A dihydroxyl imidazole biomimetic lipid compound, and a preparation method and application thereof

Abstract

The present application relates to a kind of dihydrocarbylimidazole biomimetic lipid compound and its preparation method and application, dihydrocarbylimidazole biomimetic lipid compound structure imitates natural phospholipid design, modification is not less than 10 carbon atoms alkyl on the 4th and 5th of imidazole, and modification is different alkyl chain length primary amine on 2nd position;Dihydrocarbylimidazole biomimetic lipid compound is mixed with therapeutic drug and other adjuvant, can be prepared to be loaded in the lipid nanoparticle of therapeutic drug, can be used as drug delivery system for the preparation of brain-targeted drug.Dihydrocarbylimidazole biomimetic lipid has the function of dynamic control blood-brain barrier, can realize the reversible opening of blood-brain barrier;Formed drug-loaded lipid composition can cross blood-brain barrier and better realize the brain-targeted delivery of loaded therapeutic drug;Drug-loaded lipid composition consisting of dihydrocarbylimidazole biomimetic lipid belongs to a new generation of biomimetic nanomedicine carrier, provides new strategy for realizing the brain-targeted delivery of different kinds of drugs.

Description

Technical Field

[0001] This invention belongs to the field of drug delivery technology, and in particular relates to a dialkyl imidazolium biomimetic lipid compound, its preparation method and application. Background Technology

[0002] The blood-brain barrier (BBB) ​​is a highly specialized physiological barrier system within the central nervous system, located between brain tissue and cerebral capillaries. It is primarily composed of cerebral capillary endothelial cells and their tight junctions, astrocytes, pericytes, and the basement membrane. The BBB is a selectively permeable dynamic interface, allowing essential substances such as oxygen and glucose to enter the brain while blocking harmful substances from entering the circulatory system, thus maintaining the stability of the brain's internal environment. However, 98% of small-molecule drugs and nearly 100% of large-molecule drugs are strictly restricted from entering the brain by the BBB, resulting in poor efficacy and significant side effects for most drugs used to treat brain diseases. Currently, clinically used physicochemical methods for regulating the BBB mainly include mannitol hyperosmolarity-mediated, ultrasound cavitation-mediated, and electroporation-mediated methods. However, these methods all cause irreversible damage to the structure and function of the BBB, and generally pose serious safety risks.

[0003] Lipid nanoparticles (LNPs) are a class of lipid-based, non-viral nanoscale drug delivery systems. They encapsulate drug molecules (such as small molecule drugs, peptides, and nucleic acids) through a lipid bilayer structure, protecting them from enzymatic degradation and immune clearance, and enabling efficient drug delivery. LNPs are typically formed through self-assembly in an aqueous environment from permanent cationic or ionizable cationic lipid compounds, cofactor phospholipids, cholesterol, and PEG lipids, with diameters usually ranging from 20 to 200 nm. The core advantage of LNPs lies in protecting drugs from degradation and promoting cellular uptake and intracellular release. In recent years, LNPs have attracted considerable attention due to their successful application in mRNA vaccines. Furthermore, due to their excellent biocompatibility, biodegradability, low immunogenicity, flexible customizable structure, and ease of large-scale preparation, LNPs have become the most promising carriers for clinical translation and application in various nanomedicine delivery systems.

[0004] Currently, no clinically validated LNP delivery systems capable of crossing the brain border (BBB) ​​have been reported. Therefore, there is an urgent need for a non-invasive and safe BBB modulation method and a drug delivery system capable of efficiently delivering drugs into the brain across the BBB to effectively treat brain diseases such as brain tumors, Alzheimer's disease, and Parkinson's disease. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a dialkyl imidazolium biomimetic lipid compound, its preparation method, and its applications.

[0006] The technical solution adopted in this invention is: a dialkyl imidazolium biomimetic lipid compound, the structure of which is shown in Formula 7;

[0007] Formula 7;

[0008] Where R' is C9~C 18 Straight-chain saturated or straight-chain unsaturated hydrocarbon groups, where R'' is C9~C9. 18 The straight-chain saturated or straight-chain unsaturated hydrocarbon groups, where R' and R'' are the same group or different groups;

[0009] n is an integer from 3 to 9;

[0010] X - It is an anionic group, specifically I - ,Br - Cl - BF4 - or PF6 - .

[0011] Preferably, R' and R'' are C 14 ~C 18 Straight-chain alkyl, monoalkenyl, or ynyl groups.

[0012] Preferably, R' and R'' are one or both of the following structures:

[0013]

[0014] Preferably, n is 3, 4 or 5.

[0015] Preferably, X - For I - ,Br - or Cl - .

[0016] A method for preparing dialkyl imidazolium biomimetic lipid compounds includes the following steps:

[0017] Step S01: React the compound shown in Formula 1 with the compound shown in Formula 2 to obtain the intermediate shown in Formula 3.

[0018]

[0019] Step S02: The intermediate shown in Formula 3 is oxidized to obtain the intermediate shown in Formula 4;

[0020]

[0021] Step S03: The intermediate shown in Formula 4 is reacted with the compound shown in Formula 5, ammonium acetate and HX to obtain the intermediate shown in Formula 6, wherein PG is an amino protecting group and X is I, Br or Cl.

[0022]

[0023] Step S04: Remove the protecting group PG from the intermediate shown in Formula 6 to obtain the dialkyl imidazole biomimetic lipid compound shown in Formula 7.

[0024] .

[0025] A lipid nanoparticle comprising a dialkyl imidazole biomimetic lipid compound.

[0026] The method for preparing the above-mentioned lipid nanoparticles involves mixing a dialkyl imidazole biomimetic lipid compound with natural phospholipids and cholesterol in a molar ratio of (50~75):(5~30):(35~45), and then preparing the above-mentioned lipid nanoparticles by a thin-film hydration method.

[0027] A drug-loaded lipid nanoparticle comprising a dialkyl imidazole biomimetic lipid compound and a therapeutic drug.

[0028] Preferably, the therapeutic drug is one or more of the following: synthetic small molecule chemical drugs, natural product drugs, polypeptides, proteins, and small nucleic acids.

[0029] Preferably, the synthetic small molecule chemical drug is one or more of the following: containing a free carboxyl group, containing a free hydroxyl group, and containing a free carbonyl group;

[0030] Preferably, the synthetic small molecule chemical drug is methotrexate, acyclovir, or doxorubicin;

[0031] Preferably, the natural product drug is one or more of the following: alkaloids, terpenes, flavonoids, glycosides, and phenylpropanoids.

[0032] Preferably, the small nucleic acid is one or more of siRNA, snRNA, shRNA, saRNA, and miRNA.

[0033] A method for preparing drug-loaded lipid nanoparticles involves mixing a dialkyl imidazole biomimetic lipid compound with natural phospholipids, cholesterol, and a therapeutic drug, and then preparing lipid nanoparticles in which the therapeutic drug is loaded through a thin-film hydration method.

[0034] Preferably, the molar ratio of the dialkylimidazolium biomimetic lipid compound to natural phospholipids, cholesterol and therapeutic drugs is (50~75):(5~30):(35~45):(1~15).

