CN110652494B - Low-oxygen response polyamino acid-PEG stereo drug-loaded micelle and preparation method thereof - Google Patents

Low-oxygen response polyamino acid-PEG stereo drug-loaded micelle and preparation method thereof Download PDF

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CN110652494B
CN110652494B CN201911000629.5A CN201911000629A CN110652494B CN 110652494 B CN110652494 B CN 110652494B CN 201911000629 A CN201911000629 A CN 201911000629A CN 110652494 B CN110652494 B CN 110652494B
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尹晓敏
陆舒
钱慰
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Guangzhou Dayu Chuangfu Technology Co ltd
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    • A—HUMAN NECESSITIES
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/48—Polymers modified by chemical after-treatment

Abstract

The invention discloses a hypoxia response polyamino acid-PEG stereo drug-loaded micelle and a preparation method thereof, firstly 2, 2-bis [ (2-nitro-1H-imidazole-1-yl) methyl ] -1, 3-propanediol is obtained by 2-nitroimidazole and dibromoneopentyl glycol under the catalysis of cesium carbonate, then the 2, 2-bis [ (2-nitro-1H-imidazole-1-yl) methyl ] propane-1, 3-diyl bis (4-nitrophenyl) carbonate is prepared by acid halogenation with p-nitrobenzoyl chloride under the existence of triethylamine, then the 2, 2-bis [ (2-nitro-1H-imidazole-1-yl) methyl ] propane-1, 3-diyl bis (4-nitrophenyl) carbonate reacts with paclitaxel under the catalysis of N, N-diisopropylethylamine, then poly L-amino acid-PEG or poly D-amino acid-PEG is added to respectively obtain bis 2-nitroimidazole-paclitaxel-poly L-amino acid-PEG, and bis 2-nitroimidazole-paclitaxel-poly D amino acid-PEG, mixing the two in PBS buffer solution in equal amount, homogenizing at high speed, and extruding with filter membrane with pore diameter of 100 nm. The drug-loaded micelle prepared by the invention has high entrapment rate and hypoxia response characteristic, can be effectively accumulated in tumor tissues to achieve good anti-tumor effect, and reduces the distribution of drugs in other normal tissues to reduce toxic and side effects.

Description

Low-oxygen response polyamino acid-PEG stereo drug-loaded micelle and preparation method thereof
Technical Field
The invention relates to a low-oxygen response polyamino acid-PEG stereo drug-loaded micelle and a preparation method thereof, belonging to the field of biomedical materials.
Background
The development of the nanometer technology enables the research of the drug formulation to enter a new stage, and the nanometer controlled release carrier can obviously prolong the drug effect, reduce the toxicity, and improve the activity and the bioavailability when in drug administration. The amphiphilic copolymer with a block or graft structure can spontaneously form nano-sized micelles in water due to the difference in solubility of the hydrophilic group and the hydrophobic group in a medium. The amphiphilic polymer micelle shows excellent performance in the aspects of controlled release, positioning and targeting release and the like of the drug, and has great application prospect.
At present, the study of the block copolymer which takes polyethylene glycol as a hydrophilic group and takes polylactic acid and the copolymer of the polylactic acid and glycolic acid and polycaprolactone as a hydrophobic group is the most extensive. However, the polylactic acid and the polycaprolactone follow the bulk degradation in the degradation process, so that the release of the drug is mainly diffused, the burst release effect can be generated, and meanwhile, the acidic degradation products can cause the local acidity of tissues to be excessively concentrated, and the severe inflammatory reaction is generated. Therefore, there is a strong need in the art to develop novel drug carriers to improve safety, reduce toxic side effects of drugs, or increase drug concentration in target organs, thereby exerting better therapeutic effects.
The polyamino acid as a novel biodegradable material has the advantages of good biocompatibility, more active groups and the like, and the degradation product of the polyamino acid is micromolecular amino acid without toxic or side effect, so the polyamino acid has wide application prospect in the biomedical field, such as bioseparation, tissue engineering, gene therapy, drug controlled release and the like.
