Dialkyl imidazole bionic lipid compound as well as preparation method and application thereof

Abstract

The invention relates to a dialkyl imidazole bionic lipid compound and a preparation method and application thereof.The structure of the dialkyl imidazole bionic lipid compound imitates natural phospholipid design, alkyl with no less than 10 carbon atoms is modified on the fourth site and the fifth site of imidazole, and primary amines with different alkyl chain lengths are modified on the second site; the dialkyl imidazole bionic lipid compound is mixed with a therapeutic drug and other auxiliary materials to prepare lipid nanoparticles loaded with the therapeutic drug, and the lipid nanoparticles can be used as a drug delivery system for preparation of brain-targeted drugs. The dialkyl imidazole bionic lipid has the function of dynamically regulating and controlling the blood-brain barrier, and can realize reversible opening of the blood-brain barrier; the formed drug-loaded lipid composition can pass through a blood brain barrier and well realize brain-targeted delivery of loaded therapeutic drugs; the drug-loaded lipid composition composed of the dialkyl imidazole bionic lipid belongs to a new generation of bionic nano-drug carriers, and provides a new strategy for realizing brain-targeted delivery of different types of drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of drug delivery, and particularly relates to a dihydrocarbylimidazole biomimetic lipid compound and a preparation method and application thereof. BACKGROUND

[0002] The blood-brain barrier (BBB) is a highly specialized physiological barrier system between brain tissue and brain capillaries in the central nervous system, mainly composed of brain capillary endothelial cells and their tight junctions, astrocytes, pericytes and basement membrane. The BBB is a selective permeable dynamic interface that selectively allows essential substances such as oxygen and glucose to enter the brain, while preventing the entry of harmful substances in the circulatory system, thereby maintaining the stability of the brain environment. However, 98% of small molecule drugs and nearly 100% of macromolecular drugs are strictly limited by the BBB to enter the brain, resulting in poor efficacy and significant side effects of most drugs for treating brain diseases. Currently, the physical and chemical methods for regulating the BBB in clinical practice mainly include mannitol hyperosmotic mediation, ultrasonic cavitation mediation and electric current perforation mediation. However, these methods cause irreversible damage to the structure and function of the BBB, and generally have serious safety risks.

[0003] Lipid nanoparticles (LNPs) are a class of non-viral nanoscale drug delivery systems based on lipids, which encapsulate drug molecules (such as small molecule drugs, polypeptides, nucleic acids) through a lipid bilayer structure to achieve protection from enzymatic degradation and immune clearance, as well as efficient drug delivery. LNPs are generally formed by self-assembly of permanent cationic or ionizable cationic lipid compounds, auxiliary phospholipids, cholesterol and PEG lipids in an aqueous environment, with a diameter usually ranging from 20-200 nm. The core advantage of LNPs is to protect drugs from degradation and promote cellular uptake and intracellular release. In recent years, LNPs have attracted attention due to their successful application in mRNA vaccines. In addition, due to their excellent biocompatibility, biodegradability, low immunogenicity, flexible structure customization and easy large-scale preparation, LNPs have become the most clinically transformative and promising carrier among various nanomedicine delivery systems.

[0004] Currently, there is no report on LNPs delivery systems that can cross the BBB through clinical verification. Therefore, there is an urgent need for a non-invasive and safe BBB regulation method and a drug delivery system that can efficiently deliver drugs across the BBB into the brain, effectively treating brain tumors, Alzheimer's disease, Parkinson's disease and other brain diseases. SUMMARY

[0005] To solve the above technical problems, the present application provides a dihydrocarbylimidazole biomimetic lipid compound and a preparation method and application thereof.

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

[0007] Formula 7;

[0008] Among them, R' is C9~C 18 A straight-chain saturated or straight-chain unsaturated hydrocarbon group, R'' is C9~C 18 A straight-chain saturated or straight-chain unsaturated hydrocarbon group, R' and R'' are the same group or different groups;

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

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

[0011] Preferably, R' and R'' are C 14 ~C 18 a straight-chain alkyl, monoalkenyl or alkynyl group.

[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] The method for preparing a dialkyl imidazole biomimetic lipid compound comprises the following steps:

[0017] Step S01, reacting the compound represented by Formula 1 with the compound represented by Formula 2 to obtain the intermediate represented by Formula 3;

[0018]

[0019] Step S02, oxidizing the intermediate shown in Formula 3 to obtain the intermediate shown in Formula 4;

[0020]

[0021] Step S03, reacting the intermediate represented by Formula 4 with the compound represented by Formula 5, ammonium acetate, and HX to obtain the intermediate represented by Formula 6, wherein PG is an amino protecting group and X is I, Br, or Cl;

[0022]

[0023] Step S04, removing the protecting group PG of the intermediate shown in Formula 6 to obtain the dialkyl imidazole biomimetic lipid compound shown in Formula 7;

[0024] .

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

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

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

[0028] Preferably, the therapeutic drug is one or more of 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 a small molecule chemical drug containing a free carboxyl group, a free hydroxyl group, and 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 alkaloids, terpenes, flavonoids, glycosides and phenylpropanoid natural products;

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

[0033] The invention discloses a method for preparing drug-loaded lipid nanoparticles, comprising mixing a dialkyl imidazole biomimetic lipid compound with natural phospholipids, cholesterol and therapeutic drugs, and preparing lipid nanoparticles loaded with therapeutic drugs by a thin film hydration method.