[0035] The natural phospholipids are selected from distearylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), distearylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dioleoylphosphatidylethanolamine (DOPE), and distearylphosphatidylcholine. One or more of the following: phosphatidyl DSPA, dipalmitoyl phosphatidyl DSPA, dimyristoyl phosphatidyl DSPA, dioleoyl phosphatidyl DSPA, distearyl phosphatidyl glycerol (DSPG), dipalmitoyl phosphatidyl glycerol (DPPG), dimyristoyl phosphatidyl glycerol (DMPG), dioleoyl phosphatidyl glycerol (DOPG), distearyl phosphatidyl serine (DSPS), dipalmitoyl phosphatidyl serine (DPPS), dimyristoyl phosphatidyl serine (DMPS), and dioleoyl phosphatidyl serine (DOPS).

[0036] Application of drug-loaded lipid nanoparticles in the preparation of brain-targeted drugs.

[0037] Preferably, drug-loaded lipid nanoparticles are used as a drug delivery system for the preparation of brain-targeted drugs;

[0038] Preferably, the drug-loaded lipid nanoparticle delivery system is capable of crossing the blood-brain barrier.

[0039] The advantages and positive effects of this invention are as follows: Dialkylimidazolium biomimetic lipids have the function of dynamically regulating the blood-brain barrier, enabling reversible opening of the blood-brain barrier; at the same time, the biomimetic lipid composition composed of dialkylimidazolium biomimetic lipids can cross the blood-brain barrier and achieve better brain-targeted delivery of the loaded drug; the biomimetic lipid composition composed of dialkylimidazolium biomimetic lipids can safely and effectively cross the blood-brain barrier, and its brain-targeted delivery efficiency is significantly better than that of representative ionizable cationic lipids such as ALC-0315, Dlin-MC3-DMA and SM-102 LNPs delivery systems.

[0040] Dialkylimidazolium biomimetic lipids represent a new type of biomimetic material capable of regulating the degree of blood-brain barrier opening. Drug-loaded lipid compositions composed of dialkylimidazolium biomimetic lipids belong to a new generation of biomimetic nanomedicine carriers, providing a new strategy for achieving brain-targeted delivery of different types of drugs. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of a drug delivery system for lipid nanoparticles based on dialkyl imidazole biomimetic lipid compounds.

[0042] Figure 2 This is a high-resolution mass spectrometry (HRMS) image of Example 1;

[0043] Figure 3 Transmission electron microscopy images of LNPs-1 in Example 4 and lipid nanoparticles in Comparative Examples 1-3; where A: LNPs-1; B: Comparative Example 2; C: Comparative Example 3; D: Comparative Example 1;

[0044] Figure 4 The real-time dynamic change trend of transmembrane resistance in an in vitro blood-brain barrier model mediated by the lipid nanoparticle delivery system based on partially dialkyl imidazole biomimetic lipid compounds in Example 8 and the delivery systems in Control Examples 1-3 is shown; where A: ALC-0315 (Control Example 1) and A4Z 2 15-JI / DPPC / Chol.(LNPs-1)、A5Z 2 15-JI / DPPC / Chol. (LNPs-5), A4Z ij 2 TEER variation mediated by 15-JI / DPPC / Chol. (LNPs-18) delivery system; B: ALC-0315 (Control Example 1) and A4Z 2 15-JI / DPPC / Chol.(LNPs-1)、A4Z15Z ij 15-JI / DPPC / Chol. (LNPs-16), A4Z15Z18-JI / DPPC / Chol. (LNPs-43), A4Z15Z ij TEER variation mediated by the 18-JI / DPPC / Chol. (LNPs-45) delivery system; C: SM-102 (Control Example 3) and A4Z 2 18-JI / DPPC / Chol. (LNPs-6), A5Z 2 18-JCl / DPPC / Chol. (LNPs-8), A4Z18Z ij 18-JI / DPPC / Chol. (LNPs-31), A4Z ij 2 TEER variation mediated by the 18-JI / DPPC / Chol. (LNPs-36) delivery system; D: D-Lin-MC3-DMA (Control Example 2) and A5Z 2 16-JI / DPPC / Chol. (LNPs-14), A4Z16Z ij 16-JCl / DPPC / Chol. (LNPs-21), A4Z ij 216-JCl / DPPC / Chol. (LNPs-23), A4Z17Z ij TEER variation mediated by the 17-JI / DPPC / Chol. (LNPs-26) delivery system;

[0045] Figure 5 The delivery effect of the drug-loaded lipid nanoparticle delivery system composed of dialkylimidazolium biomimetic lipid compounds in Example 9 across the in vitro blood-brain barrier model is compared with the delivery systems in Control Examples 4-6. Detailed Implementation

[0046] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0047] This invention relates to a dialkylimidazolium biomimetic lipid compound, its preparation method, and its applications. A hydrocarbon group with at least 10 carbon atoms is modified at the 4 and 5 positions of the imidazolium compound, and a primary amine with a different alkyl chain length is modified at the 2 position to form a 2-primary amine-substituted 4,5-dialkylimidazolium. The dialkylimidazolium biomimetic lipid compound possesses highly biomimetic structural features and low cytotoxicity, enabling the construction of biomimetic LNPs delivery systems, which can be used to deliver various types of small molecule chemical drugs and small nucleic acid drugs.

[0048] The structure of the dialkyl imidazolium biomimetic lipid compound is shown in Formula 7.

[0049] Formula 7;

[0050] Among them, X - It is an anionic group, specifically I - ,Br - Cl - BF4 - or PF6 - R' and R'' are each independently selected from chains of length C9~C 18 The chain consists of straight-chain saturated or unsaturated hydrocarbon groups, where R' and R'' are the same or different groups; n is an integer from 3 to 9. In some embodiments of the present invention, R' and R'' are each independently selected from chains with a chain length of C. 14 ~C 18 Straight-chain alkyl, monoalkenyl, or ynyl groups.

[0051] The preparation method of the dialkyl imidazolium biomimetic lipid compound, as shown in Formula 7, is as follows:

[0052] Step S01: React the compound shown in Formula 1 with the compound shown in Formula 2 to obtain the intermediate shown in Formula 3;

[0053]

[0054] The compound shown in Formula 1 and the compound shown in Formula 2 are mixed in solvent A at a molar ratio of 1:1 and fully dissolved. Then, organic base B and organic catalyst C are added sequentially, and the mixture is refluxed. After the reaction is completed, the intermediate shown in Formula 3 is obtained by separation. The solvent A can be anhydrous ethanol, water, or tetrahydrofuran (THF), etc.; the organic base B can be triethylamine (TEA), 4-dimethylaminopyridine (DMAP), or diisopropylethylamine (DIPEA), etc.; and the organic catalyst C can be 3-benzyl-5-(2-hydroxyethyl)-4-methylthiazole chloride (BMTC), thiamine (VB1), or 1-methyl-3-benzylimidazole chloride ([Bzmin]Cl), etc.

[0055] Step S02: Oxidize the intermediate shown in Formula 3 to obtain the intermediate shown in Formula 4;

[0056]

[0057] Oxidizing agent A is added to organic solvent B containing the intermediate shown in Formula 3, and the mixture is oxidized in an oxygen environment. After the reaction is completed, the intermediate shown in Formula 4 is obtained by separation. The organic solvent B can be acetonitrile (ACN), tetrahydrofuran (THF), or N,N-dimethylformamide (DMF), etc.; the oxidizing agent A can be vanadium oxychloride (VOCl3), vanadium pentoxide (V2O5), or pyridinium chlorochromate (PCC), etc.

