CN110790924B - A triblock amphiphilic copolymer and preparation method thereof, drug-protein co-delivery carrier and preparation method thereof - Google Patents
A triblock amphiphilic copolymer and preparation method thereof, drug-protein co-delivery carrier and preparation method thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- 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
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
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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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
- C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
- C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
- C08G69/10—Alpha-amino-carboxylic acids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/27—Endoribonucleases producing 3'-phosphomonoesters (3.1.27)
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Abstract
The invention provides a block amphiphilic copolymer, which sequentially comprises a polyethylene glycol monomethyl ether chain segment, a polyglutamic acid chain segment containing catechol side group and a polyglutamic acid chain segment containing tertiary amine side group, and has a structure shown in a formula I. The triblock copolymer provided by the invention carries a hydrophobic chemotherapeutic drug through a hydrophobic effect, and simultaneously carries a phenylboronic acid modified protein drug through reversible covalent bonding of catechol and phenylboronic acid, so that a drug/protein co-delivery carrier system is obtained. The drug/protein co-delivery carrier can transfer chemotherapeutic drugs and proteins into tumor cells, release the drugs and proteins in the acidic environment in the tumor cells, mediate endosome/lysosome escape of protein molecules through proton sponge effect generated by protonation under the acidic condition of tertiary amine, and realize the synergistic antitumor effect of the chemotherapeutic drugs and the proteins. The invention also provides a preparation method of the triblock amphiphilic copolymer and a drug/protein co-delivery carrier.
Description
Technical Field
The invention belongs to the technical field of polymers, and particularly relates to a triblock amphiphilic copolymer and a preparation method thereof, a drug-protein co-delivery carrier and a preparation method thereof.
Background
Cancer has become a major public health problem that seriously threatens the life health and social and economic development of people in China. At present, the treatment methods for cancer mainly include surgery, radiotherapy, chemotherapy, and the like. Among them, chemotherapy is a very important means in cancer treatment. Researches find that the use of a single chemotherapeutic drug easily induces the generation of drug resistance of tumor cells, and has the defects of large toxic and side effects, limited treatment scheme and the like, thereby greatly reducing the treatment effect; the drug combination, namely two or more than two drugs with different anti-cancer mechanisms are used simultaneously, can effectively overcome the defects of single chemotherapy drug treatment, reduce the generation of drug resistance of cancer cells, reduce toxic and side effects, and can enhance the treatment effect through the synergistic effect of different drugs. Therefore, the development of novel chemotherapeutic drug combination therapy has important research and clinical practical significance.
The protein drug has the advantages of high functional activity, strong specificity, small side effect, low genetic risk and the like, and the protein drug is combined with chemotherapeutic drugs to be used in basic research and clinical evaluation. However, the protein drugs commonly used in combination with chemotherapeutic drugs are mainly proteins that exert their functions outside cells, such as cytokines and monoclonal antibodies. Proteins that exert an anticancer function in cells have an anticancer mechanism different from that of cytokines and monoclonal antibodies, and thus, the use of them in combination with chemotherapeutic drugs can provide more options for the development of novel combination therapies. However, the physicochemical properties of intracellular anticancer protein drugs are significantly different from those of chemotherapeutic drugs, such as molecular weight, hydrophilicity and hydrophobicity, immunogenicity, and the like, so that the pharmacokinetics and in vivo distribution are also significantly different. Moreover, compared to chemotherapeutic drugs, protein drugs have a lower efficiency of entering cells, and are particularly likely to be retained in the endosome after being taken up by cells, and are finally degraded by proteases in lysosomes, thereby failing to reach the cytoplasm to exert its anticancer function. Therefore, with the traditional administration method, for example, the two drugs are mixed and then administered by intravenous injection, it is difficult to ensure that the chemotherapeutic drug and the intracellular anticancer protein drug reach the tumor site simultaneously according to a certain proportion, and then the chemotherapeutic drug and the intracellular anticancer protein drug are taken up by the tumor cells and successfully enter cytoplasm to play respective functions, so that the ideal combined treatment effect is difficult to obtain.
The appearance of the polymer nano-carrier provides a possible solution for the above problems, and one or more anticancer drugs can be loaded into the same nano-structure together according to a certain proportion by a physical embedding or covalent bonding mode, so that different drugs have the same pharmacokinetics, and the aggregation and cellular uptake of the drug-loaded nano-particles at tumor sites are increased by enhancing the osmotic retention (EPR) effect, thereby enhancing the treatment effect and reducing the toxic and side effects. Meanwhile, through the exquisite chemical design, the polymer nano-carrier can also realize the drug controlled release behavior of intracellular stimulation response, promote the loaded drug to be released from the nano-carrier in time, reach a specific subcellular position to play a role, and further enhance the treatment effect. However, the physicochemical properties of chemotherapeutic drugs and protein molecules are greatly different, such as stability, molecular weight, hydrophilicity and hydrophobicity, surface charge and the like, so that the development of co-delivery nano-carriers faces huge challenges. Therefore, how to effectively integrate the hydrophilic protein drug and the hydrophobic chemotherapeutic drug into the same nanocarrier is a major challenge in the development of the carrier.
Disclosure of Invention
In view of the above, the present invention aims to provide a triblock amphiphilic copolymer and a preparation method thereof, a drug-protein co-delivery carrier and a preparation method thereof, wherein the triblock amphiphilic copolymer can be used as a co-carrier of a drug and a protein to achieve a synergistic anti-tumor effect.
The invention provides a triblock amphiphilic copolymer, which has a structure shown in a formula I:
wherein x is more than or equal to 10 and less than or equal to 500; y is more than or equal to 5 and less than or equal to 100; z is more than or equal to 5 and less than or equal to 100; m is more than or equal to 5 and less than or equal to 100.
