Insoluble pharmaceutical formulations based on dialkylimidazolium biomimetic lipids, their preparation methods and applications

Abstract

This invention relates to an insoluble drug formulation based on dialkylimidazolium biomimetic lipids, its preparation method, and its application, belonging to the field of pharmaceutical formulations. First, an insoluble drug is directly coupled to the primary amine group of the dialkylimidazolium biomimetic lipid or coupled via a linker to form a lipid-drug conjugate. Then, the lipid-drug conjugate, along with other lipid excipients, is dissolved in the dialkylimidazolium biomimetic lipid, and self-assembled in an aqueous phase to form a drug-loaded lipid nanoparticle delivery system with significant drug solubilizing effect. This drug delivery system exhibits good biocompatibility and plasma stability. It can cross the blood-brain barrier and selectively deliver drugs to glioma cells, achieving sustained release of the conjugated insoluble drug in the tumor microenvironment. This provides a new strategy for improving the pharmacokinetic properties of insoluble small-molecule antitumor drugs, including brain targeting, half-life, and bioavailability.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparations, and in particular relates to insoluble pharmaceutical preparations based on dialkyl imidazole biomimetic lipids, their preparation methods and applications. Background Technology

[0002] Malignant gliomas (GBMs) are the most common malignant tumors of the central nervous system, characterized by high invasiveness, heterogeneity, and recurrence rate. Traditional surgical resection combined with radiotherapy and chemotherapy has limited efficacy, resulting in extremely low 5-year survival rates. Targeted glioma formulations refer to drugs that selectively accumulate in glioma tissue and reduce systemic toxicity through specific carriers or modification technologies. Their core objective is to overcome the blood-brain barrier (BBB) ​​and increase the effective drug concentration at the tumor site. In recent years, significant progress has been made in the development of glioma targeted formulations based on insoluble drugs. For example, liposomes loaded with vincristine and paclitaxel nanoparticles modified with transferrin have entered preclinical or early clinical trial stages, and some formulations have significantly improved the survival rate of glioma animal models.

[0003] However, glioma-targeting agents based on insoluble drugs still face several technical challenges: First, the BBB penetration efficiency is limited; even with ligand modification, drug delivery systems loaded with insoluble drugs struggle to effectively cross the BBB, resulting in low drug concentrations at the lesion site. Second, there is an imbalance between drug stability and release controllability; some carrier materials degrade too quickly, leading to drug leakage and premature exposure in the body for enzymatic metabolism, inducing systemic toxicity, while delayed release by the carrier affects drug efficacy. These problems severely restrict the clinical translation of small-molecule brain-targeting agents and urgently require targeted drug carrier and drug delivery technologies to address. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides an insoluble pharmaceutical formulation based on dialkyl imidazolium biomimetic lipids, its preparation method, and its application.

[0005] The technical solution adopted in this invention is: a lipid nanoparticle based on dialkylimidazolium biomimetic lipid loading insoluble drugs, comprising dialkylimidazolium biomimetic lipid-drug conjugate and dialkylimidazolium biomimetic lipid.

[0006] Preferably, the drug is a small molecule antitumor drug with a solubility of less than 0.1 mg / mL; the small molecule antitumor drug is a natural product drug or a synthetic drug.

[0007] Preferably, the insoluble natural product drug is paclitaxel or vincristine; the insoluble synthetic drug is pemetrexed or pazopanib.

[0008] Preferably, it also includes phospholipids and cholesterol.

[0009] Preferably, the phospholipid is one or more of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearate phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC).

[0010] Preferably, the structure of the dialkyl imidazolium biomimetic lipid is shown in general formula 1;

[0011] General Formula 1;

[0012] Where R' is C9~C 18 A straight-chain saturated or unsaturated hydrocarbon group, where R'' is C9~C9. 18 The straight-chain saturated or unsaturated hydrocarbon group, where R' and R'' are the same or different groups; n is an integer from 3 to 9; X - It is an anionic group, specifically I - ,Br - Cl - BF4 - or PF6 - .

[0013] This invention also provides a method for preparing lipid nanoparticles loaded with insoluble drugs based on dialkyl imidazolium biomimetic lipids, comprising the following steps:

[0014] Step S01: Mix the dialkylimidazolium biomimetic lipid with an insoluble drug or an insoluble drug linked with a linker, and add a coupling adjuvant to form a dialkylimidazolium biomimetic lipid-drug conjugate.

[0015] Step S02: Preheat the dialkyl imidazolium biomimetic lipid to form a pure liquid;

[0016] Step S03: Add the dialkylimidazolium biomimetic lipid-drug conjugate, phospholipids and cholesterol obtained in step S01 to the liquid dialkylimidazolium biomimetic lipid obtained in step S02 and dissolve it completely to form a drug-loaded mixture;

[0017] Step S04: Add the drug-loaded mixture to the buffer solution to form a drug-loaded emulsion;

[0018] Step S05: The drug-loaded emulsion is treated with ultrasound and then sterilized by passing it through a filter membrane to obtain lipid nanoparticles loaded with insoluble drugs.

[0019] Preferably, the preheating temperature in step S02 is 30~35℃.

[0020] Preferably, in step S03, the molar ratio of the dialkylimidazolium biomimetic lipid-drug conjugate, the dialkylimidazolium biomimetic lipid, the phospholipid, and the cholesterol in the drug-loaded mixture is (15~30):(60~90):(10~45):(25~45).

[0021] Preferably, in step S04, the drug-loaded mixture is injected into the buffer solution at a rate of 50-120 μL / s at 45-60°C.

[0022] Preferably, the buffer solution is a phosphate buffer, a sodium acetate buffer, or a citrate buffer.

[0023] The present invention also provides a brain-targeted sustained-release formulation comprising lipid nanoparticles based on dialkyl imidazole biomimetic lipids loaded with insoluble drugs.

[0024] Preferably, it also includes pharmaceutically acceptable excipients.

[0025] This invention provides the application of lipid nanoparticles loaded with insoluble drugs based on dialkylimidazolium biomimetic lipids, brain-targeted sustained-release formulations, and methods for preparing lipid nanoparticles loaded with insoluble drugs based on dialkylimidazolium biomimetic lipids in the preparation of drugs for treating glioma.

[0026] The advantages and positive effects of this invention are as follows: The insoluble drug brain-targeting sustained-release formulation provided by this invention comprises brain-targeting sustained-release lipid nanoparticles. These brain-targeting sustained-release lipid nanoparticles are formed by encapsulating a lipid-drug conjugate with lipid nanoparticles formed by the self-assembly of dialkylimidazolium biomimetic lipids, phospholipids, and cholesterol. In this lipid-drug conjugate, the insoluble small-molecule antitumor drug is directly coupled to the primary amine group of the dialkylimidazolium biomimetic lipid via a covalent bond, or coupled to the primary amine group of the dialkylimidazolium biomimetic lipid via a linker. The lipid-drug conjugate is further dissolved with other lipids in a pure liquid dialkylimidazolium biomimetic lipid, and then self-assembled in a buffer solution to form drug-loaded lipid nanoparticles, which can significantly improve the solubility of insoluble small molecule antitumor drugs. Furthermore, the dialkylimidazolium biomimetic lipid in these brain-targeting sustained-release lipid nanoparticles can enhance the delivery efficiency of the loaded small molecule antitumor drugs across the blood-brain barrier by regulating the blood-brain barrier. More significantly, the dialkylimidazolium biomimetic lipid in these brain-targeting sustained-release lipid nanoparticles can escape from the endosomes in the glioma microenvironment through the "proton sponge effect," releasing the encapsulated lipid-drug conjugate. In the lysosomes and cytoplasm, the protease and esterase systems gradually hydrolyze the covalent bonds in the lipid-drug conjugate, achieving sustained release of the conjugated small molecule antitumor drug. Therefore, this invention not only improves the brain targeting and brain tissue distribution of insoluble small molecule antitumor drugs, but also improves their half-life and bioavailability, prolongs the drug's duration of action, reduces drug toxicity, and effectively kills glioma cells. The brain-targeted sustained-release lipid nanoparticles exhibit significantly better brain-targeted delivery performance than the LNPs delivery system composed of the representative ionizable cationic lipid Dlin-MC3-DMA (CAS:1224606-06-7) currently used in the drug delivery field. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the structure of insoluble drug-targeted sustained-release lipid nanoparticles based on dialkyl imidazole biomimetic lipids.