[0058] Step S03: React the intermediate shown in Formula 4 with the compound shown in Formula 5, ammonium acetate and HX to obtain the intermediate shown in Formula 6, wherein PG is an amino protecting group and X is I, Br or Cl;

[0059]

[0060] To an organic solvent C containing the intermediate shown in Formula 4, the compound shown in Formula 5, ammonium acetate, and hydrohalic acid are added sequentially, and the mixture is refluxed. After the reaction is completed, the intermediate shown in Formula 6 is obtained by separation. The organic solvent C can be anhydrous ethanol, acetonitrile (ACN), or tetrahydrofuran (THF); the hydrohalic acid can be hydroiodic acid (HI), hydrochloric acid (HCl), or hydrobromic acid (HBr), etc.

[0061] Step S04: Remove the protecting group PG from the intermediate shown in Formula 6 to obtain the dialkyl imidazole biomimetic lipid compound shown in Formula 7.

[0062]

[0063] Add deprotecting agent A to an organic solvent D containing the intermediate shown in Formula 6, and after the reaction is complete, separate to obtain the compound shown in Formula 7; wherein, the organic solvent D can be dichloromethane (DCM), dioxane (Diox), or ethanol, etc.; the deprotecting agent A can be trifluoroacetic acid (TFA), hydrochloric acid, etc.

[0064] Furthermore, unless otherwise defined, all terms used in this invention (including technical and scientific terms) have their ordinary meanings as understood by those skilled in the art. The letters "A," "B," "C," "D," and "E" appearing in this invention are codes for reagents or solvents, used only to distinguish similar reagents or solvents, and do not indicate or imply the relative importance or order of the reagents or solvents represented. Moreover, the reagents or solvents represented by the letters "A," "B," "C," "D," and "E" used in this invention can be interchanged where appropriate. It should be noted that all terms used in this invention are merely for describing specific embodiments and are not intended to limit the invention.

[0065] In some embodiments of the present invention, in the structure of Formula 7, R' and R'' are the same group or different groups. When R' and R'' are different groups, they can be groups with the same carbon chain length or groups with different carbon chain lengths; preferably, R' and R'' are each independently selected from groups with a chain length of C 14 ~C 18 Straight-chain alkyl, monoalkenyl, or alkynyl groups; specifically, one or two of the following structural formulas:

[0066]

[0067] The carbon chain lengths of R' and R'' can modulate the biomimeticity of dialkyl imidazole biomimetic lipid compounds, thereby affecting their biocompatibility and the stability of the lipid compositions they form, thus improving their drug loading and delivery efficiency.

[0068] In some embodiments of the present invention, the dialkyl imidazolium biomimetic lipid compound may be one of the following formulas:

[0069]

[0070] Alkylated imidazolium salts (AISs) are a class of bioactive alkyl-aluminum heterocyclic azole cations. Monoalkyl imidazolium salts exhibit surface activity, effectively disrupting bacterial cell membranes and demonstrating significant bactericidal effects. The imidazolium ring, as a histidine side chain, is widely found in peptides and proteins, with a pKa of approximately 6.0, exhibiting significant buffering capacity. Furthermore, its pH responsiveness in endosomes is superior to commercially available ionizable amino-cationic lipids (such as ALC-0315), demonstrating excellent endosome escape capabilities and drug delivery potential. In addition, the chemical modifiability of the imidazolium ring provides ample room for the design of drug delivery carrier materials, potentially improving drug loading rates and targeted delivery efficiency. The π-π stacking effect of the imidazolium ring also helps enhance the encapsulation efficiency of hydrophobic drug molecules (such as aromatic natural product drugs). This invention fuses the parent structure of AISs with the lipid double-tailed chain structure of natural phospholipids to form AISs with biomimetic structural features, namely, dialkylimidazolium biomimetic lipid compounds. Due to their biomimetic properties, their biocompatibility with cell membranes is significantly better than that of monoalkyl imidazole salts. Therefore, dialkyl imidazole biomimetic lipid compounds exhibit good biocompatibility and can be used to prepare novel biomimetic LNP delivery systems.

[0071] Furthermore, the present invention also relates to a drug-loaded lipid composition comprising a dialkylimidazolium biomimetic lipid compound of Formula 7 or a pharmaceutically acceptable solvate thereof; further comprising a therapeutic drug and other auxiliary components. The therapeutic drug is one or more of synthetic small molecule chemical drugs, natural product drugs, peptides, proteins, and small nucleic acids; wherein, the synthetic small molecule chemical drug is one or more of methotrexate containing free carboxyl groups, acyclovir containing free hydroxyl groups, and doxorubicin containing free carbonyl groups; the natural product drug is selected from one or more of alkaloids, terpenes, flavonoids, glycosides, and phenylpropanoid natural products; the small nucleic acid is selected from one or more of siRNA, snRNA, shRNA, saRNA, and miRNA. The auxiliary components may be natural phospholipids and cholesterol. The natural phospholipids are selected from distearylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), distearylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dioleoylphosphatidylethanolamine (DOPE), and distearylphosphatidylcholine. One or more of the following: phosphatidyl DSPA, dipalmitoyl phosphatidyl DSPA, dimyristoyl phosphatidyl DSPA, dioleoyl phosphatidyl DSPA, distearyl phosphatidyl glycerol (DSPG), dipalmitoyl phosphatidyl glycerol (DPPG), dimyristoyl phosphatidyl glycerol (DMPG), dioleoyl phosphatidyl glycerol (DOPG), distearyl phosphatidyl serine (DSPS), dipalmitoyl phosphatidyl serine (DPPS), dimyristoyl phosphatidyl serine (DMPS), and dioleoyl phosphatidyl serine (DOPS).

[0072] In this invention, the term "solvent" refers to a complex formed by a dialkylimidazolium biomimetic lipid compound of Formula 7 and a solvent (e.g., ethanol or water). For example, the term "hydrate" refers to the case where the solvent in the aforementioned term "solvent" is water. Furthermore, the dialkylimidazolium biomimetic lipid compound of Formula 7 can be separated into solvates, and any solvate is included within the scope of protection of this invention.

[0073] This dialkylimidazolium biomimetic lipid was combined with natural phospholipids and cholesterol in different molar ratios to form biomimetic lipid compositions with diameters of 50-500 nm in a buffer solution. The dialkylimidazolium biomimetic lipid compound shown in Formula 7 can be mixed with natural phospholipids and cholesterol, and lipid nanoparticles (LNPs) can be prepared using a thin-film hydration method. The molar ratio of the dialkylimidazolium biomimetic lipid compound, natural phospholipids, and cholesterol is (50-75):(5-30):(35-45).

[0074] Furthermore, these LNPs can be used as delivery systems to load drugs for targeted drug delivery. This invention also employs a thin-film hydration method to prepare drug-loaded LNPs. A dialkylimidazolium biomimetic lipid compound is mixed with natural phospholipids, cholesterol, and a therapeutic drug in a molar ratio of (50-75):(5-30):(35-45):(1-15) to prepare a drug-loaded LNP delivery system based on the dialkylimidazolium biomimetic lipid compound.