At present, the research on the drug loading mode of physical encapsulation is relatively extensive, the polymer can be self-assembled into nano-carriers such as micelles, vesicles and liposomes in water, the polymer nano-micelle has the advantages of controllable particle size, long in vivo circulation time, capability of carrying out targeted modification and the like, but the research on the environment-responsive polymer micelle responding to the external environment stimulation is less.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a stereo drug-loaded micelle capable of responding to the environment aiming at the defects of the prior art, and the stereo drug-loaded micelle can respond to the hypoxic environment of tumors and release active drugs.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
a preparation method of a hypoxia response polyamino acid-PEG stereo drug-loaded micelle comprises the following steps:
(1) in DMF, 2-nitroimidazole and dibromo neopentyl glycol are catalyzed by cesium carbonate to obtain 2, 2-bis [ (2-nitro-1H-imidazol-1-yl) methyl ] -1, 3-propanediol;
(2) in DCM, the 2, 2-bis [ (2-nitro-1H-imidazol-1-yl) methyl ] -1, 3-propanediol obtained in step (1) and p-nitrobenzoyl chloride are acid-halogenated in the presence of triethylamine to prepare 2, 2-bis [ (2-nitro-1H-imidazol-1-yl) methyl ] propane-1, 3-diylbis (4-nitrophenyl) carbonate;
(3) in DCM, 2-bis [ (2-nitro-1H-imidazol-1-yl) methyl ] propane-1, 3-diylbis (4-nitrophenyl) carbonate obtained in step (2) reacts with paclitaxel under catalysis of N, N-diisopropylethylamine, and then poly L-amino acid-PEG or poly D-amino acid-PEG is added to obtain bis 2-nitroimidazole-paclitaxel-poly L-amino acid-PEG and bis 2-nitroimidazole-paclitaxel-poly D-amino acid-PEG respectively;
(4) and (3) mixing the bis 2-nitroimidazole-paclitaxel-poly L-amino acid-PEG prepared in the step (3) and the bis 2-nitroimidazole-paclitaxel-poly D-amino acid-PEG in PBS buffer solution in equal amount, homogenizing at high speed, and extruding by using a filter membrane with the pore diameter of 100nm to obtain the product.
The amphiphilic copolymer can form a multi-level self-assembly structure such as micelle, physical gel and the like in an aqueous solution, and the crystallization and the stereocomplex of the hydrophobic chain segment are important factors influencing the formation process, the structure and the performance of the micelle and the physical gel. Polyamino acid is a typical bio-based/biodegradable polymer, and 2 enantiomers of the polyamino acid, namely poly-L-amino acid and poly-D-amino acid, can form stereo complex crystals. Because of strong interchain interaction in the stereocomplex crystal, the uptake of macrophages can be effectively reduced, and the purpose of long circulation is achieved.
Specifically, in the step (1), the using amount ratio of DMF, 2-nitroimidazole, dibromoneopentyl glycol and cesium carbonate is (5-8ml): (0.45-0.67g): (0.26-0.52g): (0.65-0.97 g).
Preferably, the temperature of the reaction in step (1) is 30-60 ℃, preferably 50 ℃, and the reaction time is 4-24h, preferably 12 h.
Specifically, in the step (2), the dosage ratio of DCM, 2-bis [ (2-nitro-1H-imidazol-1-yl) methyl ] -1, 3-propanediol, p-nitrobenzoyl chloride and triethylamine is (30-60ml): 0.32-0.65g): 0.37-0.74g): 0.40-0.80 ml.
Preferably, the reaction temperature in the step (2) is room temperature, and the reaction time is 4-24h, preferably 12 h.
Specifically, in step (3), the poly-L-amino acid-PEG and the poly-D-amino acid-PEG are isomer polymers of the same amino acid, and include any one of poly-L-phenylalanine-PEG and poly-D-phenylalanine-PEG, poly-L-leucine-PEG and poly-D-leucine-PEG, poly-L-valine-PEG and poly-D-valine-PEG, and poly-L-isoleucine-PEG and poly-D-isoleucine-PEG.
Specifically, in the step (3), the dosage ratio of dichloromethane, 2-bis [ (2-nitro-1H-imidazole-1-yl) methyl ] propane-1, 3-diylbis (4-nitrophenyl) carbonate, paclitaxel, N-diisopropylethylamine and poly L-amino acid-PEG is (2-8ml), (0.16-0.65g), (0.21-0.85g), (0.12-0.25ml) and (0.82-1.64 g); the dosage ratio of dichloromethane, 2-bis [ (2-nitro-1H-imidazole-1-yl) methyl ] propane-1, 3-diylbis (4-nitrophenyl) carbonate, paclitaxel, N-diisopropylethylamine and poly D-amino acid-PEG is (2-8ml), (0.16-0.65g), (0.21-0.85g), (0.12-0.25ml) and (0.82-1.64 g). After the paclitaxel medicament reacts with acyl halide, one acyl halide group can be left for subsequent grafting reaction because the molecule has larger size and can form steric hindrance.
