CN116178699A - Drug delivery carrier material capable of promoting drug to enter cells, and preparation method and application thereof - Google Patents

Drug delivery carrier material capable of promoting drug to enter cells, and preparation method and application thereof Download PDF

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CN116178699A
CN116178699A CN202211104370.0A CN202211104370A CN116178699A CN 116178699 A CN116178699 A CN 116178699A CN 202211104370 A CN202211104370 A CN 202211104370A CN 116178699 A CN116178699 A CN 116178699A
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polyethylene glycol
docosahexaenoic acid
drug
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peg
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王杭祥
陈晓娜
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First Affiliated Hospital of Zhejiang University School of Medicine
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    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/331—Polymers modified by chemical after-treatment with organic compounds containing oxygen
    • C08G65/332—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing carboxyl groups, or halides, or esters thereof
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    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
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    • C08G65/3332—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing carboxamide group
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    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The invention discloses a drug delivery carrier material capable of promoting drug to enter cells, and a preparation method and application thereof, and belongs to the technical field of nano drug carrier materials. The drug delivery carrier material is a docosahexaenoic acid-polyethylene glycol conjugate and comprises a hydrophilic segment and a hydrophobic segment, wherein the hydrophilic segment is a methoxy polyethylene glycol molecule with the molecular weight of 500-10000, and the hydrophobic segment is 1-4 docosahexaenoic acid molecules. According to the invention, polyethylene glycol and docosahexaenoic acid are directly or indirectly covalently coupled, so that the interaction between PEG nanoparticles and tumor cell membranes can be regulated, and the cell entry speed of the nanoparticles is remarkably promoted. The docosahexaenoic acid-polyethylene glycol conjugate can be used for preparing drug-loaded nano-micelles, modifying nano self-assemblies to improve stability, preparing liposome and the like, and a new way is opened up for in-vivo delivery of nano-drugs and construction of green nano-drugs with high dispersion, good stability and guaranteed safety.

Description

Drug delivery carrier material capable of promoting drug to enter cells, and preparation method and application thereof
Technical Field
The invention relates to the technical field of nano drug carrier materials, in particular to a drug delivery carrier material capable of promoting drug to enter cells, and a preparation method and application thereof.
Background
The incidence and mortality of malignant tumors have increased over the last decade, and have become the second leading cause of death worldwide. The current tumor treatment methods are as follows: surgery, radiation therapy, chemotherapy, targeted therapy, and the like. The targeting drug is a corresponding therapeutic drug designed aiming at a tumor target point, and the drug can specifically select cancerogenic sites to combine with each other to act when entering the body, so that tumor cells can specifically die, normal tissue cells around the tumor can not be affected, and the targeting drug is accurate and mild and has small side effect.
However, most of the current clinical medicines for treating malignant tumors have the problems of off-target, drug resistance, poor toxicity of different degrees and the like. In addition, clinical first-line anticancer drugs including taxanes, camptothecins, various molecular targeted drugs and the like often have extremely low water solubility, so that the drugs need to be administered orally or other auxiliary materials to assist in vivo delivery, and the physical burden of patients is increased. Therefore, new strategies are urgently needed to improve the in vivo performance of drugs.
Drug delivery systems provide methods for improving the pharmacokinetics and modulating the biodistribution of therapeutic drugs, delivering the drugs into target cells or tumor microenvironments to increase their efficacy while reducing systemic adverse effects. Various biomaterial-based delivery systems, such as conjugated drugs, liposomes, nanoparticles, and cell-derived systems, have been developed in succession.
Development and functional engineering of polyethylene glycol (PEG) based materials is one of the key developments in nano-delivery systems in recent years. The polyethylene glycol material is used for carrying out surface modification on the nano particles, so as to improve the water solubility of the medicine, and the formed PEG layer can prevent the absorption of serum proteins and the ingestion of mononuclear phagocyte systems, so that the internal circulation time is prolonged. Polyethylene glycol phospholipid is a nano material which is widely applied at present, such as 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-MPEG 2000), has amphipathy of phospholipid and hydrophilic high molecular characteristics of PEG, and has been rapidly developed in recent years in drug carriers such as long-circulating liposome, high molecular micelle, long-circulating nanoparticle and the like.
However, polyethylene glycol (PEG) -modified nanoparticles, although helping to avoid the clearance of macrophages, also reduce the uptake of drugs into target cells, such as tumor cells, which is unfavorable for the rapid arrival of anticancer drugs at the site of action, and cannot achieve the effect of rapidly killing tumor cells. Furthermore, the presence of phospholipids in the pegylated phospholipids tends to place the nanoparticle with a strong negative charge, further impeding the entry of the nanoparticle into the cell by reducing the interaction of the cell membrane with the nanoparticle. Therefore, how to improve the drug carrier material, while maintaining the advantages of PEG, accelerate the uptake of tumor cells into nano particles, and finally, actually improve the treatment effect of anticancer drugs is a problem to be solved by the technicians in the field.
Disclosure of Invention
The invention aims to provide a novel drug carrier material which is used for drug delivery, can improve the water solubility of a hydrophobic drug, prolong the in vivo circulation time of the drug, and improve the uptake of target cells such as tumor cells to the drug, thereby actually improving the therapeutic effect of the drug.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides a docosahexaenoic acid-polyethylene glycol conjugate, which comprises a hydrophilic segment and a hydrophobic segment, wherein the hydrophilic segment is methoxy polyethylene glycol molecules with the molecular weight of 500-10000, and the hydrophobic segment is 1-4 docosahexaenoic acid molecules.