[0075] A dialkylimidazolium biomimetic lipid compound, natural phospholipids, and cholesterol were dissolved in a MeOH / CHCl3 (1:1, v / v) mixture. After removing the organic solvent, a translucent mixed lipid film was formed on the inner wall of a container. An equal volume of PBS buffer containing a therapeutic drug was added to the container, and the mixture was incubated at 50°C for 5-10 min with vortexing for 1-3 min, repeated 3-10 times to fully hydrate the lipid film, resulting in a turbid mixed lipid colloidal solution. After sonication, the colloidal solution was repeatedly extruded through a 100 nm pore size PC filter membrane 30-40 times. The resulting drug-loaded lipid composition after separation and purification constitutes the LNPs drug delivery system based on the dialkylimidazolium biomimetic lipid compound. Figure 1 As shown.

[0076] In dialkyl imidazolium biomimetic lipid compounds, the introduction of a primary amine with a certain alkyl chain length at the 2-position of the imidazolium ring can effectively regulate the degree of blood-brain barrier opening, achieving reversible opening of the blood-brain barrier. Drug-loaded lipid compositions composed of dialkyl imidazolium biomimetic lipid compounds can load synthetic small molecule chemical drugs, natural product drugs, peptides, proteins, or small nucleic acids, and can cross the blood-brain barrier to achieve brain-targeted drug delivery. When preparing brain-targeted drug-loaded lipid compositions, in the compound structure of Formula 7, n is preferably 3, 4, or 5.

[0077] The dialkylimidazolium biomimetic lipid compound shown in Formula 7 differs significantly in structure from representative ionizable cationic lipids in related delivery technologies, such as ALC-0315, Dlin-MC3-DMA, and SM-102, and exhibits highly biomimetic structural features, demonstrating good biocompatibility and low cytotoxicity. Furthermore, the dialkylimidazolium biomimetic lipid compound shown in Formula 7 possesses the function of dynamically regulating the blood-brain barrier, enabling reversible opening of the blood-brain barrier. By constructing a biomimetic LNP delivery system using dialkylimidazolium biomimetic lipids with highly biomimetic structural features and low cytotoxicity, it can be used to prepare drug delivery systems and formulations that cross the blood-brain barrier, achieving safe and effective brain-targeted drug delivery across the blood-brain barrier.

[0078] Biomimetic lipid compositions composed of dialkyl imidazolium-based biomimetic lipids can safely and effectively cross the blood-brain barrier, exhibiting significantly higher brain-targeted delivery efficiency than LNPs delivery systems composed of representative ionizable cationic lipids such as ALC-0315, Dlin-MC3-DMA, and SM-102. These biomimetic drug-carrying lipid compositions represent a new generation of biomimetic nanomedicine carriers, beneficial for improving the delivery efficiency of small molecule chemical drugs and small nucleic acid drugs across the blood-brain barrier.

[0079] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in the accompanying drawings are performed according to the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies. It should be noted that, unless otherwise defined, the technical or scientific terms used in this invention should have the ordinary meaning understood by those skilled in the art.

[0080] Example 1: Dialkylimidazolium biomimetic lipid compound A4Z 2 Synthesis of 15-JI

[0081] A4Z 2 The synthesis process of 15-JI is shown in the following formula:

[0082]

[0083] The specific synthesis process is as follows:

[0084] (1) Preparation of 17-hydroxytridodecane-16-one (I-1)

[0085] 2.0 g (8.32 mmol, 1.0 eq.) hexadecaldehyde (I-0) was dissolved in 20 mL of anhydrous ethanol. Then, 0.35 mL of triethylamine (2.50 mmol, 0.3 eq.) and 0.11 g of 3-benzyl-5-(2-hydroxyethyl)-4-methylthiazole chloride (0.42 mmol, 0.05 eq.) were added sequentially. The mixture was refluxed at 80 °C and stirred for 3 h. After the reaction was completed by TLC monitoring, the reaction solution was poured into ice water, the precipitated white solid was collected by filtration, and recrystallized from ethanol to obtain white solid I-1 (900 mg, 1.87 mmol), with a yield of 45.0%.

[0086] (2) Preparation of tris(dodecane-16,17-dione) (I-2)

[0087] 200 mg (0.42 mmol, 1.0 eq.) of 17-hydroxy-16-hydroxy-16-dione (I-1) was added to 2.0 mL of acetonitrile, dissolved, and then 0.72 mg of VOCl3 (4.2 μmol, 0.01 eq.) was added. The mixture was stirred overnight at room temperature under oxygen conditions, and then the reaction was continued at 60 °C for 3 h. After the reaction was completed by TLC monitoring, the reaction solution was quenched with saturated NaHCO3. The organic phase was extracted three times with PE / EA (1:1) solvent, and then washed once each with saturated ammonium chloride and saturated sodium chloride solutions. After washing, the product was dried with anhydrous sodium sulfate and rotary evaporated to obtain the crude product. Finally, the crude product was recrystallized from ethanol to obtain 160 mg (0.33 mmol) of 16,17-dione (I-2), with a yield of 80.3%.

[0088] (3) Preparation of 2-[5-(tert-butoxycarbonylamino)pentyl]-4,5-bispentadecanyl-1H-imidazol-3-onium salt (I-3)

[0089] 70 mg (0.15 mmol, 1.0 eq.) of dodecane-16,17-dione (I-2) was dissolved in 2 mL of anhydrous ethanol. Then, 38 mg of tert-butoxycarbonyl-6-aminohexanal (0.18 mmol, 1.2 eq.), 27 mg of NH4OAc (0.35 mmol, 2.4 eq.), and 19 mg of hydroiodic acid (0.15 mmol, 1.0 eq.) were added sequentially. The mixture was refluxed and stirred at 90 °C for 3 h. After the reaction was completed by TLC monitoring, the reaction solution was quenched with saturated NaHCO3 (pH > 12). The organic phase was extracted three times with DCM and dried over anhydrous sodium sulfate. After the solvent was evaporated, the crude product was purified by column chromatography (DCM:MeOH = 90:10) to obtain a pale yellow solid I-3 (30 mg, 0.04 mmol), with a yield of 30.4%.

[0090] (4) Preparation of 2-(5-aminopentyl)-4,5-bispentadecanyl-1H-imidazol-3-onium salt (A4Z) 2 15-JI)

[0091] 30 mg (0.04 mmol) of intermediate I-3 was dissolved completely in 2 mL of DCM, followed by the addition of 0.4 mL of TFA. The mixture was stirred at room temperature for 3 h. After the reaction was completed by TLC monitoring, the reaction solution was diluted with 10 mL of DCM and 10 mL of water. The organic phase was then washed three times each with 1 M NaOH solution and saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The concentrated product yielded a yellow solid, A4Z. 2 15-JI (8 mg, 0.01 mmol), yield 25%.