Preferably, in the step (3), the temperature of the reaction of the 2, 2-bis [ (2-nitro-1H-imidazol-1-yl) methyl ] propane-1, 3-diylbis (4-nitrophenyl) carbonate and the paclitaxel under the catalysis of N, N-diisopropylethylamine is room temperature, and the reaction time is 2-6H, preferably 4H; after adding poly L-amino acid-PEG or poly D-amino acid-PEG, the reaction temperature is room temperature, and the reaction time is 4-12h, preferably 8 h.
Specifically, in the step (4), the dosage ratio of the bis 2-nitroimidazole-paclitaxel-poly L-amino acid-PEG, the bis 2-nitroimidazole-paclitaxel-poly D-amino acid-PEG and the PBS buffer solution is (1-4g): 100-.
The hypoxia-responsive polyamino acid-PEG stereo drug-loaded micelle prepared by the method is also in the protection scope of the invention.
Has the advantages that:
1. the polyamino acid-PEG stereo-composite micelle prepared by the invention is a novel polymer micelle, and has low CMC value and good dispersion and dissolution performance. Meanwhile, under the action of EPR effect, the micelle can be effectively accumulated in tumor tissues to achieve good anti-tumor effect, and the distribution of the drug in other normal tissues is reduced to reduce toxic and side effects.
2. The invention can respond to the hypoxic environment of the tumor through the 2-nitroimidazole group. The drug-loaded micelle prepared by the invention carries 2-nitroimidazole groups and paclitaxel coupled prodrugs, and can be reduced by specific reductase in hypoxic tissues to release active drugs.
Drawings
The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
FIG. 1 is a synthetic circuit diagram of the hypoxia-responsive polyamino acid-PEG stereo drug-loaded micelle of the present invention.
FIG. 2 is a transmission electron microscope image of hypoxia responsive polyamino acid-PEG stereodrug loaded micelle prepared in example 1 before and after hypoxia treatment.
FIG. 3 is an in vitro release profile of example and comparative materials under normoxic and hypoxic conditions, respectively.
FIG. 4 is a graph of survival curves for example and comparative example materials and paclitaxel on C57BL/6 solid tumor model mice.
Detailed Description
The invention will be better understood from the following examples.
Example 1
The hypoxia-responsive polyamino acid-PEG stereo drug-loaded micelle is prepared according to the synthetic route shown in figure 1:
the method comprises the following steps: adding 0.5g of 2-nitroimidazole, 0.4g of dibromoneopentyl glycol and 0.8g of cesium carbonate into 6ml of DMF, reacting at 50 ℃ for 12H, concentrating by reduced pressure distillation after the reaction is finished, and separating the concentrate by silica gel column chromatography to obtain 2, 2-bis [ (2-nitro-1H-imidazol-1-yl) methyl ] -1, 3-propanediol;
step two: adding 0.5g of 2, 2-bis [ (2-nitro-1H-imidazol-1-yl) methyl ] -1, 3-propanediol, 0.6g of p-nitrobenzoyl chloride and 0.6ml of triethylamine into 50ml of DCM, reacting for 12H at room temperature, concentrating by reduced pressure distillation after the reaction is finished, and separating the concentrate by silica gel column chromatography to obtain 2, 2-bis [ (2-nitro-1H-imidazol-1-yl) methyl ] propane-1, 3-diylbis (4-nitrophenyl) carbonate;
step three: adding 0.5g of 2, 2-bis [ (2-nitro-1H-imidazole-1-yl) methyl ] propane-1, 3-diylbis (4-nitrophenyl) carbonate, 0.5g of paclitaxel and 0.2ml of N, N-diisopropylethylamine into 5ml of DCM, reacting for 4H at room temperature, then adding 1.28g of poly-L-valine-PEG or poly-D-valine-PEG, reacting for 8H at room temperature, concentrating by reduced pressure distillation after the reaction is finished, and separating the concentrate by silica gel column chromatography to obtain bis-2-nitroimidazole-paclitaxel-poly-L-valine-PEG and bis-2-nitroimidazole-paclitaxel-poly-D-valine-PEG;
step four: and (3) mixing 2g of bis 2-nitroimidazole-paclitaxel-poly L-valine-PEG prepared in the third step and 2g of bis 2-nitroimidazole-paclitaxel-poly D-valine-PEG in 200ml of PBS buffer solution, homogenizing at a high speed, and extruding by using a filter membrane with the aperture of 100nm to obtain the hypoxia response poly valine-PEG stereo drug-loaded micelle.