According to the invention, a non-toxic polyethylene glycol chain (PEG) with good biocompatibility and biodegradability is directly connected with polyunsaturated fatty acid (DHA) or coupled through a linker, so that the DHA-PEG conjugate is obtained. The conjugate is coupled by chemical crosslinking method, such as esterification, amidation, etc.
Preferably, the structural formula of the DHA-PEG conjugate is shown in any one of formulas (I) to (X),
Figure BDA0003840915830000021
Figure BDA0003840915830000031
wherein R is 1 The structural formula is as follows:
Figure BDA0003840915830000032
R 2 the structural formula is as follows:
Figure BDA0003840915830000033
n is an integer of 10 to 230.
Preferably, n is an integer of 23 to 227, and more preferably, n is 23, 45, 114, 227.
The invention also provides a preparation method of the docosahexaenoic acid-polyethylene glycol (DHA-PEG) conjugate, which comprises the following steps: under the action of a condensing agent and a catalyst, performing condensation reaction on docosahexaenoic acid and methoxy polyethylene glycol with a structural formula shown as formula (XI) and R being hydroxyl or amino to obtain DHA-PEG conjugate;
or under the action of condensing agent and catalyst, firstly, making docosahexaenoic acid and a connector compound with two or more connecting groups, wherein the connecting groups are selected from hydroxyl, amino and carboxyl; then the reaction product is condensed with methoxy polyethylene glycol with structural formula as shown in formula (XI) and R is hydroxyl, amino or carboxyl to prepare DHA-PEG conjugate,
Figure BDA0003840915830000034
wherein n is an integer of 10 to 230.
Preferably, the linker compound is one of ethylene glycol, propylene glycol, butylene glycol, ethylene diamine, propylene diamine, butylene diamine, lysine, and isopropylidene glycerol.
Further, the structural formula of the linker compound is as follows:
Figure BDA0003840915830000041
further preferably, the DHA-PEG conjugate with the structural formulas (III) - (V) is prepared by performing esterification reaction on docosahexaenoic acid and equivalent ethylene glycol, propylene glycol or butanediol under the action of a condensing agent and a catalyst, and then performing esterification reaction on the docosahexaenoic acid and equivalent methoxypolyethylene glycol-carboxyl (mPEG-COOH) with the structural formula (XI).
Or under the action of condensing agent and catalyst, first making docosahexaenoic acid and equivalent ethylenediamine, propylenediamine or butylenediamine undergo the process of amidation reaction, then making them undergo the process of amidation reaction with equivalent methoxypolyethylene glycol-carboxyl group (mPEG-COOH) whose structural formula is shown in formula (XI) so as to obtain the DHA-PEG conjugate whose structural formula is shown in formulas (VI) - (VIII).
Or under the action of condensing agent and catalyst, firstly, the docosahexaenoic acid is reacted with N-hydroxysuccinimide to synthesize the docosahexaenoic acid modified by succinimidyl, and then the docosahexaenoic acid is reacted with D-lysine in a ratio of 2:1, and then reacting with methoxypolyethylene glycol-amino group (mPEG-NH) having the structural formula (XI) 2 ) Carrying out amidation reaction to obtain DHA-PEG conjugate with structural formula shown In (IX).
Or under the action of condensing agent and catalyst, firstly synthesizing methoxy polyethylene glycol-carboxyl (mPEG-COOH) with structural formula as shown in formula (XI) and isopropylidene glycerol through esterification reaction to obtain polyethylene glycol-isopropylidene glycerol, then removing protecting groups under the catalysis of organic acid to expose two hydroxyl sites, and further reacting with two times of equivalent docosahexaenoic acid to obtain DHA-PEG conjugate with structural formula as shown in formula (X).
Preferably, the condensing agent is 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC), and the catalyst is 4-Dimethylaminopyridine (DMAP) or N, N-Diisopropylethylamine (DIEA).
Preferably, the reaction solvent is dichloromethane or chloroform.
Preferably, the reaction temperature is 40 to 50 ℃. The reaction time is 2-72 hours.
Studies of the present invention have shown that the above-described docosahexaenoic acid-polyethylene glycol conjugates as drug carrier materials can be used in a variety of applications, including but not limited to: preparing a drug-loaded nano-micelle by encapsulating a hydrophobic anticancer drug; modifying the prodrug self-assembly body to play a role in stabilizing and optimizing the particle performance; as a liposome constituent, a liposome is prepared by liposome technology.
The endocytosis experiment shows that the endocytosis speed of the nanoparticle prepared by the docosahexaenoic acid-polyethylene glycol conjugate is far faster than that of the commercial material DSPE-PEG 2k The prepared nano particles prove that the DHA-PEG conjugate has the effect of promoting the medicine to enter cells. The cytotoxicity experimental result further shows that the DHA-PEG conjugate modified prodrug self-assembly body has higher anti-tumor activity. Animal toxicity experiment results show that the DHA-PEG conjugate has biological safety as a drug carrier.
Thus, the present invention provides the use of a docosahexaenoic acid-polyethylene glycol conjugate as a drug delivery carrier material in the preparation of a pharmaceutical formulation.