[0092] The prepared dialkyl imidazolium biomimetic lipid compound A4Z 2 15-JI (compound 1) was characterized, such as Figure 2 The image shown is A4Z. 2 15-JI High Resolution Mass Spectrometry (HRMS) [MI] + : C 38 H 76 N3 + Theoretical m / z value: 574.60, measured value: 574.6034. The proton NMR spectrum results are as follows:

[0093] 1 H NMR (400 MHz, CDCl3) δ 8.90-9.20 (s, 1H), 7.45-7.50 (s, 1H), 3.95-4.00 (t, J = 7.2, 2H), 2.72 (t, J = 7.0, 2H), 1.70-1.75 (m, 4H), 1.55-1.62 (m, 4H), 1.22-1.27 (m, 88H), 0.85-0.89 (t, J = 6.8, 6H).

[0094] Example 2: Synthesis of dialkylimidazolium biomimetic lipid compounds 2-45

[0095] Dialkylimidazolium biomimetic lipid compound A3Z was synthesized according to the method in Example 1. 2 15-JI, A4Z 2 15-JCl, A4Z 2 15-JBr, A5Z 2 15-JI, A4Z 2 18-JI, A3Z 2 18-JBr, A5Z 2 18-JCl, A4Z2 17-JI、A5Z 2 17-JCl、A4Z 2 14-JI、A5Z 2 14-JI、A4Z 2 16-JI、A5Z 2 16-JI、A5Z14Z ij 14-JI、A4Z15Z ij 15-JI、A5Z15Z ij 15-JI、A4Z ij 2 15-JI、A5Z15Z de 15-JI、A5Z de 2 15-JI、A4Z16Z ij 16-JCl、A5Z16Z ij 16-JI、A4Z ij 2 16-JCl、A5Z16Z de 16-JI、A5Z de 2 16-JI、A4Z17Z ij 17-JI、A5Z17Z ij 17-JI、A4Z17Z de 17-JI、A5Z17Z de 17-JI、A4Z18Z bc '18-JI、A4Z18Z ij 18-JI、A5Z18Z ij 18-JI、A4Z18Z de 18-JI、A5Z18Z de 18-JI、A4Z18Z ij '18-JI、A4Z ij 2 18-JI、A5Z ij 2 18-JI、A4Z de 2 18-JI、A5Z de 2 18-JI、A4Z15Z de 15-JI、A4Z de 2 15-JI、A5Z ij 2 15-JI、A4Z15Z18-JI、A4Z15Z de 18-JI、A4Z15Z ij18-JI (compounds 2~45).

[0096] The above-mentioned compound synthesis method is the same as that of A4Z. 2 Similar to 15-JI (compound 1), the difference lies in that in step (1), hexadecaldehyde (I-0) can be replaced with saturated and / or unsaturated aliphatic aldehydes of different chain lengths; in step (3), tert-butoxycarbonyl 6-aminohexanal can be replaced with tert-butoxycarbonyl 5-aminopentanal or tert-butoxycarbonyl 7-aminoheptanal, and hydroiodic acid can be replaced with hydrochloric acid or hydrobromic acid. The starting materials (I-0) and reaction conditions of each step for the above dialkyl imidazolium biomimetic lipid compounds are shown in Table 1.

[0097] Table 1

[0098]

[0099] Example 3: Preparation of LNPs based on dialkylimidazolium biomimetic lipid compounds

[0100] The A4Z prepared in Example 1 was subjected to thin-film hydration. 2 15-JI (compound 1) was further prepared to form LNPs.

[0101] A4Z 2 15-JI, natural phospholipids DPPC, and cholesterol were dissolved in a mixed solvent of MeOH / CHCl3 (1:1, v / v) at a molar ratio of 70:15:36. After removing the organic solvent by nitrogen blowing or rotary evaporation at 50°C, a translucent mixed lipid film was formed on the inner wall of a container. Then, an equal volume of PBS buffer was added to the container, and the mixture was incubated at 50°C for 5 min, followed by vortexing for 1–3 min to fully hydrate the lipid film. This hydration process was repeated 5 times to obtain a turbid mixed lipid colloidal solution. After sonication for 30 min, the colloidal solution was repeatedly extruded through a 100 nm pore size PC filter membrane 30–40 times to obtain an A4Z-based product. 2 15-JI lipid nanoparticle (LNPs-1) delivery system.

[0102] The corresponding LNPs were prepared using compounds 2-45 as raw materials according to the above method. The dialkyl imidazolium biomimetic lipid compound A4Z was then used. 2 15-JI was replaced with the corresponding dialkyl imidazole biomimetic lipid compounds in Table 1, and other conditions remained unchanged to prepare LNPs-2 to LNPs-45.

[0103] Comparative Example 1:

[0104] The dialkyl imidazole biomimetic lipid compound A4Z from Example 3 2 15-JI was replaced with cationic lipid compounds ALC-0315 (CAS:2036272-55-4), D-Lin-MC3-DMA (CAS:1224606-06-7), and SM-102 (CAS:2089251-47-6) for drug delivery, respectively, and other conditions remained unchanged to obtain the LNPs of Control Examples 1 to 3.

[0105] Example 4: Physicochemical and cytotoxic characterization of LNPs

[0106] The physicochemical properties and cytotoxicity of LNPs-1 to LNPs-45 prepared in Example 3 and LNPs of Control Examples 1 to 3 prepared in Comparative Example 1 were characterized.

[0107] The morphology of the prepared LNPs-1 to LNPs-45 and the LNPs of control examples 1 to 3 was characterized by transmission electron microscopy (TEM). Figure 3 The image shows a morphological comparison of LNPs-1 and three control examples. All LNPs in each group exhibited a relatively complete globular vesicle structure with no significant changes. The structures of LNPs-2 to LNPs-45 were similar to those of LNPs-1, also possessing a complete globular vesicle structure.

[0108] In addition, the average particle size and zeta potential of LNPs-1 to LNPs-45 and control examples 1 to 3 were characterized by nanoflow cytometry (NanoFCM) and zeta potential analyzer (ZetaPALS), respectively. The characterization results are shown in Table 2.

[0109] Table 2

[0110]

[0111] As shown in Table 2, the LNPs-1 to LNPs-45 have relatively uniform particle sizes, with average particle sizes ranging from 118.9 to 141.2 nm and median zeta potentials ranging from 32.1 to 41.4 mV. In contrast, the LNPs in Control Examples 1 to 3 have average particle sizes of 187.4 nm, 171.0 nm, and 182.1 nm, respectively, and median zeta potentials of 12.1 mV, 10.8 mV, and 14.8 mV, respectively. Compared to the LNPs based on dialkylimidazolium biomimetic lipid compounds, the control LNPs have larger particle sizes and lower median zeta potentials.

[0112] The cytotoxicity of LNPs-1 to LNPs-45 and controls 1 to 3 in human glioma cells U-118MG was further evaluated using the MTT assay. U-118MG cells were cultured in DMEM medium containing 10% fetal bovine serum and seeded at a density of 3000 cells / well in 96-well plates, incubated for 24 h at 5% CO2 and 37°C. Then, different concentrations of various LNPs were added to each well, with a final concentration of 1 × 10⁻⁶. 7 LNPs / mL, 5×10 7 LNPs / mL, 1×10 8 LNPs / mL, 5×10 8 LNPs / mL, 1×10 9 LNPs / mL, 2×10 9 LNPs / mL and 5×10 9 LNPs were added at a concentration of 1 / mL, and incubation continued for 24 h. Afterward, 5 mg / mL MTT reagent was added to each well, and incubation continued for 4 h. The culture medium was then discarded, and 150 μL of DMSO was added to each well. After shaking for 10 min, the absorbance of each well was measured at 570 nm using a microplate reader. U-118MG cells without added LNPs were used as a negative control, and cell viability was calculated. If cell viability was greater than 80%, this type of LNP was considered non-cytotoxic.