[0034] Preferably, the molar ratio of the dialkyl imidazole biomimetic lipid compound to the natural phospholipid, cholesterol and therapeutic drug is (50-75): (5-30): (35-45): (1-15).

[0035] Among them, natural phospholipids are selected from distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dioleoylphosphatidylethanolamine (DOPE), ... One or more of daptomycin, daptomycin, daptomycin (DPSA), daptomycin (DPPA), daptomycin (DMPA), daptomycin (DOPA), distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG), dioleoylphosphatidylglycerol (DOPG), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidylserine (DPPS), dimyristoylphosphatidylserine (DMPS), and dioleoylphosphatidylserine (DOPS).

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

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

[0038] Preferably, the drug-loaded lipid nanoparticle delivery system is able to cross the blood-brain barrier.

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

[0040] Dialkyl imidazole biomimetic lipids represent a new type of biomimetic material that can regulate the degree of openness of the blood-brain barrier. The drug-loaded lipid composition composed of dialkyl imidazole biomimetic lipids belongs to a new generation of biomimetic nanodrug carriers, providing a new strategy for achieving brain-targeted delivery of different types of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram of the structure of the drug-loaded lipid nanoparticle delivery system based on dialkyl imidazole biomimetic lipid compounds;

[0042] Figure 2 is a high resolution mass spectrum (HRMS) diagram of Example 1;

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

[0044] Figure 4 The real-time dynamic change trend of the transmembrane resistance of the in vitro blood-brain barrier model mediated by the lipid nanoparticle delivery system based on some dialkyl imidazole biomimetic lipid compounds in Example 8 and the delivery system in Control Examples 1 to 3; wherein, 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 changes 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 changes mediated by 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 changes mediated by 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 Changes in TEER mediated by the 17-JI / DPPC / Chol. (LNPs-26) delivery system;

[0045] Figure 5 The delivery effects of the drug-loaded lipid nanoparticle delivery system composed of the dialkyl imidazole biomimetic lipid compound in Example 9 and the delivery systems in Control Examples 4 to 6 across the in vitro blood-brain barrier model. DETAILED DESCRIPTION

[0046] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0047] The present invention relates to a dialkyl imidazole biomimetic lipid compound, its preparation method, and application. The 4- and 5-positions of the imidazole are modified with a hydrocarbon group having no fewer than 10 carbon atoms, and the 2-position is modified with a primary amine of varying alkyl chain lengths to form a 2-primary amine-substituted 4,5-dialkyl imidazole. The dialkyl imidazole biomimetic lipid compound has highly biomimetic structural characteristics and low cytotoxicity, enabling the construction of a biomimetic LNPs delivery system for the delivery of various types of small molecule chemical drugs and small nucleic acid drugs.

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

[0049] Formula 7;

[0050] Among them, X - is an anionic group, specifically I - Br - 、Cl - 、BF4 - or PF6 - ; R' and R'' are each independently selected from a chain length of C9~C 18 In certain embodiments of the present invention, R' and R'' are each independently selected from a group having a chain length of C 14 ~C 18 a straight-chain alkyl, monoalkenyl or alkynyl group.

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

[0052] Step S01: reacting the compound represented by Formula 1 with the compound represented by Formula 2 to obtain the intermediate represented by Formula 3;

[0053]

[0054] The compound represented by Formula 1 and the compound represented by Formula 2 are mixed in a solvent A in a molar ratio of 1:1 and fully dissolved, and then an organic base B and an organic catalyst C are added in sequence to carry out a reflux reaction. After completion, the intermediate represented by Formula 3 is obtained by separation; wherein, 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.; 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: oxidizing the intermediate represented by Formula 3 to obtain the intermediate represented by Formula 4;

[0056]

[0057] An oxidant A is added to an organic solvent B containing the intermediate shown in Formula 3, and oxidation is carried out in an oxygen environment. After the reaction is completed, the intermediate shown in Formula 4 is obtained by separation; wherein, the organic solvent B can be acetonitrile (ACN), tetrahydrofuran (THF) or N,N-dimethylformamide (DMF), etc.; the oxidant A can be vanadium trichloride (VOCl3), vanadium pentoxide (V2O5) or pyridinium chlorochromate (PCC), etc.

[0058] Step S03: reacting the intermediate represented by Formula 4 with the compound represented by Formula 5, ammonium acetate, and HX to obtain the intermediate represented by 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 represented by Formula 4, a compound represented by Formula 5, ammonium acetate and a hydrohalic acid are added in sequence for reflux reaction. After completion, the intermediate represented by Formula 6 is obtained by separation; wherein, 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: removing the protecting group PG of the intermediate shown in Formula 6 to obtain the dialkyl imidazole biomimetic lipid compound shown in Formula 7;

[0062]

[0063] A deprotecting agent A is added to an organic solvent D containing the intermediate represented by Formula 6. After the reaction is completed, the compound represented by Formula 7 is obtained by separation; wherein the organic solvent D can be dichloromethane (DCM), dioxane (Diox) or ethanol; and the deprotecting agent A can be trifluoroacetic acid (TFA) or hydrochloric acid.