[0028] Figure 2 This is a transmission electron microscope (TEM) image of insoluble drug-targeting sustained-release lipid nanoparticles based on dialkyl imidazolium biomimetic lipids. Among them, Figure 2 A represents lipid nanoparticles PTX@A4Z loaded with paclitaxel (PTX). 2 15-JI / DMPC / Chol.; Figure 2 B represents lipid nanoparticles VCR@A5Z loaded with vincristine (VCR). 2 18-JI / DMPC / Chol.; Figure 2 C represents lipid nanoparticles PMX@A4Z loaded with pemetrexed (PMX). 2 15-JI / DMPC / Chol.; Figure 2 D represents lipid nanoparticles PAZ@A4Z loaded with pazopanib (PAZ). 2 15-JI / DMPC / Chol.;

[0029] Figure 3 This is a dynamic light scattering (DLS) image of insoluble drug-targeting sustained-release lipid nanoparticles based on dialkyl imidazole biomimetic lipids. Among them, Figure 3 A represents lipid nanoparticles PTX@A4Z loaded with paclitaxel (PTX). 2 15-JI / DMPC / Chol.; Figure 3 B represents lipid nanoparticles VCR@A5Z loaded with vincristine (VCR). 2 18-JI / DMPC / Chol.; Figure 3 C represents lipid nanoparticles PMX@A4Z loaded with pemetrexed (PMX). 2 15-JI / DMPC / Chol.; Figure 3 D represents lipid nanoparticles PAZ@A4Z loaded with pazopanib (PAZ). 2 15-JI / DMPC / Chol.;

[0030] Figure 4 This shows the drug-time curves of insoluble drug-targeting sustained-release lipid nanoparticles based on dialkyl imidazole biomimetic lipids administered via tail vein in a rat model within 24 hours. Figure 4 A represents lipid nanoparticles PTX@A4Z loaded with paclitaxel (PTX). 2 15-JI / DMPC / Chol. and PTX technical grade injection; Figure 4 B represents lipid nanoparticles VCR@A5Z loaded with vincristine (VCR). 2 18-JI / DMPC / Chol. and VCR original drug injection; Figure 4 C represents lipid nanoparticles PMX@A4Z loaded with pemetrexed (PMX). 2 15-JI / DMPC / Chol. and PMX technical grade injection; Figure 4 D represents lipid nanoparticles PAZ@A4Z loaded with pazopanib (PAZ). 2 15-JI / DMPC / Chol. and PAZ technical grade injection;

[0031] Figure 5 PTX@A4Z lipid nanoparticles loaded with paclitaxel (PTX) for use in mouse models 2The graphs show the brain tissue-to-plasma concentration ratio (B / P) and tumor weight changes at different time points after the delivery of the lipid nanoparticles PTX@MC3 / DMPC / Chol. in Comparative Example 1 and the PTX technical grade injection across the blood-brain barrier. Figure 5 A is a B / P relationship diagram; Figure 5 B is a graph showing the relationship between tumor weight changes;

[0032] Figure 6 Lipid nanoparticles VCR@A5Z loaded with vincristine (VCR) in mouse models 2 The graphs show the brain tissue-to-plasma concentration ratio (B / P) and tumor weight changes at different time points after the lipid nanoparticles VCR@MC3 / DMPC / Chol. in Comparative Example 2 and the VCR original drug injection were delivered across the blood-brain barrier. Figure 6 A is a B / P relationship diagram; Figure 6 B is a graph showing the relationship between tumor weight changes;

[0033] Figure 7 Lipid nanoparticles PMX@A4Z loaded with pemetrexed (PMX) for use in mouse models 2 The graphs show the brain tissue-to-plasma concentration ratio (B / P) and tumor weight changes at different time points after drug delivery across the blood-brain barrier in 15-JI / DMPC / Chol., Comparative Example 3, and the lipid nanoparticles PMX@MC3 / DMPC / Chol. and PMX technical grade injection. Figure 7 A is a B / P relationship diagram; Figure 7 B is a graph showing the relationship between tumor weight changes;

[0034] Figure 8 Lipid nanoparticles PAZ@A4Z loaded with pazopanib (PAZ) for use in mouse models 2 The graphs show the brain tissue-to-plasma concentration ratio (B / P) and tumor weight changes at different time points after drug delivery across the blood-brain barrier using lipid nanoparticles PAZ@MC3 / DMPC / Chol. (Comparative Example 4) and PAZ technical grade injection. Figure 8 A is a B / P relationship diagram; Figure 8 B is a graph showing the relationship between changes in tumor weight. Detailed Implementation

[0035] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0036] This invention relates to an insoluble drug formulation based on dialkylimidazolium biomimetic lipids, its preparation method, and its application. The insoluble small molecule drug is coupled with dialkylimidazolium biomimetic lipids, and then dissolved with other lipids in pure liquid dialkylimidazolium biomimetic lipids. The mixture is then self-assembled in a buffer solution to prepare lipid nanoparticles loaded with insoluble drugs. This enables the solubilization, brain-targeted delivery, and sustained release of insoluble small molecule antitumor drugs in the tumor microenvironment.

[0037] Bio-mimetic dialkylimidazolium lipids (BDILs) are a class of imidazole-based cationic lipid compounds with biomimetic structural features. The hydrophilic head of BDILs consists of an imidazole ring and a substituent at position 2, while the hydrophobic tail consists of flexible lipid chains linked at positions 4 and 5 of the imidazole ring. Their structure is designed to mimic natural phospholipids, exhibiting both amphiphilicity and good biocompatibility. Furthermore, the introduction of primary amine groups of varying alkyl chain lengths at position 2 of the imidazole ring enables the mediating of temporary opening of the BBB tight junction through interactions with BBB tight junction functional proteins, thereby enhancing drug delivery efficiency across the BBB to target brain tissue.

[0038] The structure of the dialkyl imidazolium biomimetic lipid is shown in general formula 1;

[0039] General Formula 1;

[0040] Among them, R' and R'' are each independently selected from chains with lengths C9 to C1. 18 The groups are straight-chain saturated or unsaturated hydrocarbon groups, where R' and R'' are the same or different groups. In some embodiments of the present invention, R' and R'' are each independently selected from C. 14 ~C 18 A straight-chain alkyl or monoalkenyl group; preferably, R' and R'' are one or both of the following structures:

[0041]

[0042] The carbon chain length and unsaturation of R' and R'' can modulate the biocompatibility and self-assembly polymorphism of dialkylimidazolium biomimetic lipids, thereby affecting the stability of the lipid nanoparticles and their drug loading and delivery efficiency.

[0043] In general formula 1, n is an integer from 3 to 9; n is preferably 4 or 5. X - It is an anionic group, specifically I - ,Br - Cl - BF4 - or PF6 - ;X - Preferably I -or Cl - .

[0044] In some embodiments of the present invention, the above-mentioned dialkyl imidazolium biomimetic lipid is one of the following compounds:

[0045]

[0046] The insoluble drug is an insoluble small-molecule antitumor drug with a solubility in water of <0.1 mg / mL. The insoluble small-molecule antitumor drug includes insoluble natural product drugs or insoluble synthetic drugs. In some embodiments of this invention, the insoluble natural product drug is paclitaxel or vincristine; the insoluble synthetic drug is pemetrexed or pazopanib. Based on the biomimetic nature of BDILs and their "on / off" regulation of the blood-brain barrier (BBB), drug carriers composed of BDILs, natural phospholipids, and lipid excipients such as cholesterol are expected to overcome a series of technical bottlenecks, such as the poor blood-brain barrier permeability and brain-targeted delivery efficiency of insoluble drugs, and effectively treat malignant gliomas.

[0047] The preparation method for the above-mentioned lipid nanoparticles based on dialkyl imidazolium biomimetic lipids loaded with insoluble drugs is as follows:

[0048] Step S01: The dialkylimidazolium biomimetic lipid is mixed with an insoluble drug or an insoluble drug linked with a linker (a linker derivative of the insoluble drug) in a certain proportion, and a coupling adjuvant is added to complete the coupling of the insoluble drug with the dialkylimidazolium biomimetic lipid, forming a dialkylimidazolium biomimetic lipid-drug conjugate. The linker derivative of the insoluble drug is a carboxylic acid derivative, phosphate derivative, primary alcohol derivative, thiol derivative, or aldehyde (ketone) derivative with a certain degree of lipid solubility. The linker derivative of the insoluble drug is preferably a carboxylic acid derivative or a primary alcohol derivative; the coupling adjuvant includes an activator and / or a catalyst, the activator being carbodiimide or carbonyl diimidazole, preferably dicyclohexylcarbodiimide (DCC) or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC); the catalyst is an organic base, preferably triethylamine (TEA), 4-dimethylaminopyridine (DMAP), or diisopropylethylamine (DIPEA).

[0049] Step S02: The dialkylimidazolium biomimetic lipid is preheated at a certain temperature to form a pure liquid. The preheating temperature of the dialkylimidazolium biomimetic lipid is 30~35℃, preferably 35℃.

[0050] Step S03: The dialkylimidazolium biomimetic lipid-drug conjugate, phospholipids, and cholesterol obtained in step S01 are added to the liquid dialkylimidazolium biomimetic lipid obtained in step S02 in a certain proportion and fully dissolved to form a drug-loaded mixture. The phospholipid is one or more of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearate phosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), preferably DMPC; the molar ratio of the dialkylimidazolium biomimetic lipid-drug conjugate, dialkylimidazolium biomimetic lipid, phospholipids, and cholesterol in the drug-loaded mixture is (15~30):(60~90):(10~45):(25~45), preferably 25:85:15:30.

[0051] Step S04: The drug-loaded mixture obtained in step S03 is added to a buffer solution at a certain rate and temperature to form a drug-loaded emulsion. The drug-loaded mixture is injected at a rate of 50-120 μL / s, preferably 80 μL / s; the temperature is controlled at 45-60℃, preferably 50℃; the buffer solution is phosphate buffer, sodium acetate buffer, or citrate buffer, preferably phosphate buffer.

