CN-121987822-A - Cationic liposome nanoparticle with multistage trigger system, and preparation method and application thereof

CN121987822ACN 121987822 ACN121987822 ACN 121987822ACN-121987822-A

Abstract

The invention provides a cationic liposome nanoparticle with a multistage trigger system, a preparation method and application thereof. The method comprises the steps of preparing liposome nano particles, introducing cationic lipid, loading targeting functional peptide, covalent coupling reaction and introducing functional lipid. The stability of the liposome nano particles in blood circulation is improved by optimizing the composition and the surface charge of the liposome nano particles, and the targeting peptide is utilized to be combined with a specific receptor on a blood brain barrier, so that the nano particles can be actively mediated to pass through the blood brain barrier and directionally released to cognition related brain areas such as hippocampus, prefrontal cortex and the like, and the memory decline or the movement dysfunction of the Parkinson disease related to the Alzheimer disease are improved.

Inventors

CUI MINGQIANG
CHEN KEKE
LI DEQIANG
HUANG HONGYAN
CHEN RONGRONG

Assignees

中煜福新能源科技(深圳)有限公司

Dates

Publication Date: 20260508
Application Date: 20260211

Claims (10)

1. A method for preparing cationic liposome nanoparticles with a multi-stage trigger system, comprising the steps of: Step S1, preparing liposome nano particles, namely mixing structural phospholipid and cholesterol according to a preset molar ratio to form a stable lipid bilayer structure, and then fusing the lipid bilayer structure with water to form the liposome nano particles; step S2, introducing cationic lipid, namely mixing cationic lipid with quaternary ammonium salt head group or tertiary amine structure with the liposome nano-particles obtained in the step S1 to form cationic liposome nano-particles; Step S3, loading the targeting functional peptide, namely mixing the targeting peptide with the cationic liposome nanoparticle obtained in the step S2, keeping away from light at room temperature, and stirring and incubating for 30-60 min at the speed of 300-600 rpm to obtain the cationic liposome nanoparticle loaded with the targeting functional peptide; Step S4, covalent coupling reaction, namely reducing the cationic liposome nano particles loaded with the targeting functional peptide obtained in the step S3, then adding a polyethylene glycol lipid derivative with a reactive functional group, incubating for 2-4 h at room temperature under the condition of a pH value of 6.5-7.2 in a dark place, and removing free peptide in a reaction system to obtain the functional cationic liposome nano particles; and S5, introducing functional lipid, namely adding at least one functional lipid with environmental response characteristics into the functional cationic liposome nano-particles obtained in the step S4, and finally forming the cationic liposome nano-particles with a multi-stage triggering system.
2. The method for preparing cationic liposome nanoparticles with multi-stage triggering system as set forth in claim 1, The structural phospholipid comprises hydrogenated phosphatidylcholine or dioleoyl phosphatidylethanolamine, and cholesterol accounts for 20-30 mol% of the total reactant; The cationic lipid comprises 1, 2-dioleoyl-3-trimethylammonium propane or 3 beta- [ N- (N ', N' -dimethylaminoethyl) carbamoyl ] cholesterol, and the dosage of the cationic lipid is 5-15 mol% of the total system.
3. The method of preparing cationic liposome nanoparticles with a multi-stage triggering system according to claim 1, wherein the liposome nanoparticles are formed using a thin film hydration method, comprising the steps of: s1-1, dissolving the lipid bilayer structure in an organic solvent to obtain a lipid solution; Step S1-2, placing the lipid solution in a glass container, heating to evaporate the organic solvent so as to form a uniform lipid film on the inner wall of the glass container; S1-3, adding a buffer water phase into a glass container, and then placing the glass container into a constant-temperature oscillator to hydrate for 60-90 min at 60-65 ℃ to obtain hydrated crude suspension; s1-4, treating the crude suspension for 5-10 min under the ultrasonic action of a pulse mode under the power of 100-150W; and step S1-5, repeatedly passing through the porous ultrafiltration membrane, thereby obtaining the liposome nano particles.
4. The method of preparing cationic liposome nanoparticles with a multi-stage triggering system according to claim 1, wherein the liposome nanoparticles are formed using microfluidic technology, comprising the steps of: And dissolving the lipid bilayer structure in absolute ethyl alcohol, respectively introducing the obtained lipid bilayer structure ethanol solution and a buffer water phase into two sample injection channels of a microfluidic chip, and adjusting the sample injection flow rate ratio of the two solutions to 3:1 and the total flow rate to 12 mL/min, thereby obtaining the liposome nanoparticle.
5. The method of preparing cationic liposome nanoparticles with a multistage trigger system according to claim 1, wherein the targeting peptide is a peptide sequence having arginine and/or lysine residues.
6. The method of claim 1, wherein the reactive functional group-containing pegylated lipid derivative is one of DSPE-PEG-Mal, DSPE-PEG-NHS or DSPE-PEG-DBCO, wherein the molecular weight of the PEG is in the range of 2000-3400 Da.
7. The method of preparing cationic liposome nanoparticles with a multi-stage trigger system of claim 1, wherein the environmental response characteristic is selected from pH response and/or enzyme response.
8. A cationic liposome nanoparticle with a multi-stage trigger system prepared using the preparation method according to any one of claims 1 to 7.
9. Use of a cationic liposome nanoparticle with a multi-level trigger system prepared according to the preparation method of any one of claims 1 to 8 or a cationic liposome nanoparticle with a multi-level trigger system according to claim 8 in the preparation of a medicament for treating brain lesions.
10. The use according to claim 9, wherein the drug for treating brain lesions is microfluidically encapsulated on the cationic liposome nanoparticle with a multi-stage trigger system.