Specifically, the invention provides application of the docosahexaenoic acid-polyethylene glycol conjugate to preparation of drug-loaded nano-micelle, and the drug-loaded nano-micelle is prepared by encapsulating hydrophobic drugs (such as anti-tumor drugs such as cabazitaxel, and the like), so that the dissolving capacity of the hydrophobic drugs can be remarkably improved, intravenous injection and the like of the drugs can be realized, and the bioavailability of the drugs can be improved.
The application comprises: and respectively dissolving the docosahexaenoic acid-polyethylene glycol conjugate and the hydrophobic drug in an organic solvent, mixing the two, and injecting the mixture into an aqueous phase to form the drug-loaded nano micelle. Alternatively, the nanoparticles are separated from the mixed liquor.
Preferably, in the method, the concentration of the docosahexaenoic acid-polyethylene glycol conjugate in the organic solvent is 0.01-80.00 mg/mL, the concentration of the hydrophobic drug is 0.01-2.00 mg/mL, and the mass ratio is 1-40: 1, self-assembling to prepare nano particles.
The nano particles with average particle diameter of 5-200nm can be prepared by the method.
The invention also provides an application of the docosahexaenoic acid-polyethylene glycol conjugate modified prodrug self-assembly body, which comprises the following steps: and respectively dissolving the docosahexaenoic acid-polyethylene glycol conjugate and the prodrug modified by fatty acid in an organic solvent, mixing the two, and injecting the mixture into an aqueous phase to form a self-assembly body of the prodrug modified by the docosahexaenoic acid-polyethylene glycol conjugate.
The fatty acid modified prodrug has self-assembly capability, and the docosahexaenoic acid-polyethylene glycol conjugate participates in the self-assembly process of forming the nanoparticle and is modified on the nanoparticle, so that the effect of stabilizing and optimizing the particle performance is achieved.
Preferably, the fatty acid is an unsaturated fatty acid, such as docosahexaenoic acid (DHA).
Preferably, in the method, the concentration of the docosahexaenoic acid-polyethylene glycol conjugate in the organic solvent is 0.01-60.00 mg/ml, the concentration of the prodrug is 0.01-3.00 mg/ml, and the mass ratio is 1-20: 1 preparing nano particles.
The nano particles with average particle diameter of 30-300nm can be prepared by the method.
The invention also provides a liposome prepared by using the docosahexaenoic acid-polyethylene glycol conjugate as a liposome composition through a liposome technology. The liposome is formed by docosahexaenoic acid-polyethylene glycol conjugate, lecithin and cholesterol.
The application comprises: lecithin, cholesterol and a docosahexaenoic acid-polyethylene glycol conjugate are added into a solution containing drug molecules, and self-assembly is carried out to form the drug-entrapped liposome.
The raw materials dissolved in the organic solvent are mixed and injected into the water phase, and then the uniformly dispersed drug-carrying liposome is obtained by utilizing the organic solvent evaporation method or the film dispersion method.
Preferably, the mass ratio of lecithin, cholesterol, DHA-PEG and the medicine is 10-40: 1 to 8:3:1 to 10.
The nano particles with average particle diameter of 20-1000nm can be prepared by the method.
In the preparation process of the nano particles, the organic solvent is selected from one or more of acetone, methanol, ethanol, methylene dichloride and dimethyl sulfoxide.
Furthermore, the drug preparation is a drug preparation for encapsulating an anti-tumor drug, and a nano drug delivery system for targeting tumor cells is formed by constructing carrier materials such as the anti-tumor drug and the docosahexaenoic acid-polyethylene glycol conjugate.
The docosahexaenoic acid-polyethylene glycol conjugate prepared by the invention can balance the effect of PEG on tumor cell membranes and promote the cell entry speed of nano particles, thereby increasing the accumulation of the drug at the tumor part and better playing the anti-tumor effect.
The invention has the beneficial effects that:
(1) The invention directly or indirectly covalently couples polyethylene glycol and docosahexaenoic acid, and the obtained docosahexaenoic acid-polyethylene glycol conjugate can be used as a nano carrier for various purposes, including but not limited to preparing nano micelle to improve the solubility of hydrophobic drugs, modifying nano self-assembly to improve the stability, preparing liposome and the like. Opens up a new way for in vivo delivery of nano-drugs and construction of green nano-drugs with high dispersion, good stability and guaranteed safety.
(2) Docosahexaenoic acid has unique advantages in tumor drug delivery: as a nutrient, the nanoparticle can promote the uptake of the nanoparticle by tumor cells with high energy consumption. Therefore, the chain segment is used for balancing the interaction between PEG and tumor cell membrane, can effectively promote the cell entering speed of nano particles and improve the anti-tumor effect of the medicine.
(3) The polyethylene glycol is cheap and easy to obtain, can be produced in large scale, has easily controlled molecular weight and good physicochemical properties, and the preparation process is relatively simple, easy to realize large-scale production and has high product yield.
(4) The docosahexaenoic acid-polyethylene glycol conjugate is metabolized in the human body to release only the metabolite of the docosahexaenoic acid and the polyethylene glycol. Docosahexaenoic acid is used as polyunsaturated fatty acid, is a necessary nutrient substance for human body, and is harmless to human body. Polyethylene glycol is a polymer which is nontoxic and has good biocompatibility and biodegradability. Therefore, the in vivo safety of the docosahexaenoic acid-polyethylene glycol conjugate is guaranteed, the safety burden brought by a carrier material is reduced, and the method is beneficial to clinical transformation.