Preferably, the triblock amphiphilic copolymer is in particular:
mPEG2000-b-PL(GA19CA6)-b-PGTA20、mPEG2000-b-PL(GA6CA19)-b-PGTA20、mPEG2000-b-PL(GA38CA12)-b-PGTA40、mPEG2000-b-PL(GA12CA38)-b-PGTA40、mPEG2000-b-PL(GA76CA24)-b-PGTA80、mPEG2000-b-PL(GA24CA76)-b-PGTA80、mPEG5000-b-PL(GA19CA6)-b-PGTA20、mPEG5000-b-PL(GA6CA19)-b-PGTA20、mPEG5000-b-PL(GA38CA12)-b-PGTA40、mPEG5000-b-PL(GA12CA38)-b-PGTA40、mPEG5000-b-PL(GA76CA24)-b-PGTA80、mPEG5000-b-PL(GA24CA76)-b-PGTA80、mPEG10000-b-PL(GA19CA6)-b-PGTA20、mPEG10000-b-PL(GA6CA19)-b-PGTA20、mPEG10000-b-PL(GA38CA12)-b-PGTA40、mPEG10000-b-PL(GA12CA38)-b-PGTA40、mPEG10000-b-PL(GA76CA24)-b-PGTA80or mPEG10000-b-PL(GA24CA76)-b-PGTA80。
The invention provides a preparation method of the triblock amphiphilic copolymer in the technical scheme, which comprises the following steps:
in the presence of a solvent and a catalyst, polyethylene glycol monomethyl ether-b-poly (L-glutamic acid) -b-poly (gamma-1Carrying out amidation reaction on the N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) block copolymer and dopamine with a structure of a formula III to obtain a triblock amphiphilic copolymer with a structure of a formula I;
preferably, the temperature of the amidation reaction is 15-80 ℃; the amidation reaction time is 12-96 h.
Preferably, the polyethylene glycol monomethyl ether-b-poly (A) having the structure of formula IIL-glutamic acid) -bPoly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) block copolymer and dopamine having the structure of formula III in a molar ratio of 1: 1 to 500.
Preferably, the polyethylene glycol monomethyl ether-b-poly (A) having the structure of formula IIL-glutamic acid) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-LThe-glutamate) block copolymer is prepared from polyethylene glycol monomethyl ether-b-poly (I) (having a structure of formula IVL-glutamic acid) -b-poly (gamma-propargyl-L-glutamate) block copolymer and 2-ethyl azide-N, N-diisopropylamine having the structure of formula V:
preferably, the polyethylene glycol monomethyl ether-b-poly (A) having the structure of formula IVL-glutamic acid) -b-poly (gamma-propargyl-LThe-glutamate) block copolymer is prepared from polyethylene glycol monomethyl ether-b-poly (gamma-benzyl-L-glutamate) -b-poly (gamma-propargyl-L-glutamate) is subjected to a benzyl deprotection reaction to obtain:
in the formula VI, x is more than or equal to 10 and less than or equal to 500; y is more than or equal to 5 and less than or equal to 100; z is more than or equal to 5 and less than or equal to 100.
Preferably, the polyethylene glycol monomethyl ether-b-poly (gamma-benzyl-L-glutamate) -b-poly (gamma-propargyl-L-glutamate) is prepared according to the following method:
under the initiation of amination-terminated polyethylene glycol monomethyl ether, sequentially carrying out gamma-benzyl-glutamate-N-carboxyl in a solventRing-opening polymerization in the cyclic anhydride and the gamma-propargyl-glutamate-N-carboxyl cyclic anhydride, and the acetic anhydride is adopted to seal the terminal amino group to obtain the polyethylene glycol monomethyl ether-b-poly (gamma-benzyl-L-glutamate) -b-poly (gamma-propargyl-L-glutamate).
The invention provides a drug/protein co-delivery carrier, which comprises a hydrophobic chemotherapeutic drug, a phenylboronic acid modified protein drug and a carrier;
the carrier is the triblock amphiphilic copolymer with the structure of the formula I in the technical scheme;
the molar ratio of the carrier to the hydrophobic chemotherapeutic drug is 1: 0.1 to 500; the molar ratio of the carrier to the phenylboronic acid modified protein drug is 1: 0.1 to 500.
The invention provides a preparation method of the drug/protein co-delivery carrier in the technical scheme, which comprises the following steps:
mixing a hydrophobic chemotherapeutic drug, a triblock amphiphilic copolymer with a structure shown in a formula I and a solvent, dripping the mixture into deionized water, stirring the mixture for reaction, and dialyzing and freeze-drying the mixture to obtain an independent drug carrier;
dissolving the phenylboronic acid modified protein drug and the single drug carrier in a neutral phosphate buffer solution, stirring for reaction, and performing ultrafiltration purification to obtain a drug/protein co-delivery carrier;
the molar ratio of the carrier to the hydrophobic chemotherapeutic drug is 1: 0.1 to 500; the molar ratio of the carrier to the phenylboronic acid modified protein drug is 1: 0.1 to 500.
The invention provides a triblock copolymer and a preparation method thereof, and a drug-protein carrier system and a preparation method thereof. The triblock copolymer sequentially comprises a polyethylene glycol monomethyl ether chain segment, a polyglutamic acid chain segment containing catechol side group and a polyglutamic acid chain segment containing tertiary amine side group. The triblock copolymer provided by the invention is used as a carrier to carry hydrophobic chemotherapeutic drugs and phenylboronic acid modified protein drugs, so that a drug/protein co-delivery carrier system is obtained. The obtained drug/protein co-delivery carrier can transfer chemotherapeutic drugs and protein drugs into tumor cells, release protein through dissociation of the binding effect of phenylboronic acid and catechol in an acidic environment in the tumor cells, release the chemotherapeutic drugs through protonation of tertiary amine side groups to become hydrophilic, mediate endosome/lysosome escape of protein molecules through proton sponge effect generated by protonation of tertiary amine, and realize a synergistic antitumor effect with the chemotherapeutic drugs. Experimental results show that the drug/protein co-delivery carrier provided by the invention can effectively co-carry chemotherapeutic drugs and protein drugs, has pH-responsive chemotherapeutic drug and protein drug release behaviors, and can synergistically inhibit tumor cell proliferation in vitro.
