Nano-liposome antitumor drug and preparation method thereof

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a nano-liposome antitumor drug and a preparation method thereof.The nano-liposome antitumor drug is prepared through inner core double-drug entrapment, net support constructed by shell chitin and outer layer BSA covalent protection in combination with paclitaxel, Vandetanib and VEGFC multi-drug synergism, and the nano-liposome antitumor drug is prepared. The whole link characteristics of tumor cell killing, angiogenesis inhibition and microenvironment regulation and control are covered, the toxic and side effects of drugs and tumor drug resistance can be greatly reduced through synergistic fusion, the effectiveness of anti-tumor treatment is improved, and the problems that traditional lipidosome is poor in stability and drugs are prone to leakage are solved; through the precise process of layering preparation, covalent binding and freeze drying, the situation that due to the fact that a traditional liposome is unstable in structure, a drug is released in advance or a carrier is agglomerated is avoided, meanwhile, it is ensured that the drug is efficiently enriched at a tumor site, and the stable and efficient anti-tumor performance is kept in the whole period of patient treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a nanoliposome antitumor drug and its preparation method. Background Technology

[0002] Currently, paclitaxel is a natural small-molecule anti-tumor drug with excellent anti-tumor activity and good inhibitory effect on a variety of tumors. However, paclitaxel has poor water solubility, low absorption efficiency, and lacks selectivity for tumor cells. While treating tumors, it also causes great damage to the patient's normal tissue cells. In order to reduce the toxic side effects of paclitaxel, polysaccharide compounds are chosen as drug carriers.

[0003] However, in existing technologies, using chitosan or bovine serum albumin as a single drug carrier is still not suitable for the complex metabolic environment of the human body. During the circulation process in the body, particle size deformation and membrane structure damage are prone to occur, leading to premature leakage of the encapsulated drug, which reduces the drug concentration at the tumor site and increases the toxicity to normal tissues. Summary of the Invention

[0004] The purpose of this invention is to provide a nanoliposome antitumor drug and its preparation method, aiming to solve the technical problem that the existing technology, which uses chitosan or bovine serum albumin as a single drug carrier, still cannot adapt to the complex metabolic environment of the human body. During the circulation process in vivo, particle size deformation and membrane structure damage are prone to occur, resulting in premature leakage of the encapsulated drug, which reduces the drug concentration at the tumor site and increases the toxicity to normal tissues.

[0005] To achieve the above objectives, the present invention provides a method for preparing a nanoliposome antitumor drug, comprising the following steps:

[0006] Synthetic imidazole can ionize lipids;

[0007] Preparation of kernel dispersion;

[0008] Imidazole-ionizable lipids, PEG2000-modified phospholipids, FA-PEG2000-phospholipids, RGD-PEG2000-phospholipids, chitin, and VEGFCs were dissolved in sterile ethanol to obtain solvent A. Solvent A was injected into the core dispersion at a rate of 0.5-1.0 mL / min under a stirring speed of 400-600 rpm and incubated at 37-40℃ for 1-2 h. Then, the mixture was repeatedly extruded 8-12 times under a pressure of 10-15 MPa using a sterile microliposome extruder with a pore size of 0.22 μm to obtain complex solution B.

[0009] Complex solution B was dialyzed for 12-16 h using a sterile dialysis bag with a molecular weight cutoff of 3.5 kDa and sterile PBS at pH 7.4. After dialyzing, a 0.6-1.0% BSA aqueous solution was added to complex solution B. The mixture of BSA aqueous solution and complex solution B was sonicated at 300 W for 15-30 min. The mixture was then stirred at 30-35 °C for 1-1.5 h. After filtration through a 0.45 μm sterile filter membrane, the filtrate was freeze-dried under sterile conditions to obtain the nanoliposome antitumor drug.

[0010] The synthesis of imidazole-ionizable lipids includes the following steps:

[0011] 1,2-dodecanediol and succinic anhydride were mixed at a molar ratio of 1:2.2, and dichloromethane was added as a solvent and 4-dimethylaminopyridine as a catalyst. The mixture was stirred at 23-27°C for 4 hours to obtain a dicarboxylic acid intermediate.

[0012] The dicarboxylic acid intermediate was mixed with imidazole-3-propionic acid at a molar ratio of 1:1.1. N,N'-dicyclohexylcarbodiimide was added to activate the carboxyl group, and the reaction was carried out at 30°C for 6 h to obtain an intermediate containing an imidazole ring.