[0064] Further, unless otherwise defined, all terms (including technical and scientific terms) used in the present application are to be interpreted as is customary in the art. The letters "A, B, C, D, E" and the like appearing in the present application are the designations of reagents or solvents, and are used only to distinguish similar reagents or solvents, and do not indicate or imply relative importance, order of precedence, or the like. In addition, the reagents or solvents represented by the letters "A, B, C, D, E" and the like used in the present application can be interchanged as appropriate. It should be noted that all terms used in the present application are merely used to describe specific embodiments, and are not intended to limit the present application.

[0065] In some embodiments of the present application, in the structure of Formula 7, R' and R" are the same or different groups, and 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 linear alkyl, mono-alkenyl or alkynyl groups with a chain length of C 14 18 In particular, they can be one or both of the following structures:

[0066]

[0067] The carbon chain length of R' and R" can adjust the biomimicry of the dihydroxy imidazole biomimetic lipid compound, thereby affecting the biocompatibility thereof and the stability of the lipid composition composed thereof, and thus improving the drug loading and delivery efficiency thereof.

[0068] In some embodiments of the present application, the dihydroxy imidazole biomimetic lipid compound can be one of the following formulas:

[0069]

[0070] ​Alkylated imidazolium salts (AISs) are a class of biologically active alkylnitrogen heterocyclic azole cations. Monoalkyl imidazolium salts exhibit a certain surface activity and can effectively disrupt bacterial cell membranes, demonstrating significant bactericidal effects. The imidazole ring, as a side chain of histidine, is commonly found in peptides and proteins. Its pKa is approximately 6.0, exhibiting significant buffering properties. Its pH responsiveness within endosomes is superior to that of commercially available ionizable amino cationic lipids (such as ALC-0315), demonstrating excellent endosomal escape and drug delivery potential. Furthermore, the chemical modifiability of the imidazole ring provides ample opportunities for the design of drug delivery carrier materials, potentially improving drug loading efficiency and targeted delivery. The π-π stacking effect of the imidazole ring also helps enhance the encapsulation efficiency of hydrophobic drug molecules, such as aromatic natural products. This invention fuses the parent structure of AISs with the lipid double-tail structure of natural phospholipids to form AISs with biomimetic structural features, namely dialkyl imidazole biomimetic lipid compounds. Due to its biomimetic properties, its biocompatibility with cell membranes is significantly better than that of monoalkyl imidazole salts. Therefore, dialkyl imidazole biomimetic lipid compounds exhibit good biosafety and can be prepared to form a new LNPs biomimetic delivery system.

[0071] Furthermore, the present invention also relates to a drug-loaded lipid composition comprising a dialkylimidazole biomimetic lipid compound represented by Formula 7 or a pharmaceutically acceptable solvate thereof; a therapeutic drug and other auxiliary components. The therapeutic drug is one or more of a synthetic small molecule chemical drug, a natural product drug, a peptide, a protein, and a small nucleic acid. The synthetic small molecule chemical drug is one or more of a small molecule containing a free carboxyl group such as methotrexate, a free hydroxyl group such as acyclovir, and a free carbonyl group such as doxorubicin. The natural product drug is one or more of alkaloids, terpenes, flavonoids, glycosides, and phenylpropanoids. The small nucleic acid is one or more of siRNA, snRNA, shRNA, saRNA, and miRNA. The auxiliary components may be natural phospholipids and cholesterol. Among them, natural phospholipids are selected from distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dioleoylphosphatidylethanolamine (DOPE), ... One or more of daptomycin, daptomycin, daptomycin (DPSA), daptomycin (DPPA), daptomycin (DMPA), daptomycin (DOPA), distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG), dioleoylphosphatidylglycerol (DOPG), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidylserine (DPPS), dimyristoylphosphatidylserine (DMPS), and dioleoylphosphatidylserine (DOPS).

[0072] In the present invention, the term "solvate" refers to a complex formed by the dialkyl imidazole biomimetic lipid compound shown in Formula 7 and a solvent (e.g., ethanol or water). For example, the term "hydrate" refers to the case where the solvent in the above-mentioned term "solvate" is water. Furthermore, the dialkyl imidazole biomimetic lipid compound shown in Formula 7 can be isolated as a solvate, and any solvate is included within the scope of protection of the present invention.

[0073] This biomimetic dialkyl imidazole lipid was combined with natural phospholipids and cholesterol at varying molar ratios in a buffer solution to form biomimetic lipid compositions with diameters ranging from 50 to 500 nm. The biomimetic dialkyl imidazole lipid compound (Formula 7) was mixed with natural phospholipids and cholesterol to prepare lipid nanoparticles (LNPs) using a thin film hydration method. The molar ratio of the biomimetic dialkyl imidazole lipid compound, natural phospholipid, and cholesterol was (50-75):(5-30):(35-45).