Example 2
The method comprises the following steps: adding 0.45g of 2-nitroimidazole, 0.26g of dibromoneopentyl glycol and 0.65g of cesium carbonate into 5ml of DMF, reacting at 50 ℃ for 12 hours, concentrating by reduced pressure distillation after the reaction is finished, and separating the concentrate by silica gel column chromatography to obtain 2, 2-bis [ (2-nitro-1H-imidazol-1-yl) methyl ] -1, 3-propanediol;
step two: adding 0.32g of 2, 2-bis [ (2-nitro-1H-imidazol-1-yl) methyl ] -1, 3-propanediol, 0.37g of p-nitrobenzoyl chloride and 0.40ml of triethylamine into 30ml of DCM, reacting for 12H at room temperature, concentrating by reduced pressure distillation after the reaction is finished, and separating the concentrate by silica gel column chromatography to obtain 2, 2-bis [ (2-nitro-1H-imidazol-1-yl) methyl ] propane-1, 3-diylbis (4-nitrophenyl) carbonate;
step three: adding 0.16g of 2, 2-bis [ (2-nitro-1H-imidazole-1-yl) methyl ] propane-1, 3-diylbis (4-nitrophenyl) carbonate, 0.21g of paclitaxel and 0.12ml of N, N-diisopropylethylamine into 2ml of DCM, reacting for 4 hours at room temperature, then adding 0.82g of poly-L-phenylalanine-PEG or poly-D-phenylalanine-PEG, reacting for 8 hours at room temperature, concentrating by reduced pressure distillation after the reaction is finished, and separating the concentrate by silica gel column chromatography to obtain bis-2-nitroimidazole-paclitaxel-poly-L-phenylalanine-PEG and bis-2-nitroimidazole-paclitaxel-poly-D-phenylalanine-PEG;
step four: and (3) mixing 1g of bis 2-nitroimidazole-paclitaxel-poly L-phenylalanine-PEG prepared in the step three and 1g of bis 2-nitroimidazole-paclitaxel-poly D-phenylalanine-PEG in 100ml of PBS buffer solution, homogenizing at a high speed, and extruding by using a filter membrane with the pore diameter of 100nm to obtain the hypoxia response poly phenylalanine-PEG stereo drug-loaded micelle.
Example 3
The method comprises the following steps: adding 0.67g of 2-nitroimidazole, 0.52g of dibromoneopentyl glycol and 0.97g of cesium carbonate into 8ml of DMF, reacting at 50 ℃ for 12H, concentrating by reduced pressure distillation after the reaction is finished, and separating the concentrate by silica gel column chromatography to obtain 2, 2-bis [ (2-nitro-1H-imidazol-1-yl) methyl ] -1, 3-propanediol;
step two: adding 0.65g of 2, 2-bis [ (2-nitro-1H-imidazol-1-yl) methyl ] -1, 3-propanediol, 0.74g of p-nitrobenzoyl chloride and 0.80ml of triethylamine into 60ml of DCM, reacting for 12H at room temperature, concentrating by reduced pressure distillation after the reaction is finished, and separating the concentrate by silica gel column chromatography to obtain 2, 2-bis [ (2-nitro-1H-imidazol-1-yl) methyl ] propane-1, 3-diylbis (4-nitrophenyl) carbonate;
step three: adding 0.65g of 2, 2-bis [ (2-nitro-1H-imidazole-1-yl) methyl ] propane-1, 3-diylbis (4-nitrophenyl) carbonate, 0.85g of paclitaxel and 0.25ml of N, N-diisopropylethylamine into 8ml of DCM, reacting for 4 hours at room temperature, then adding 1.64g of poly L-isoleucine-PEG or poly D-isoleucine-PEG, reacting for 8 hours at room temperature, concentrating by reduced pressure distillation after the reaction is finished, and separating the concentrate by silica gel column chromatography to obtain bis 2-nitroimidazole-paclitaxel-poly L-isoleucine-PEG and bis 2-nitroimidazole-paclitaxel-poly D-isoleucine-PEG;
step four: and (3) mixing 4g of bis 2-nitroimidazole-paclitaxel-poly L-isoleucine-PEG prepared in the step three and 4g of bis 2-nitroimidazole-paclitaxel-poly D-isoleucine-PEG in 400ml of PBS buffer solution, homogenizing at a high speed, and extruding by using a filter membrane with the pore diameter of 100nm to obtain the low-oxygen response poly isoleucine-PEG stereo drug-loaded micelle.