[0113] The test results are shown in Table 2. No significant cytotoxicity was observed in LNPs-1 to LNPs-45 and in Control Examples 1 to 3.

[0114] Example 5: Construction of a drug delivery system for LNPs based on dialkyl imidazole biomimetic lipid compounds – loading small molecule chemical drugs

[0115] The LNPs-1 to LNPs-45 prepared in Example 3 can be used as delivery systems to load small molecule chemical drugs and achieve delivery functions. In this example, methotrexate (MTX) was used as the target drug, and MTX was loaded into LNPs-1 to LNPs-45 to construct the corresponding drug-loaded LNPs delivery system.

[0116] MTX was dissolved in DMSO with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) in a molar ratio of 1:1:1 and reacted at 25°C for 30 min to form an MTX activator, which was then diluted with PBS buffer.

[0117] The A4Z synthesized in Example 1 215-JI was dissolved thoroughly in a mixed solvent of MeOH / CHCl3 (1:1, v / v) with natural phospholipids DPPC and cholesterol at a molar ratio of 70:15:36. After removing the organic solvent by nitrogen blowing or rotary evaporation at 50°C, a semi-transparent film of mixed lipids was formed on the inner wall of the container. Then, an equal volume of the above-mentioned PBS buffer diluted with MTX activator (A4Z) was added to the container. 2 The molar ratio of 15-JI to MTX was 14:1. After incubation at 50℃ for 5 min, the mixture was vortexed for 1–3 min to fully hydrate the lipid film. This hydration process was repeated 10 times to obtain a turbid mixed lipid colloidal solution. After sonication for 30 min, the colloidal solution was repeatedly extruded through a 100 nm pore size PC filter membrane 30–40 times. Finally, 0.1 M NaOH was added, and the mixture was incubated at 37℃ for 30 min. Purification was then performed by column chromatography to obtain an A4Z-based solution. 2 15-JI drug-loaded LNPs for MTX loading: MTX@A4Z 2 15-JI·LNPs (MTX@LNPs-1).

[0118] A4Z 2 Replace 15-JI with the dialkylimidazolium biomimetic lipid compounds in Table 1, and keep other conditions unchanged to obtain drug-loaded LNPs loaded with MTX: MTX@LNPs-2~MTX@LNPs-45.

[0119] Comparative Example 2:

[0120] The dialkyl imidazole biomimetic lipid compound A4Z from Example 5 2 15-JI was replaced with cationic lipid compounds ALC-0315, D-Lin-MC3-DMA and SM-102 respectively, while other conditions remained unchanged, to obtain drug-loaded LNPs loaded with MTX as in Control Examples 4-6.

[0121] Example 6: Construction of a drug delivery system for LNPs based on dialkyl imidazolium biomimetic lipid compounds – loading small nucleic acid drugs

[0122] The LNPs-1 to LNPs-45 prepared in Example 3 can be used as a delivery system to load small nucleic acid drugs and achieve delivery. In this example, the small nucleic acid drug miR-206 is used as the target drug, and miR-206 is loaded into LNPs-1 to LNPs-45 to construct the corresponding drug-loaded LNPs delivery system.

[0123] The A4Z synthesized in Example 1 215-JI was dissolved thoroughly in a mixed solvent of MeOH / CHCl3 (1:1, v / v) with natural phospholipids DPPC and cholesterol at a molar ratio of 70:15:36. After removing the organic solvent by nitrogen blowing or rotary evaporation at 50°C, a semi-transparent film of mixed lipids was formed on the inner wall of the container. Then, an equal volume of PBS buffer (A4Z) containing the small nucleic acid miR-206 was added to the container. 2 The lipid membrane was incubated at 50°C for 5 min with a mass ratio of 15-JI to miR-206 of 9:1, followed by vortexing for 1–3 min to fully hydrate the lipid membrane. This hydration process was repeated 5 times to obtain a turbid mixed lipid colloidal solution. After sonication for 30 min, the colloidal solution was repeatedly extruded through a 100 nm pore size PC filter membrane 30–40 times. Finally, it was purified by column chromatography to obtain an A4Z-based solution. 2 15-JI-loaded LNPs containing miR-206: miR-206@A4Z 2 15-JI·LNPs (miR-206@LNPs-1).

[0124] A4Z 2 Replace 15-JI with the dialkylimidazolium biomimetic lipid compounds in Table 1, and keep other conditions unchanged to obtain drug-loaded LNPs loaded with miR-206: miR-206@LNPs-2~miR-206@LNPs-45.

[0125] Comparative Example 3:

[0126] The dialkyl imidazole biomimetic lipid compound A4Z from Example 6 2 By replacing 15-JI with cationic lipid compounds ALC-0315, D-Lin-MC3-DMA and SM-102 respectively, and keeping other conditions unchanged, drug-loaded LNPs loaded with miR-206 as in Control Examples 7-9 can be obtained.

[0127] Example 7: Encapsulation efficiency characterization of drug-loaded LNPs delivery system

[0128] The residual MTX content in the drug-loaded LNPs of MTX@LNPs-1 to MTX@LNPs-45 and Control Examples 4 to 6 was determined by HPLC, and the encapsulation efficiency of MTX in each group of LNPs was calculated. The results are shown in Table 3.

[0129] miR-206 was labeled with 5-FAM. The residual miR-206 content in the drug-loaded LNPs of miR-206@LNPs-1 to miR-206@LNPs-45 and Control Examples 7 to 9 was estimated by fluorescence spectrophotometry. The encapsulation efficiency of miR-206 in each group of LNPs was calculated. The results are shown in Table 3.

[0130] Table 3

[0131]

[0132] Table 3 shows that the encapsulation efficiency of MTX in the MTX@LNPs-1 to MTX@LNPs-45 small molecule drug delivery systems ranged from 81.6% to 98.1%. In contrast, the encapsulation efficiencies of MTX-loaded LNPs prepared in Control Examples 4 to 6 were 64.2%, 57.3%, and 40.8%, respectively, significantly lower than those in MTX@LNPs-1 to MTX@LNPs-45. The encapsulation efficiency of miR-206 in the miR-206@LNPs-1 to miR-206@LNPs-45 small nucleic acid drug delivery systems ranged from 91.8% to 99.1%. In contrast, the miR-206-loaded drug-loaded LNPs prepared in Comparative Examples 7-9 had miR-206 encapsulation efficiencies of 78.4%, 79.9%, and 84.5%, respectively, which were much lower than miR-206@LNPs-1 to miR-206@LNPs-45.

[0133] Table 3 demonstrates that the dialkyl imidazole biomimetic lipid compounds prepared in Examples 1 and 2, based on their structural characteristics and advantages, can be used to prepare LNPs delivery systems with better drug loading performance, significantly improving drug encapsulation efficiency; this characteristic is universal and shows good drug loading performance for other types of small molecule chemical drugs and small nucleic acid drugs.

