(s, 1H), 6.98(s, 1H).

[0046] Synthesis of V: ​​355.8 mg of 2-octyl-1-dodecanoic acid was dissolved in 2 mL of anhydrous methanol, and 133.38 mg of 2-(1H-imidazol-1-yl)ethylamine was added. After mixing thoroughly, the mixture was reacted at 40 °C for 1 h, and the reaction progress was monitored by TLC. After the aldehyde and amine reacted completely, 336.54 mg of linoleic acid was added to the reaction solution, and the mixture was reacted at 40 °C for 30 min. Finally, 118.9 mg of methyl isonitrile acetate was added to the reaction solution, and the mixture was reacted overnight at 40 °C, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was evaporated to dryness and dissolved in 20 mL of a DCM:MeOH mixture (v / v = 10:1). The solution was washed with saturated sodium bicarbonate and saturated brine, respectively, and the organic phase was enriched. The target product V was collected by column purification, yielding 246 mg of product.

[0047] 1 HNMR(400 MHz, CDCl3): δ0.860.91(m, 9H), 2.00-2.07(m, 6H), 2.75-2.78(m, 2H), 3.73(s, 3H), 5.31-5.39(m, 4H), 6.95(s, 1H), 7.07(s,1H), 7.52(s,1H).

[0048] Synthesis of VI: 355.8 mg of 2-octyl-1-dodecanoic acid was dissolved in 2 mL of anhydrous methanol, and 133.4 mg of histamine was added. After mixing thoroughly, the mixture was reacted at 40 °C for 1 h, and the reaction progress was monitored by TLC. After the aldehyde and amine reacted completely, 336.54 mg of linoleic acid was added to the reaction solution, and the mixture was reacted at 40 °C for 30 min. Finally, 118.9 mg of methyl isonitrile acetate was added to the reaction solution, and the mixture was reacted overnight at 40 °C, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was evaporated to dryness and dissolved in 20 mL of a DCM:MeOH mixture (v / v = 10:1). The solution was washed with saturated sodium bicarbonate and saturated brine, respectively, and the organic phase was enriched. The target product VI was collected by column purification, yielding 106 mg of product.

[0049] 1 HNMR(400 MHz, CDCl3): δ0.790.84(m, 9H), 1.02-1.30(m, 44H), 1.30-1.49(m, 3H), 1.95-2.0(m, 4H), 2.68-2.71(m, 3H), 3.66(s, 3H), 5.27-5.30(m, 4H), 6.77(s, 1H), 7.54(s, 1H).

[0050] Synthesis of VII: 355.8 mg of 2-octyl-1-dodecanoic acid was dissolved in 2 mL of anhydrous methanol, and 150.2 mg of 1-(3-aminopropyl)imidazolium was added. After mixing thoroughly, the mixture was reacted at 40 °C for 1 h, and the reaction progress was monitored by TLC. After the aldehyde and amine reacted completely, 336.54 mg of linoleic acid was added to the reaction solution, and the mixture was reacted at 40 °C for 30 min. Finally, 118.9 mg of methyl isonitrile acetate was added to the reaction solution, and the mixture was reacted overnight at 40 °C, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was evaporated to dryness and dissolved in 20 mL of a DCM:MeOH mixture (v / v):10:1). The solution was washed with saturated sodium bicarbonate and saturated brine, respectively, and the organic phase was enriched. The target product VII was collected by column purification, yielding 322 mg of product.

[0051] 1 HNMR(400 MHz, CDCl3): δ0.880.91(m, 9H), 2.04-2.07(m, 8H), 2.77-2.80(m, 2H), 3.24-3.28(m, 2H), 3.71(s, 3H), 5.32-5.42(m, 4H), 6.98(s, 1H), 7.10(s, 1H), 7.56(s, 1H).