Drawings
FIG. 1 is the NMR spectrum of 2-ethyl azide-N, N-diisopropylamine obtained in example 1 of the present invention;
FIG. 2 shows mPEG prepared in example 5 of the present invention5000-b-PBLG25-b-PPLG20The nuclear magnetic resonance hydrogen spectrum of (a);
FIG. 3 shows mPEG prepared in example 14 of the present invention5000-b-PLGA25-b-PPLG20The nuclear magnetic resonance hydrogen spectrum of (a);
FIG. 4 shows mPEG prepared in example 23 of the present invention5000-b-PLGA25-b-PGTA20The nuclear magnetic resonance hydrogen spectrum of (a);
FIG. 5 shows mPEG prepared in example 35 of the present invention5000-b-PL(GA19CA6)-b-PGTA20The nuclear magnetic resonance hydrogen spectrum of (a);
FIG. 6 is a graph of dynamic light scattering of nanoparticles assembled from individual drug carriers prepared in comparative example 7 of the present invention in an aqueous phase;
FIG. 7 is a dynamic light scattering diagram of nanoparticles assembled by individual protein carriers prepared in comparative example 25 of the present invention in an aqueous phase;
FIG. 8 is a dynamic light scattering diagram of nanoparticles assembled by drug/protein carriers prepared in example 53 of the present invention in aqueous phase;
FIG. 9 is a TEM image of nanoparticles assembled from the drug/protein carrier prepared in example 53 of the present invention in an aqueous phase;
FIG. 10 is a graph showing the cumulative release of drugs at different pH conditions for drug/protein carriers prepared in example 53 of the present invention;
FIG. 11 is a graph showing the cumulative protein release at different pH conditions for drugs/protein carriers prepared in example 53 of the present invention;
fig. 12 is a graph of cytotoxicity experiments after incubation of the drug-carrier-only nanoparticles prepared in comparative example 7, the protein-carrier-only nanoparticles prepared in comparative example 25, and the drug/protein-carrier nanoparticles prepared in example 53 with B16F10 tumor cells for 72 h.
Detailed Description
The invention provides a triblock amphiphilic copolymer, which has a structure shown in a formula I:
wherein x is more than or equal to 10 and less than or equal to 500; y is more than or equal to 5 and less than or equal to 100; z is more than or equal to 5 and less than or equal to 100; m is more than or equal to 5 and less than or equal to 100.
In the present invention, preferably, 40. ltoreq. x.ltoreq.250; y is more than or equal to 25 and less than or equal to 100; z is more than or equal to 20 and less than or equal to 80; m is more than or equal to 5 and less than or equal to 25.
More specifically, the triblock amphiphilic copolymer is specifically:
mPEG2000-b-PL(GA19CA6)-b-PGTA20、mPEG2000-b-PL(GA6CA19)-b-PGTA20、mPEG2000-b-PL(GA38CA12)-b-PGTA40、mPEG2000-b-PL(GA12CA38)-b-PGTA40、mPEG2000-b-PL(GA76CA24)-b-PGTA80、mPEG2000-b-PL(GA24CA76)-b-PGTA80、mPEG5000-b-PL(GA19CA6)-b-PGTA20、mPEG5000-b-PL(GA6CA19)-b-PGTA20、mPEG5000-b-PL(GA38CA12)-b-PGTA40、mPEG5000-b-PL(GA12CA38)-b-PGTA40、mPEG5000-b-PL(GA76CA24)-b-PGTA80、mPEG5000-b-PL(GA24CA76)-b-PGTA80、mPEG10000-b-PL(GA19CA6)-b-PGTA20、mPEG10000-b-PL(GA6CA19)-b-PGTA20、mPEG10000-b-PL(GA38CA12)-b-PGTA40、mPEG10000-b-PL(GA12CA38)-b-PGTA40、mPEG10000-b-PL(GA76CA24)-b-PGTA80or mPEG10000-b-PL(GA24CA76)-b-PGTA80。
The invention provides a preparation method of the triblock amphiphilic copolymer in the technical scheme, which comprises the following steps:
in the presence of a solvent and a catalyst, polyethylene glycol monomethyl ether-b-poly (L-glutamic acid) -b-poly (gamma-1Carrying out amidation reaction on the N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) block copolymer and dopamine with a structure of a formula III to obtain a triblock amphiphilic copolymer with a structure of a formula I;
in the present invention, in order to distinguish from the catalyst in the following technical scheme, polyethylene glycol monomethyl ether-b-poly (I) having a structure of formula IIL-glutamic acid) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) block copolymer and dopamine having the structure of formula III was subjected to amidation reaction using a catalyst designated as the first catalyst. The first catalyst is preferably a mixture of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) and N-hydroxysuccinimide (NHS); in the EDC and NHS mixtureThe molar ratio of EDC to NHS is preferably 1 (1-10), more preferably 1 (1-4).
In the present invention, in order to distinguish from the solvent in the following technical scheme, polyethylene glycol monomethyl ether-b-poly (I) having a structure of formula IIL-glutamic acid) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) block copolymer and dopamine having the structure of formula III to perform amidation reaction using a solvent named first solvent. In the present invention, the first solvent is preferably dimethyl sulfoxide (DMSO).
The invention preferably uses polyethylene glycol monomethyl ether-b-poly (I) having the structure of formula IIL-glutamic acid) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) in a first solvent, removing oxygen in the reaction system by bubbling nitrogen, adding dopamine having the structure of formula III to the solution, removing oxygen in the reaction system by bubbling nitrogen, adding NHS to the solution, removing oxygen in the reaction system by bubbling nitrogen, adding EDC to the solution, removing oxygen in the reaction system by bubbling nitrogen, and stirring for reaction. And after the reaction is finished, dialyzing and freeze-drying the reaction product to obtain the triblock amphiphilic copolymer with the structure of the formula I.
The source of the dopamine is not particularly limited in the invention, and commercially available dopamine can be used, or the dopamine can be prepared by itself according to a technical scheme for preparing dopamine, which is well known to those skilled in the art.
In the present invention, the polyethylene glycol monomethyl ether-b-poly (I) having the structure of formula IIL-glutamic acid) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) block copolymer and dopamine having the structure of formula III preferably in a molar ratio of 1: (1-500), more preferably 1: (1-100), most preferably 1: (1-10).
In the invention, the temperature of the amidation reaction is preferably 15-80 ℃, more preferably 15-50 ℃, and most preferably 25 ℃; the amidation reaction time is 12-96 h, more preferably 24-72 h, and most preferably 48 h.