[0013] The intermediate containing the imidazole ring was mixed with glycine at a molar ratio of 1:1.2, and the pH was adjusted to 8.0 with triethylamine. The mixture was then reacted at 40°C for 5 hours to obtain the reaction solution.

[0014] The reaction solution was concentrated under reduced pressure and then purified by silica gel column chromatography. The eluent was a mixture of chloroform and methanol in a molar ratio of 10:1. The target fraction was collected and dried under vacuum to obtain imidazole-ionizable lipids.

[0015] The preparation of the kernel dispersion includes the following steps:

[0016] Paclitaxel and vandetanil were mixed in a molar ratio of 1:(0.5~2) to obtain mixture α;

[0017] Dichloromethane and methanol were mixed in a mass ratio of 1:1.5 to obtain a mixed solvent β;

[0018] Mixture α is dissolved in mixed solvent β to obtain mixed solution γ, with a mixing mass ratio of 1:(55~65);

[0019] Phospholipids and cholesterol are added to the mixed solution γ and stirred until homogeneous. The mixture is then sonicated at 300-500W for 5-15 minutes to form a homogeneous emulsion. The total mass of phospholipids and cholesterol is 100-300 times the total mass of the mixture α, and the mass ratio of phospholipids to cholesterol is 0.8:1.

[0020] The emulsion was distilled under reduced pressure at 37~45℃ and -0.08~-0.1MPa to remove the mixed solvent β, forming a dense solid film;

[0021] Add 250mM ammonium sulfate buffer solution at pH 5.4 to a dense solid membrane, hydrate at 65-70℃ for 10-20 min, and then sonicate with a 600W probe in a sonication mode of 2s running and 3s stopping, repeating the sonication for 10-20 min to obtain a core dispersion with a particle size of 80-150nm.

[0022] The phospholipids used are either distearate phosphatidylcholine or lecithin; the chitin has a molecular weight of 50-200 kDa and a degree of deacetylation ≥85%; the BSA aqueous solution is inactivated by incubation at 60°C for 10 hours, with exogenous factors being negative; the acute toxicity test LD50 of the imidazole-ionizable lipids is... 50 >5000mg / kg.

[0023] Wherein, the drug solubility of the mixture α in the mixed solvent β is ≥99.5%.

[0024] The molar ratio of PEG2000 modified phospholipid, FA-PEG2000-phospholipid, and RGD-PEG2000-phospholipid is (1.7~12):1:(1~2), and the volume ratio of solvent A to the core dispersion is 1:(3~5).

[0025] The volume ratio of the BSA aqueous solution to the complex solution B is 1:(1~2).

[0026] The freeze-drying process is as follows: the temperature is lowered to -50℃ to -60℃ at a rate of 5℃ / min, and after holding at this temperature for 2 to 3 hours, the vacuum is drawn to 0.01 to 0.02 mbar, and the drying continues for 12 to 16 hours.

[0027] The present invention also provides a nanoliposome antitumor drug, which is prepared by the nanoliposome antitumor drug preparation method described above, wherein the nanoliposome antitumor drug comprises a three-layer spherical structure consisting of a core, a shell, and an outer layer;

[0028] The core is a hydrophobic core formed by paclitaxel and vandetanil encapsulated by a bilayer membrane of phospholipids and cholesterol.

[0029] The shell is composed of imidazole ionizable lipids, PEG2000 modified phospholipids, FA-PEG2000-phospholipids, RGD-PEG2000-phospholipids and chitin, with VEGFCs encapsulated within the shell and chitin uniformly dispersed in the shell to form a network support structure.

[0030] The outer layer is a protective layer formed by the covalent bonding of amino groups and lipid carboxyl groups in a BSA aqueous solution.

[0031] Among them, the encapsulation efficiency of the nanoliposome antitumor drugs for paclitaxel is ≥92%, for vandetanib it is ≥88%, and for VEGFC it is ≥85%.

[0032] The present invention provides a nanoliposome antitumor drug and its preparation method, which has the following beneficial effects:

[0033] 1. Overcoming the limitations of single-carrier or single-drug therapy, it utilizes a dual-drug core, a chitinous shell for network support, and an outer BSA for covalent protection. Combined with the synergistic effects of paclitaxel, vandetanib, and VEGFC, it covers the entire spectrum of tumor cell killing, angiogenesis inhibition, and microenvironment regulation. This synergistic fusion can significantly reduce drug toxicity and tumor drug resistance, improve the effectiveness of anti-tumor therapy, and solve the problems of poor stability and easy drug leakage of traditional liposomes.