Drawings
FIG. 1 shows the synthetic route for docosahexaenoic acid-polyethylene glycol conjugate 1 of example 1.
FIG. 2 is a nuclear magnetic resonance spectrum of docosahexaenoic acid-polyethylene glycol conjugate 1 of example 1.
FIG. 3 shows the synthetic route for the docosahexaenoic acid-polyethylene glycol conjugate I-VI of example 2.
FIG. 4 is a synthetic route for docosahexaenoic acid-polyethylene glycol conjugate 2 of example 3.
FIG. 5 is a nuclear magnetic resonance spectrum of a lysine-docosahexaenoic acid conjugate of example 3;
FIG. 6 is a nuclear magnetic resonance spectrum of docosahexaenoic acid-polyethylene glycol conjugate 2 of example 3.
FIG. 7 shows the synthetic route for docosahexaenoic acid-polyethylene glycol conjugate 3 of example 4.
Fig. 8 is a particle size distribution of the drug-loaded nano-micelle of example 5.
Fig. 9 is a transmission electron microscope image of the drug-loaded nano-micelle of example 5.
FIG. 10 shows the synthetic route for DHA-FL118 in example 6.
FIG. 11 is a nuclear magnetic resonance spectrum of DHA-FL118 in example 6.
FIG. 12 shows DHA in example 6 2 Particle size distribution of PEG-modified prodrug self-assemblies.
FIG. 13 is DH of example 6A 2 Transmission electron microscopy of PEG-modified prodrug self-assemblies.
FIG. 14 is a particle size distribution of the drug-loaded liposome of example 7.
FIG. 15 is a transmission electron microscope image of the drug-loaded liposome of example 7.
FIG. 16 shows DF and DF/DHA in example 8 2 In vitro stability test of PEG.
FIG. 17 is an endocytosis assay of fluorescent-labeled nanomicelles in tumor cells in example 9.
FIG. 18 is an endocytosis assay of the fluorescently labeled prodrug self-assemblies of example 10 in tumor cells.
FIG. 19 shows DHA in example 11 2 In vitro cytotoxicity assay of PEG-modified prodrug self-assemblies.
FIG. 20 is an in vitro toxicity test of docosahexaenoic acid-polyethylene glycol conjugate 2 of example 12.
FIG. 21 is a graph showing the effect of docosahexaenoic acid-polyethylene glycol conjugate 2 of example 13 on mouse body weight.
FIG. 22 is a graph showing the effect of docosahexaenoic acid-polyethylene glycol conjugate 2 of example 13 on blood cells.
Detailed Description
The invention will be further illustrated with reference to specific examples. The following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
mPEG 2k -NH 2 Purchased from Shanghai Asia also Biotechnology Co., ltd;
docosahexaenoic acid is available from sigma-aldrich, U.S. CAS:6217-54-5;
n-hydroxysuccinimide was purchased from TCI Techniai (Shanghai) chemical industry development Co., ltd., CAS:6066-82-6;
d-lysine was purchased from TCI Techniaria (Shanghai) chemical industry development Co., ltd., CAS:923-27-3;
cabazitaxel is purchased from Shanghai Tao Su Biochemical technology Co., ltd., CAS:183133-96-2;
FL118 is purchased from Shanghai Yao pharmaceutical technology Co., ltd., CAS:135415-73-5.
EXAMPLE 1 Synthesis of docosahexaenoic acid-polyethylene glycol conjugate 1 (DHA-PEG)
The synthetic route is shown in FIG. 1:
polyethylene glycol (mPEG) was added sequentially to a 100ml round bottom flask equipped with a bulb condenser 2k -NH 2 2.00g,0.95 mmol), docosahexaenoic acid (DHA, 0.33g,1.00 mmol) was dissolved in 4ml of anhydrous dichloromethane and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC, 0.15g,1.00 mmol) and N, N-diisopropylethylamine (DIEA, 0.13g,1.00 mmol) were added dropwise rapidly. Stirred overnight at 43℃and the reaction was observed by thin layer chromatography (developer: DCM: meOH=20:1). When the reaction was substantially completed, the reaction solution was cooled, the solvent was removed by rotary evaporation of the filtrate, and precipitation was performed three times with glacial ethyl ether to obtain a brown yellow precipitate. The resulting precipitate was collected and purified by column chromatography (DCM: meoh=50:1) to give docosahexaenoic acid-polyethylene glycol conjugate 1 (yellowish solid, 1.60g, yield 72.9%).
Product 1 1 The H NMR spectrum and the assignment of peaks are shown in FIG. 2, which proves that covalent connection of polyethylene glycol and docosahexaenoic acid is successful, and the obtained product is confirmed to be a target product with high purity.
Example 2 Synthesis of docosahexaenoic acid-polyethylene glycol conjugate I-VI (DHA-PEG) the procedure for synthesizing the conjugate I-VI is shown in FIG. 3.