In the present invention, the polyethylene glycol monomethyl ether-b-poly (I) having the structure of formula IIL-glutamic acid) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-LThe-glutamate) block copolymer is preferably prepared from polyethylene glycol monomethyl ether-b-poly(s) (having the structure of formula IVL-glutamic acid) -b-poly (gamma-propargyl-L-glutamate) block copolymer and 2-ethyl azide-N, N-diisopropylamine having the structure of formula V are prepared by a click chemistry reaction;
more specifically, polyethylene glycol monomethyl ether-b-poly (A) having the structure of formula IVL-glutamic acid) -b-poly (gamma-propargyl-L-glutamate) block copolymer and 2-ethyl azide-N, N-diisopropylamine having a structure of formula V are dissolved in a first solvent to obtain a mixture solution;
mixing the mixture solution with a second catalyst, deoxidizing, and carrying out click chemical reaction to obtain polyethylene glycol monomethyl ether-b-poly (A-methyl glycol) with a structure of formula IIL-glutamic acid) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) block copolymer.
In the present invention, the 2-ethyl azide-N, N-diisopropylamine having the structure of formula V is preferably as described in document 1: J.Polym.Sci.part A: Polym.Chem.2014,52: 671-679.
In the present invention, the second catalyst is preferably a mixture of copper sulfate pentahydrate and sodium ascorbate; the mole ratio of the copper sulfate pentahydrate to the sodium ascorbate in the mixture of the copper sulfate pentahydrate and the sodium ascorbate is preferably 1 (2-10), and more preferably 1 (2-5).
In the present invention, the polyethylene glycol monomethyl ether-b-poly (A) having the structure of formula IVL-glutamic acid) -b-poly (gamma-propargyl-L-glutamate) block copolymer and 2-ethyl azide-N, N-diisopropylamine having the structure of formula V, preferably in a molar ratio of 1: (1-500), more preferably 1: (1-100), most preferably 1: 20.
in the invention, the temperature of the click chemistry reaction is preferably 15-80 ℃, more preferably 30-50 ℃, and most preferably 40 ℃; the click chemistry reaction time is preferably 12-96 h, more preferably 24-96 h, and most preferably 72 h.
After the click chemical reaction is finished, the reaction product solution is preferably dialyzed and lyophilized to obtain the polyethylene glycol monomethyl ether-b-poly (I) with the structure of formula IIL-glutamic acid) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) block copolymer.
In the present invention, the polyethylene glycol monomethyl ether-b-poly (A) having the structure of formula IVL-glutamic acid) -b-poly (gamma-propargyl-LThe-glutamate) block copolymer is prepared from polyethylene glycol monomethyl ether-b-poly (gamma-benzyl-L-glutamate) -b-poly (gamma-propargyl-L-glutamate) is deprotected by benzyl groups to give:
in the formula VI, x is more than or equal to 10 and less than or equal to 500; y is more than or equal to 5 and less than or equal to 100; z is more than or equal to 5 and less than or equal to 100.
The invention preferably uses the polyethylene glycol monomethyl ether-b-poly (gamma-benzyl-L-glutamate) -b-poly (gamma-propargyl-L-glutamate) is dissolved in a second solvent, then a third catalyst is added into the solution, deprotection reaction is carried out under stirring, ether is settled, the settled product is dissolved by a third solvent, and the polyethylene glycol monomethyl ether-b-poly (A) (I) with the structure of formula IV is obtained by dialysis and freeze-dryingL-glutamic acid) -b-poly (gamma-propargyl-L-glutamate) block copolymer.
In the present invention, the third catalyst is preferably hydrogen bromide, the second solvent is preferably dichloroacetic acid, and the third solvent is preferably N, N-Dimethylformamide (DMF).
In the invention, the temperature of the deprotection reaction is preferably 15-80 ℃, more preferably 20-40 ℃, and most preferably 30 ℃; the deprotection reaction time is preferably 0.5-10 h, more preferably 1-2 h, and most preferably 1.5 h.
In the present invention, the polyethylene glycol monomethyl ether-b-poly (gamma-benzyl-L-glutamate) -b-poly (gamma-propargyl-L-glutamate) is prepared according to the following method:
under the initiation of end-aminated polyethylene glycol monomethyl ether, sequentially carrying out ring-opening polymerization in gamma-benzyl-glutamate-N-carboxyl cyclic internal anhydride and gamma-propargyl-glutamate-N-carboxyl cyclic internal anhydride in a solvent, and adopting acetic anhydride to seal the end amino to obtain polyethylene glycol monomethyl ether-b-poly (gamma-benzyl-L-glutamate) -b-poly (gamma-propargyl-L-glutamate).
The solvent used for the ring-opening polymerization is preferably the third solvent described in the above technical scheme; the third solvent is preferably DMF. The ring-opening polymerization is preferably carried out in the presence of a fourth catalyst; the fourth catalyst is preferably 4-Dimethylaminopyridine (DMAP).
In the present invention, the terminally aminated polyethylene glycol monomethyl ether, γ -benzyl-glutamate-N-carboxycyclic anhydride and γ -propargyl-glutamate-N-carboxycyclic anhydride are preferably described in document 2: prepared by the synthesis method described in Polym.chem.2015,6: 3807-3815.
The source of the acetic anhydride is not particularly limited in the present invention, and commercially available acetic anhydride may be used, or the acetic anhydride may be prepared by itself according to a technical scheme for preparing acetic anhydride known to those skilled in the art.
In the present invention, the molar ratio of the aminoated polyethylene glycol monomethyl ether, γ -benzyl-glutamate-N-carboxyanhydride, γ -propargyl-glutamate-N-carboxyanhydride and acetic anhydride is preferably 1: (5-100): (5-100): (1-500), more preferably 1: (10-50): (10-40): (10-100), most preferably 1: 25: 20: 50.
in the invention, the temperature of the ring-opening polymerization and the terminal amino-group blocking reaction is preferably 15-80 ℃, more preferably 20-50 ℃, and most preferably 20-25 ℃; the time for the ring-opening polymerization of the gamma-benzyl-glutamate-N-carboxyl cyclic internal anhydride is preferably 1-168 hours, more preferably 48-120 hours, and most preferably 72 hours; the time for the ring-opening polymerization of the gamma-propargyl-glutamate-N-carboxyl cyclic internal anhydride is preferably 1-168 hours, more preferably 48-120 hours, and most preferably 72 hours; the time of the terminal amino group blocking reaction is preferably 1-168 hours, more preferably 12-48 hours, and most preferably 24 hours.