[0052] Step S05: The drug-loaded emulsion obtained in step S04 is ultrasonically treated and then sterilized by passing it through a filter membrane with uniform pore size. The preferred pore size of the sterilization filter membrane is 0.22 μm, resulting in lipid nanoparticles loaded with insoluble drugs. A schematic diagram of their structure is shown below. Figure 1 As shown.

[0053] Dialkyl imidazole biomimetic lipids (BDILs) are designed to mimic the structure of natural phospholipids. They feature a hydrophilic head with a 2-amino-substituted imidazole ring and a hydrophobic tail with two flexible lipid chains, classifying them as novel, highly biomimetic ionizable cationic lipids. Therefore, lipid nanoparticles formed by the self-assembly of BDILs with phospholipids and cholesterol exhibit good biocompatibility with the lipid bilayer of biological membranes, showing no significant cytotoxicity and demonstrating excellent biosafety. Furthermore, BDILs belong to the imidazole ionic liquid category, thus possessing good solvent properties and can be used to dissolve structurally similar or lipid-soluble drug molecules and their derivatives.

[0054] The primary amine group at the hydrophilic head of BDILs can condense with the carboxyl or carbonyl groups of insoluble drugs to form amide bonds or Schiff bases, forming lipid-drug conjugates (LDCs). BDILs can also be coupled to insoluble drugs via linkers to form LDCs. LDCs coupled to BDILs via amide bonds can be hydrolyzed by proteases in the tumor microenvironment, releasing the original insoluble drug; LDCs coupled to BDILs via Schiff bases can be hydrolyzed in a weakly acidic tumor microenvironment, releasing the original insoluble drug; LDCs coupled to BDILs via carbamates can be hydrolyzed by acid and enzymatic reactions in the tumor microenvironment, releasing the original insoluble drug. Therefore, LDCs not only enable controlled loading of insoluble drugs, significantly improving their solubility, but also allow for gradual degradation in the tumor microenvironment via acid and / or enzymatic responses, achieving sustained release of insoluble drugs, thereby improving pharmacokinetic aspects such as half-life and bioavailability.

[0055] Furthermore, the primary amino group at the 2-position of the imidazole ring in BDILs can effectively regulate the opening and closing of the blood-brain barrier. Therefore, drug-loaded lipid nanoparticles composed of BDILs can cross the blood-brain barrier, improving the permeability of insoluble drugs to the blood-brain barrier and their distribution in brain tissue. Once inside the brain, the drug-loaded lipid nanoparticles composed of BDILs are taken up in large quantities by glioma cells due to the high permeability and retention effect (EPR effect) of the blood-brain barrier to glioma tissue, entering the tumor microenvironment. In endosome (pH 5.0–6.5) and lysosomal (pH 4.5–5.0) environments, BDILs, being divalent cationic lipids, will trigger the "proton sponge effect," releasing encapsulated LDCs. The LDCs will further trigger acid and / or enzyme responses in the tumor microenvironment, gradually degrading and releasing the insoluble drug to exert its therapeutic effect. Therefore, drug-loaded lipid nanoparticles encapsulating LDCs with BDILs can not only effectively cross the blood-brain barrier and improve the brain-targeted delivery efficiency of insoluble drugs, but also prolong the drug's duration of action, reduce drug toxicity, and effectively kill glioma cells. In this invention, the brain-targeted delivery efficiency of insoluble drug brain-targeted sustained-release lipid nanoparticles based on dialkyl imidazole biomimetic lipids is significantly superior to that of representative ionizable cationic lipid LNPs delivery systems such as Dlin-MC3-DMA.

[0056] 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 instructions. Unless otherwise specified, the instruments, reagents, and consumables used in the examples 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. The structural characteristics and preparation method of the dialkylimidazolium biomimetic lipid involved in this invention are disclosed in patent CN120757505B.

[0057] Example 1: Preparation of a brain-targeted sustained-release formulation of paclitaxel based on dialkyl imidazolium biomimetic lipids

[0058] This embodiment uses the insoluble natural product paclitaxel (PTX, CAS: 33069-62-4) as the target drug, and utilizes the dialkyl imidazole biomimetic lipid A4Z. 2 15-JI (A0), A4Z ij 2 15-JCl(A1), A4Z 2 18-JI (A18), A5Z 2 18-JI (A19) and A5Z ij 2 18-JCl (A22) was combined with natural phospholipid DMPC and cholesterol (Chol.) to form brain-targeted sustained-release lipid nanoparticles loaded with PTX, thus forming a PTX brain-targeted sustained-release formulation.

[0059] First, select the dialkyl imidazole biomimetic lipid A4Z. 2 15-JI (A0), natural phospholipid DMPC, and cholesterol (Chol.) are the three components constituting lipid nanoparticles. The specific preparation method of PTX-loaded brain-targeted sustained-release lipid nanoparticles is as follows:

[0060] The C2'-hydroxyl group of PTX was modified with 8-hydroxyoctanoic acid linker (CAS: 764-89-6) to obtain a primary alcohol derivative of PTX (PTX-C8-OH). PTX-C8-OH was then reacted with the activator carbonyl diimidazole (CDI, CAS: 530-62-1) and A4Z. 2 15-JI (A0) was fully dissolved in anhydrous tetrahydrofuran (THF) at a molar ratio of 1:1.3:1 and reacted at 25°C for ~12 h, A4Z 2 15-JI(A0) is coupled with PTX-C8-OH via carbamate to form A4Z. 2 The coupling compound A0-C8-PTX of 15-JI(A0) and PTX-C8-OH. Then A4Z... 215-JI (A0) was preheated to 35°C to become a pure liquid. A0-C8-PTX, A0, DMPC, and Chol were then thoroughly mixed and dissolved in a molar ratio of 25:85:15:30 to form a drug-loaded mixture. Using a glass syringe, the drug-loaded mixture was steadily injected into PBS buffer (pH 7.4) at a constant rate of 80 μL / s, and stirred at 50°C for 10–15 min to form a drug-loaded emulsion. Finally, after sonication, the emulsion was sterilized using a 0.22 μm filter membrane to obtain PTX@A4Z brain-targeted sustained-release lipid nanoparticles loaded with PTX. 2 15-JI / DMPC / Chol., further formulated with pharmaceutically acceptable excipients to contain PTX@A4Z 2 A brain-targeted sustained-release formulation of PTX from 15-JI / DMPC / Chol.

[0061] A4Z 2 Replace 15-JI (A0) with A4Z respectively. ij 2 15-JCl(A1), A4Z 2 18-JI (A18), A5Z 2 18-JI (A19) and A5Z ij 2 By using 18-JCl(A22) and keeping other conditions unchanged, lipid nanoparticles PTX@A4Z loaded with PTX can be obtained. ij 2 15-JCl / DMPC / Chol.、PTX@A4Z 2 18-JI / DMPC / Chol.、PTX@A5Z 2 18-JI / DMPC / Chol. with PTX@A5Z ij 2 18-JCl / DMPC / Chol. and PTX brain-targeted sustained-release formulations containing the above-mentioned lipid nanoparticles.

[0062] Comparative Example 1:

[0063] A4Z in Example 1 2 15-JI was replaced with the cationic lipid compound Dlin-MC3-DMA (MC3). MC3, DMPC and Chol were mixed in a molar ratio of 85:15:30 and lipid nanoparticles loaded with PTX, PTX@MC3 / DMPC / Chol, were prepared by thin-film hydration.

[0064] Example 2: Preparation of a vincristine brain-targeted sustained-release formulation based on dialkyl imidazolium biomimetic lipids

[0065] This embodiment uses the insoluble natural product vincristine (VCR, CAS: 57-22-7) as the target drug, and utilizes the dialkyl imidazole biomimetic lipid A4Z. 2 15-JI (A0), A4Z ij 2 15-JCl(A1), A4Z 2 18-JI (A18), A5Z 2 18-JI (A19) and A5Z ij 2 18-JCl (A22) was combined with natural phospholipid DMPC and cholesterol (Chol.) to form brain-targeted sustained-release lipid nanoparticles loaded with VCR, thus forming a VCR brain-targeted sustained-release formulation.

[0066] First, select the dialkyl imidazole biomimetic lipid A4Z. 2 15-JI (A0), natural phospholipid DMPC, and cholesterol (Chol.) are the three components constituting lipid nanoparticles. The specific preparation method of brain-targeted sustained-release lipid nanoparticles loaded with VCRs is as follows:

[0067] Connect VCR and A4Z 2 15-JI (A0) and the catalyst triethylamine (TEA) were fully dissolved in anhydrous tetrahydrofuran (THF) at a molar ratio of 1:1.3:0.3, and the mixture was refluxed at 60°C for 12–24 h. A4Z 2 15-JI(A0) is coupled to VCR via Schiff base coupling to form A4Z. 2 The coupling compound A0-VCR of 15-JI (A0) and VCR. Then A4Z... 2 15-JI (A0) was preheated to 35°C to become a pure liquid. A0-VCR, A0, DMPC, and Chol were then thoroughly mixed and dissolved in a molar ratio of 25:85:15:30 to form a drug-loaded mixture. Using a glass syringe, the drug-loaded mixture was steadily injected into PBS buffer (pH 7.4) at a constant rate of 80 μL / s, and stirred at 50°C for 10–15 min to form a drug-loaded emulsion. Finally, after sonication, the mixture was sterilized using a 0.22 μm filter membrane to obtain brain-targeted sustained-release lipid nanoparticles VCR@A4Z loaded with VCR. 2 15-JI / DMPC / Chol., further formulated with pharmaceutically acceptable excipients to contain VCR@A4Z 2 VCR brain-targeted sustained-release formulation of 15-JI / DMPC / Chol.