[0052] Synthesis of VIII: 355.8 mg of 2-octyl-1-dodecaldehyde was dissolved in 2 mL of anhydrous methanol, and 167 mg of 3-(2-methyl-1H-imidazol-1-yl)prop-1-amine was added. After thorough mixing, the mixture was reacted at 40 °C for 1 h, and the reaction progress was monitored by TLC. After the aldehyde and amine had reacted completely, 336.54 mg of linoleic acid was added to the reaction solution, and the mixture was reacted at 40 °C for 30 min. Finally, 118.9 mg of methyl isonitrile acetate was added to the reaction solution, and the mixture was reacted overnight at 40 °C, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was evaporated to dryness and dissolved in 20 mL of a DCM:MeOH mixture (v / v):10:1). The solution was washed with saturated sodium bicarbonate and saturated brine, respectively, and the organic phase was enriched. The target product VIII was collected by column purification, yielding 104 mg of the product.

[0053] 1 HNMR(400 MHz, CDCl3): δ0.880.93(m, 9H), 2.42(s, 3H), 2.77-2.81(m,2H), 3.27-3.31(m, 2H), 3.71(s, 3H), 5.34-5.41(m, 4H), 6.88-6.89(d, 1H), 6.96(d, 1H).

[0054] Synthesis of IX: 355.8 mg of 2-octyl-1-dodecanoic acid was dissolved in 2 mL of anhydrous methanol, and 133.4 mg of 2-(1H-pyrazol-1-yl)ethylamine was added. After mixing thoroughly, the mixture was reacted at 40 °C for 1 h, and the reaction progress was monitored by TLC. After the aldehyde and amine reacted completely, 336.54 mg of linoleic acid was added to the reaction solution, and the mixture was reacted at 40 °C for 30 min. Finally, 118.9 mg of isonitrile acetate was added to the reaction solution, and the mixture was reacted overnight at 40 °C, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was evaporated to dryness and dissolved in 20 mL of a DCM:MeOH mixture (v / v):10:1). The solution was washed with saturated sodium bicarbonate and saturated brine, respectively, and the organic phase was enriched. The target product IX was collected by column purification, yielding 72 mg of product.

[0055] 1 HNMR(400 MHz, CDCl3): δ0.880.93(m, 9H), 1.89-2.10(m, 6H), 2.78-2.81(m, 2H), 3.73-3.82(m, 5H), 5.33-5.40(m, 4H), 6.23-6.25(m, 1H), 7.47(s, 1H),7.56(s, 1H).

[0056] Synthesis of X: 355.8 mg of 2-octyl-1-dodecanoic acid was dissolved in 2 mL of anhydrous methanol, and 157.3 mg of 1-(3-aminopropyl)-4-methylpiperazine was added. After mixing thoroughly, the mixture was reacted at 40 °C for 1 h, and the reaction progress was monitored by TLC. After the aldehyde and amine reacted completely, 336.54 mg of linoleic acid was added to the reaction solution, and the mixture was reacted at 40 °C for 30 min. Finally, 118.9 mg of methyl isonitrile acetate was added to the reaction solution, and the mixture was reacted overnight at 40 °C, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was evaporated to dryness and dissolved in 20 mL of a DCM:MeOH mixture (v / v):10:1). The solution was washed with saturated sodium bicarbonate and saturated brine, respectively, and the organic phase was enriched. The target product X was collected by column purification, yielding 390.8 mg of product.

[0057] 1 HNMR(400 MHz, CDCl3): δ0.880.91(m, 9H), 2.06-2.08(m, 4H), 2.77-2.80(m, 2H), 3.34(m, 2H),3.72(m, 3H), 3.93-3.98(m, 2H), 5.33-5.41(m, 4H).

[0058] Synthesis of XI: 355.8 mg of 2-octyl-1-dodecanoic acid was dissolved in 2 mL of anhydrous methanol, and 122.2 mg of 3-(2-aminoethyl)pyridine was added. After mixing thoroughly, the mixture was reacted at 40 °C for 1 h, and the reaction progress was monitored by TLC. After the aldehyde and amine reacted completely, 336.54 mg of linoleic acid was added to the reaction solution, and the mixture was reacted at 40 °C for 30 min. Finally, 118.9 mg of methyl isonitrile acetate was added to the reaction solution, and the mixture was reacted overnight at 40 °C, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was evaporated to dryness and dissolved in 20 mL of a DCM:MeOH mixture (v / v):10:1). The solution was washed with saturated sodium bicarbonate and saturated brine, respectively, and the organic phase was enriched. The target product XI was collected by column chromatography, yielding 482.6 mg of the product.