[0034] 2. The stability of the carrier, drug utilization rate and tumor targeting are quantified into comprehensive therapeutic advantages. Through dynamic structural adaptation and precise target identification, the influence of complex factors such as individual differences and tumor heterogeneity can be avoided, ensuring stable therapeutic performance in different tumor treatment scenarios.

[0035] 3. Overcoming the limitations of traditional liposome preparation processes and drug encapsulation, through a precise process of "layered preparation - covalent bonding - freeze drying", it adapts to the stability requirements of nanoliposomes throughout the entire process of storage, transportation, and in vivo circulation. This avoids the premature drug release or carrier aggregation caused by the structural instability of traditional liposomes, while ensuring the efficient accumulation of drugs at the tumor site and maintaining stable and efficient anti-tumor performance throughout the entire treatment cycle of patients. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 This is a flowchart of the steps in preparing a nanoliposome antitumor drug according to the present invention. Detailed Implementation

[0038] Please see Figure 1 This invention provides a method for preparing a nanoliposome antitumor drug, comprising the following steps:

[0039] S1: Synthesize imidazole-ionizable lipids;

[0040] S2: Preparation of kernel dispersion;

[0041] S3: Imidazole-ionizable lipids, PEG2000-modified phospholipids, FA-PEG2000-phospholipids, RGD-PEG2000-phospholipids, chitin, and VEGFC were dissolved in sterile ethanol to obtain solvent A. Solvent A was injected into the core dispersion at a rate of 0.5-1.0 mL / min under a stirring speed of 400-600 rpm and incubated at 37-40℃ for 1-2 h. Then, the mixture was repeatedly extruded 8-12 times under a pressure of 10-15 MPa using a sterile microliposome extruder with a pore size of 0.22 μm to obtain complex solution B.

[0042] S4: Dialyze complex solution B using a sterile dialysis bag with a molecular weight cutoff of 3.5 kDa and sterile PBS at pH 7.4 for 12-16 h. Add 0.6-1.0% BSA aqueous solution to complex solution B after dialysis. Sonicate the mixture of BSA aqueous solution and complex solution B at 300 W for 15-30 min. Stir the mixture of BSA aqueous solution and complex solution B at 30-35 °C for 1-1.5 h. Then filter through a 0.45 μm sterile filter membrane. Freeze-dry the filtrate under sterile conditions to obtain the nanoliposome antitumor drug.

[0043] Furthermore, the synthesis of the imidazole-ionizable lipid includes the following steps:

[0044] 1,2-dodecanediol and succinic anhydride were mixed at a molar ratio of 1:2.2, and dichloromethane was added as a solvent and 4-dimethylaminopyridine as a catalyst. The mixture was stirred at 23-27°C for 4 hours to obtain a dicarboxylic acid intermediate.

[0045] The dicarboxylic acid intermediate was mixed with imidazole-3-propionic acid at a molar ratio of 1:1.1. N,N'-dicyclohexylcarbodiimide was added to activate the carboxyl group, and the reaction was carried out at 30°C for 6 h to obtain an intermediate containing an imidazole ring.

[0046] The intermediate containing the imidazole ring was mixed with glycine at a molar ratio of 1:1.2, and the pH was adjusted to 8.0 with triethylamine. The mixture was then reacted at 40°C for 5 hours to obtain the reaction solution.

[0047] The reaction solution was concentrated under reduced pressure and then purified by silica gel column chromatography. The eluent was a mixture of chloroform and methanol in a molar ratio of 10:1. The target fraction was collected and dried under vacuum to obtain imidazole-ionizable lipids.

[0048] Further, the kernel dispersion is prepared by the following steps:

[0049] Paclitaxel and vandetanil were mixed in a molar ratio of 1:(0.5~2) to obtain mixture α;

[0050] Dichloromethane and methanol were mixed in a mass ratio of 1:1.5 to obtain a mixed solvent β;

[0051] Mixture α is dissolved in mixed solvent β to obtain mixed solution γ, with a mixing mass ratio of 1:(55~65);

[0052] Phospholipids and cholesterol are added to the mixed solution γ and stirred until homogeneous. The mixture is then sonicated at 300-500W for 5-15 minutes to form a homogeneous emulsion. The total mass of phospholipids and cholesterol is 100-300 times the total mass of the mixture α, and the mass ratio of phospholipids to cholesterol is 0.8:1.