Description

Cationic liposome nanoparticle with multistage trigger system, and preparation method and application thereof Technical Field The invention relates to the technical field of biology, in particular to cationic liposome nano particles with a multi-stage triggering system, a preparation method and application thereof. Background Currently, liposome nanoparticles are receiving a great deal of attention as drug delivery systems in clinical applications, especially in the in vivo delivery of nucleic acid drugs, showing great potential. The traditional liposome preparation method mostly adopts a simple film hydration or solvent injection technology, and realizes drug loading through passive encapsulation. However, the problems of wide particle size distribution, low encapsulation efficiency, unstable drug loading and the like generally exist, and the requirements of accurate medical treatment on the uniformity and the controllability of a delivery system are difficult to meet. The conventional formula is generally composed of phospholipid and cholesterol, the formed lipid bilayer structure has limited mechanical strength, is easy to adsorb protein and clear by reticuloendothelial system in blood, and has short circulation half-life and low bioavailability. In addition, the traditional liposome lacks effective targeting recognition capability, mainly relies on enrichment of passive targeting effects (such as EPR effect) in tumor tissues, has low cell uptake efficiency, cannot distinguish normal cells from pathological cells, and limits the therapeutic effect. In order to improve targeting, the targeting ligand is usually connected to the surface of the liposome by physical adsorption or random chemical coupling, but the coupling site is uncontrollable, so that the active region of the functional peptide is easily shielded or spatially oriented, the specific binding capacity of the functional peptide and a target receptor is seriously influenced, and the targeting efficiency is low. Meanwhile, most liposome systems do not have environmental response characteristics, the drug release behavior mainly depends on passive diffusion of membrane permeability, programmed trigger release cannot be realized according to the change of focus microenvironment, advanced leakage of drugs at non-target positions is easily caused, and systemic toxicity risk is increased. Although some studies have attempted to introduce cationic lipids to enhance cell membrane affinity and nucleic acid entrapment capacity, serum protein aggregation, accelerated clearance or cytotoxicity problems are often caused by too high charge density, and fine regulation of the targeting ligand immobilization pattern is lacking. In addition, although PEGylation modifications improve the stability of circulation, their "stealth" effects are often accompanied by a decrease in the efficiency of endocytosis, resulting in the so-called PEG DILEMMA. The existing preparation process has remarkable limitations in aspects of component proportion control, accurate regulation and control of self-assembly process and multifunctional integration, and is difficult to realize unification of multiple performances such as high-stability, high-efficiency targeted identification, stimulus-responsive drug release and the like in a synergistic manner, so that further application of liposome nano-particles in accurate treatment is restricted. In addition, the research shows that the existing neuroprotectant is faced with key bottlenecks of poor blood-brain barrier penetration capability, weak brain tissue targeting, low drug delivery efficiency and the like when treating central nervous system diseases such as Alzheimer disease, parkinsonism and the like. Although the traditional liposome has certain biocompatibility and drug carrying capacity, the surface of the liposome lacks a specific recognition function, so that the liposome is difficult to actively cross a blood brain barrier, and a plurality of preparations are insufficient in stability and easy to clear in blood circulation, so that the concentration of the drug in a brain region cannot reach an effective treatment level. In addition, in the prior art, the targeting ligand is mostly modified on the surface of the nanoparticle in a physical adsorption mode, so that the targeting ligand is unstable in combination and easy to fall off, and the targeting precision and the delivery efficiency are seriously affected. Meanwhile, most of drug release behaviors are passive diffusion, lack of responsiveness to pathological change microenvironment, and limit the accurate release capacity of the drug at focus positions. Disclosure of Invention Aiming at the defects of the prior art, the invention provides the cationic liposome nanoparticle with a multistage trigger system, and the preparation method and the application thereof, so that the encapsulation rate of the liposome nanoparticle is improved, and the targeting and the stability