[0059] 1 HNMR(400 MHz, CDCl3): δ0.880.91(m, 9H), 2.04-2.09(m, 4H), 2.77-2.81(m, 4H), 3.49-3.51(m, 2H), 5.32-5.43(m, 4H), 7.61-7.63(d,1H), 8.50-8.52(d,2H).

[0060] Synthesis of XII: 355.8 mg of 2-octyl-1-dodecanoic acid was dissolved in 2 mL of anhydrous methanol, and 128.2 mg of 1-(3-aminopropyl)pyrrolidine was added. After mixing thoroughly, the mixture was reacted at 40 °C for 1 h, and the reaction progress was monitored by TLC. After the aldehyde and amine reacted completely, 336.54 mg of linoleic acid was added to the reaction solution, and the mixture was reacted at 40 °C for 30 min. Finally, 118.9 mg of methyl isonitrile acetate was added to the reaction solution, and the mixture was reacted overnight at 40 °C, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was evaporated to dryness and dissolved in 20 mL of a DCM:MeOH mixture (v / v):10:1). The solution was washed with saturated sodium bicarbonate and saturated brine, respectively, and the organic phase was enriched. The target product XII was collected by column purification, yielding 42 mg of the product.

[0061] 1 HNMR (400 MHz, CDCl3): δ0.880.93(m, 9H), 2.77-2.81(m, 2H), 3.37-3.50(m, 2H), 3.97(s, 2H), 5.31-5.42(m, 4H).

[0062] Synthesis of XIII: 355.8 mg of 2-octyl-1-dodecanoic acid was dissolved in 2 mL of anhydrous methanol, and 144.2 mg of N-(3-aminopropyl)morpholine was added. After mixing thoroughly, the mixture was reacted at 40 °C for 1 h, and the reaction progress was monitored by TLC. After the aldehyde and amine reacted completely, 336.54 mg of linoleic acid was added to the reaction solution, and the mixture was reacted at 40 °C for 30 min. Finally, 118.9 mg of methyl isonitrile acetate was added to the reaction solution, and the mixture was reacted overnight at 40 °C, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was evaporated to dryness and dissolved in 20 mL of a DCM:MeOH mixture (v / v):10:1). The solution was washed with saturated sodium bicarbonate and saturated brine, respectively, and the organic phase was enriched. The target product XIII was collected by column purification, yielding 393.7 mg of the product.

[0063] 1 HNMR(400 MHz, CDCl3): δ0.880.92(m, 9H), 2.06-2.07(m, 4H), 2.77-2.80(m, 2H), 3.72(m, 6H), 3.96-3.97(s, 2H), 5.34-5.39(m, 4H).

[0064] Synthesis of XIV: 355.8 mg of 2-octyl-1-dodecanoic acid was dissolved in 2 mL of anhydrous methanol, and 130.2 mg of 3-diethylaminopropylamine was added. After thorough mixing, the mixture was reacted at 40 °C for 1 h, and the reaction progress was monitored by TLC. After the aldehyde and amine had reacted completely, 336.54 mg of linoleic acid was added to the reaction solution, and the mixture was reacted at 40 °C for 30 min. Finally, 118.9 mg of methyl isonitrile acetate was added to the reaction solution, and the mixture was reacted overnight at 40 °C, with the reaction progress monitored by TLC. After the reaction was completed, the reaction solution was evaporated to dryness and dissolved in 20 mL of a DCM:MeOH mixture (v / v):10:1). The solution was washed with saturated sodium bicarbonate and saturated brine, respectively, and the organic phase was enriched. The target product XIV was collected by column purification, yielding 208.7 mg of the product.