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

[0075] Dialkyl imidazole biomimetic lipid compounds, natural phospholipids and cholesterol are added to a mixed solvent of MeOH / CHCl3 (1:1, v / v) in proportion and fully dissolved. After removing the organic solvent, a mixed lipid translucent film is formed on the inner wall of the container. PBS buffer containing therapeutic drugs in an equal volume to the organic mixed solvent is added to the container, incubated at 50°C for 5-10 minutes and vortexed for 1-3 minutes, and repeated 3-10 times to fully hydrate the lipid film and obtain a turbid mixed lipid colloidal solution. After ultrasonic treatment, the colloidal solution is repeatedly extruded 30-40 times using a PC filter membrane with a pore size of 100 nm. The drug-loaded lipid composition obtained after separation and purification is the LNPs drug delivery system based on dialkyl imidazole biomimetic lipid compounds, such as Figure 1 shown.

[0076] In the dialkyl imidazole biomimetic lipid compound, the introduction of a primary amine with a certain alkyl chain length at the 2-position of the imidazole ring can effectively regulate the degree of opening of the blood-brain barrier and achieve reversible opening of the blood-brain barrier. The drug-loaded lipid composition composed of the dialkyl imidazole biomimetic lipid compound can be loaded with synthetic small molecule chemical drugs, natural product drugs, polypeptides, proteins or small nucleic acids, and can cross the blood-brain barrier to achieve brain-targeted drug delivery. When used to prepare a brain-targeted drug-loaded lipid composition, in the compound structure of formula 7, n is preferably 3, 4 or 5.

[0077] The dialkyl imidazole biomimetic lipid compound shown in Formula 7 has significant structural differences compared to representative ionizable cationic lipids in related delivery technologies, such as ALC-0315, Dlin-MC3-DMA, and SM-102, and has highly biomimetic structural characteristics, exhibiting good biosafety and low cytotoxicity. In addition, the dialkyl imidazole biomimetic lipid compound shown in Formula 7 has the function of dynamically regulating the blood-brain barrier and can achieve reversible opening of the blood-brain barrier. By constructing a biomimetic LNPs delivery system using dialkyl imidazole biomimetic lipids with highly biomimetic structural characteristics and low cytotoxicity, it can be used to prepare drug delivery systems and preparations that cross the blood-brain barrier, thereby achieving safe and effective brain-targeted drug delivery across the blood-brain barrier.

[0078] A biomimetic lipid composition composed of dialkylimidazole biomimetic lipids can safely and effectively cross the blood-brain barrier, with significantly better brain-targeted delivery efficiency than LNPs delivery systems composed of representative ionizable cationic lipids such as ALC-0315, Dlin-MC3-DMA, and SM-102. This biomimetic drug-loaded lipid composition represents a new generation of biomimetic nanodrug carriers, potentially improving the delivery efficiency of small molecule drugs and small nucleic acid drugs across the blood-brain barrier.

[0079] The present invention is described below with reference to the accompanying drawings. Experimental methods not specifically described were performed according to the corresponding commercial specifications. Instruments, reagents, and consumables used in the examples were all commercially available unless otherwise specified. It should be noted that, unless otherwise defined, technical or scientific terms used herein shall have the same meanings as those commonly understood by those skilled in the art to which the present invention pertains.

[0080] Example 1: Dialkylimidazole 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-hydroxytriacontane-16-one (I-1)

[0085] 2.0 g (8.32 mmol, 1.0 eq.) of hexadecanal (I-0) was added to 20 mL of anhydrous ethanol and fully dissolved. 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 in sequence. The mixture was refluxed and stirred at 80°C for 3 h. After the reaction was completed by TLC monitoring, the reaction solution was poured into ice water, and the precipitated white solid was collected by filtration and recrystallized from ethanol to obtain a white solid I-1 (900 mg, 1.87 mmol) with a yield of 45.0%.

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

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

[0088] (3) Preparation of 2-[5-(tert-butyloxycarbonylamino)pentyl]-4,5-dipentadecyl-1H-imidazol-3-ium iodide (I-3)

[0089] 70 mg (0.15 mmol, 1.0 eq.) of dotriacontane-16,17-dione (I-2) was dissolved in 2 mL of anhydrous ethanol, followed by the addition of 38 mg of tert-butyloxycarbonyl 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.). The mixture was refluxed and stirred at 90°C for 3 h. After completion of the reaction, the reaction solution was quenched with saturated NaHCO3 (pH>12) and the organic phase was extracted three times with DCM and dried over anhydrous sodium sulfate. The solvent was evaporated, and the crude product was separated and purified by column chromatography (DCM:MeOH=90:10) to obtain I-3 (30 mg, 0.04 mmol) as a light yellow solid in a yield of 30.4%.

[0090] (4) Preparation of 2-(5-aminopentyl)-4,5-dipentadecyl-1H-imidazol-3-ium iodide (A4Z 2 15-JI)

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

[0092] The prepared dialkyl imidazole biomimetic lipid compound A4Z 2 15-JI (compound 1) was characterized, as Figure 2 A4Z shown 2 15-JI high-resolution mass spectrometry (HRMS) [MI] + : C 38 H 76 N3 + , m / z theoretical calculated value: 574.60, measured value: 574.6034. The hydrogen 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 dialkyl imidazole biomimetic lipid compounds 2 to 45

[0095] According to the method of Example 1, dialkyl imidazole biomimetic lipid compounds A3Z were synthesized respectively. 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 synthesis method of the above compounds is similar to that of A4Z 2 The reaction is similar to 15-JI (Compound 1), except that hexadecanal (I-0) in step (1) can be replaced by saturated fatty aldehydes and / or unsaturated fatty aldehydes of different chain lengths; tert-butyloxycarbonyl 6-aminohexanal can be replaced by tert-butyloxycarbonyl 5-aminopentanal or tert-butyloxycarbonyl 7-aminoheptanal in step (3), and hydroiodic acid can be replaced by hydrochloric acid or hydrobromic acid. The raw materials (I-0) used in the above-mentioned dialkyl imidazole biomimetic lipid compounds and the reaction conditions of each step are shown in Table 1.