[0134] Example 8: Regulation of the blood-brain barrier model by LNP delivery system

[0135] This embodiment characterizes the dynamic regulatory effect of LNPs-1 to LNPs-45 prepared in Example 3 and LNPs of Control Examples 1 to 3 prepared in Comparative Example 1 as a delivery system on an in vitro blood-brain barrier model.

[0136] HCMEC / D3 immortalized human brain microvascular endothelial cells were cultured in ECM medium containing 5% fetal bovine serum and seeded at a density of 50,000 cells / well in Transwell chambers. TM Collagen-coated polycarbonate membrane (0.4 µm pore size and 0.33 cm² growth area) in No. 3413 Costar. 2Replace the medium with fresh ECM medium containing 5% fetal bovine serum and continue culturing for 2-3 days. Then replace the medium with DMEM / F12 serum-free medium containing 550 nM hydrocortisone and continue culturing for two more days at 5% CO2 and 37°C until the HCMEC / D3 cell monolayers fuse to form a barrier model.

[0137] The transmembrane resistance (TEER) of the HCMEC / D3 cell barrier formed above was measured using a CellZscope II real-time cell dynamics analyzer (nanoAnalytic, Germany). The test module and serum-free DMEM / F12 medium containing 550 nM hydrocortisone were preheated at 5% CO2 and 37°C for 1 h. 1.2 mL of DMEM / F12 serum-free medium was added to each metal chamber of the test module. After transferring the transwell chambers to the metal chambers of the test module, the TEER in each transwell chamber was measured at a rate of one chamber / min under 5% CO2 and 37°C. The results showed that the TEER of each chamber was greater than 300 Ω·cm. 2 This indicates that the fused HCMEC / D3 cell monolayer possesses barrier function, and the in vitro blood-brain barrier model has been successfully constructed, which can be used for experiments on the dynamic regulation of the blood-brain barrier.

[0138] The LNPs-1 to LNPs-45 prepared in Example 3 and the LNPs of Control Examples 1 to 3 prepared in Comparative Example 1 were added to each Transwell chamber, and the TEER of each chamber was measured according to the above method. Real-time changes within 24 hours were observed. The maximum magnitude of TEER downregulation and the corresponding time for each group were recorded, and the results are shown in Table 4. Among them, A4Z 2 15-JI / DPPC / Chol.(LNPs-1)、A5Z 2 15-JI / DPPC / Chol. (LNPs-5), A4Z 2 18-JI / DPPC / Chol. (LNPs-6), A5Z 2 18-JCl / DPPC / Chol. (LNPs-8), A5Z 2 16-JI / DPPC / Chol. (LNPs-14), A4Z15Z ij 15-JI / DPPC / Chol. (LNPs-16), A4Z ij 2 15-JI / DPPC / Chol. (LNPs-18), A4Z16Z ij 16-JCl / DPPC / Chol. (LNPs-21), A4Z ij 216-JCl / DPPC / Chol. (LNPs-23), A4Z17Z ij 17-JI / DPPC / Chol. (LNPs-26), A4Z18Z ij 18-JI / DPPC / Chol. (LNPs-31), A4Z ij 2 18-JI / DPPC / Chol.(LNPs-36)、A4Z15Z18-JI / DPPC / Chol.(LNPs-43)、A4Z15Z ij The real-time dynamic changes of TEER in 18-JI / DPPC / Chol. (LNPs-45) and control examples 1-3 are as follows: Figure 4 As shown.

[0139] Table 4

[0140]

[0141] As shown in Table 4, all LNPs-1 to LNPs-45 delivery systems mediated a downregulation of TEER in the in vitro blood-brain barrier model constructed from HCMEC / D3 cells, with TEER reduction ranging from 33.8% to 87.2%. In contrast, the LNPs delivery systems prepared in Control Examples 1 to 3 did not show significant TEER downregulation. The results in Table 4 demonstrate that the LNPs-1 to LNPs-45 delivery systems composed of dialkylimidazolium biomimetic lipid compounds prepared in Examples 1 and 2 possess the potential to dynamically regulate the opening degree of the blood-brain barrier.

[0142] like Figure 4 As shown, this is A4Z. 2 15-JI / DPPC / Chol.(LNPs-1)、A5Z 2 15-JI / DPPC / Chol. (LNPs-5), A4Z 2 18-JI / DPPC / Chol. (LNPs-6), A5Z 2 18-JCl / DPPC / Chol. (LNPs-8), A5Z 2 16-JI / DPPC / Chol. (LNPs-14), A4Z15Z ij 15-JI / DPPC / Chol. (LNPs-16), A4Z ij 2 15-JI / DPPC / Chol. (LNPs-18), A4Z16Z ij 16-JCl / DPPC / Chol. (LNPs-21), A4Zij 2 16-JCl / DPPC / Chol. (LNPs-23), A4Z17Z ij 17-JI / DPPC / Chol. (LNPs-26), A4Z18Z ij 18-JI / DPPC / Chol. (LNPs-31), A4Z ij 2 18-JI / DPPC / Chol.(LNPs-36)、A4Z15Z18-JI / DPPC / Chol.(LNPs-43)、A4Z15Z ij Changes in TEER mediated by the 18-JI / DPPC / Chol. (LNPs-45) delivery system. As shown in the figures, all constructed LNPs in each experimental group mediated the downregulation of TEER in the in vitro blood-brain barrier model constructed from HCMEC / D3 cells, and achieved almost complete recovery of TEER levels within 24 h. Similar results were observed in other groups of the LNPs-1 to LNPs-45 delivery systems. This indicates that the dialkylimidazolium biomimetic lipid compounds (compounds 1-45) synthesized in Examples 1 and 2 possess the potential to mediate reversible opening of the blood-brain barrier.

[0143] Example 9: Delivery performance of drug-loaded LNPs delivery system across an in vitro blood-brain barrier model

[0144] An in vitro blood-brain barrier-glioma co-culture model (in vitro blood-brain barrier model) was constructed, consisting of HCMEC / D3 cells and U-118MG cells. Drug-loaded LNPs containing MTX, prepared from MTX@LNPs-1, MTX@LNPs-5, MTX@LNPs-6, MTX@LNPs-8, MTX@LNPs-14, MTX@LNPs-16, MTX@LNPs-18, MTX@LNPs-21, MTX@LNPs-23, MTX@LNPs-26, MTX@LNPs-31, MTX@LNPs-36, MTX@LNPs-43, MTX@LNPs-45, and controls 4-6, were selected. The delivery efficiency of each selected group of drug-loaded LNPs across the in vitro blood-brain barrier in the in vitro blood-brain barrier-glioma co-culture model was assessed.

[0145] U-118MG cells were cultured in DMEM medium containing 10% fetal bovine serum and seeded at a density of 15,000 cells / well in 24-well plates for 24 h. Transwell chambers containing an in vitro blood-brain barrier model constructed from HCMEC / D3 cells (as described in Example 8) were transferred to the wells of the 24-well plates. Selected drug-loaded LNPs were added to each Transwell chamber. A control group containing the same amount of MTX (10 nmol) was also included. The cells were cultured for another 24 h at 5% CO2 and 37°C. Using untreated U-118MG cells as a negative control, cell viability was calculated using the MTT assay for each example and control group. The results are as follows: Figure 5 As shown.