1. Into a 100ml round bottom flask equipped with a bulb condenser were successively added ethylene glycol (or one of propylene glycol, butylene glycol, ethylenediamine, propylenediamine, butylenediamine; 52mg,0.846 mmol), docosahexaenoic acid (DHA, 300mg,0.705 mmol) and 4-dimethylaminopyridine (DMAP, 103mg,0.846 mmol) dissolved in 4ml anhydrous dichloromethane and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC, 131mg,0.846 mmol) was added dropwise rapidly. Stirred overnight at 43℃and the reaction was observed by thin layer chromatography (developer: DCM: meOH=20:1). When the reaction was substantially completed, the reaction solution was cooled, and then washed with 5% citric acid, saturated sodium bicarbonate and saturated brine, respectively. The organic layer was dried over anhydrous sodium sulfate, and after drying was completed, it was filtered. The filtrate was distilled off to remove the solvent. Purification by column chromatography (DCM: meoh=20:1) afforded ethylene glycol-docosahexaenoic acid conjugate (or intermediate product of propylene glycol, butylene glycol, ethylene diamine, propylene diamine, butylene diamine coupling).
2. Polyethylene glycol (mPEG) was added sequentially to a 100ml round bottom flask equipped with a bulb condenser 2k -COOH, 0.5538 g,0.279 mmol), ethylene glycol-docosahexaenoic acid conjugate (0.125 g,0.335 mmol) or other intermediate and 4-dimethylaminopyridine (DMAP, 0.041g,0.335 mmol) were dissolved in 4ml anhydrous dichloromethane and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC, 0.052g,0.335 mmol) was added dropwise. Stirred overnight at 43℃and the reaction was observed by thin layer chromatography (developer: DCM: meOH=10:1). When the reaction was substantially completed, the reaction solution was cooled, the solvent was removed by rotary evaporation of the filtrate, and precipitation was performed three times with glacial ethyl ether to obtain a brown yellow precipitate. The precipitate obtained was isolated and purified by column chromatography (DCM: meoh=10:1) to give the docosahexaenoic acid-polyethylene glycol conjugate I-vi.
Example 3 docosahexaenoic acid-polyethylene glycol conjugate 2 (DHA 2 -PEG) synthesis
The synthetic route is shown in FIG. 4:
1. synthesis of succinimidyl modified docosahexaenoic acid
N-hydroxysuccinimide (NHS, 207mg,1.8 mmol), docosahexaenoic acid (DHA, 500mg,1.5 mmol) were added sequentially to a 100ml round bottom flask equipped with a bulb condenser, dissolved in 4ml anhydrous dichloromethane, followed by rapid dropwise addition of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC, 279mg,1.8 mmol) and N, N-diisopropylethylamine (DIEA, 233mg,1.8 mmol). The reaction was observed by thin layer chromatography (developer: DCM: meoh=100:1). When the reaction was substantially completed, the reaction solution was cooled, and then washed with 5% citric acid, saturated sodium bicarbonate and saturated brine, respectively. The organic layer was dried over anhydrous sodium sulfate, and after drying was completed, it was filtered. The filtrate was distilled off to remove the solvent. Isolation and purification by column chromatography (DCM: meoh=100:1) afforded the product succinimidyl modified docosahexaenoic acid (yellow liquid, 588mg, yield 92.1%).
2. Synthesis of lysine-docosahexaenoic acid conjugate
Lysine (Lysine, 49mg,0.336 mmol), succinimidyl modified docosahexaenoic acid (DHA-NHS, 300mg,0.705 mmol) were added sequentially to a 100ml round bottom flask equipped with a bulb condenser, dissolved in 4ml anhydrous dichloromethane, followed by rapid dropwise addition of N, N-diisopropylethylamine (DIEA, 91mg, 0.704 mmol). Stirred overnight at 43℃and the reaction was observed by thin layer chromatography (developer: DCM: meOH=10:1). When the reaction was substantially completed, the reaction solution was cooled, and then washed with 5% citric acid, saturated sodium bicarbonate and saturated brine, respectively. The organic layer was dried over anhydrous sodium sulfate, and after drying was completed, it was filtered. The filtrate was distilled off to remove the solvent. Purification by column chromatography (DCM: meoh=10:1) afforded the product lysine-docosahexaenoic acid conjugate (yellow liquid, 211.2mg, yield 81.9%).
The product is 1 The H NMR spectrum and the assignment of peaks are shown in fig. 5, demonstrating the equivalent covalent linkage between lysine and docosahexaenoic acid, and the resulting product is confirmed to be a target product of high purity.
3. Docosahexaenoic acid-polyethylene glycol conjugate 2 (DHA) 2 -PEG) Synthesis of polyethylene glycol (mPEG) was added sequentially to a 100ml round bottom flask equipped with a bulb condenser 2k -NH 2 0.5538 g,0.279 mmol), lysine-docosahexaenoic acid conjugate (Lysine-DHA) 2 0.257g,0.335 mmol) was dissolved in 4ml of anhydrous dichloromethane and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC, 0.052g,0.335 mmol) and N, N-diisopropylethylamine (DIEA, 0.043g,0.335 mmol) was added dropwise rapidly. Stirred overnight at 43℃and the reaction was observed by thin layer chromatography (developer: DCM: meOH=10:1). When the reaction was substantially completed, the reaction solution was cooled, the solvent was removed by rotary evaporation of the filtrate, and the mixture was precipitated three times with glacial ethyl etherA brown-yellow precipitate was obtained. The resulting precipitate was collected and purified by column chromatography (DCM: meoh=10:1) to give docosahexaenoic acid-polyethylene glycol conjugate 2 (yellowish solid, 0.580g, yield 74.2%).