[0068] A4Z 2 Replace 15-JI (A0) with A4Z respectively. ij 215-JCl(A1), A4Z 2 18-JI (A18), A5Z 2 18-JI (A19) and A5Z ij 2 By using 18-JCl(A22) and keeping other conditions unchanged, lipid nanoparticles VCR@A4Z loaded with VCR can be obtained. ij 2 15-JCl / DMPC / Chol.、VCR@A4Z 2 18-JI / DMPC / Chol.、VCR@A5Z 2 18-JI / DMPC / Chol. with VCR@A5Z ij 2 18-JCl / DMPC / Chol. and VCR brain-targeted sustained-release formulations containing the above-mentioned lipid nanoparticles.

[0069] Comparative Example 2:

[0070] A4Z in Example 2 2 15-JI was replaced with the cationic lipid compound Dlin-MC3-DMA (MC3). MC3, DMPC and Chol were mixed in a molar ratio of 85:15:30 and lipid nanoparticles VCR@MC3 / DMPC / Chol were prepared by thin-film hydration.

[0071] Example 3: Preparation of a brain-targeted sustained-release formulation of pemetrexed based on dialkyl imidazole biomimetic lipids

[0072] This embodiment uses the insoluble synthetic drug pemetrexed (PMX, CAS: 137281-23-3) as the target drug, and utilizes the dialkyl imidazole biomimetic lipid A4Z. 2 15-JI (A0), A4Z ij 2 15-JCl(A1), A4Z 2 18-JI (A18), A5Z 2 18-JI (A19) and A5Z ij 2 18-JCl (A22) was combined with natural phospholipid DMPC and cholesterol (Chol.) to form PMX-loaded brain-targeted sustained-release lipid nanoparticles, thus forming a PMX brain-targeted sustained-release formulation.

[0073] First, select the dialkyl imidazole biomimetic lipid A4Z. 2 15-JI (A0), natural phospholipid DMPC, and cholesterol (Chol.) are the three components constituting lipid nanoparticles. The specific preparation method of PMX-loaded brain-targeted sustained-release lipid nanoparticles is as follows:

[0074] PMX and A4Z 2 15-JI (A0), activator 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and catalyst 4-dimethylaminopyridine (DMAP) were fully dissolved in dichloromethane in a molar ratio of 1:1.1:1:0.1 and reacted at 30°C for 5-7 h. A4Z 2 15-JI(A0) is coupled with PMX via an amide bond to form A4Z. 2 The conjugate A0-PMX of 15-JI (A0) and PMX. Then A4Z... 2 15-JI (A0) was preheated to 35°C to become a pure liquid. A0-PMX, A0, DMPC, and Chol were then thoroughly mixed and dissolved in a molar ratio of 25:85:15:30 to form a drug-loaded mixture. Using a glass syringe, the drug-loaded mixture was steadily injected into PBS buffer (pH 7.4) at a constant rate of 80 μL / s, and stirred at 50°C for 10–15 min to form a drug-loaded emulsion. Finally, after sonication, the emulsion was sterilized using a 0.22 μm filter membrane to obtain PMX@A4Z brain-targeted sustained-release lipid nanoparticles loaded with PMX. 2 15-JI / DMPC / Chol., further formulated with pharmaceutically acceptable excipients containing PMX@A4Z 2 PMX brain-targeted sustained-release formulation of 15-JI / DMPC / Chol.

[0075] A4Z 2 Replace 15-JI (A0) with A4Z respectively. ij 2 15-JCl(A1), A4Z 2 18-JI (A18), A5Z 2 18-JI (A19) and A5Z ij 2 By using 18-JCl(A22) and keeping other conditions unchanged, lipid nanoparticles loaded with PMX, PMX@A4Z, can be obtained. ij 2 15-JCl / DMPC / Chol.、PMX@A4Z 2 18-JI / DMPC / Chol.、PMX@A5Z 2 18-JI / DMPC / Chol. with PMX@A5Z ij 2 18-JCl / DMPC / Chol. and PMX brain-targeted sustained-release formulations containing the above-mentioned lipid nanoparticles.

[0076] Comparative Example 3:

[0077] A4Z in Example 3 2 15-JI was replaced with the cationic lipid compound Dlin-MC3-DMA (MC3). MC3, DMPC and Chol were mixed in a molar ratio of 85:15:30 and lipid nanoparticles loaded with PMX, PMX@MC3 / DMPC / Chol, were prepared by thin-film hydration.

[0078] Example 4: Preparation of Pazopanib Brain-Targeted Sustained-Release Formulation Based on Dialkylimidazolium Biomimetic Lipids

[0079] This embodiment uses the insoluble synthetic drug pazopanib (PAZ, CAS: 444731-52-6) as the target drug, and utilizes the dialkyl imidazole biomimetic lipid A4Z. 2 15-JI (A0), A4Z ij 2 15-JCl(A1), A4Z 2 18-JI (A18), A5Z 2 18-JI (A19) and A5Z ij 2 18-JCl (A22) was combined with natural phospholipid DMPC and cholesterol (Chol.) to form PAZ-loaded brain-targeted sustained-release lipid nanoparticles, thus forming a PAZ brain-targeted sustained-release formulation.

[0080] First, select the dialkyl imidazole biomimetic lipid A4Z. 2 15-JI (A0), natural phospholipid DMPC, and cholesterol (Chol.) are the three components constituting lipid nanoparticles. The specific preparation method of brain-targeted sustained-release lipid nanoparticles loaded with PAZ is as follows:

[0081] PAZ was modified with benzenesulfonamide via succinic acid semialdehyde Linker (CAS: 692-29-5) to obtain a carboxylic acid derivative of PAZ (PAZ-C3-COOH). PAZ-C3-COOH was then reacted with A4Z. 2 15-JI (A0), EDC, and DMAP were fully dissolved in dichloromethane at a molar ratio of 1:1.5:1:0.1 and reacted at 30°C for 2-5 hours. A4Z 2 15-JI(A0) is coupled with PAZ-C3-COOH via an amide bond to form A4Z. 2 The coupling compound A0-C4-PAZ of 15-JI (A0) and PAZ-C3-COOH. Subsequently, A4Z... 215-JI (A0) was preheated to 35°C to become a pure liquid. A0-C4-PAZ, A0, DMPC, and Chol were then thoroughly mixed and dissolved in a molar ratio of 25:85:15:30 to form a drug-loaded mixture. Using a glass syringe, the drug-loaded mixture was steadily injected into PBS buffer (pH 7.4) at a constant rate of 80 μL / s, and stirred at 50°C for 10–15 min to form a drug-loaded emulsion. Finally, after sonication, the mixture was sterilized using a 0.22 μm filter membrane to obtain PAZ@A4Z brain-targeting sustained-release lipid nanoparticles loaded with PAZ. 2 15-JI / DMPC / Chol., further formulated with pharmaceutically acceptable excipients to contain PAZ@A4Z. 2 15-JI / DMPC / Chol. PAZ brain-targeted sustained-release formulation.

[0082] A4Z 2 Replace 15-JI (A0) with A4Z respectively. ij 2 15-JCl(A1), A4Z 2 18-JI (A18), A5Z 2 18-JI (A19) and A5Z ij 2 By using 18-JCl(A22) and keeping other conditions unchanged, lipid nanoparticles PAZ@A4Z loaded with PAZ can be obtained. ij 2 15-JCl / DMPC / Chol.、PAZ@A4Z 2 18-JI / DMPC / Chol.、PAZ@A5Z 2 18-JI / DMPC / Chol. and PAZ@A5Z ij 2 18-JCl / DMPC / Chol. and PAZ brain-targeted sustained-release formulations containing the above-mentioned lipid nanoparticles.

[0083] Comparative Example 4:

[0084] A4Z in Example 4 2 15-JI was replaced with the cationic lipid compound Dlin-MC3-DMA (MC3). MC3, DMPC and Chol were mixed in a molar ratio of 85:15:30 and lipid nanoparticles PAZ@MC3 / DMPC / Chol were prepared by thin-film hydration.

[0085] Example 5: Encapsulation efficiency characterization of insoluble drug-targeting sustained-release lipid nanoparticles based on dialkyl imidazolium biomimetic lipids

[0086] 1% Triton X-100 was added to each group of lipid nanoparticles in Example 1 and Comparative Example 1, and the mixture was incubated in PBS buffer at 25°C for 10 min to release the contents encapsulated by the lipid nanoparticles. Then, 0.1 M NaOH was added to each group of contents, and the mixture was incubated at 25°C for another 30 min. The PTX content in the contents was determined by HPLC, and the encapsulation efficiency of PTX in each group of lipid nanoparticles was calculated. The results are shown in Table 1. The PMX content in each group of lipid nanoparticles in Example 3 and Comparative Example 3 was determined using the same method, and the PMX encapsulation efficiency was calculated. The results are shown in Table 1.