[0065] 1 HNMR (400 MHz, CDCl3): δ0.880.93(m, 9H), 2.04-2.09(m, 6H), 2.77-2.80(m, 6H), 3.72(m, 3H), 5.31-5.42(m, 4H).

[0066] Synthesis of XV: 355.8 mg of 2-octyl-1-dodecanoic acid was dissolved in 2 mL of anhydrous methanol, and 143.2 mg of 4-thiazolylacetic acid was added. After mixing thoroughly, the mixture was reacted at 40 °C for 1 h, and the reaction progress was monitored by TLC. After the aldehyde and amine reacted completely, 336.54 mg of linoleic acid was added to the reaction solution, and the mixture was reacted at 40 °C for 30 min. Finally, 118.9 mg of methyl isonitrile acetate was added to the reaction solution, and the mixture was reacted overnight at 40 °C, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was evaporated to dryness and dissolved in 20 mL of a DCM:MeOH mixture (v / v):10:1). The solution was washed with saturated sodium bicarbonate and saturated brine, respectively, and the organic phase was enriched. The target product XV was collected by column purification, yielding 765.7 mg of the product.

[0067] 1 HNMR(400 MHz, CDCl3): δ0.880.93(m, 9H), 1.97-2.03(m, 4H), 2.36-2.37(s, 1H), 3.89-4.02(m, 2H), 5.32-5.40(m, 2H), 8.78-8.79(s, 1H), 9.04-9.05(s, 1H).

[0068] Effect test: (1) Lipid nanoparticles containing the lipid compounds provided in the examples encapsulate mRNA Spleen-targeting lipid molecules (the spleen-targeting lipid molecule selected in this patent is the N24-O10(3T) molecule in patent CN117534585A), DOPE (dilinoleylphospholipid ethanolamine, purchased from Aivitol Pharmaceutical Technology Co., Ltd.), CHOL (cholesterol, purchased from Aivitol Pharmaceutical Technology Co., Ltd.), and DMG-PEG2000 (1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000, purchased from Aivitol Pharmaceutical Technology Co., Ltd.) were prepared in a molar ratio of 45:15:38.5:1.5 as the organic phase, and Lucferase mRNA (LucRNA) was dissolved in an aqueous solution at pH=4 as the aqueous phase. A nanoparticle suspension was prepared using a nanomedicine fabrication instrument at a volume ratio of 3:1 between the aqueous and organic phases. After preparation, the suspension was concentrated by ultrafiltration to obtain the final spleen-targeting lipid nanoparticles LucRNA LNP, which were stored at 2-8℃ for later use. Spleen-targeting ionizable lipid molecules, lipid compounds VI, DOPE, CHOL, and DMG-PEG2000 were prepared in a molar ratio of 35:10:15:38.5:1.5 using ethanol as the organic phase. Lucferase mRNA (LucRNA) was dissolved in an aqueous solution at pH 4 as the aqueous phase. The volume ratio of the aqueous phase to the organic phase was 3:1. A nanoparticle suspension was then prepared using a nanomedicine fabrication system. After preparation, the suspension was concentrated by ultrafiltration to obtain the final VI-introduced lipid nanoparticles, LucRNA VI@LNP, which were stored at 2–8°C for later use. Lipid compounds I, II, III, IV, V, VII, VIII, IX, X, XI, XII, XIII, XIV, and XV correspond to the lipid nanoparticles LucRNA I@LNP, LucRNA II@LNP, LucRNA III@LNP, LucRNA IV@LNP, LucRNA V@LNP, LucRNA VII@LNP, LucRNA VIII@LNP, LucRNA IX@LNP, LucRNA X@LNP, LucRNA XI@LNP, LucRNA XII@LNP, LucRNA XIII@LNP, LucRNA XIV@LNP, and LucRNA XV@LNP, respectively. The preparation method of the lipid nanoparticles is the same as that of LucRNA VI@LNP, except that VI is replaced with the corresponding lipid compound, while the other components and proportions remain unchanged.