[0097] Table 1

[0098] Example 3: Preparation of LNPs based on dialkyl imidazole biomimetic lipid compounds

[0099] The A4Z prepared in Example 1 was prepared by thin film hydration method. 2 15-JI (Compound 1) was further prepared to form LNPs.

[0100] A4Z 2 15-JI, natural phospholipid DPPC and cholesterol were added to a mixed solvent of MeOH / CHCl3 (1:1, v / v) in a molar ratio of 70:15:36 and fully dissolved. 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. Thereafter, PBS buffer of equal volume to the organic mixed solvent was added to the container, incubated at 50°C for 5 minutes, and then vortexed for 1-3 minutes to fully hydrate the lipid film. The above hydration process was repeated 5 times to obtain a turbid mixed lipid colloidal solution. After ultrasonic treatment for 30 minutes, the colloidal solution was repeatedly extruded 30-40 times using a PC filter membrane with a pore size of 100 nm to obtain A4Z-based 2 Lipid nanoparticle (LNPs-1) delivery system of 15-JI.

[0101] According to the above method, compounds 2 to 45 were used as raw materials to prepare the corresponding LNPs. 2 15-JI was replaced by the corresponding dialkyl imidazole biomimetic lipid compounds in Table 1, and other conditions remained unchanged to prepare LNPs-2~LNPs-45.

[0102] Comparative Example 1:

[0103] The dialkyl imidazole biomimetic lipid compound A4Z in Example 3 2 15-JI was replaced by 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, while other conditions remained unchanged, and the LNPs of Control Examples 1 to 3 were obtained.

[0104] Example 4: Physicochemical and cytotoxicity characterization of LNPs

[0105] The physicochemical properties and cytotoxicity of LNPs-1 to LNPs-45 prepared in Example 3 and the LNPs of Control Examples 1 to 3 prepared in Comparative Example 1 were characterized respectively.

[0106] 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 figure shows a morphological comparison of LNPs-1 and three control LNPs. All LNPs exhibited relatively complete spherical vesicle structures, with no significant changes observed. LNPs-2 through LNPs-45 exhibited similar structures to LNPs-1, also possessing complete spherical vesicle structures.

[0107] In addition, the average particle size and Zeta potential of LNPs-1 to LNPs-45 and the LNPs of 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.

[0108] Table 2

[0109] As shown in Table 2, the particle sizes of LNPs-1 to LNPs-45 are relatively uniform, 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 comparison, the average particle sizes of the LNPs in Control Examples 1 to 3 were 187.4 nm, 171.0 nm, and 182.1 nm, respectively, and their median zeta potentials were 12.1 mV, 10.8 mV, and 14.8 mV, respectively. Compared to LNPs based on dialkylimidazole biomimetic lipid compounds, the LNPs in the Control Examples had larger particle sizes and lower median zeta potentials.

[0110] The cytotoxicity of LNPs-1 to LNPs-45 and the LNPs in control examples 1 to 3 was further evaluated using the MTT assay on human glioma U-118MG cells. 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. The cells were incubated at 5% CO2 and 37°C for 24 h. Then, different concentrations of various LNPs were added to each well to a final concentration of 1×10 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 / mL were added to each well and incubated for a further 24 hours. Subsequently, 5 mg / mL of MTT reagent was added to each well. After a 4-hour incubation, the culture medium was discarded and 150 μL of DMSO was added to each well. After shaking for 10 minutes, the absorbance of each well was measured at 570 nm using a microplate reader. U-118MG cells without LNPs were used as a negative control to calculate cell viability. If the cell viability was greater than 80%, the LNPs were considered non-cytotoxic.

[0111] The test results are shown in Table 2. No significant cytotoxicity was observed in LNPs-1 to LNPs-45 and the LNPs in control examples 1 to 3.

[0112] Example 5: Construction of a drug-loaded LNPs delivery system based on dialkyl imidazole biomimetic lipid compounds - loading small molecule chemical drugs

[0113] The LNPs-1 to LNPs-45 prepared in Example 3 can be used as a delivery system to load and deliver small molecule drugs. In this example, methotrexate (MTX) was used as the target drug. LNPs-1 to LNPs-45 were used to load MTX and construct the corresponding drug-loaded LNP delivery system.

[0114] MTX, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), and N-hydroxysuccinimide (NHS) were dissolved in DMSO at 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.