Product 2 1 H NMR spectra and peak assignment are shown in fig. 6, demonstrating 1 between polyethylene glycol and docosahexaenoic acid: 2 equivalents of covalent linkage, and the resulting product is confirmed to be the target product of high purity.
Example 4 docosahexaenoic acid-polyethylene glycol conjugate 3 (DHA 2 -PEG) synthesis
The synthesis of conjugate VIII is shown in FIG. 7.
1. Synthesis of polyethylene glycol-isopropylidenediol
Polyethylene glycol (mPEG) was added sequentially to a 100ml round bottom flask equipped with a bulb condenser 2k -COOH, 0.5538 g,0.279 mmol), isopropylidene glycerol (0.044 g,0.335 mmol) and 4-dimethylaminopyridine (DMAP, 0.041g,0.335 mmol) were dissolved in 4ml anhydrous dichloromethane and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide 4' amine (EDC, 0.052g,0.335 mmol) was added dropwise. Stirred overnight at 43℃and the reaction was observed by thin layer chromatography (developer: DCM: meOH=20:1). When the reaction was substantially completed, the reaction solution was cooled, the solvent was removed by rotary evaporation of the filtrate, and precipitation was performed three times with glacial ethyl ether to obtain a precipitate. The dried precipitate, 1mL of methylene chloride and 0.25mL of glacial trifluoroacetic acid, was added to a 100mL round bottom flask equipped with a bulb-shaped condenser, reacted at-20℃for 30 minutes, and neutralized by adding 2mol/L sodium hydroxide solution. Then, the cells were washed with 5% citric acid, saturated sodium bicarbonate and saturated brine, respectively. The organic layer was dried over anhydrous sodium sulfate, and after drying was completed, it was filtered. The filtrate was distilled off to remove the solvent. Purification by column chromatography (DCM: meoh=10:1) afforded the product.
2. Docosahexaenoic acid-polyethylene glycol conjugate 3 (DHA) 2 -PEG) synthesis
In a 100ml round bottom flask equipped with a bulb condenser were successively added polyethylene glycol-isopropylidenediol (0.500 g,0.235 mmol), docosahexaenoic acid conjugate (DHA, 0.170g,0.516 mmol) and 4-dimethylaminopyridine (DMAP, 29mg,0.516 mmol) dissolved in 4ml anhydrous dichloromethane and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC, 0.080g,0.516 mmol) added dropwise. Stirred overnight at 43℃and the reaction was observed by thin layer chromatography (developer: DCM: meOH=10:1). When the reaction was substantially completed, the reaction solution was cooled, the solvent was removed by rotary evaporation of the filtrate, and precipitation was performed three times with glacial ethyl ether to obtain a brown yellow precipitate. The collected precipitate was isolated and purified by column chromatography (DCM: meoh=10:1) to give docosahexaenoic acid-polyethylene glycol conjugate 3.
Example 5 preparation of drug-loaded nanomicelle
7.6mg of docosahexaenoic acid-polyethylene glycol conjugate 2 (DHA) 2 -PEG) in 0.1mL dmso to prepare a first solution of 76 mg/mL; dissolving 0.4mg of cabazitaxel in 0.1mL of DMSO to prepare a second solution of 4 mg/mL; uniformly mixing the first solution and the second solution according to the volume of 1:1, slowly injecting 0.2mL of DMSO solution mixed with the two drugs into 4mL of ultrapure water under ultrasonic vibration, and uniformly mixing the solutions and rapidly assembling the micelles to form the drug-loaded nano micelle drug. Residual dimethyl sulfoxide was removed by dialysis. The particle size distribution and the transmission electron microscope of the nano particles are shown in figures 8 and 9, the nano micelles are uniformly distributed in a monodispersion mode, the particle diameter is about 12nm, and the morphology is regular spherical.
EXAMPLE 6DHA 2 Preparation of PEG-modified prodrug self-assemblies
1. The synthesis route of the prodrug DHA-FL118 is shown in FIG. 10, and the synthesis process is as follows:
FL118 (100 mg,0.25 mmol), docosahexaenoic acid (DHA, 92mg,0.28 mmol) dissolved in 4ml anhydrous dichloromethane and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC, 43mg,0.28 mmol) and N, N-diisopropylethylamine (DIEA, 36mg,0.28 mmol) were added in sequence to a 100ml round bottom flask equipped with a bulb condenser. The reaction was observed by thin layer chromatography (developer: DCM: meoh=20:1). When the reaction was substantially completed, the reaction solution was cooled, and then washed with 5% citric acid, saturated sodium bicarbonate and saturated brine, respectively. The organic layer was dried over anhydrous sodium sulfate, and after drying was completed, it was filtered. The filtrate was distilled off to remove the solvent. Purification by column chromatography (DCM: meoh=20:1) afforded the product DHA-FL118 (yellow solid, 122mg, 67.5% yield).
The product is 1 The H NMR spectrum and peak assignment are shown in fig. 11, demonstrating the equivalent covalent linkage between FL118 and docosahexaenoic acid, and the resulting product was confirmed to be the target product of high purity.