In a specific embodiment, the synthesis method of the triblock amphiphilic copolymer with the structure of the formula I comprises the following steps of dissolving polyethylene glycol monomethyl ether with amino group at the end into DMF, firstly adding gamma-benzyl-glutamate-N-carboxyanhydride, stirring and reacting at room temperature for 72h, then adding gamma-propargyl-glutamate-N-carboxyanhydride, stirring and reacting at room temperature for 72h, then adding DMAP into the mixed solution, adding acetic anhydride, reacting at room temperature for 24h, settling with diethyl ether, and performing vacuum rotary evaporation to obtain polyethylene glycol monomethyl ether-b-poly (gamma-benzyl-one-chain) with the structure of the formula VIL-glutamate) -b-poly (gamma-propargyl-L-glutamate). Polyethylene glycol monomethyl ether-b-poly (gamma-benzyl-L-glutamate) -b-poly (gamma-propargyl-L-glutamate) is dissolved in dichloroacetic acid, then hydrogen bromide is added into the solution, after reaction for 1.5h at 30 ℃, after ether sedimentation, the settled product is dissolved by DMF, and after dialysis and freeze-drying, the polyethylene glycol monomethyl ether-b-poly (A) (with the structure of formula IV) is obtainedL-glutamic acid) -b-poly (gamma-propargyl-L-glutamate); polyethylene glycol monomethyl ether-b-poly (A) with a structure of formula IVL-glutamic acid) -b-poly (gamma-propargyl-L-glutamate) block copolymer and 2-ethyl azide-N, N-diisopropylamine having a structure of formula V were dissolved in DMSO to obtain a mixture solution. Then adding the blue vitriod and sodium ascorbate into the mixed solution, removing oxygen in the reaction system by bubbling nitrogen flow, and then stirring for reaction. Stirring at 40 deg.C for 72 hr, dialyzing the reaction product solutionFreeze-drying to obtain polyethylene glycol monomethyl ether-b-poly (I) with a structure shown in formula IIL-glutamic acid) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate). Polyethylene glycol monomethyl ether-b-poly (I) with a structure of formula IIL-glutamic acid) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) was dissolved in DMSO, oxygen in the reaction system was removed by bubbling nitrogen, then dopamine having the structure of formula III was added to the above solution, oxygen in the reaction system was removed by bubbling nitrogen, NHS was added to the above solution, oxygen in the reaction system was removed by bubbling nitrogen, finally EDC was added to the above solution, oxygen in the reaction system was removed by bubbling nitrogen, and then the reaction was stirred. After stirring and reacting for 48h at 25 ℃, dialyzing and freeze-drying a reaction product to obtain the triblock amphiphilic copolymer mPEG-b-PL (GA/CA) -b-PGTA with the structure of the formula I.
The invention also provides a drug/protein co-delivery carrier, which comprises a chemotherapeutic drug, a protein drug and a carrier; the carrier is the triblock amphiphilic copolymer with the structure shown in the formula I in the technical scheme or the triblock amphiphilic copolymer with the structure shown in the formula I obtained by the preparation method in the technical scheme. The drug protein complex carrier system described in the present invention can be expressed as a protein/drug carrier.
In the present invention, the chemotherapeutic agent preferably comprises one or more of doxorubicin, paclitaxel and camptothecin, more preferably comprises doxorubicin and paclitaxel, and most preferably comprises doxorubicin; the protein drug preferably comprises ribonuclease A, granzyme B and cytochrome C, more preferably comprises ribonuclease A and granzyme B, and most preferably comprises ribonuclease A. In the present invention, the molar ratio of the carrier to the chemotherapeutic agent is preferably 1: (0.1 to 500), more preferably 1: (0.1 to 300), most preferably 1: (0.1 to 50); the molar ratio of the carrier to the protein drug is preferably 1: (0.1 to 500), more preferably 1: (0.1 to 300), most preferably 1: (0.1 to 50).
The invention also provides a preparation method of the drug/protein carrier, which comprises the following steps:
mixing a chemotherapeutic drug, the triblock amphiphilic copolymer with the structure of the formula I and a first solvent, dripping the mixture into deionized water, stirring for reaction, and dialyzing and freeze-drying to obtain an independent drug carrier;
and dissolving the phenylboronic acid modified protein drug and the single drug carrier in neutral Phosphate Buffer Solution (PBS), stirring for reaction, and performing ultrafiltration purification to obtain the drug/protein carrier.
In the present invention, the kind of the chemotherapeutic drug is the same as that of the chemotherapeutic drug in the above technical scheme, and is not described herein again; the types of the protein medicines in the invention are consistent with those of the protein medicines in the technical scheme, and are not described again; the preparation method of the phenylboronic acid modified protein drug in the invention is preferably as follows document 3: the method described in Angew. chem. int. Ed.2014,53: 13444-13448.
In the present invention, the molar ratio of the carrier to the chemotherapeutic agent is preferably 1: (0.1 to 500), more preferably 1: (0.1 to 300), most preferably 1: (0.1 to 50); the molar ratio of the carrier to the protein drug is preferably 1: (0.1 to 500), more preferably 1: (0.1 to 300), most preferably 1: (0.1 to 50).
In the present invention, the neutral phosphate buffer is not particularly limited, and may be prepared according to a conventional preparation method in a laboratory.
In the invention, the reaction of the chemotherapeutic drug and the triblock amphiphilic copolymer with the structure shown in the formula I is preferably carried out under a dark condition, and the reaction time is preferably 6-72 hours, more preferably 10-24 hours, and most preferably 12 hours; the temperature of the reaction is preferably 10 to 40 ℃ and more preferably 25 ℃. The reaction of the phenylboronic acid modified protein drug and the single drug carrier is preferably carried out under the condition of keeping out of the sun; the reaction time is preferably 1 to 24 hours, more preferably 1 to 12 hours, and most preferably 2 hours; the reaction temperature is preferably 10-40 ℃, and more preferably 25 ℃.