[0115] A4Z synthesized in Example 1 215-JI, natural phospholipid DPPC and cholesterol were added to a mixed solvent of MeOH / CHCl3 (1:1, v / v) in a molar ratio of 70:15:36 to fully dissolve. 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 MTX activator (A4Z) diluted with the above PBS buffer was added to the container. 2 The 15-JI to MTX molar ratio was 14:1. The lipid film was fully hydrated by incubation at 50°C for 5 minutes and then vortexed for 1-3 minutes. This hydration process was repeated 10 times to obtain a turbid mixed lipid colloidal solution. After ultrasonic treatment for 30 minutes, the colloidal solution was repeatedly extruded 30-40 times using a 100 nm pore size PC filter membrane. Finally, 0.1 M NaOH was added, and the mixture was incubated at 37°C for 30 minutes. Purification by centrifugal column chromatography yielded the A4Z-based 2 15-JI-loaded MTX-loaded LNPs: MTX@A4Z 2 15-JI·LNPs (MTX@LNPs-1).

[0116] A4Z 2 By replacing 15-JI with the dialkyl imidazole biomimetic lipid compounds listed in Table 1 and keeping other conditions unchanged, MTX-loaded LNPs were obtained: MTX@ LNPs-2~MTX@ LNPs-45.

[0117] Comparative Example 2:

[0118] The dialkyl imidazole biomimetic lipid compound A4Z in Example 5 2 By replacing 15-JI with the cationic lipid compounds ALC-0315, D-Lin-MC3-DMA and SM-102, respectively, and keeping other conditions unchanged, the MTX-loaded LNPs of control examples 4 to 6 were obtained.

[0119] Example 6: Construction of a drug-loaded LNPs delivery system based on dialkyl imidazole biomimetic lipid compounds - loading small nucleic acid drugs

[0120] The LNPs-1 to LNPs-45 prepared in Example 3 can be used as a delivery system to load and deliver small nucleic acid drugs. In this example, miR-206, a small nucleic acid drug, was used as the target drug. LNPs-1 to LNPs-45 were used to load miR-206 and construct a corresponding drug-loaded LNPs delivery system.

[0121] A4Z synthesized in Example 1 215-JI, natural phospholipid DPPC and cholesterol were added to a mixed solvent of MeOH / CHCl3 (1:1, v / v) in a molar ratio of 70:15:36 to fully dissolve. 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, PBS buffer (A4Z) containing small nucleic acid miR-206 was added to the container with an equal volume of the organic mixed solvent. 2 The mass ratio of 15-JI to miR-206 was 9:1. After incubation at 50°C for 5 minutes, the lipid film was vortexed for 1-3 minutes to fully hydrate the lipid film. The above hydration process was repeated 5 times to obtain a turbid mixed lipid colloidal solution. After ultrasonic treatment for 30 minutes, the colloidal solution was repeatedly extruded 30-40 times using a 100 nm pore size PC filter membrane. Finally, it was purified by centrifugal column chromatography to obtain A4Z-based 2 15-JI miR-206-loaded LNPs: miR-206@A4Z 2 15-JI·LNPs (miR-206@LNPs-1).

[0122] A4Z 2 By replacing 15-JI with the dialkyl imidazole biomimetic lipid compounds listed in Table 1 and keeping other conditions unchanged, miR-206-loaded LNPs were obtained: miR-206@LNPs-2~miR-206@LNPs-45.

[0123] Comparative Example 3:

[0124] The dialkyl imidazole biomimetic lipid compound A4Z in Example 6 2 15-JI was replaced by cationic lipid compounds ALC-0315, D-Lin-MC3-DMA and SM-102, respectively, and other conditions remained unchanged to obtain the miR-206-loaded LNPs of Control Examples 7 to 9.

[0125] Example 7: Characterization of the encapsulation efficiency of the drug-loaded LNPs delivery system

[0126] 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 MTX encapsulation efficiency of each group of LNPs was calculated. The results are shown in Table 3.

[0127] miR-206 was labeled with 5-FAM, and 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.

[0128] Table 3

[0129] As shown in Table 3, the MTX encapsulation efficiency in the MTX@LNPs-1 to MTX@LNPs-45 small molecule chemical drug delivery systems ranged from 81.6% to 98.1%. In comparison, the MTX-loaded LNPs prepared in Control Examples 4 to 6 had MTX encapsulation efficiencies of 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 comparison, the miR-206 encapsulation efficiencies of the miR-206-loaded LNPs prepared in control examples 7 to 9 were 78.4%, 79.9%, and 84.5%, respectively, which were much lower than those of miR-206@LNPs-1 to miR-206@LNPs-45.

[0130] Table 3 demonstrates that the dialkyl imidazole biomimetic lipid compounds prepared in Example 1 and Example 2, based on their structural characteristics and advantages, can be used to prepare LNPs delivery systems with better drug loading performance, which can significantly improve the drug encapsulation efficiency; this characteristic is universal and exhibits better drug loading performance for other types of small molecule chemical drugs and small nucleic acid drugs.

[0131] Example 8: Regulation of the in vitro blood-brain barrier model by the LNPs delivery system

[0132] This example characterizes the dynamic regulatory effects of LNPs-1 to LNPs-45 prepared in Example 3 and LNPs of Control Examples 1 to 3 prepared in Comparative Example 1 as delivery systems on an in vitro blood-brain barrier model.