2、DHA 2 The preparation process of the PEG-modified prodrug self-assembly is as follows:
10mg of docosahexaenoic acid-polyethylene glycol conjugate 2 (DHA) 2 -PEG) in 0.5mL DMSO to make up a 20mg/mL first solution; 18.4mg of DHA-FL118 (containing FL118 in a mass of 10 mg) was dissolved in 0.5mL of DMSO to prepare a second solution of 20 mg/mL; uniformly mixing the first solution and the second solution according to the volume of 1:1, slowly injecting 1mL of DMSO solution mixed with the two drugs into 10mL of ultrapure water under ultrasonic vibration, uniformly mixing the solutions, and rapidly assembling micelles to form DHA 2 PEG-modified prodrug self-assemblies (DF/DHA 2 -PEG). Residual dimethyl sulfoxide was removed by dialysis. The particle size distribution and the transmission electron microscope of the nanoparticles are shown in figures 12 and 13, the nanoparticles are uniformly distributed in a monodispersed mode, the particle diameter is about 145nm, and the morphology is in a regular sphere shape.
EXAMPLE 7 preparation of FL118 prodrug liposomes
To prepare FL118 prodrug lipid nanoparticles with a drug loading of 5% for FL118 and a final drug concentration of 1mg/mL, lecithin, cholesterol and DHA were first prepared 2 The lipid mixture with the PEG mass ratio of 14:2:3 was dissolved in 60. Mu.L of ethanol, which was then thoroughly mixed with DHA-FL118 prodrug molecule dissolved in 1mL of acetone, the mixture was slowly dropped into water while stirring, after stirring for 10 minutes, the remaining acetone was removed by a rotary evaporator to obtain uniformly dispersed lipid particles. The mixture was extruded through a 400nm polycarbonate film using an extruder to prepare FL118 prodrug liposomes. The particle size distribution and transmission electron microscope of the nanoparticles are shown in figures 14 and 15, the nanoparticles are uniformly distributed in a monodispersed mode, the particle diameter is about 150nm, the morphology is a vesicle structure, and the prepared liposome is a large single-chamber liposome.
Example 8DF and DF/DHA 2 In vitro stability test of PEG
To verify DHA 2 The modification of the PEG plays a role in increasing the stability of the prodrug self-assembly body, and DHA-free is prepared respectively 2 PEG-modified DHA-FL118 prodrug self-assembly (DF) and DHA-bearing 2 PEG-modified DHA-FL118 prodrug self-assembly (DF/DHA 2 -PEG)。
The nanoparticles were stored in 37 ℃ environment and particle size was measured daily with a dynamic light scatterometer, three replicates each time, as shown in figure 16.
The results show that DF/DHA 2 PEG remains stable for at least 30 days, whereas DF has a pronounced precipitation on day 7, which is much less stable than DF/DHA 2 PEG, demonstrating DHA 2 -destabilization of nanoparticles by PEG.
Example 9 endocytosis of different DiI-labeled nanomicelles
Because the polyethylene glycol on the particle surface is easy to reduce the interaction between tumor cell membrane and nano-particles, the drug-loaded nano-particles are prevented from entering the tumor cells to play a killing role. DHA in example 5 was labeled with DiI as fluorescent molecule 2 PEG micelle (DHA) 2 Pegmiceles); commercial material DSPE-PEG is selected 2k DHA in alternative example 5 2 -PEG, preparation of DiI-labeled DSPE-PEG 2k Micelle (DSPE-PEG) 2k micoles). To investigate DHA 2 -pegmiceles and DSPE-PEG 2k In vitro endocytosis of micoles, the particle entry experiments were further examined.
HCT116 cells were seeded in six well plates at a density of 2X 10 5 Well, incubated overnight at 37 ℃. DiI-labeled DHA 2 -pegmiceles and DSPE-PEG 2k Microles (500 nmol/LDiI) were added to the cells and incubated at 37℃for various times. Cells incubated with normal culture served as controls. Cells were collected by digestion, washed three times with PBS, and analyzed for endocytosis by flow cytometry.
As shown in FIG. 17, DHA 2 In vitro endocytosis of pegmiceles is significantly higher than DSPE-PEG 2k micoles suggested that it has a faster and stronger cell killing ability.
Example 10 endocytosis of different DiI-labeled self-assembled nanoparticles
Because the polyethylene glycol on the particle surface is easy to reduce the interaction between tumor cell membrane and nano-particles, the drug-loaded nano-particles are prevented from entering the tumor cells to play a killing role. Labelling of DF/DHA in example 6 with DiI as fluorescent molecule 2 -PEG; commercial material DSPE-PEG is selected 2k DHA in alternative example 6 2 -PEG, preparation of DiI-labeled DSPE-PEG 2k Modified self-assembled nanoparticles (DF/DSPE-PEG 2k ). To examine DF, DF/DSPE-PEG 2k 、DF/DHA 2 In vitro endocytosis of PEG, nanoparticle entry experiments were further examined.
HCT116 cells were seeded in six well plates at a density of 2X 10 5 Well, incubated overnight at 37 ℃. DiI-labeled DF, DF/DSPE-PEG 2k 、DF/DHA 2 PEG (500 nmol/L DiI) was added to the cells and incubated at 37℃for various times. Cells incubated with normal culture served as controls. Cells were collected by digestion, washed three times with PBS, and analyzed for endocytosis by flow cytometry.
As shown in FIG. 18, DF/DHA 2 In vitro endocytosis of PEG is significantly higher than DF/DSPE-PEG 2k Suggesting that it has a faster and stronger cell killing ability.