Comparative example 1
The method comprises the following steps: adding 0.5g of imidazole, 0.4g of dibromoneopentyl glycol and 0.8g of cesium carbonate into 6ml of DMF, reacting for 12h at 50 ℃, concentrating by reduced pressure distillation after the reaction is finished, and separating the concentrate by silica gel column chromatography to obtain 2, 2-bis [ (imidazole-1-yl) methyl ] -1, 3-propanediol;
step two: adding 0.5g of 2, 2-bis [ (imidazol-1-yl) methyl ] -1, 3-propanediol, 0.6g of p-nitrobenzoyl chloride and 0.6ml of triethylamine into 50ml of DCM, reacting at room temperature for 12h, concentrating by reduced pressure distillation after the reaction is finished, and separating the concentrate by silica gel column chromatography to obtain 2, 2-bis [ (imidazol-1-yl) methyl ] propane-1, 3-diylbis (4-nitrophenyl) carbonate;
step three: adding 0.5g of 2, 2-bis [ (imidazole-1-yl) methyl ] propane-1, 3-diylbis (4-nitrophenyl) carbonate, 0.5g of paclitaxel and 0.2ml of N, N-diisopropylethylamine into 5ml of DCM, reacting for 4 hours at room temperature, then adding 1.28g of poly-L-valine-PEG or poly-D-valine-PEG, reacting for 8 hours at room temperature, concentrating by reduced pressure distillation after the reaction is finished, and separating the concentrate by silica gel column chromatography to obtain the diimidazole-paclitaxel-poly-L-valine-PEG and the diimidazole-paclitaxel-poly-D-valine-PEG;
step four: and (3) mixing 2g of bisimidazole-paclitaxel-poly L-valine-PEG prepared in the third step and 2g of bisimidazole-paclitaxel-poly D-valine-PEG in 200ml of PBS buffer solution, homogenizing at a high speed, and extruding by using a filter membrane with the pore diameter of 100nm to obtain the poly valine-PEG stereo drug-loaded micelle.
Comparative example 2
The method comprises the following steps: 0.5g of paclitaxel, 1.28g of poly-L-valine-PEG and 1.28g of poly-D-valine-PEG are mixed in 200ml of PBS buffer solution, homogenized at high speed and extruded by a filter membrane with the pore diameter of 100nm to obtain the polyamino acid-PEG stereo drug-loaded micelle.
The method for measuring the encapsulation efficiency comprises the following steps: 100mg of the material is taken and added with 10ml of PBS buffer solution, after full stirring, the material is centrifuged at 15000r/min for 30min, and the precipitate is taken and completely dissolved by DMF, and then the content of paclitaxel is measured by HPLC.
In the following examples, the HPLC detection method for paclitaxel is as follows:
the chromatographic column used was Inersil ODS-3C18(250 mm. times.4.6 mm, 5mm), the mobile phase was acetonitrile/water, the flow rate ratio was 60:40, the flow rate was 1mL/min, the column temperature was 30 ℃, the detection wavelength was 227nm, and the sample injection amount was 20. mu.l.
Wherein the encapsulation efficiency is calculated by adopting the following formula:
Figure GDA0002290963390000061
wherein WGeneral assemblyThe amount of paclitaxel added to prepare 100mg of material; wMaterialIs the mass of paclitaxel entrapped in the material.
TABLE 1 encapsulation efficiency of materials prepared in examples and comparative examples
Figure GDA0002290963390000071
As can be seen from Table 1, the encapsulation efficiency of the material prepared by the invention is higher.