[0133] HCMEC / D3 immortalized human brain microvascular endothelial cells were cultured in ECM medium containing 5% fetal bovine serum and seeded into Transwell chambers at a density of 50,000 cells / well. TM No. 3413 Costar) with collagen-coated polycarbonate membrane (pore size 0.4 µm and membrane growth area 0.33 cm 2) and replace with fresh ECM culture medium containing 5% fetal bovine serum for another 2-3 days. The culture medium was then replaced with serum-free DMEM / F12 medium containing 550 nM hydrocortisone and cultured for another two days in an environment of 5% CO2 and 37°C until the HCMEC / D3 cell monolayer became confluent and formed a barrier model.

[0134] The transmembrane electrical 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 for 1 hour at 5% CO₂ and 37°C. 1.2 mL of serum-free DMEM / F12 medium was then added to each metal chamber of the test module. The Transwell chambers were then transferred to each metal chamber of the test module. The TEER of each Transwell chamber was measured at a rate of one chamber / min at 5% CO₂ and 37°C. The TEER of each chamber was greater than 300 Ω·cm. 2 This indicates that the fused HCMEC / D3 cell monolayer has barrier function, and the in vitro blood-brain barrier model has been constructed, which can be used for dynamic regulation experiments of the blood-brain barrier.

[0135] LNPs-1 to LNPs-45 prepared in Example 3 and 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, and its real-time changes within 24 hours were observed. The maximum amplitude and corresponding time of TEER reduction in each group were recorded, and the results are shown in Table 4. 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 of 18-JI / DPPC / Chol. (LNPs-45) and control examples 1 to 3 are shown in Figure 2. Figure 4 shown.

[0136] Table 4

[0137] As shown in Table 4, the LNPs-1 to LNPs-45 delivery systems were able to mediate a decrease in the TEER of the in vitro blood-brain barrier model constructed with HCMEC / D3 cells, with TEER reductions ranging from 33.8% to 87.2%. In contrast, no significant decrease in TEER was observed after the LNPs delivery systems prepared in Control Examples 1 to 3. The results in Table 4 demonstrate that the LNPs-1 to LNPs-45 delivery systems composed of the dialkylimidazole biomimetic lipid compounds prepared in Examples 1 and 2 have the potential to dynamically regulate the openness of the blood-brain barrier.

[0138] like Figure 4 As shown, it 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), A4Z ij2 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 TEER changes mediated by the 18-JI / DPPC / Chol. (LNPs-45) delivery system. As shown in the figures, the LNPs in each experimental group were able to downregulate the TEER of an in vitro blood-brain barrier model constructed using HCMEC / D3 cells, achieving near-complete restoration of TEER levels within 24 hours. Similar results were observed in the other groups in the LNPs-1 to LNPs-45 delivery systems. This demonstrates that the dialkylimidazole biomimetic lipid compounds (Compounds 1-45) synthesized in Examples 1 and 2 have the potential to mediate reversible opening of the blood-brain barrier.

[0139] Example 9: Delivery effect of drug-loaded LNPs delivery system across the in vitro blood-brain barrier model

[0140] An in vitro blood-brain barrier-glioma co-culture model (in vitro blood-tumor barrier model) consisting of HCMEC / D3 cells and U-118MG cells was established. MTX-loaded LNPs prepared in Examples 4-6, including 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, were selected. The drug-loaded LNPs were tested for their efficacy across the blood-brain barrier in the in vitro blood-brain barrier-glioma co-culture model.

[0141] U-118MG cells were cultured in DMEM medium containing 10% fetal bovine serum, seeded in a 24-well plate at a density of 15,000 cells / well, and cultured for 24 hours. The Transwell chamber containing the in vitro blood-brain barrier model constructed by HCMEC / D3 cells obtained in Example 8 was transferred to each well of the 24-well plate, and the selected groups of drug-loaded LNPs were added to each Transwell chamber. A reference group with the same MTX dose (10 nmol) was also set up, and cultured for 24 hours in an environment of 5% CO2 and 37°C. Untreated U-118MG cells were used as a negative reference, and the cell survival rate of each embodiment and comparative example was calculated by MTT method. The results are shown as follows: Figure 5 shown.

[0142] like Figure 5 As shown in the figure, after the MTX (10 nmol)-loaded 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 were applied to the in vitro blood-tumor barrier model, the survival rates of U-118MG cells ranged from 18% to 66%. In comparison, the survival rates of U-118MG cells in the control samples 4 to 6 were 98%, 95%, and 93%, respectively. This shows that the MTX-loaded LNPs prepared from the above-mentioned dialkyl imidazole biomimetic lipid compound can effectively cross the in vitro blood-brain barrier model, and the MTX delivery efficiency is significantly better than that of control examples 4 to 6.

[0143] Example 10: Brain delivery effect of drug-loaded LNPs delivery system at the in vivo level

[0144] The brain delivery effects of the MTX@LNPs-1~MTX@LNPs-45 and miR-206@LNPs-1~miR-206@LNPs-45 delivery systems prepared in Example 5 and Example 6 were respectively detected at the in vivo level.