[0087] The 0.1 M NaOH in the above method was replaced with 1.0 M hydrochloric acid, and other steps remained unchanged. The VCR content in each group of lipid nanoparticles in Example 2 and Comparative Example 2 was determined, and the encapsulation efficiency of each group of lipid nanoparticles for VCR was calculated. The results are shown in Table 1. The PAZ content in each group of lipid nanoparticles in Example 4 and Comparative Example 4 was determined using the same method, and the encapsulation efficiency of PAZ was calculated. The results are shown in Table 1.

[0088] Table 1

[0089]

[0090] As shown in Table 1, the encapsulation efficiency of PTX by the lipid nanoparticles in Example 1 ranged from 71.2% to 91.0%, among which, PTX@A4Z 2 15-JI / DMPC / Chol. exhibited a maximum encapsulation efficiency of 91.0%, resulting in a 194.2-fold increase in PTX solubility (PTX technical solubility was 5.56 μg / mL). In contrast, the encapsulation efficiency of PTX in Comparative Example 1 was only 41.8%.

[0091] The lipid nanoparticles in Example 2 exhibited encapsulation efficiency of 69.7%–89.0% for VCR, with VCR@A5Z being one example. 2 18-JI / DMPC / Chol. exhibits a maximum encapsulation efficiency of 89.0%, and the VCR solubility can be increased by up to 25 times (VCR drug solubility is 30 μg / mL). In contrast, the VCR@MC3 / DMPC / Chol. in Comparative Example 2 only achieved an encapsulation efficiency of 37.4% for VCR.

[0092] In Example 3, the lipid nanoparticles exhibited encapsulation efficiency of 71.7%–90.9% for PMX, specifically PMX@A4Z. 215-JI / DMPC / Chol. exhibits a maximum encapsulation efficiency of 90.9%, resulting in a 19.7-fold increase in PMX solubility (PMX technical grade solubility is 45.5 μg / mL). In contrast, the encapsulation efficiency of PMX@MC3 / DMPC / Chol. in Comparative Example 3 is only 39.3%.

[0093] In Example 4, the encapsulation efficiency of PAZ by the lipid nanoparticles ranged from 82.7% to 93.2%, wherein PAZ@A4Z 2 15-JI / DMPC / Chol. exhibits a maximum encapsulation efficiency of 93.2%, with a maximum PAZ solubility increase of 28.9 times (PAZ technical solubility is 43.3 μg / mL). In contrast, the PAZ@MC3 / DMPC / Chol. in Comparative Example 4 only achieved a PAZ encapsulation efficiency of 46.4%.

[0094] Based on the results shown in Table 1, it can be seen that regardless of the type of active drug loaded, the encapsulation efficiency of the lipid nanoparticles in Examples 1-4 is greater than 69%. In contrast, the encapsulation efficiency of the lipid nanoparticles based on the ionizable cationic lipid Dlin-MC3-DMA (MC3) in Comparative Examples 1-4 is less than 50%. This indicates that, compared to the loading of insoluble drugs by lipid nanoparticles composed of MC3 through physical adsorption, the brain-targeted sustained-release lipid nanoparticles based on BDILs can achieve more efficient and controllable loading of insoluble small molecule antitumor drugs by encapsulating the LDC formed by BDILs and insoluble drugs.

[0095] Example 6: Physicochemical characterization of insoluble drug-targeting sustained-release lipid nanoparticles based on dialkyl imidazolium biomimetic lipids

[0096] Based on the results shown in Table 1 of Example 5, lipid nanoparticles with the highest encapsulation efficiency were screened from Examples 1-4, namely PTX@A4Z. 2 15-JI / DMPC / Chol.、VCR@A5Z 2 18-JI / DMPC / Chol., PMX@ A4Z 2 15-JI / DMPC / Chol. and PAZ@A4Z 2 15-JI / DMPC / Chol., and the following physicochemical characterizations were performed on these four lipid nanoparticles.

[0097] (1) Morphology

[0098] The morphology of the four types of lipid nanoparticles was characterized using transmission electron microscopy (TEM). Figure 2As shown, all four lipid nanoparticles exhibited relatively intact spherical vesicle morphology. Furthermore, the morphology of the remaining insoluble drug-targeted brain-release lipid nanoparticles based on dialkyl imidazole biomimetic lipids listed in Table 1 was similar to... Figure 2 Similarly, it also has a complete spherical vesicle structure.

[0099] (2) Particle size distribution

[0100] The average particle size of the lipid nanoparticles prepared in Examples 1-4 and Comparative Examples 1-4 was characterized by nanoflow cytometry (NanoFCM), and the characterization results are shown in Table 1.

[0101] In Example 1, the average particle size of the lipid nanoparticles loaded with PTX ranged from 125.9 to 139.5 nm, which was generally smaller than the average particle size of PTX@MC3 / DMPC / Chol. in Comparative Example 1 (176.9 nm). In Example 2, the average particle size of the lipid nanoparticles loaded with VCR ranged from 127.1 to 137.5 nm, which was generally smaller than the average particle size of VCR@MC3 / DMPC / Chol. in Comparative Example 2 (182.6 nm). In Example 3, the average particle size of the lipid nanoparticles loaded with PMX ranged from 123.9 to 136.5 nm, which was also generally smaller than the average particle size of PMX@MC3 / DMPC / Chol. in Comparative Example 3 (171.4 nm). In Example 4, the average particle size of the lipid nanoparticles loaded with PAZ ranged from 127.5 to 139.7 nm, which was also generally smaller than the average particle size of PAZ@MC3 / DMPC / Chol. in Comparative Example 4 (175.0 nm).

[0102] Among them, drug-loaded lipid nanoparticles PTX@A4Z 2 15-JI / DMPC / Chol.、VCR@A5Z 2 18-JI / DMPC / Chol.、PMX@A4Z 2 15-JI / DMPC / Chol. and PAZ@A4Z 2 The average particle sizes of 15-JI / DMPC / Chol. were 125.9 nm, 136.8 nm, 123.9 nm, and 127.5 nm, respectively, and their particle size distributions are as follows: Figure 3 As shown.

[0103] (3) Surface potential

[0104] Meanwhile, the surface potential of the lipid nanoparticles prepared in Examples 1-4 and Comparative Examples 1-4 was characterized using a Zeta potential analyzer (ZetaPALS), and the characterization results are shown in Table 1.

[0105] The median zeta potential of the lipid nanoparticles in Example 1 ranged from 29.3 to 35.2 mV, which was generally higher than the median zeta potential of PTX@MC3 / DMPC / Chol. in Comparative Example 1 (16.9 mV); the median zeta potential of the lipid nanoparticles in Example 2 ranged from 33.4 to 36.0 mV, which was generally higher than the median zeta potential of VCR@MC3 / DMPC / Chol. in Comparative Example 2 (19.0 mV); the median zeta potential of the lipid nanoparticles in Example 3 ranged from 27.1 to 37.5 mV, which was generally higher than the median zeta potential of PMX@MC3 / DMPC / Chol. in Comparative Example 3 (22.2 mV); and the median zeta potential of the lipid nanoparticles in Example 4 ranged from 31.0 to 38.3 mV, which was generally higher than the median zeta potential of PAZ@MC3 / DMPC / Chol. in Comparative Example 4 (18.6 mV).

[0106] In summary, the insoluble drug-targeting sustained-release lipid nanoparticles based on dialkylimidazolium biomimetic lipids in Examples 1-4 all exhibited intact spherical vesicle morphology, with average particle sizes generally smaller than those of the lipid nanoparticles based on the ionizable cationic lipid Dlin-MC3-DMA (MC3) in Comparative Examples 1-4. Simultaneously, their median zeta potentials were generally higher than those of the lipid nanoparticles based on Dlin-MC3-DMA (MC3) in Comparative Examples 1-4. These physicochemical properties facilitate crossing the blood-brain barrier and absorption by glioma cells.

[0107] Example 7: Plasma stability of insoluble drug-targeted sustained-release lipid nanoparticles based on dialkyl imidazolium biomimetic lipids

[0108] The lipid nanoparticles PTX@A4Z with the highest encapsulation efficiency selected from Examples 1-4 2 15-JI / DMPC / Chol.、VCR@A5Z 2 18-JI / DMPC / Chol.、PMX@A4Z 2 15-JI / DMPC / Chol. and PAZ@A4Z 2 15-JI / DMPC / Chol were added to rat plasma, and the plasma stability of each group of lipid nanoparticles was evaluated by the plasma drug concentration at different time points.