Example 4: hypoxia response study of hypoxia-responsive polyamino acid-PEG stereo drug-loaded micelle
The micelles prepared in examples 1 to 3 were treated in a water bath at 37 ℃ for 3 hours, and liver microsomes (100. mu.g/ml) and reducing coenzyme II (NADPH) (100. mu. mol/L) were added to the hypoxic group as an oxygen scavenger and a reducing agent, respectively, and NADPH alone was added to the normoxic group. Fig. 2 is a Transmission Electron Microscope (TEM) image of the hypoxia-responsive polyamino acid-PEG stereodrug-loaded micelle prepared in example 1 before and after hypoxia treatment, and the result shows that the micelle prepared in example 1 (fig. 2A) is spherical and has a particle size of about 100 nm. After the hypoxic treatment, the spherical structure of the micelles had been completely destroyed (fig. 2B), and the components in the micelles were released.
The examples and comparative materials were tested for in vitro release profiles under normoxic and hypoxic conditions, respectively:
the materials prepared in examples and comparative examples were diluted with PBS and sealed in quartz cuvettes, and paclitaxel in vitro release experiments were performed at 37 ℃. Among them, the hypoxic group was added rat liver microsomes (100. mu.g/ml) and NADPH (100. mu. mol/L), and the normoxic group was added NADPH only (100. mu. mol/L). Samples were taken at 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 4h, 5h, 7h, 9h, and 12h, respectively, and the paclitaxel concentration was determined by HPLC.
The results are shown in fig. 3, the micelles prepared in the examples release more than 50% of paclitaxel in 12h under the hypoxic condition at 37 ℃, and only about 20% of paclitaxel in the normoxic condition, and the micelles prepared in the comparative examples release less than 20% of paclitaxel in both normoxic and hypoxic conditions, which indicates that the micelles prepared in the examples can accelerate the release of paclitaxel in the hypoxic condition, and the non-hypoxia-responsive materials can hardly release paclitaxel in both hypoxic and normoxic conditions.
Examples and comparative example materials and therapeutic effects of paclitaxel on C57BL/6 solid tumor model mice:
the specific implementation process is as follows: after inoculation of RM-1 cells by subcutaneous injection, the tumor volume is allowed to grow to approximately 50mm3Solid tumor model mice were randomly divided into seven groups (n ═ 8), and injected tail vein with PBS buffer, paclitaxel, and the micelles prepared in examples 1-3 and comparative examples 1-2 (paclitaxel dose 5mg/kg), respectively, with the first administration being recorded as day 0 and the second administration on day 5.
The results are shown in fig. 4, fig. 4 is a survival curve of seven mice in the treatment groups, the survival time of the mice evaluated by PBS, paclitaxel and comparative examples 1-2 is obviously shorter than that of the mice evaluated by the example groups, most of the mice in the example groups survive after 40 days, and the materials of the example can remarkably prolong the survival time of the mice, thereby showing that the treatment effect is the best.
The invention provides a thought and a method for a hypoxia-responsive polyamino acid-PEG stereo drug-loaded micelle and a preparation method thereof, and a plurality of methods and ways for realizing the technical scheme are provided. All the components not specified in the present embodiment can be realized by the prior art.

Claims (9)

1. A preparation method of a hypoxia response polyamino acid-PEG stereo drug-loaded micelle is characterized by comprising the following steps:
(1) in N, N-dimethylformamide, 2-nitroimidazole and dibromoneopentyl glycol are catalyzed by cesium carbonate to obtain 2, 2-bis [ (2-nitro-1H-imidazol-1-yl) methyl ] -1, 3-propanediol;
(2) in dichloromethane, the 2, 2-bis [ (2-nitro-1H-imidazole-1-yl) methyl ] -1, 3-propanediol obtained in the step (1) and paranitrobenzoyl chloride are subjected to acid halogenation in the presence of triethylamine to prepare 2, 2-bis [ (2-nitro-1H-imidazole-1-yl) methyl ] propane-1, 3-diylbis (4-nitrophenyl) carbonate;
(3) reacting the 2, 2-bis [ (2-nitro-1H-imidazole-1-yl) methyl ] propane-1, 3-diylbis (4-nitrophenyl) carbonate obtained in the step (2) with paclitaxel in dichloromethane under the catalysis of N, N-diisopropylethylamine, and then adding poly-L-amino acid-PEG or poly-D-amino acid-PEG to respectively obtain bis-2-nitroimidazole-paclitaxel-poly-L-amino acid-PEG and bis-2-nitroimidazole-paclitaxel-poly-D-amino acid-PEG;
(4) and (3) mixing the bis 2-nitroimidazole-paclitaxel-poly L-amino acid-PEG prepared in the step (3) and the bis 2-nitroimidazole-paclitaxel-poly D-amino acid-PEG in PBS buffer solution in equal amount, homogenizing at high speed, and extruding by using a filter membrane with the pore diameter of 100nm to obtain the product.