EXAMPLE 11 in vitro cytotoxicity experiments of different drugs
For comparison with the common PEG-phospholipid material, the commercial material DSPE-PEG is selected 2k DHA in alternative example 6 2 -PEG, preparation of DSPE-PEG 2k Modified self-assembled nanoparticles (DF/DSPE-PEG 2k ). The killing effect of different nanoparticles on tumor cells HCT116 and LoVo was examined by CCK8, using free FL118 as a reference. The results are shown in Table 1.
TABLE 1 determination of cell viability IC after 72 hours of drug action 50 ±SD(nM)
Figure BDA0003840915830000141
Figure BDA0003840915830000151
Table 1 shows that the prodrug nanoparticle DF/DHA prepared in example 6 2 The antitumor activity of the PEG is higher than DF and DF/DSPE-PEG 2k 。
EXAMPLE 12DHA 2 In vitro cytotoxicity assay of PEG
Investigation of DHA by CCK8 2 The killing effect of PEG on normal cell lines Raw264.7, HUVEC and 293T is shown in FIG. 20. The results show that DHA obtained in example 3 2 PEG has very low cytotoxicity to normal cell lines.
EXAMPLE 13DHA 2 In vivo toxicity test of PEG
DHA was evaluated using ICR mice (about 25g each) as a subject 2 -in vivo toxicity of PEG. A total of 2 groups of 4 mice each. Experimental group 300 mu LDHA by tail vein injection 2 PEG solution, control group injected with the same volume of PBS. 1 injection every 3 days for a total of 3 injections. The changes in body weight and blood cell number of the mice were measured and recorded within 14 days after administration.
The changes in body weight and blood cell number of mice are shown in FIGS. 21 and 22, which show DHA obtained in example 3 2 PEG was not significantly toxic in vivo.

Claims (9)

1. The docosahexaenoic acid-polyethylene glycol conjugate is characterized by comprising a hydrophilic segment and a hydrophobic segment, wherein the hydrophilic segment is a methoxy polyethylene glycol molecule with the molecular weight of 500-10000, and the hydrophobic segment is 1-4 docosahexaenoic acid molecules.
2. The docosahexaenoic acid-polyethylene glycol conjugate according to claim 1, wherein the structural formula of the conjugate is represented by any one of formulae (I) to (X),
Figure FDA0003840915820000011
wherein R is 1 The structural formula is as follows:
Figure FDA0003840915820000012
R 2 the structural formula is as follows:
Figure FDA0003840915820000013
n is an integer of 10 to 230.
3. The docosahexaenoic acid-polyethylene glycol conjugate of claim 2, wherein n is 23, 45, 114 or 227.
4. The method of preparing a docosahexaenoic acid-polyethylene glycol conjugate according to claim 1, comprising: under the action of a condensing agent and a catalyst, performing condensation reaction on docosahexaenoic acid and methoxy polyethylene glycol with a structural formula shown as formula (XI) and R being hydroxyl or amino to obtain DHA-PEG conjugate;
or under the action of condensing agent and catalyst, firstly, making docosahexaenoic acid and a connector compound with two or more connecting groups, wherein the connecting groups are selected from hydroxyl, amino and carboxyl; then the reaction product is condensed with methoxy polyethylene glycol with structural formula as shown in formula (XI) and R is hydroxyl, amino or carboxyl to prepare DHA-PEG conjugate,
Figure FDA0003840915820000021
wherein n is an integer of 10 to 230.
5. The method of claim 4, wherein the linker compound is one of ethylene glycol, propylene glycol, butylene glycol, ethylene diamine, propylene diamine, butylene diamine, lysine, isopropylidene glycerol.
6. The method for preparing a docosahexaenoic acid-polyethylene glycol conjugate according to claim 4 or 5, wherein the condensing agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, and the catalyst is 4-dimethylaminopyridine or N, N-diisopropylethylamine.
7. Use of a docosahexaenoic acid-polyethylene glycol conjugate as claimed in claim 1 as a drug delivery carrier material for the preparation of a pharmaceutical formulation.
8. The use according to claim 7, comprising: respectively dissolving the docosahexaenoic acid-polyethylene glycol conjugate and the hydrophobic drug in an organic solvent, mixing the two, and injecting the mixture into an aqueous phase to form a drug-loaded nano micelle;
or respectively dissolving the docosahexaenoic acid-polyethylene glycol conjugate and the prodrug modified by fatty acid in an organic solvent, mixing the two, and injecting the mixture into an aqueous phase to form a self-assembly body of the prodrug modified by the docosahexaenoic acid-polyethylene glycol conjugate;
alternatively, lecithin, cholesterol, and a docosahexaenoic acid-polyethylene glycol conjugate are added to a solution containing a drug molecule, and self-assembled to form drug-entrapped liposomes.
9. The use according to claim 8, wherein the mass ratio of the docosahexaenoic acid-polyethylene glycol conjugate to the hydrophobic drug is 1-40 when preparing the drug-loaded nano-micelle: 1, a step of;
preparing a self-assembly body of a prodrug modified by the docosahexaenoic acid-polyethylene glycol conjugate, wherein the mass ratio of the docosahexaenoic acid-polyethylene glycol conjugate to the prodrug is 1-20: 1, a step of;
when preparing liposome for encapsulating medicine, the mass ratio of lecithin, cholesterol, docosahexaenoic acid-polyethylene glycol conjugate and medicine is 10-40: 1 to 8:3:1 to 10.
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