[0145] U-118MG cell suspension (1×10 6 After 1-2 weeks of growth, the tumor volume was about 100-150 mm 3The immunodeficient mouse models were randomly divided into three groups, and the MTX@LNPs-1~MTX@LNPs-45 delivery systems and the LNPs loaded with MTX (18 nM) prepared in Comparative Examples 4~6 were injected through the tail vein, and the reference group was injected with the same volume of PBS buffer, with an injection volume of 200 μL and an injection interval of 24 h, for a total of 3 injections. The longest diameter (a) and the shortest diameter (b) of the tumor and the body weight of the mouse were recorded every day, the tumor volume was calculated by the formula V = 1 / 2 × a × b × b, and the tumor volumes of the experimental and reference groups were compared, and the results are shown in Table 5.

[0146] The brain of a Wistar rat with an average mass of about 200 g was implanted with a C6 glioma cell suspension (2×10 6 cells), and after 1 week of growth, a tumor-bearing rat model with a tumor volume of about 100-200 mm 3 was obtained. The rats were randomly divided into three groups, and the miR-206@LNPs-1~miR-206@LNPs-45 delivery systems and the LNPs loaded with miR-206 (50 nM) prepared in Comparative Examples 7~9 were injected through the tail vein, and the reference group was injected with the same volume of PBS buffer, with an injection interval of 72 h, for a total of 3 injections. The longest diameter (a) and the shortest diameter (b) of the tumor and the body weight of the rat were recorded every day, the tumor volume was calculated by the formula V = 1 / 2 × a × b × b, and the tumor volumes of the experimental and reference groups were compared, and the results are shown in Table 5.

[0147] Table 5

[0148] As shown in Table 5, after the MTX@LNPs-1~MTX@LNPs-45 delivery systems were injected through the tail vein, the brain glioma volume of the mouse was significantly reduced, and the tumor volume ratio of the reference group was between 10.8% and 39.7%. In contrast, the tumor volume ratios of Comparative Examples 4~6 and the reference group were 79.1%, 73.2%, and 67.9%, respectively, and the brain-targeted delivery effect was inferior to that of the MTX@LNPs-1~MTX@LNPs-45 delivery system.

[0149] Furthermore, the miR-206@LNPs-1 to miR-206@LNPs-45 delivery systems significantly reduced the volume of rat brain gliomas after tail vein injection, with tumor volume ratios ranging from 18.0% to 63.5% compared to the reference group. In comparison, the tumor volume ratios of control samples 7 to 9 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. Combined with these results, both the MTX@LNPs-1 to MTX@LNPs-45 and miR-206@LNPs-1 to miR-206@LNPs-45 delivery systems exhibited stronger brain-targeted delivery efficacy.

[0150] This shows that the drug-loaded lipid composition prepared by dialkyl imidazole biomimetic lipid compounds (compounds 1~45) can effectively cross the blood-brain barrier at the in vivo level, and better deliver the loaded small molecule chemical drug MTX or small nucleic acid drug miR-206 to brain lesions, significantly inhibiting the growth of brain gliomas.

[0151] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and should not be considered to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A dialkyl imidazole biomimetic lipid compound, characterized in that: The structure is shown in formula 7; Formula 7; Among them, R' is C9~C 18 A straight-chain saturated or straight-chain unsaturated hydrocarbon group, R'' is C9~C 18 A straight-chain saturated or straight-chain unsaturated hydrocarbon group, R' and R'' are the same group or different groups; n is an integer from 3 to 9; X - is an anionic group, specifically I - Br - 、Cl - 、BF4 - or PF6 - .

2. The dialkyl imidazole biomimetic lipid compound according to claim 1, characterized in that: R' and R'' are C 14 ~C 18 a straight-chain alkyl, monoalkenyl or alkynyl group.

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

5. A method for preparing the dialkyl imidazole biomimetic lipid compound according to any one of claims 1 to 4, characterized in that: The steps include: Step S01, reacting the compound represented by Formula 1 with the compound represented by Formula 2 to obtain the intermediate represented by Formula 3; ； Step S02, oxidizing the intermediate shown in Formula 3 to obtain the intermediate shown in Formula 4; ； Step S03, reacting the intermediate represented by Formula 4 with the compound represented by Formula 5, ammonium acetate, and HX to obtain the intermediate represented by Formula 6, wherein PG is an amino protecting group and X is I, Br, or Cl; ； Step S04, removing the protecting group PG of the intermediate shown in Formula 6 to obtain the dialkyl imidazole biomimetic lipid compound shown in Formula 7; 。 6. A lipid nanoparticle, characterized in that: The invention comprises the dialkyl imidazole biomimetic lipid compound described in any one of claims 1 to 4.

7. A drug-loaded lipid nanoparticle, characterized in that: The invention comprises the dialkyl imidazole biomimetic lipid compound described in any one of claims 1 to 4 and a therapeutic drug.

8. The drug-loaded lipid nanoparticle according to claim 7, characterized in that: Therapeutic drugs are one or more of synthetic small molecule chemical drugs, natural product drugs, peptides, proteins and small nucleic acids.

9. The method for preparing the drug-loaded lipid nanoparticles according to claim 7 or 8, characterized in that: The dialkyl imidazole biomimetic lipid compound is mixed with natural phospholipids, cholesterol and therapeutic drugs to prepare lipid nanoparticles with therapeutic drugs loaded therein.

10. Use of the drug-loaded lipid nanoparticles according to claim 7 or 8 in the preparation of brain-targeted drugs.

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