2. The method for preparing a hypoxia-responsive polyamino acid-PEG stereo drug-loaded micelle according to claim 1, wherein in the step (1), the N, N-dimethylformamide, the 2-nitroimidazole, the dibromoneopentyl glycol and the cesium carbonate are used in a ratio of (5-8ml), (0.45-0.67g), (0.26-0.52g) and (0.65-0.97 g).
3. The preparation method of the hypoxia-responsive polyamino acid-PEG stereo drug-loaded micelle according to claim 1, wherein the reaction temperature in the step (1) is 30-60 ℃, and the reaction time is 4-24 h.
4. The method for preparing a hypoxia-responsive polyamino acid-PEG stereo drug-loaded micelle as claimed in claim 1, wherein in the step (2), dichloromethane, 2-bis [ (2-nitro-1H-imidazol-1-yl) methyl ] -1, 3-propanediol, p-nitrobenzoyl chloride and triethylamine are used in a ratio of (30-60ml), (0.32-0.65g), (0.37-0.74g) and (0.40-0.80 ml).
5. The method for preparing the hypoxia-responsive polyamino acid-PEG stereo drug-loaded micelle according to claim 1, wherein in the step (3), the poly-L-amino acid-PEG and the poly-D-amino acid-PEG are isomer polymers of the same amino acid, and comprise any one combination of poly-L-phenylalanine-PEG and poly-D-phenylalanine-PEG, poly-L-leucine-PEG and poly-D-leucine-PEG, poly-L-valine-PEG and poly-D-valine-PEG, and poly-L-isoleucine-PEG and poly-D-isoleucine-PEG.
6. The method for preparing a hypoxia-responsive polyamino acid-PEG stereo drug-loaded micelle according to claim 1, wherein in the step (3), dichloromethane, 2-bis [ (2-nitro-1H-imidazol-1-yl) methyl ] propane-1, 3-diylbis (4-nitrophenyl) carbonate, paclitaxel, N-diisopropylethylamine, and poly L-amino acid-PEG are used in a ratio of (2-8ml), (0.16-0.65g), (0.21-0.85g), (0.12-0.25ml), (0.82-1.64 g); the dosage ratio of dichloromethane, 2-bis [ (2-nitro-1H-imidazole-1-yl) methyl ] propane-1, 3-diylbis (4-nitrophenyl) carbonate, paclitaxel, N-diisopropylethylamine and poly D-amino acid-PEG is (2-8ml), (0.16-0.65g), (0.21-0.85g), (0.12-0.25ml) and (0.82-1.64 g).
7. The preparation method of the hypoxia-responsive polyamino acid-PEG stereo drug-loaded micelle according to claim 1, wherein in the step (3), the temperature of the reaction of 2, 2-bis [ (2-nitro-1H-imidazol-1-yl) methyl ] propane-1, 3-diylbis (4-nitrophenyl) carbonate and paclitaxel under the catalysis of N, N-diisopropylethylamine is room temperature, and the reaction time is 2-6H; adding poly L-amino acid-PEG or poly D-amino acid-PEG, reacting at room temperature for 4-12 h.
8. The method for preparing hypoxia-responsive polyamino acid-PEG stereo drug-loaded micelle as claimed in claim 1, wherein in step (4), the amount ratio of bis 2-nitroimidazole-paclitaxel-poly L-amino acid-PEG, bis 2-nitroimidazole-paclitaxel-poly D-amino acid-PEG, PBS buffer is (1-4g): 100-.
9. The hypoxia-responsive polyamino acid-PEG stereo drug-loaded micelle prepared by any one preparation method of claim 1-8.
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