[0109] PTX@A4Z was processed using ultrapure water. 2 15-JI / DMPC / Chol.、VCR@A5Z 2 18-JI / DMPC / Chol.、PMX@A4Z 215-JI / DMPC / Chol. and PAZ@A4Z 2 Dilute 15-JI / DMPC / Chol to prepare a working solution with a drug concentration of 50 μM for test sample preparation. SD rat plasma was divided into two groups, with 490 μL from each group incubated at 37℃ for 5 min. Then, 10 μL of working solution was added and vortexed to mix (final drug concentration 1 μM), and the mixture was incubated at 37℃ for 24 h. At time points of 0, 0.0833, 0.25, 1, 2, 4, 8, 12, 18, and 24 h, 50 μL of drug-containing plasma samples were collected and placed in EP tubes containing 350 μL of ice-cold acetonitrile containing the internal standard (ethoxybenzamide). The samples were vortexed for 2 min to inactivate the drug, centrifuged at 12,000 rpm for 10 min at 4℃, and 100 μL of the supernatant was collected and 100 μL of ultrapure water was added. The mixture was vortexed again for 3 min, centrifuged at 3,000 g for 10 min at 4℃, and then injected for analysis. Three parallel experiments were set up at each time point. The blood drug concentration of lipid nanoparticles in each group after being added to plasma was quantitatively determined by LC-MS / MS (Waters Acquity I UPLC XEVO TQ-S), and the results are shown in Table 2.

[0110] Table 2

[0111]

[0112] Table 2 shows that the insoluble drug-targeting sustained-release lipid nanoparticles PTX@A4Z based on dialkyl imidazole biomimetic lipids... 2 15-JI / DMPC / Chol.、VCR@A5Z 2 18-JI / DMPC / Chol., PMX@ A4Z 2 15-JI / DMPC / Chol. and PAZ@A4Z 2 No significant drug release was observed in 15-JI / DMPC / Chol within 0–24 h, and the maximum average drug release percentage of the four lipid nanoparticles did not exceed 5%, thus demonstrating plasma stability.

[0113] Example 8: Pharmacokinetics of an insoluble drug brain-targeted sustained-release formulation based on dialkyl imidazole biomimetic lipids

[0114] This embodiment further characterizes the lipid nanoparticle PTX@A4Z using an SD rat model. 2 15-JI / DMPC / Chol.、VCR@A5Z 2 18-JI / DMPC / Chol.、PMX@A4Z 2 15-JI / DMPC / Chol. and PAZ@A4Z 2Pharmacokinetics of brain-targeted sustained-release formulation of 15-JI / DMPC / Chol.

[0115] Table 3

[0116]

[0117] (1) Includes PTX@A4Z 2 Pharmacokinetics of PTX brain-targeted sustained-release formulation of 15-JI / DMPC / Chol.

[0118] PTX@A4Z was treated with saline solution. 2 15-JI / DMPC / Chol was diluted to 1.0 mg / mL (PTX concentration), and a separate 1.0 mg / mL PTX stock solution in physiological saline was prepared for injection. SD rats (n = 30, half male and half female) aged 6-8 weeks with an average weight of 200 g were randomly divided into two groups. Each group received a single tail vein injection of 5.0 mg / kg. Whole blood (300 μL) was collected at 5 min, 15 min, 30 min, 1, 2, 4, 8, 12, 18, and 24 h after administration, transferred to EDTA-K2 anticoagulant tubes, and immediately placed on ice. Within 30 min after collection, the plasma sample was centrifuged at 12,000 rpm for 5 min at 4℃. 50 μL of plasma was collected and 1% Triton X-100 was added. After vortexing for 3 min, 50 μL of methanol solution was added, followed by 100 μL of internal standard working solution (verapamil, diluted with acetonitrile). The sample was then centrifuged at 3,000 g for 15 min at 4℃. 100 μL of the supernatant was collected and 100 μL of ultrapure water was added. After vortexing again, the sample was centrifuged at 3,000 g for 5 min at 4℃ before injection for analysis. Three parallel experiments were set up at each time point. Analysis was performed using LC-MS / MS (AB SCIEX TRIPLE QUAD). TM The plasma drug concentrations of the two groups were quantitatively determined at each time point using a 5500+AB SCIEX Exion LC AD assay, and drug-time curves were plotted. The results are as follows: Figure 4 As shown in Figure A, the half-life (t) was calculated using the statistical moments method of a non-compartmental model with the software Phoenix WinNonlin 8.1.0. 1 / 2 The results of the curve analysis and the area under the curve (AUC) are shown in Table 3.

[0119] Depend on Figure 4 As shown in Table A and Table 3, PTX is rapidly metabolized after entering the bloodstream, with a half-life of approximately 5.70 h, and complete metabolism occurs in about 12 hours. In contrast, PTX@A4Z... 2After 15-JI / DMPC / Chol. entered the bloodstream, the total plasma concentration remained at a high level for 24 hours. Combined with the data listed in Table 2, this further demonstrates its good plasma stability. Furthermore, PTX@A4Z 2 AUC of 15-JI / DMPC / Chol. 0-∞ It is significantly larger than the original PTX drug, with a bioavailability increase of approximately 4.1 times and a half-life extension of approximately 5.0 times.

[0120] (2) Includes VCR@A5Z 2 Pharmacokinetics of VCR brain-targeted sustained-release formulation of 18-JI / DMPC / Chol.

[0121] VCR@A5Z was treated with saline solution. 2 18-JI / DMPC / Chol. was diluted to 1.5 mg / mL (VCR concentration), and a 1.5 mg / mL concentration of VCR original drug in physiological saline injection was prepared separately. SD rats with an average weight of 200 g at 6-8 weeks of age (n = 30, half male and half female) were randomly divided into two groups. Each group was given a single tail vein injection at a dose of 7.5 mg / kg, and other experimental methods were the same as in (1). The blood drug concentrations of the two groups at each time point were measured, and the drug-time curves were plotted. The results are as follows. Figure 4 As shown in B, the half-life (t) was calculated using the statistical moments method of the non-compartmental model with the software Phoenix WinNonlin 8.1.0. 1 / 2 The results of the curve analysis and the area under the curve (AUC) are shown in Table 3.

[0122] Depend on Figure 4 As shown in B and Table 3, the metabolic rate of VCR (vitamin C) after entering the bloodstream is faster than that of PTX, with a half-life of only 1.48 h and complete metabolism in about 8 hours. In contrast, VCR@A5Z... 2 After 18-JI / DMPC / Chol. enters the bloodstream, the total plasma concentration remains at a high level for 24 hours. (Compared to PTX@A4Z) 2 Similarly, the data listed in Table 2 further demonstrate its good plasma stability. Furthermore, VCR@A5Z 2 AUC of 18-JI / DMPC / Chol. 0-∞ It is significantly larger than the original VCR drug, with bioavailability increased by about 7.0 times and half-life extended by about 8.6 times.

[0123] (3) Includes PMX@A4Z 2 Pharmacokinetics of the PMX brain-targeted sustained-release formulation of 15-JI / DMPC / Chol.

[0124] PMX@A4Z was treated with saline solution.2 15-JI / DMPC / Chol. was diluted to 1.0 mg / mL (PMX concentration), and a 1.0 mg / mL concentration of PMX raw drug in physiological saline injection was prepared separately. SD rats with an average weight of 200 g at 6-8 weeks of age (n = 30, half male and half female) were randomly divided into two groups. Each group was given a single tail vein injection at a dose of 5.0 mg / kg, and other experimental methods were the same as in (1). The blood drug concentrations of the two groups at each time point were measured, and the drug-time curves were plotted. The results are as follows. Figure 4 As shown in C. The half-life (t) was calculated using the statistical moments method of the non-compartmental model with the software Phoenix WinNonlin 8.1.0. 1 / 2 The results of the curve analysis and the area under the curve (AUC) are shown in Table 3.

[0125] Depend on Figure 4 As shown in C and Table 3, the metabolic rate of PMX after entering the bloodstream is similar to that of VCR, with a half-life of only 1.51 h, and complete metabolism occurs in approximately 8 hours. In contrast, PMX@A4Z... 2 Following administration of 15-JI / DMPC / Chol, the total plasma concentration remained at a high level for 24 hours. This is consistent with PTX@A4Z. 2 Similarly, the data listed in Table 2 further demonstrate its good plasma stability. Additionally, PMX@A4Z 2 AUC of 15-JI / DMPC / Chol. 0-∞ It is significantly larger than the original PMX drug, with bioavailability increased by approximately 8.2 times and half-life extended by approximately 9.7 times.

[0126] (4) Includes PAZ@A4Z 2 Pharmacokinetics of PAZ brain-targeted sustained-release formulation of 15-JI / DMPC / Chol.

[0127] PAZ@A4Z was treated with saline solution. 2 15-JI / DMPC / Chol. was diluted to 1.0 mg / mL (PAZ concentration), and a 1.0 mg / mL concentration of PAZ raw drug in physiological saline injection was prepared separately. SD rats with an average weight of 200 g at 6-8 weeks of age (n = 30, half male and half female) were randomly divided into two groups. Each group was given a single tail vein injection at a dose of 5.0 mg / kg, and other experimental methods were the same as in (1). The blood drug concentrations of the two groups at each time point were measured, and the drug-time curves were plotted. The results are as follows. Figure 4 As shown in D. The half-life (t) was calculated using the statistical moments method of a non-compartmental model with the software Phoenix WinNonlin 8.1.0. 1 / 2The results of the curve analysis and the area under the curve (AUC) are shown in Table 3.