[0053] The emulsion was distilled under reduced pressure at 37~45℃ and -0.08~-0.1MPa to remove the mixed solvent β, forming a dense solid film;

[0054] Add 250mM ammonium sulfate buffer solution at pH 5.4 to a dense solid membrane, hydrate at 65-70℃ for 10-20 min, and then sonicate with a 600W probe in a sonication mode of 2s running and 3s stopping, repeating the sonication for 10-20 min to obtain a core dispersion with a particle size of 80-150nm.

[0055] Further, the phospholipid is either distearate or lecithin; the chitin has a molecular weight of 50-200 kDa and a degree of deacetylation ≥85%; the BSA aqueous solution is inactivated by incubation at 60°C for 10 hours, with exogenous factors being negative; the acute toxicity test LD50 of the imidazole-ionizable lipid is [not specified]. 50 >5000mg / kg.

[0056] Furthermore, the drug solubility of the mixture α in the mixed solvent β is ≥99.5%.

[0057] Furthermore, the molar ratio of the PEG2000 modified phospholipid, FA-PEG2000-phospholipid, and RGD-PEG2000-phospholipid is (1.7~12):1:(1~2), and the volume ratio of solvent A to the core dispersion is 1:(3~5).

[0058] Furthermore, the volume ratio of the BSA aqueous solution to the complex solution B is 1:(1~2).

[0059] Furthermore, the freeze-drying process is as follows: the temperature is lowered to -50℃ to -60℃ at a rate of 5℃ / min, and after holding at this temperature for 2 to 3 hours, a vacuum is applied to 0.01 to 0.02 mbar, and the drying continues for 12 to 16 hours.

[0060] The present invention also provides a nanoliposome antitumor drug, which is prepared by the nanoliposome antitumor drug preparation method described above, wherein the nanoliposome antitumor drug comprises a three-layer spherical structure consisting of a core, a shell, and an outer layer;

[0061] The core is a hydrophobic core formed by paclitaxel and vandetanil encapsulated by a bilayer membrane of phospholipids and cholesterol.

[0062] The shell is composed of imidazole ionizable lipids, PEG2000 modified phospholipids, FA-PEG2000-phospholipids, RGD-PEG2000-phospholipids and chitin, with VEGFCs encapsulated within the shell and chitin uniformly dispersed in the shell to form a network support structure.

[0063] The outer layer is a protective layer formed by the covalent bonding of amino groups and lipid carboxyl groups in a BSA aqueous solution.

[0064] Furthermore, the encapsulation efficiency of the nanoliposome antitumor drugs is ≥92% for paclitaxel, ≥88% for vandetanib, and ≥85% for VEGFC.

[0065] In this embodiment,

[0066] The zeta potential of the core dispersion is -20 to -30 mV, and the particle size variation coefficient is ≤15%.

[0067] When extruding complex solution B using a micro liposome extruder, the system temperature is maintained at 37~39℃ during the extrusion process using a constant temperature water bath; after ultrasonication of the BSA aqueous solution and complex solution B, the BSA binding rate on the surface of the complex is ≥90%;

[0068] During the freeze-drying process, 2% mannitol was added to the filtrate as a freeze-drying protectant. The mass ratio of mannitol to filtrate was 1:1. The reconstitution rate of the dry powder after freeze-drying was ≥95%, and the bacterial endotoxin in the reconstituted solution was ≤0.5 EU / mL.

[0069] In an environment with pH 5.0–5.5, the protonation rate of imidazole-ionized lipids is ≥80%, the cumulative release rate of paclitaxel within 24 hours is ≥60%, and the cumulative release rate of vandetanib is ≥55%; in an environment with pH 7.4, the cumulative drug release rate within 24 hours is ≤10%; the long-term stability (25℃ / 60%RH) is ≤10% in particle size change within 6 months.

[0070] The chitin constitutes 2% to 5% of the mass of the shell;

[0071] Collecting the target fraction refers to collecting the eluent fraction that contains only the target product (imidazolium-ionizable lipid) after separation by silica gel column chromatography.

[0072] In the preparation process of nanoliposome antitumor drugs: Paclitaxel and vandetanib are dissolved in a mixed solvent of dichloromethane and methanol, and then phospholipids and cholesterol are added. The mixture is stirred and sonicated to form an emulsion, which lays the basis for the core components. The emulsion is then distilled under reduced pressure to remove the dichloromethane and methanol solvents, forming a dense solid film. This dense solid film is a "phospholipid-cholesterol dense solid film". The dense solid film aggregates due to solvent evaporation, forming a bilayer membrane precursor that can encapsulate the drug. Ammonium sulfate buffer is added to the dense solid film to allow it to redisperse into a bilayer membrane structure. The phospholipid-cholesterol dense solid film spontaneously encapsulates the hydrophobic paclitaxel and vandetanib to form independent core particles, which are the innermost layers of the three-layer structure.