[0069] LNP particle size and zeta potential were characterized using a Zetasizer Pro nanoparticle potentiometer (Malvin Panaco). The encapsulation efficiency of the LNPs was determined using a Ribogreen F-280 fluorescence spectrophotometer (Tianjin Gangdong). The results are shown in Table 1.

[0070] Table 1 The results above show that the particle size of lipid nanoparticles is around 100 nm before and after the addition of I-XV molecules, the particle size distribution of nanoparticles is relatively narrow (PDI is small), and the encapsulation efficiency of nucleic acids is over 90%.

[0071] In addition, the transfection efficiency of the prepared LucRNA LNPs and LucRNA I@LNP, LucRNA II@LNP, LucRNA III@LNP, LucRNA IV@LNP, LucRNA V@LNP, LucRNA VI@LNP, LucRNA VII@LNP, LucRNA VIII@LNP, LucRNA IX@LNP, LucRNA X@LNP, LucRNA XI@LNP, LucRNA XII@LNP, LucRNA XIII@LNP, LucRNA XIV@LNP, and LucRNA XV@LNP in Jurkat cells was detected using a multi-functional microplate reader (BioTek, model SLXFATS) with fluorescein reporter gene assay. The in vitro transcription method for LucRNA was as follows: Jurkat cells were plated at a density of 6 x 10⁵ cells / mL, and transfection was performed after plating. Protein expression levels were detected using a multi-functional microplate reader 24 h after transfection. The negative control was cell culture medium without lipid nanoparticles. The in vitro cell transfection efficiency is as follows: Figure 1 As shown, this indicates that mRNA encapsulated by LNP containing lipid compound VI has a high transfection efficiency in Jurkat cells.

[0072] The results above show that the lipid nanoparticles LucRNA VI@LNP containing lipid compound VI have better physicochemical characteristics and their in vitro cell transfection efficiency is about 1.5 times higher than that of LucRNA LNP.

[0073] (2) Lipid nanoparticles containing the lipid compounds provided in the examples encapsulate paclitaxel (PTX). Spleen-targeting ionizable lipid molecules, DOPE, CHOL, and DMG-PEG2000 were prepared in a molar ratio of 45:15:38.5:1.5 to form an ethanol solution as the organic phase. PTX was dissolved in anhydrous ethanol as the organic phase, and an aqueous solution with pH=4 was used as the aqueous phase. A nanoparticle suspension was prepared using a nanomedicine fabrication system at a volume ratio of 3:1 for the aqueous and organic phases. After preparation, the suspension was concentrated by ultrafiltration to obtain the final spleen-targeting lipid nanoparticles PTX LNP, which were stored at 2-8℃ for later use.

[0074] Spleen-targeting ionizable lipid molecules, lipid compounds VI, DOPE, CHOL, and DMG-PEG2000 were prepared in a molar ratio of 35:10:15:38.5:1.5 as the organic phase, with PTX dissolved in anhydrous ethanol as the organic phase and an aqueous solution at pH 4 as the aqueous phase. A nanoparticle suspension was prepared using a nanomedicine fabrication system at a volume ratio of 3:1 for the aqueous and organic phases. After preparation, the suspension was concentrated by ultrafiltration to obtain the final VI-introduced lipid nanoparticles, PTX VI@LNP, which were stored at 2–8°C for later use.

[0075] The LNP particle size and zeta potential were characterized using a Zetasizer Pro nanoparticle potentiometer (Malvin Panaco). The results are shown in Table 2. The lipid nanoparticles PTX VI@LNP prepared by the novel lipid compound formulation have a particle size of approximately 45 nm and a narrow particle size distribution (small PDI).

[0076] Table 2 The cytotoxic effects of the prepared PTX VI@LNP and PTXLNP on Jurkat cells were detected using a BioTek SLXFATS multi-mode microplate reader (CCK8 assay). The in vitro cytotoxicity assay was performed as follows: Jurkat cells were plated at a density of 6 x 10⁻⁶ cells / cells. 5 Cells were cultured at a density of 100 cells / mL and transfected after plating. Cell viability was assessed using a multi-functional microplate reader 24 h after transfection. The positive control was direct administration of the same concentration of PTX to cells, while the negative control was cell culture medium without LNP. In vitro cell-killing effects were as follows: Figure 2 As shown, PTX VI@LNP containing lipid compound VI has a good cytotoxic effect on T lymphoma cells.