[0128] Depend on Figure 4 As shown in Table D and Table 3, the metabolic rate of PAZ after entering the bloodstream is relatively slow, with a half-life of 7.28 h, and complete metabolism occurs in approximately 24 hours. In contrast, PAZ@A4Z... 2 15-JI / DMPC / Chol. After entering the bloodstream, it exhibits a higher total blood drug concentration level within 24 hours, and its AUC 0-∞ Significantly greater than the PAZ original drug, with approximately 3.1 times higher bioavailability and approximately 4.0 times longer half-life. Furthermore, compared to PTX@A4Z... 2 Similarly, the data in Table 2 further demonstrate its good plasma stability.

[0129] In summary, pharmacokinetic characterization of a single tail vein injection in an SD rat model demonstrates that the lipid nanoparticles PTX@A4Z contain [the desired active ingredient / product]. 2 15-JI / DMPC / Chol.、VCR@A5Z 2 18-JI / DMPC / Chol.、PMX@A4Z 2 15-JI / DMPC / Chol. and PAZ@A4Z 2 The brain-targeted sustained-release formulation of 15-JI / DMPC / Chol. can improve the bioavailability and half-life of insoluble drugs to some extent. Furthermore, in... Figure 4 In AD, the total blood drug concentration of the four drug-loaded lipid nanoparticles mentioned above was significantly reduced at 4 h, which may be related to the optimal delivery window for crossing the blood-brain barrier.

[0130] Example 9: Brain delivery efficacy of paclitaxel brain-targeting sustained-release formulation based on dialkyl imidazole biomimetic lipids in vivo.

[0131] Based on the above results, further evaluation of PTX@A4Z containing lipid nanoparticles was conducted. 2 The brain delivery efficacy of the PTX brain-targeted sustained-release formulation of 15-JI / DMPC / Chol. in vivo.

[0132] 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 (cells / mL), after 1-2 weeks of growth, a tumor volume of approximately 100-150 mm was obtained. 3 An immunodeficient mouse model was established. These mice were randomly divided into four groups of three each. PTX@A4Z was then injected into each group. 2The PTX brain-targeted sustained-release formulation of 15-JI / DMPC / Chol., PTX@MC3 / DMPC / Chol. (Comparative Example 1), and PTX technical grade injection were administered via tail vein injection (dose: 10 mg / kg for all formulations), with an injection volume of 200 μL. The control group received an equal volume of physiological saline. Brain tissue was collected from mice at 2, 4, 8, 12, and 24 h post-injection. The free PTX content in brain tissue and the total PTX content in plasma were quantitatively determined by LC-MS / MS at each time point. The brain tissue-to-plasma concentration ratio (B / P) for each group was calculated. Figure 5 As shown in Figure A. The above experiment was repeated, except that brain tissue from the mice was removed one week after injection, and the tumor weight of each group was compared. The results are shown in Figure A. Figure 5 As shown in B.

[0133] Depend on Figure 5 As can be seen from A, injecting lipid nanoparticles PTX@A4Z into the tail vein of a mouse model... 2 Following 15-JI / DMPC / Chol. injection, the mean B / P ratios at 2, 4, 8, 12, and 24 h were approximately 0.75, 1.54, 1.75, 1.88, and 1.94, respectively. In contrast, following tail vein injection of PTX@MC3 / DMPC / Chol. in Comparative Example 1, the mean B / P ratios at each time point were approximately 0.02, 0.13, 0.18, 0.26, and 0.32, respectively, all below 1.0 and significantly lower than those of PTX@A4Z. 2 15-JI / DMPC / Chol. Furthermore, the average B / P ratio at all time points in the PTX technical injection group was below 0.20. This indicates that the PTX@A4Z-containing... 2 The PTX brain-targeted sustained-release formulation of 15-JI / DMPC / Chol. can cross the blood-brain barrier, significantly improving PTX blood-brain barrier permeability and brain tissue distribution in mouse models. Its brain delivery effect is far superior to Comparative Example 1 and the injectable PTX drug. Notably, the average B / P ratio exceeding 1 (B / P = 1.54) first appeared at 4 hours, indicating an optimal delivery window for crossing the blood-brain barrier around 4 hours. Figure 4 The results shown in A are consistent. Furthermore, the injection contained PTX@A4Z. 2 After formulation with 15-JI / DMPC / Chol., the average B / P ratio gradually increased over time, demonstrating the achievement of sustained release of PTX.

[0134] Figure 5 In case B, lipid nanoparticles PTX@A4Z were injected via tail vein. 2One week after injection of 15-JI / DMPC / Chol., the average tumor weight was 0.7 g, while the average tumor weight after injection of PTX@MC3 / DMPC / Chol. in Comparative Example 1 was 2.3 g, and the average tumor weight after injection of PTX stock was 2.4 g. Simultaneously, the average tumor weight of the blank group (saline group) one week after injection was measured to be 2.1 g. Therefore, under the same PTX dosage, injection of PTX@A4Z... 2 One week after injection of 15-JI / DMPC / Chol., the average tumor weight decreased by approximately 67%. However, one week after injection of PTX@MC3 / DMPC / Chol. (Comparative Example 1) and the original PTX, the average tumor weight did not decrease, indicating that the PTX@A4Z containing lipid nanoparticles... 2 The brain-targeted sustained-release formulation of PTX 15-JI / DMPC / Chol. showed superior brain delivery compared to PTX@MC3 / DMPC / Chol. and the PTX technical injection in Comparative Example 1. Figure 5 The results shown in A are consistent.

[0135] Example 10: In vivo brain delivery effect of vincristine brain-targeting sustained-release formulation based on dialkyl imidazole biomimetic lipids.

[0136] The experimental method described in Example 9 was used to compare the contents of VCR@A5Z. 2 The B / P ratios of the VCR brain-targeted sustained-release formulation of 18-JI / DMPC / Chol., VCR@MC3 / DMPC / Chol. in Comparative Example 2, and the VCR original drug injection at 2, 4, 8, 12, and 24 h post-injection ( Figure 6 A) and changes in tumor weight one week after injection ( Figure 6 B).

[0137] Depend on Figure 6 As can be seen from A, injecting lipid nanoparticles VCR@A5Z into the tail vein of a mouse model... 2 Following 18-JI / DMPC / Chol. injection, the mean B / P ratios at 2, 4, 8, 12, and 24 h were approximately 0.69, 1.23, 1.48, 1.66, and 1.84, respectively. In contrast, following tail vein injection of VCR@MC3 / DMPC / Chol. in Comparative Example 2, the mean B / P ratios at each time point were approximately 0.05, 0.19, 0.21, 0.26, and 0.37, respectively, all below 1.0 and significantly lower than those of VCR@A5Z. 2 18-JI / DMPC / Chol. Furthermore, the average B / P ratio of the VCR active pharmaceutical ingredient injection group was below 0.20 at all time points. This indicates that the VCR@A5Z-containing... 2The brain-targeted sustained-release formulation of 18-JI / DMPC / Chol. can cross the blood-brain barrier, significantly improving the blood-brain barrier permeability of VCR and its distribution in the brain tissue of mouse models. Its brain delivery effect is far superior to Comparative Example 2 and the original VCR injectable formulation. Notably, the average B / P ratio exceeding 1 (B / P = 1.23) first appeared at 4 hours, indicating an optimal delivery window for crossing the blood-brain barrier around 4 hours. Figure 4 The results shown in B are consistent. Furthermore, the injection contained VCR@A5Z. 2 After formulation with 18-JI / DMPC / Chol., the average B / P ratio gradually increased over time, demonstrating the achievement of sustained release of VCR.

[0138] Figure 6 In case B, lipid nanoparticles VCR@A5Z were injected via tail vein. 2 One week after injection of 18-JI / DMPC / Chol., the average tumor weight was 0.6 g, while the average tumor weight after injection of VCR@MC3 / DMPC / Chol. in Comparative Example 2 was 1.5 g, and the average tumor weight after injection of the original VCR was 1.6 g. Simultaneously, the average tumor weight of the blank group (saline group) one week after injection was measured to be 2.1 g. Therefore, under the same VCR dosage, injection of VCR@A5Z... 2 One week after injection of 18-JI / DMPC / Chol., the average tumor weight decreased by approximately 71%, while one week after injection of VCR@MC3 / DMPC / Chol. (Comparative Example 2) and the original VCR drug, the average tumor weight decreased by only 29% and 24%, respectively. This indicates that VCR@A5Z containing lipid nanoparticles... 2 The brain-targeted sustained-release formulation of 18-JI / DMPC / Chol. showed superior brain delivery compared to VCR@MC3 / DMPC / Chol. and the original VCR injectable formulation in Comparative Example 2. Figure 6 The results shown in A are consistent.

[0139] Example 11: In vivo brain delivery efficacy of pemetrexed brain-targeting sustained-release formulation based on dialkyl imidazole biomimetic lipids.

[0140] The experimental method described in Example 9 was used to compare the contents of PMX@A4Z. 2 The B / P ratios of the PMX brain-targeted sustained-release formulation of 15-JI / DMPC / Chol., PMX@MC3 / DMPC / Chol. in Comparative Example 3, and the PMX technical grade injection at 2, 4, 8, 12, and 24 h post-injection ( Figure 7 A) and changes in tumor weight one week after injection ( Figure 7 B).