[0073] Solvent A contains all the functional components of the shell layer. Solvent A is injected into the core dispersion. Ethanol is used as a good solvent to promote the migration of lipid components (imidazolium-ionizable lipids, PEG2000 modified phospholipids, FA-PEG2000-phospholipids, RGD-PEG2000-phospholipids) in solvent A to the surface of the dense phospholipid-cholesterol solid membrane of the core layer. The lipophilic ends of the lipids bind to the core layer, while the hydrophilic ends are exposed. At the same time, chitin dissolves in ethanol and enters the aqueous phase, uniformly dispersing between the lipids to form a network structure. The lipids and chitin together form a network structure. VEGFCs are "retained" and wrapped in the network formed by the lipids and chitin, initially forming a composite particle of "core layer + shell layer". After repeated extrusion, the composite particles are homogenized to form a stable "core layer + shell layer" bilayer structure, which is the complex solution B.

[0074] Complex solution B was dialyzed using a dialysis bag to remove unbound free components, while simultaneously exposing the lipid carboxyl groups on the shell surface to create conditions for subsequent binding with BSA. BSA was added to complex solution B and subjected to sonication to promote contact between BSA and the shell surface. Complex solution B was then stirred with BSA. BSA contains a large number of amino groups, which can undergo amidation (covalent bonding) with the carboxyl groups of lipids on the shell surface, allowing BSA molecules to uniformly form a coating layer on the "core-shell" surface. After filtration through a filter membrane, the solution was freeze-dried to allow the BSA protective layer to stably adhere to the shell surface, ultimately forming a three-layer nanoliposome antitumor drug consisting of a "core-shell-outer layer".

[0075] Implementation Example Design:

[0076] Common materials (reagents): 1,2-dodecanediol (≥98%), succinic anhydride (analytical grade), 4-dimethylaminopyridine (≥99%), imidazole-3-propionic acid (≥98%), N,N'-dicyclohexylcarbodiimide (analytical grade), glycine (analytical grade), paclitaxel (≥99%), vandetanil (≥98%), phospholipids (≥99%), cholesterol (analytical grade), PEG2000 modified phospholipids (≥98%), FA-PEG2000-phospholipids (≥97%), RGD-PEG2000-phospholipids (≥97%), chitin (50~200kDa, degree of deacetylation ≥85%), VEGFC (≥95%), BSA (analytical grade), mannitol (analytical grade);

[0077] Common materials (instruments): magnetic stirrer, ultrasonic probe (600W), micro liposome extruder (0.22μm), dialysis bag (3.5kDa), freeze dryer, particle size / Zeta potential analyzer, HPLC, small animal tumor model (Balb / c nude mice, inoculated with A549 lung cancer cells);

[0078] Common detection indicators:

[0079] Basic performance: particle size (nm), zeta potential (mV), drug encapsulation efficiency (paclitaxel / vandetanil / VEGFC, %), reconstitution rate (%), bacterial endotoxin (EU / mL).

[0080] Stability: Particle size change rate (%) after 6 months of storage at 25℃ / 60%RH, and drug leakage rate (%) after 12 months of storage at 4℃;

[0081] Functional performance: Cumulative drug release rate (%) in pH 5.2 environment over 24 hours, cumulative drug release rate (%) in pH 7.4 environment over 24 hours, tumor inhibition rate in nude mice (%, after 21 days of administration), acute toxicity in mice (LD50). 50 (mg / kg).

[0082] Example 1:

[0083] 1,2-dodecanediol and succinic anhydride were mixed at a molar ratio of 1:2.2, and dichloromethane was added as a solvent and 4-dimethylaminopyridine as a catalyst. The mixture was stirred at 25°C for 4 hours to obtain a dicarboxylic acid intermediate.

[0084] The dicarboxylic acid intermediate was mixed with imidazole-3-propionic acid at a molar ratio of 1:1.1. N,N'-dicyclohexylcarbodiimide was added to activate the carboxyl group, and the reaction was carried out at 30°C for 6 h to obtain an intermediate containing an imidazole ring.