[0077] The results above show that the lipid nanoparticles PTX VI@LNP containing lipid VI have good physicochemical characteristics and have a good cell killing effect on T lymphoma cells in vitro. The cell survival rate is only about 15% under 24 h co-incubation conditions.

[0078] (3) In vivo targeted transfection of mouse spleen with lipid nanoparticles containing the lipid compounds provided in the examples. LucRNA LNP and LucRNA VI@LNP lipid nanoparticles were prepared according to the method described in (1).

[0079] The distribution of LNPs in mice and different organs was determined using a small animal optical 3D in vivo imaging system (PerkinElmer). The in vivo distribution was measured as follows: Different LNP samples loaded with Luc mRNA were injected into mice via the tail vein at a concentration of 0.5 mg / kg, with a final volume of 200 µL. Six hours later, D-Luciferin was injected into the mice, and approximately 10 minutes later, the mice were anesthetized with isoflurane. The distribution of LNPs in mice and in the heart, liver, spleen, lungs, and kidneys was observed using the in vivo imaging system. The distribution in organs is shown below. Figure 3 As shown, the addition of lipid VI can increase the targeting and transfection intensity of LNP in mouse spleen.

[0080] The results above show that the addition of lipid compound VI increased the transfection intensity of LNP in mouse spleen by about 10 times.

[0081] (4) In vivo targeted transfection of T cells with lipid nanoparticles containing the lipid compounds provided in the examples. Spleen-targeting lipid molecules, DOPE, CHOL, and DMG-PEG2000 were prepared in an ethanol solution at a molar ratio of 45:15:38.5:1.5 as the organic phase, and GFP mRNA was dissolved in an aqueous solution at pH 4 as the aqueous phase. A nanoparticle suspension was prepared using a nanomedicine fabrication system at a volume ratio of 3:1 for the aqueous phase to the organic phase. After preparation, the suspension was concentrated by ultrafiltration to obtain the final spleen-targeting lipid nanoparticles GFP mRNA LNPs, which were stored at 2–8℃ for later use.

[0082] Spleen-targeting ionizable lipid molecules, lipid compounds VI, DOPE, CHOL, and DMG-PEG2000 were prepared in a molar ratio of 35:10:15:38.5:1.5 using ethanol as the organic phase and GFP mRNA dissolved in an aqueous solution at pH 4 as the aqueous phase. The nanoparticle suspension was prepared using a nanomedicine fabrication system with a volume ratio of aqueous to organic phase of 3:1. After preparation, the suspension was concentrated by ultrafiltration to obtain the final VI-introduced lipid nanoparticles GFP mRNA VI@LNP, which were stored at 2–8°C for later use.

[0083] The LNP particle size and zeta potential were characterized using a Zetasizer Pro nanoparticle potentiometer (Malvin Panaco). The results are shown in Table 3. The lipid nanoparticles GFP mRNA VI@LNP prepared by the novel lipid compound formulation had a particle size of approximately 50 nm.