The invention provides a triblock amphiphilic copolymer with a structure shown in formula I and an application of a drug/protein carrier system in preparation of an anti-tumor drug, wherein the triblock amphiphilic copolymer is prepared by the preparation method in the technical scheme or the technical scheme.
In the present invention, the tumor cell is preferably a B16F10 cell. The drug/protein carrier obtained by using the triblock amphiphilic copolymer with the structure shown in the formula I as the carrier can transfer chemotherapeutic drugs and protein drugs into tumor cells, mediate the controllable release of the two drugs in the cells and the escape of endosome lysosomes of the protein drugs, and show an obvious killing effect on the tumor cells through the synergistic effect of the two drugs.
To further illustrate the present invention, a triblock amphiphilic copolymer and a method for preparing the same, a drug-protein co-delivery vehicle and a method for preparing the same, which are provided by the present invention, will be described in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1:
20g of diisopropylamino ethyl chloride hydrochloride and 19.48g of sodium azide were weighed out, and 60mL of water was added to the reaction flask. The reaction was stirred at 80 ℃ overnight, and the resulting mixture was adjusted to pH 10 with potassium hydroxide and extracted three times with 50ml of diethyl ether, respectively. The organic phase was collected and dried over anhydrous magnesium sulfate, and the ether was removed by rotary evaporation to give 2-ethyl azide-N, N-diisopropylamine as a pale yellow oily product.
The obtained 2-ethyl azide-N, N-diisopropylamine is detected, and FIG. 1 is a nuclear magnetic resonance hydrogen spectrogram of the 2-ethyl azide-N, N-diisopropylamine prepared in the embodiment 1 of the invention, and the result shows that the 2-ethyl azide-N, N-diisopropylamine has a structure shown in a formula V.
Example 2:
1.6g of terminally aminated polyethylene glycol monomethyl ether (molecular weight 2000) synthesized in reference 2 was weighed, subjected to azeotropic dehydration with toluene, and dissolved in 70ml of anhydrous DMF. Further weighing 5.3g of the γ -benzyl-glutamate-N-carboxyanhydride synthesized in reference 1, adding the weighed γ -benzyl-glutamate-N-carboxyanhydride to the above-mentioned terminally aminated polyethylene glycol monomethyl ether solution at one time, reacting while stirring at 25 ℃ for 72 hours, further weighing 3.4g of the γ -propargyl-glutamate-N-carboxyanhydride synthesized in reference 1, dissolving in 30ml of anhydrous DMF, adding the resulting mixture to the above-mentioned mixed solution at one time, reacting while stirring at 25 ℃ for 72 hours, and weighingAdding DMAP 117.3mg into the mixed solution, then adding 5mL of acetic anhydride into the mixed solution, stirring and reacting for 24h at 25 ℃, dialyzing and freeze-drying to obtain a white powder product, namely the polyethylene glycol monomethyl ether-b-poly (gamma-benzyl-L-glutamate) -b-poly (gamma-propargyl-L-glutamate) block copolymer mPEG2000-b-PBLG25-b-PPLG20。
Example 3:
1.6g of terminally aminated polyethylene glycol monomethyl ether (molecular weight 2000) synthesized in reference 2 was weighed, subjected to azeotropic dehydration with toluene, and dissolved in 70ml of anhydrous DMF. Weighing 10.6g of gamma-benzyl-glutamate-N-carboxyanhydride synthesized by reference 1, adding the gamma-benzyl-glutamate-N-carboxyanhydride into the polyethylene glycol monomethyl ether solution with aminated tail end at one time, stirring and reacting at 25 ℃ for 72h, weighing 6.8g of gamma-propargyl-glutamate-N-carboxyanhydride synthesized by reference 1, dissolving the gamma-propargyl-glutamate-N-carboxyanhydride in 30mL of anhydrous DMF, adding the anhydrous DMF into the mixed solution at one time, stirring and reacting at 25 ℃ for 72h, weighing 234.6mg of DMAP, adding the DMAP into the mixed solution, adding 10mL of acetic anhydride into the mixed solution, stirring and reacting at 25 ℃ for 24h, dialyzing, and freeze-drying to obtain a white powder product, namely polyethylene glycol monomethyl ether-b-poly (gamma-benzyl-poly-one-hydroxy-ethyl ether) with the structure of formula VIL-glutamate) -b-poly (gamma-propargyl-L-glutamate) block copolymer mPEG2000-b-PBLG50-b-PPLG40。
Example 4:
1.6g of terminally aminated polyethylene glycol monomethyl ether (molecular weight 2000) synthesized in reference 2 was weighed, subjected to azeotropic dehydration with toluene, and dissolved in 70ml of anhydrous DMF. Further weighing 21.2g of γ -benzyl-glutamate-N-carboxyanhydride synthesized in reference 1, adding it to the above-mentioned terminally aminated polyethylene glycol monomethyl ether solution at one time, reacting while stirring at 25 ℃ for 72 hours, further weighing 13.6g of γ -propargyl-glutamate-N-carboxyanhydride synthesized in reference 1, dissolving it in 30mL of anhydrous DMF, adding it to the above-mentioned mixed solution at one time, reacting while stirring at 25 ℃ for 72 hours, weighing 469.2mg of DMAP, adding it to the above-mentioned mixed solution, and adding 20mL of ethyl acetateAdding anhydride into the mixed solution, stirring and reacting for 24h at 25 ℃, dialyzing, and freeze-drying to obtain a white powder product of polyethylene glycol monomethyl ether-b-poly (gamma-benzyl-L-glutamate) -b-poly (gamma-propargyl-L-glutamate) block copolymer mPEG2000-b-PBLG100-b-PPLG80。
Example 5:
4g of terminally aminated polyethylene glycol monomethyl ether (molecular weight: 5000) synthesized in reference 2 was weighed, subjected to azeotropic dehydration with toluene, and dissolved in 70ml of anhydrous DMF. Weighing 5.3g of gamma-benzyl-glutamate-N-carboxyanhydride synthesized by reference 1, adding the gamma-benzyl-glutamate-N-carboxyanhydride into the polyethylene glycol monomethyl ether solution with aminated terminal at one time, stirring and reacting at 25 ℃ for 72h, weighing 3.4g of gamma-propargyl-glutamate-N-carboxyanhydride synthesized by reference 1, dissolving the gamma-propargyl-glutamate-N-carboxyanhydride in 30mL of anhydrous DMF, adding the anhydrous DMF into the mixed solution at one time, stirring and reacting at 25 ℃ for 72h, weighing 117.3mg of DMAP, adding the DMAP into the mixed solution, adding 5mL of acetic anhydride into the mixed solution, stirring and reacting at 25 ℃ for 24h, dialyzing, and freeze-drying to obtain a white powder product, namely polyethylene glycol monomethyl ether-b-poly (gamma-benzyl-poly-one-hydroxy-ethyl ether) with the structure of formula VIL-glutamate) -b-poly (gamma-propargyl-L-glutamate) block copolymer mPEG5000-b-PBLG25-b-PPLG20。
For mPEG obtained in the above5000-b-PBLG25-b-PPLG20Detection is carried out, and FIG. 2 shows mPEG prepared in example 5 of the present invention5000-b-PBLG25-b-PPLG20The result shows that mPEG5000-b-PBLG25-b-PPLG20Has a structure of formula VI.