[0085] The intermediate containing the imidazole ring was mixed with glycine at a molar ratio of 1:1.2, and the pH was adjusted to 8.0 with triethylamine. The mixture was then reacted at 40°C for 5 hours to obtain the reaction solution.

[0086] The reaction solution was concentrated under reduced pressure and then purified by silica gel column chromatography. The eluent was a mixture of chloroform and methanol in a molar ratio of 10:1. The target fraction was collected and dried under vacuum to obtain imidazole-ionizable lipids.

[0087] Paclitaxel and vandetanil were mixed in a molar ratio of 1:1 to obtain mixture α;

[0088] Dichloromethane and methanol were mixed in a mass ratio of 1:1.5 to obtain a mixed solvent β;

[0089] The mixture α was dissolved in the mixed solvent β to obtain the mixed solution γ, with a mixing mass ratio of 1:60;

[0090] Phospholipids and cholesterol were added to the mixed solution γ and stirred until homogeneous. The mixture was then sonicated at 400W for 10 minutes to form a homogeneous emulsion. The total mass of phospholipids and cholesterol was 200 times the total mass of the mixture α, and the mass ratio of phospholipids to cholesterol was 0.8:1.

[0091] The emulsion was distilled under reduced pressure at 41℃ and -0.09MPa to remove the mixed solvent β, forming a dense solid film;

[0092] Add 250mM ammonium sulfate buffer solution at pH 5.4 to a dense solid membrane, hydrate at 68℃ for 20 min, and then sonicate with a 600W probe. The sonication mode is 2s run, 3s stop, and 20 min cycle sonication to obtain a core dispersion with a particle size of 80~150nm.

[0093] Imidazole-ionizable lipids, PEG2000-modified phospholipids, FA-PEG2000-phospholipids, RGD-PEG2000-phospholipids, chitin, and VEGFC were dissolved in sterile ethanol to obtain solvent A. The molar ratio of PEG2000-modified phospholipids, FA-PEG2000-phospholipids, and RGD-PEG2000-phospholipids was 5:1:1.5. The molecular weight of chitin was 100 kDa (accounting for 3% of the shell layer). Solvent A was injected into the core dispersion at a rate of 1.0 mL / min under a stirring speed of 600 rpm. After incubation at 40 °C for 2 h, the mixture was repeatedly extruded 10 times under a pressure of 12 MPa using a sterile microliposome extruder with a pore size of 0.22 μm to obtain complex solution B.

[0094] Complex solution B was dialyzed for 16 h using a sterile dialysis bag with a molecular weight cutoff of 3.5 kDa and sterile PBS at pH 7.4. After dialyzing, 0.8% BSA aqueous solution was added to complex solution B, with a volume ratio of complex solution B to BSA of 1.5:1. The mixture of BSA aqueous solution and complex solution B was sonicated at 300 W for 30 min, and then stirred at 35 °C for 1.5 h. After filtration through a 0.45 μm sterile filter membrane, the filtrate was freeze-dried under sterile conditions (-55 °C for 2.5 h, 0.015 mbar vacuum) to obtain the nanoliposome antitumor drug.

[0095] Test results: Particle size change rate ≤8% over 6 months; tumor inhibition rate (21 days) 78.3%; LD 50 >5000mg / kg.

[0096] Example 2:

[0097] Key differences:

[0098] The molar ratio of PEG2000 modified phospholipids, FA-PEG2000-phospholipids, and RGD-PEG2000-phospholipids is 2:1:2.

[0099] The molar ratio of paclitaxel to vandetanil is 1:2;

[0100] The remaining parameters and procedures are the same as in Example 1.

[0101] Test results: Particle size change rate ≤9% over 6 months; tumor inhibition rate (21 days) 82.5%; LD 50 >5000mg / kg.

[0102] Example 3:

[0103] Key differences:

[0104] Chitin has a molecular weight of 200 kDa (accounting for 5% of the shell).

[0105] The BSA concentration is 1.0%, and the volume ratio of complex solution B to BSA is 1:1;

[0106] The freeze-drying conditions were: -60℃ for 3 hours, vacuum 0.01 mbar;

[0107] The remaining parameters and procedures are the same as in Example 1.

[0108] Test results: Particle size change rate ≤5% over 6 months; drug leakage rate ≤3% over 12 months; tumor inhibition rate (21 days) 76.9%.

[0109] Comparative Example 1:

[0110] Key differences:

[0111] The imidazole-ionizable lipid in solvent A was replaced with a common cationic lipid (DOTAP), and the remaining parameters and procedures were the same as in Example 1.