[0084] Table 3 The transfection efficiency of LNPs on T cells in mice was determined by flow cytometry (FACS). The method was as follows: Different LNP samples loaded with GFP mRNA were injected into mice via the tail vein at a concentration of 0.5 mg / kg, with a final volume of 200 µL. A second injection was administered at the same dose 24 h later. 48 h later, the mice were sacrificed, and the spleens were harvested and soaked in PBS. The spleens were then homogenized using a sieve into 50 mL sterile, enzyme-free centrifuge tubes. During homogenization, the sieve was washed with PBS to ensure the spleen was homogenized as much as possible into the centrifuge tubes, maintaining a final volume of approximately 5 mL. The homogenate was centrifuged at 4°C, the supernatant was discarded, and the homogenate was resuspended in 3 mL of ACK to lyse the erythrocytes. The mixture was then co-incubated at room temperature for 5 min. After co-incubation, lysis was terminated with 10 mL of PBS, and the mixture was centrifuged at 4°C, with the supernatant discarded. Splenic cells were reselected using 5 mL of PBS, and then 1 mL of the resuspended cells were transferred to a 1.5 mL centrifuge tube. The cells were centrifuged again at 4°C, and the supernatant was discarded. Cells were stained with antibody, washed twice with PBS, and then resuspended in Flow Staining Buffer. After passing through a sieve, the samples were analyzed by flow cytometry. The transfection status of LNP samples on spleen immune cells in mice is as follows. Figure 4 As shown, the addition of lipid compound VI can increase the transfection efficiency of LNP in mouse spleen immune cells. Figure 5 This indicates that the addition of lipid compound VI can increase the specific transfection of LNP into mouse spleen T cells.

[0085] The results above show that the addition of lipid compound VI increased the transfection intensity and specificity of LNP on mouse spleen T cells by about 3 times.

[0086] The applicant declares that the above embodiments illustrate the lipid compounds and their applications, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0087] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0088] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A lipid compound, characterized in that, The results for the lipid compounds are shown in Formula I: Formula I R1, R2, R3, and R4 are independently selected from hydrogen, substituted or unsubstituted straight-chain or branched C1-30 alkyl groups, substituted or unsubstituted straight-chain or branched C2-30 alkenyl groups, substituted or unsubstituted straight-chain or branched C2-30 alkynyl groups, -CH2CH(OH)R5, and -CH2COOR5. , or Any one of R1, R2, R3, and R4, and any one of them is At least one of them is or In which the carbon atoms on the alkyl, alkenyl, and alkynyl groups are not substituted or at least one carbon atom is independently substituted by an oxygen atom, a sulfur atom, or a nitrogen atom; R5 is selected from hydrogen, substituted or unsubstituted straight-chain or branched C1-30 alkyl, substituted or unsubstituted straight-chain or branched C2-30 alkenyl, substituted or unsubstituted straight-chain or branched C2-30 alkynyl, wherein the carbon atom on the alkyl, alkenyl, or alkynyl group is not substituted or at least one carbon atom is independently substituted by an oxygen atom, a sulfur atom, or a nitrogen atom. R6, R7, and R8 are independently selected from hydrogen, C1-3 alkyl, or C1-3 alkoxy. R9 is selected from hydrogen, C1-3 alkyl, C1-3 alkoxy, or hydroxyl groups; n is an integer independently selected from 1 to 8, m is an integer independently selected from 0 to 8, and m being 0 indicates that the group does not exist here.

2. The lipid compound according to claim 1, characterized in that, The substituents are independently selected from any one of halogens, hydroxyl groups, straight-chain or branched C1-10 alkyl groups, and straight-chain or branched C1-10 alkoxy groups.

3. The lipid compound according to claim 1 or 2, characterized in that, Any one of R1, R2, R3, and R4 is And at least one of them is R6, R7, R8, n, and m have the same scope as in claim 1 or 2.

4. The lipid compound according to any one of claims 1-3, characterized in that, R6, R7, and R8 are selected from hydrogen; Preferably, R9 is selected from hydrogen.

5. The lipid compound according to any one of claims 1-4, characterized in that, The n is independently selected from integers from 4 to 8; Preferably, m is independently selected from an integer between 4 and 8.

6. The lipid compound according to any one of claims 1-5, characterized in that, The lipid compound is selected from any one of the following structural compounds: 。 7. The use of a lipid compound according to any one of claims 1-6 in the preparation of a bioactive substance delivery system.

8. A bioactive substance delivery system, characterized in that, The bioactive substance delivery system comprises the lipid compound according to any one of claims 1-6.

9. The bioactive substance delivery system according to claim 8, characterized in that, The bioactive substance delivery system includes either nanoparticles or microbubbles.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes the bioactive substance delivery system of claim 8 or 9.

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