[0146] like Figure 5 As shown, after MTX@LNPs-1, MTX@LNPs-5, MTX@LNPs-6, MTX@LNPs-8, MTX@LNPs-14, MTX@LNPs-16, MTX@LNPs-18, MTX@LNPs-21, MTX@LNPs-23, MTX@LNPs-26, MTX@LNPs-31, MTX@LNPs-36, MTX@LNPs-43, and MTX@LNPs-45 loaded with MTX (10 nmol) were applied to an in vitro hematoma barrier model, the survival rate of U-118MG cells ranged from 18% to 66%. In contrast, the survival rates of U-118MG cells under the treatment of controls 4-6 were 98%, 95%, and 93%, respectively. This indicates that the drug-loaded LNPs loaded with MTX prepared from the above-mentioned dialkylimidazolium biomimetic lipid compounds can effectively cross the in vitro blood-brain barrier model, and the delivery efficiency of MTX is significantly better than that of controls 4-6.

[0147] Example 10: Drug delivery system for LNPs in vivo for brain delivery

[0148] The in vivo brain delivery effects of the MTX@LNPs-1~MTX@LNPs-45 and miR-206@LNPs-1~miR-206@LNPs-45 delivery systems prepared in Examples 5 and 6 were tested, respectively.

[0149] U-118MG cell suspension (1×10⁻⁶) was implanted into the brains of immunodeficient female nude mice with an average weight of approximately 25 g at 4-8 weeks of age. 6 (each cell) and after 1-2 weeks of growth, a tumor volume of approximately 100-150 mm is obtained. 3An immunodeficient mouse model was established. These mice were randomly divided into three groups. The mice were administered MTX@LNPs-1 to MTX@LNPs-45 delivery systems and LNPs loaded with MTX (18 nM) prepared in Control Examples 4 to 6 via tail vein injection, respectively. The control group received an equal volume of PBS buffer. The injection volume was 200 μL, with injections spaced 24 h apart, for a total of three injections. The longest diameter (a) and shortest diameter (b) of the tumor, as well as the mouse's body weight, were recorded daily. The tumor volume was calculated using the formula V = 1 / 2 × a × b × b. The tumor volumes of the experimental and control groups were compared, and the results are shown in Table 5.

[0150] The brains of Wistar rats with an average weight of approximately 200 g were implanted with a suspension of C6 glioma cells (2 × 10⁻⁶). 6 (1 cell), after 1 week of growth, a tumor volume of approximately 100-200 mm was obtained. 3 A rat model bearing tumor was established. These rats were randomly divided into three groups. The miR-206@LNPs-1 to miR-206@LNPs-45 delivery systems and LNPs loaded with miR-206 (50 nM) prepared in Control Examples 7-9 were administered via tail vein injection, respectively. The control group received an equal volume of PBS buffer. Injections were given at 72-hour intervals, for a total of three injections. The longest diameter (a) and shortest diameter (b) of the tumor, as well as the rat's body weight, were recorded daily. Tumor volume was calculated using the formula V = 1 / 2 × a × b × b. The tumor volumes of the experimental and control groups were compared, and the results are shown in Table 5.

[0151] Table 5

[0152]

[0153] As shown in Table 5, the MTX@LNPs-1 to MTX@LNPs-45 delivery systems significantly reduced the volume of gliomas in mice after tail vein injection, with tumor volumes ranging from 10.8% to 39.7% compared to the control group. In contrast, the tumor volume ratios of controls 4 to 6 compared to the control group were 79.1%, 73.2%, and 67.9%, respectively, indicating that the brain-targeted delivery effect was inferior to that of the MTX@LNPs-1 to MTX@LNPs-45 delivery systems.

[0154] Furthermore, after tail vein injection, the miR-206@LNPs-1 to miR-206@LNPs-45 delivery systems significantly reduced the volume of rat gliomas, with tumor volumes ranging from 18.0% to 63.5% compared to the control group. In contrast, the tumor volume ratios of controls 7-9 to the control group were 69.7%, 72.8%, and 75.8%, respectively, indicating inferior brain-targeted delivery efficacy compared to the miR-206@LNPs-1 to miR-206@LNPs-45 delivery systems. These results suggest that both the MTX@LNPs-1 to MTX@LNPs-45 and miR-206@LNPs-1 to miR-206@LNPs-45 delivery systems exhibit stronger brain-targeted delivery efficacy.

[0155] This indicates that the drug-loaded lipid compositions prepared from dialkylimidazolium biomimetic lipid compounds (compounds 1-45) can effectively cross the blood-brain barrier at the in vivo level and deliver the loaded small molecule chemical drug MTX or small nucleic acid drug miR-206 to brain lesions, significantly inhibiting the growth of glioma.

[0156] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A dialkyl imidazolium biomimetic lipid compound, characterized in that: The structure is shown in Equation 7; Formula 7; Where R' is C 14 ~C 18 Straight-chain alkyl or monoalkenyl, where R'' is C 14 ~C 18 The straight-chain alkyl or monoalkenyl group, where R' and R'' are the same group or different groups; n is an integer from 3 to 9; X - It is an anionic group, specifically I - ,Br - Cl - BF4 - or PF6 - .

2. The dialkyl imidazolium biomimetic lipid compound according to claim 1, characterized in that: R' and R'' can be one or both of the following structures: 。 3. The dialkyl imidazolium biomimetic lipid compound according to claim 1, characterized in that: n is 3, 4, or 5.

4. A method for preparing the dialkylimidazolium biomimetic lipid compound according to any one of claims 1-3, characterized in that: Includes the following steps: Step S01: React the compound shown in Formula 1 with the compound shown in Formula 2 to obtain the intermediate shown in Formula 3. ； Step S02: The intermediate shown in Formula 3 is oxidized to obtain the intermediate shown in Formula 4; ； Step S03: The intermediate shown in Formula 4 is reacted with the compound shown in Formula 5, ammonium acetate and HX to obtain the intermediate shown in Formula 6, wherein PG is an amino protecting group and X is I, Br or Cl. ； Step S04: Remove the protecting group PG from the intermediate shown in Formula 6 to obtain the dialkyl imidazole biomimetic lipid compound shown in Formula 7. 。 5. A lipid nanoparticle, characterized in that: Including the dialkylimidazolium biomimetic lipid compound as described in any one of claims 1-3.

6. A drug-loaded lipid nanoparticle, characterized in that: Includes the dialkylimidazolium biomimetic lipid compound, therapeutic drug, natural phospholipid, and cholesterol as described in any of claims 1-3; Therapeutic drugs are synthetic small molecule chemical drugs or small nucleic acids; synthetic small molecule chemical drugs are one or more of the following: small molecule chemical drugs containing free carboxyl groups, free hydroxyl groups, and free carbonyl groups; small nucleic acids are one or more of the following: siRNA, snRNA, shRNA, saRNA, and miRNA.

7. The method for preparing the drug-loaded lipid nanoparticles according to claim 6, characterized in that: Dialkyl imidazole biomimetic lipid compounds are mixed with natural phospholipids, cholesterol, and therapeutic drugs to prepare lipid nanoparticles in which therapeutic drugs are loaded.

8. The use of the drug-loaded lipid nanoparticles according to claim 6 in the preparation of brain-targeting drugs.

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