[0112] Test results: Tumor inhibition rate (21 days) 52.1%; Particle size change rate ≤10% over 6 months.

[0113] Comparative Example 2:

[0114] Key differences:

[0115] Chitin was removed from solution A, and the remaining parameters and procedures were the same as in Example 1.

[0116] Test results:

[0117] Tumor inhibition rate (21 days) 70.2%; particle size change rate (6 months) 28.5%.

[0118] Comparative Example 3:

[0119] Key differences:

[0120] The BSA modification step was removed, and the cells were directly freeze-dried after dialysis. The remaining parameters and procedures were the same as in Example 1.

[0121] Test results: Tumor inhibition rate (21 days) 61.5%; Particle size change rate ≤9% over 6 months.

[0122] Comparative Example 4:

[0123] Key differences:

[0124] The core contains only paclitaxel (molar amount is the total molar amount of paclitaxel + vandetanib in Example 1), and the other parameters and procedures are the same as in Example 1.

[0125] Test results: Tumor inhibition rate (21 days) 56.8%; Particle size change rate ≤7% over 6 months.

[0126] Summary of conclusions:

[0127] Group Differentiation technology Tumor inhibition rate (%) 6-month stability (particle size change rate %) Example 1 pH responsiveness, stability, and targeting synergy 78.3 ≤8 Example 2 High tumor-targeting enrichment rate and strong anti-angiogenic effect 82.5 ≤9 Example 3 Optimal long-term storage stability and low drug leakage rate 76.9 ≤5 Comparative Example 1 Loss of pH response, increased toxicity, and decreased inhibition rate 52.1 ≤10 Comparative Example 2 The shell is unsupported, the particles are agglomerated, and the encapsulation efficiency of VEGFCs is low. 70.2 28.5 Comparative Example 3 Poor reconstitution, short circulation, and high toxicity to normal tissues. 61.5 ≤9 Comparative Example 4 No synergistic effect, high tumor recurrence rate 56.8 ≤7

[0128] As can be seen from the above three embodiments and four comparative examples, the "three-layer structure (core dual-drug + shell network support + BSA outer layer) + imidazole ionizable lipid pH response" of this application is the core to achieve high stability, low toxicity and strong synergistic anti-tumor effect. The absence of any key component or process will lead to a significant decrease in performance.

[0129] The above description discloses only one preferred embodiment of the present invention, and should not be construed as limiting the scope of the present invention. Those skilled in the art will understand that all or part of the processes of the above embodiments can be implemented, and equivalent changes made in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. A method for preparing a nanoliposome antitumor drug, characterized in that, Includes the following steps: Synthetic imidazole can ionize lipids; Preparation of kernel dispersion; Imidazole-ionizable lipids, PEG2000-modified phospholipids, FA-PEG2000-phospholipids, RGD-PEG2000-phospholipids, chitin, and VEGFCs were dissolved in sterile ethanol to obtain solvent A. Solvent A was injected into the core dispersion at a rate of 0.5-1.0 mL / min under a stirring speed of 400-600 rpm and incubated at 37-40℃ for 1-2 h. Then, the mixture was repeatedly extruded 8-12 times under a pressure of 10-15 MPa using a sterile microliposome extruder with a pore size of 0.22 μm to obtain complex solution B. Complex solution B was dialyzed for 12-16 h using a sterile dialysis bag with a molecular weight cutoff of 3.5 kDa and sterile PBS at pH 7.

4. After dialyzing, a 0.6-1.0% BSA aqueous solution was added to complex solution B. The mixture of BSA aqueous solution and complex solution B was sonicated at 300 W for 15-30 min. The mixture was then stirred at 30-35 °C for 1-1.5 h. After filtration through a 0.45 μm sterile filter membrane, the filtrate was freeze-dried under sterile conditions to obtain the nanoliposome antitumor drug.

2. The method for preparing a nanoliposome antitumor drug according to claim 1, wherein the synthesized imidazole-ionizable lipid is characterized in that, Includes the following steps: 1,2-dodecanediol and succinic anhydride were mixed at a molar ratio of 1:2.2, and dichloromethane was added as a solvent and 4-dimethylaminopyridine as a catalyst. The mixture was stirred at 23-27°C for 4 hours to obtain a dicarboxylic acid intermediate. The dicarboxylic acid intermediate was mixed with imidazole-3-propionic acid at a molar ratio of 1:1.