[0141] Depend on Figure 7As can be seen from A, injecting lipid nanoparticles PMX@A4Z into the tail vein of a mouse model... 2 Following 15-JI / DMPC / Chol. injection, the mean B / P ratios at 2, 4, 8, 12, and 24 h were approximately 0.55, 1.34, 1.59, 1.72, and 1.81, respectively. In contrast, following tail vein injection of PMX@MC3 / DMPC / Chol. in Comparative Example 3, the mean B / P ratios at each time point were approximately 0.01, 0.06, 0.23, 0.34, and 0.33, respectively, all below 1.0 and significantly lower than those of PMX@A4Z. 2 15-JI / DMPC / Chol. Furthermore, the average B / P ratio of the PMX technical grade injection group was below 0.25 at all time points. This indicates that the PMX@A4Z formulation... 2 The PMX brain-targeted sustained-release formulation of 15-JI / DMPC / Chol. can cross the blood-brain barrier, significantly improving the blood-brain barrier permeability of PMX and its distribution in mouse brain tissue. Its brain delivery effect is far superior to Comparative Example 3 and the injectable PMX drug. Notably, the average B / P ratio exceeding 1 (B / P = 1.34) first appeared at 4 hours, indicating an optimal delivery window for crossing the blood-brain barrier around 4 hours. Figure 4 The results shown in C are consistent. Furthermore, the injection contained PMX@A4Z. 2 After formulation with 15-JI / DMPC / Chol., the average B / P ratio gradually increased over time, demonstrating the achievement of sustained release of PMX.

[0142] Figure 7 In case B, lipid nanoparticles PMX@A4Z were injected via tail vein. 2 One week after injection of 15-JI / DMPC / Chol., the average tumor weight was 0.9 g, while the average tumor weight after injection of PMX@MC3 / DMPC / Chol. in Comparative Example 3 was 1.7 g, and the average tumor weight after injection of PMX undiluted was 1.8 g. Simultaneously, the average tumor weight of the blank group (saline group) one week after injection was measured to be 2.1 g. Therefore, under the same PMX dosage, injection of PMX@A4Z... 2 One week after injection of 15-JI / DMPC / Chol., the average tumor weight decreased by approximately 57%, while one week after injection of PMX@MC3 / DMPC / Chol. (Comparative Example 3) and the original PMX, the average tumor weight decreased by only 19% and 14%, respectively. This indicates that the lipid nanoparticle-containing PMX@A4Z... 2 The PMX brain-targeted sustained-release formulation of 15-JI / DMPC / Chol. showed superior brain delivery compared to PMX@MC3 / DMPC / Chol. and the PMX technical grade injectable in Comparative Example 3. Figure 7 The results shown in A are consistent.

[0143] Example 12: In vivo brain delivery efficacy of pazopanib brain-targeted sustained-release formulation based on dialkyl imidazole biomimetic lipids.

[0144] The experimental method described in Example 9 was used to compare the contents of PAZ@A4Z. 2 The B / P ratios of PAZ brain-targeted sustained-release formulation of 15-JI / DMPC / Chol., PAZ@MC3 / DMPC / Chol. in Comparative Example 4, and PAZ technical grade injection at 2, 4, 8, 12, and 24 h post-injection ( Figure 8 A) and changes in tumor weight one week after injection ( Figure 8 B).

[0145] Depend on Figure 8 As can be seen from A, injecting lipid nanoparticles PAZ@A4Z into the tail vein of a mouse model... 2 Following 15-JI / DMPC / Chol. injection, the mean B / P ratios at 2, 4, 8, 12, and 24 h were approximately 0.63, 1.42, 1.62, 1.84, and 1.90, respectively. In contrast, following tail vein injection of PAZ@MC3 / DMPC / Chol. in Comparative Example 4, the mean B / P ratios at each time point were approximately 0.08, 0.15, 0.22, 0.32, and 0.32, respectively, all below 1.0 and significantly lower than those of PAZ@A4Z. 2 15-JI / DMPC / Chol. Furthermore, the average B / P ratio of the PAZ technical grade injection group was below 0.25 at all time points. This indicates that the PAZ@A4Z formulation... 2 The PAZ brain-targeting sustained-release formulation of 15-JI / DMPC / Chol. can cross the blood-brain barrier, significantly improving PAZ's blood-brain barrier permeability and brain tissue distribution in mouse models. Its brain delivery effect is far superior to Comparative Example 4 and the injectable PAZ drug. Notably, the average B / P ratio exceeding 1 (B / P = 1.42) first appeared at 4 hours, indicating an optimal delivery window for crossing the blood-brain barrier around 4 hours. Figure 4 The results shown in D are consistent. Furthermore, the injection contained PAZ@A4Z. 2 After formulation with 15-JI / DMPC / Chol., the average B / P ratio gradually increased over time, demonstrating the achievement of sustained release of PAZ.

[0146] Figure 8 In case B, lipid nanoparticles PAZ@A4Z were injected via the tail vein. 2One week after injection of 15-JI / DMPC / Chol., the average tumor weight was 0.6 g, while the average tumor weight after injection of PAZ@MC3 / DMPC / Chol. in Comparative Example 4 was 1.7 g, and the average tumor weight after injection of undiluted PAZ was 2.2 g. Simultaneously, the average tumor weight of the blank group (saline group) one week after injection was measured to be 2.1 g. Therefore, under the same PAZ dosage, injection of PAZ@A4Z... 2 One week after injection of 15-JI / DMPC / Chol., the average tumor weight decreased by approximately 71%. In contrast, one week after injection of PAZ@MC3 / DMPC / Chol. in Comparative Example 4, the average tumor weight decreased by only 19%; and one week after injection of the original PAZ, the average tumor weight did not decrease. This indicates that PAZ@A4Z containing lipid nanoparticles... 2 The brain-targeted sustained-release formulation of 15-JI / DMPC / Chol. showed superior brain delivery compared to PAZ@MC3 / DMPC / Chol. and the PAZ technical grade injectable in Comparative Example 4. Figure 8 The results shown in A are consistent.

[0147] In summary, including PTX@A4Z 2 15-JI / DMPC / Chol.、VCR@A5Z 2 18-JI / DMPC / Chol.、PMX@A4Z 2 15-JI / DMPC / Chol. and PAZ@A4Z 2 The brain-targeted sustained-release formulation of insoluble drugs based on dialkylimidazolium biomimetic lipids of 15-JI / DMPC / Chol. can achieve solubilization of insoluble drugs, effectively cross the blood-brain barrier for delivery, thereby improving the distribution of insoluble drugs in brain tissue, improving their half-life and bioavailability, and significantly inhibiting the growth of gliomas. Its in vivo brain delivery effect is far superior to that of lipid nanoparticles based on ionizable cationic lipid Dlin-MC3-DMA in the comparative example.

[0148] 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 method for preparing lipid nanoparticles loaded with insoluble drugs based on dialkyl imidazolium biomimetic lipids, characterized in that: Includes the following steps: Step S01: Mix the dialkylimidazolium biomimetic lipid with an insoluble drug or an insoluble drug linked with a linker, and add a coupling adjuvant to form a dialkylimidazolium biomimetic lipid-drug conjugate. Step S02: Preheat the dialkyl imidazolium biomimetic lipid to form a pure liquid; Step S03: Add the dialkylimidazolium biomimetic lipid-drug conjugate, phospholipids and cholesterol obtained in step S01 to the liquid dialkylimidazolium biomimetic lipid obtained in step S02 and dissolve it completely to form a drug-loaded mixture; the molar ratio of the dialkylimidazolium biomimetic lipid-drug conjugate, dialkylimidazolium biomimetic lipid, phospholipids and cholesterol in the drug-loaded mixture is 15~30:60~90:10~45:25~45; Step S04: Add the drug-loaded mixture to the buffer solution to form a drug-loaded emulsion; Step S05: The drug-loaded emulsion is ultrasonically treated and then sterilized by passing it through a filter membrane to obtain lipid nanoparticles loaded with insoluble drugs; The structure of the dialkyl imidazolium biomimetic lipid is shown in general formula 1; General Formula 1; Where R' and R'' are C 14 ~C 18 Straight-chain alkyl or monoalkenyl, R' and R'' are the same 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 method for preparing lipid nanoparticles based on dialkyl imidazole biomimetic lipids loaded with insoluble drugs according to claim 1, characterized in that: In step S02, the preheating temperature is 30~35℃.

3. The method for preparing lipid nanoparticles based on dialkyl imidazolium biomimetic lipids loaded with insoluble drugs according to claim 1, characterized in that: In step S04, the drug-loaded mixture is injected into the buffer solution at a rate of 50-120 μL / s at 45-60°C.

4. The lipid nanoparticles loaded with insoluble drugs prepared by the method for preparing lipid nanoparticles based on dialkylimidazolium biomimetic lipids according to any one of claims 1-3.

5. A brain-targeted sustained-release formulation comprising the lipid nanoparticles based on dialkylimidazolium biomimetic lipids loaded with insoluble drugs as described in claim 4.

6. The application of lipid nanoparticles prepared by the method of preparing lipid nanoparticles based on dialkylimidazolium biomimetic lipids loaded with insoluble drugs according to claim 4, the brain-targeted sustained-release formulation according to claim 5, and the lipid nanoparticles based on dialkylimidazolium biomimetic lipids loaded with insoluble drugs according to any one of claims 1-3 in the preparation of drugs for treating glioma.

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