1. N,N'-dicyclohexylcarbodiimide was added to activate the carboxyl group, and the reaction was carried out at 30°C for 6 h to obtain an intermediate containing an imidazole ring. The intermediate containing the imidazole ring was mixed with glycine at a molar ratio of 1:1.2, and the pH was adjusted to 8.0 with triethylamine. The mixture was then reacted at 40°C for 5 hours to obtain the reaction solution. The reaction solution was concentrated under reduced pressure and then purified by silica gel column chromatography. The eluent was a mixture of chloroform and methanol in a molar ratio of 10:

1. The target fraction was collected and dried under vacuum to obtain imidazole-ionizable lipids.

3. The method for preparing a nanoliposome antitumor drug as described in claim 1, wherein the core dispersion is prepared, characterized in that, Includes the following steps: Paclitaxel and vandetanil were mixed in a molar ratio of 1:(0.5~2) to obtain mixture α; Dichloromethane and methanol were mixed in a mass ratio of 1:1.5 to obtain a mixed solvent β; Mixture α is dissolved in mixed solvent β to obtain mixed solution γ, with a mixing mass ratio of 1:(55~65); Phospholipids and cholesterol are added to the mixed solution γ and stirred until homogeneous. The mixture is then sonicated at 300-500W for 5-15 minutes to form a homogeneous emulsion. The total mass of phospholipids and cholesterol is 100-300 times the total mass of the mixture α, and the mass ratio of phospholipids to cholesterol is 0.8:

1. The emulsion was distilled under reduced pressure at 37~45℃ and -0.08~-0.1MPa to remove the mixed solvent β, forming a dense solid film; Add 250mM ammonium sulfate buffer solution at pH 5.4 to a dense solid membrane, hydrate at 65-70℃ for 10-20 min, and then sonicate with a 600W probe in a sonication mode of 2s running and 3s stopping, repeating the sonication for 10-20 min to obtain a core dispersion with a particle size of 80-150nm.

4. The method for preparing a nanoliposome antitumor drug as described in claim 1, characterized in that, The phospholipid is either distearate phosphatidylcholine or lecithin; the chitin has a molecular weight of 50-200 kDa and a degree of deacetylation ≥85%; the BSA aqueous solution inactivation process involves incubation at 60°C for 10 hours, with exogenous factors being negative; the acute toxicity test LD50 of the imidazole-ionizable lipid is... 50 >5000mg / kg.

5. The method for preparing a nanoliposome antitumor drug as described in claim 3, characterized in that, The drug solubility of the mixture α in the mixed solvent β is ≥99.5%.

6. The method for preparing a nanoliposome antitumor drug as described in claim 1, characterized in that, The molar ratio of the PEG2000 modified phospholipid, FA-PEG2000-phospholipid, and RGD-PEG2000-phospholipid is (1.7~12):1:(1~2), and the volume ratio of solvent A to the core dispersion is 1:(3~5).

7. The method for preparing a nanoliposome antitumor drug as described in claim 1, characterized in that, The volume ratio of the BSA aqueous solution to the complex solution B is 1:(1~2).

8. The method for preparing a nanoliposome antitumor drug as described in claim 1, characterized in that, The freeze-drying process is as follows: the temperature is lowered to -50℃ to -60℃ at a rate of 5℃ / min, and after holding at this temperature for 2 to 3 hours, the vacuum is drawn to 0.01 to 0.02 mbar, and the drying is continued for 12 to 16 hours.

9. A nanoliposome antitumor drug, prepared by the method for preparing nanoliposome antitumor drugs according to any one of claims 1-6, characterized in that, The nanoliposome antitumor drug comprises a three-layered spherical structure consisting of a core, a shell, and an outer layer. The core is a hydrophobic core formed by paclitaxel and vandetanil encapsulated by a bilayer membrane of phospholipids and cholesterol. The shell is composed of imidazole ionizable lipids, PEG2000 modified phospholipids, FA-PEG2000-phospholipids, RGD-PEG2000-phospholipids and chitin, with VEGFCs encapsulated within the shell and chitin uniformly dispersed in the shell to form a network support structure. The outer layer is a protective layer formed by the covalent bonding of amino groups and lipid carboxyl groups in a BSA aqueous solution.

10. The nanoliposome antitumor drug as described in claim 9, characterized in that, The nanoliposome antitumor drugs have an encapsulation efficiency of ≥92% for paclitaxel, ≥88% for vandetanib, and ≥85% for VEGFC.

Images