Example 6:
4g of terminally aminated polyethylene glycol monomethyl ether (molecular weight: 5000) synthesized in reference 2 was weighed, subjected to azeotropic dehydration with toluene, and dissolved in 70ml of anhydrous DMF. Further, 10.6g of gamma-benzyl-glutamate-N-carboxyanhydride synthesized in reference 1 was weighed and added to the above-mentioned terminally aminated polyethylene glycol monomethyl ether solution at a time, 25Stirring and reacting for 72h, then weighing 6.8g of gamma-propargyl-glutamate-N-carboxyanhydride synthesized by reference 1, dissolving in 30mL of anhydrous DMF, then adding the anhydrous DMF into the mixed solution at one time, stirring and reacting for 72h at 25 ℃, weighing 234.6mg of DMAP, adding the DMAP into the mixed solution, then adding 10mL of acetic anhydride into the mixed solution, stirring and reacting for 24h at 25 ℃, dialyzing and freeze-drying to obtain a white powder product, namely the polyethylene glycol monomethyl ether-b-poly (gamma-benzyl-one-chain-type) with the structure of formula VIL-glutamate) -b-poly (gamma-propargyl-L-glutamate) block copolymer mPEG5000-b-PBLG50-b-PPLG40。
Example 7:
4g of terminally aminated polyethylene glycol monomethyl ether (molecular weight: 5000) synthesized in reference 2 was weighed, subjected to azeotropic dehydration with toluene, and dissolved in 70ml of anhydrous DMF. Weighing 21.2g of gamma-benzyl-glutamate-N-carboxyanhydride synthesized by reference 1, adding the gamma-benzyl-glutamate-N-carboxyanhydride into the polyethylene glycol monomethyl ether solution with the aminated tail end at one time, stirring and reacting at 25 ℃ for 72h, weighing 13.6g of gamma-propargyl-glutamate-N-carboxyanhydride synthesized by reference 1, dissolving the gamma-propargyl-glutamate-N-carboxyanhydride in 30mL of anhydrous DMF, adding the anhydrous DMF into the mixed solution at one time, stirring and reacting at 25 ℃ for 72h, weighing DMAP 469.2mg, adding the DMAP into the mixed solution, adding 20mL of acetic anhydride into the mixed solution, stirring and reacting at 25 ℃ for 24h, dialyzing, and freeze-drying to obtain a white powder product, namely polyethylene glycol monomethyl ether-b-poly (gamma-benzyl-poly-one-hydroxy-ethyl ether) with the structure of formula VIL-glutamate) -b-poly (gamma-propargyl-L-glutamate) block copolymer mPEG5000-b-PBLG100-b-PPLG80。
Example 8:
8g of terminally aminated polyethylene glycol monomethyl ether (molecular weight: 10000) synthesized in reference 2 was weighed, subjected to azeotropic dehydration with toluene, and dissolved in 70ml of anhydrous DMF. Further, 5.3g of gamma-benzyl-glutamate-N-carboxyanhydride synthesized in reference 1 was weighed, and the resulting mixture was added to the above-mentioned terminally aminated polyethylene glycol monomethyl ether solution at one time, stirred at 25 ℃ for reaction for 72 hours, and then, gamma-propargyl was weighed and synthesized in reference 1Dissolving 3.4g of base-glutamate-N-carboxyanhydride in 30mL of anhydrous DMF (dimethyl formamide), adding the mixture into the mixed solution at one time, stirring and reacting at 25 ℃ for 72h, weighing 117.3mg of DMAP (dimethyl formamide), adding the DMAP into the mixed solution, adding 5mL of acetic anhydride into the mixed solution, stirring and reacting at 25 ℃ for 24h, dialyzing, and freeze-drying to obtain a white powder product, namely the polyethylene glycol monomethyl ether-b-poly (gamma-benzyl-one-component) with the structure of formula VIL-glutamate) -b-poly (gamma-propargyl-L-glutamate) block copolymer mPEG10000-b-PBLG25-b-PPLG20。
Example 9:
8g of terminally aminated polyethylene glycol monomethyl ether (molecular weight: 10000) synthesized in reference 2 was weighed, subjected to azeotropic dehydration with toluene, and dissolved in 70ml of anhydrous DMF. Weighing 10.6g of gamma-benzyl-glutamate-N-carboxyanhydride synthesized by reference 1, adding the gamma-benzyl-glutamate-N-carboxyanhydride into the polyethylene glycol monomethyl ether solution with aminated tail end at one time, stirring and reacting at 25 ℃ for 72h, weighing 6.8g of gamma-propargyl-glutamate-N-carboxyanhydride synthesized by reference 1, dissolving the gamma-propargyl-glutamate-N-carboxyanhydride in 30mL of anhydrous DMF, adding the anhydrous DMF into the mixed solution at one time, stirring and reacting at 25 ℃ for 72h, weighing 234.6mg of DMAP, adding the DMAP into the mixed solution, adding 10mL of acetic anhydride into the mixed solution, stirring and reacting at 25 ℃ for 24h, dialyzing, and freeze-drying to obtain a white powder product, namely polyethylene glycol monomethyl ether-b-poly (gamma-benzyl-poly-one-hydroxy-ethyl ether) with the structure of formula VIL-glutamate) -b-poly (gamma-propargyl-L-glutamate) block copolymer mPEG10000-b-PBLG50-b-PPLG40。
Example 10:
8g of terminally aminated polyethylene glycol monomethyl ether (molecular weight: 10000) synthesized in reference 2 was weighed, subjected to azeotropic dehydration with toluene, and dissolved in 70ml of anhydrous DMF. Further, 21.2g of γ -benzyl-glutamate-N-carboxyanhydride synthesized in reference 1 was weighed, and added to the above-mentioned terminally aminated polyethylene glycol monomethyl ether solution at one time, followed by reaction with stirring at 25 ℃ for 72 hours, and then 13.6g of γ -propargyl-glutamate-N-carboxyanhydride synthesized in reference 1 was weighed and dissolved in 30ml of anhydrous DMFThen adding the obtained product into the mixed solution at one time, stirring and reacting for 72h at 25 ℃, weighing DMAP 469.2mg, adding the DMAP 469.2mg into the mixed solution, adding 20mL of acetic anhydride into the mixed solution, stirring and reacting for 24h at 25 ℃, dialyzing and freeze-drying to obtain a white powder product, namely the polyethylene glycol monomethyl ether-b-poly (gamma-benzyl-L-glutamate) -b-poly (gamma-propargyl-L-glutamate) block copolymer mPEG10000-b-PBLG100-b-PPLG80。
Example 11:
5g of mPEG prepared in example 2 were weighed2000-b-PBLG25-b-PPLG20Dissolving in 50mL of dichloroacetic acid, adding 15mL of hydrogen bromide, stirring at 30 ℃ for reaction for 1.5h, settling by diethyl ether, dissolving the settled product in 15mL of DMF, stirring at room temperature overnight, dialyzing, and freeze-drying to obtain a white powder product, namely polyethylene glycol monomethyl ether-b-poly (ethylene glycol mono-methyl ether-b-poly (ethylene glycol) (PEG)) (IV) with a structure of formula IVL-glutamic acid) -b-poly (gamma-propargyl-L-glutamate) triblock copolymer mPEG2000-b-PLGA25-b-PPLG20。
Example 12:
5g of mPEG prepared in example 3 were weighed2000-b-PBLG50-b-PPLG40Dissolving in 50mL of dichloroacetic acid, adding 15mL of hydrogen bromide, stirring at 30 ℃ for reaction for 1.5h, settling by diethyl ether, dissolving the settled product in 15mL of DMF, stirring at room temperature overnight, dialyzing, and freeze-drying to obtain a white powder product, namely polyethylene glycol monomethyl ether-b-poly (ethylene glycol mono-methyl ether-b-poly (ethylene glycol) (PEG)) (IV) with a structure of formula IVL-glutamic acid) -b-poly (gamma-propargyl-L-glutamate) triblock copolymer mPEG2000-b-PLGA50-b-PPLG40。
Example 13:
5g of mPEG from example 4 were weighed2000-b-PBLG100-b-PPLG80Dissolving in 50mL of dichloroacetic acid, adding 15mL of hydrogen bromide, stirring at 30 ℃ for reaction for 1.5h, settling by diethyl ether, dissolving the settled product in 15mL of DMF, stirring at room temperature overnight, dialyzing, and freeze-drying to obtain a white powder product, namely polyethylene glycol monomethyl ether-b-poly (ethylene glycol mono-methyl ether-b-poly (ethylene glycol) (PEG)) (IV) with a structure of formula IVL-glutamic acid) -b-poly (gamma-propargyl-L-glutamate) triblock copolymer mPEG2000-b-PLGA100-b-PPLG80。
Example 14:
5g of mPEG prepared in example 5 were weighed5000-b-PBLG25-b-PPLG20Dissolving in 50mL of dichloroacetic acid, adding 15mL of hydrogen bromide, stirring at 30 ℃ for reaction for 1.5h, settling by diethyl ether, dissolving the settled product in 15mL of DMF, stirring at room temperature overnight, dialyzing, and freeze-drying to obtain a white powder product, namely polyethylene glycol monomethyl ether-b-poly (ethylene glycol mono methyl ether-b-poly (ethylene glycol) (A-methyl ether)), (N-methyl ether-b-poly (ethylene glycol) (AL-glutamic acid) -b-poly (gamma-propargyl-L-glutamate) triblock copolymer mPEG5000-b-PLGA25-b-PPLG20。
For mPEG obtained in the above5000-b-PLGA25-b-PPLG20Detection is carried out, and FIG. 3 shows mPEG prepared in example 14 of the present invention5000-b-PLGA25-b-PPLG20The result shows that mPEG5000-b-PLGA25-b-PPLG20Has a structure shown in formula IV.
Example 15:
5g of mPEG prepared in example 6 were weighed5000-b-PBLG50-b-PPLG40Dissolving in 50mL of dichloroacetic acid, adding 15mL of hydrogen bromide, stirring at 30 ℃ for reaction for 1.5h, settling by diethyl ether, dissolving the settled product in 15mL of DMF, stirring at room temperature overnight, dialyzing, and freeze-drying to obtain a white powder product, namely polyethylene glycol monomethyl ether-b-poly (ethylene glycol mono-methyl ether-b-poly (ethylene glycol) (PEG)) (IV) with a structure of formula IVL-glutamic acid) -b-poly (gamma-propargyl-L-glutamate) triblock copolymer mPEG5000-b-PLGA50-b-PPLG40。
Example 16:
5g of mPEG prepared in example 7 were weighed5000-b-PBLG100-b-PPLG80Dissolving in 50mL of dichloroacetic acid, adding 15mL of hydrogen bromide, stirring at 30 ℃ for reaction for 1.5h, settling by diethyl ether, dissolving the settled product in 15mL of DMF, stirring at room temperature overnight, dialyzing, and freeze-drying to obtain a white powder product, namely polyethylene glycol monomethyl ether-b-poly (ethylene glycol mono-methyl ether-b-poly (ethylene glycol) (PEG)) (IV) with a structure of formula IVL-glutamic acid) -b-poly (gamma-propargyl-L-glutamate) triblock copolymer mPEG5000-b-PLGA100-b-PPLG80。
Example 17:
5g of mPEG from example 8 were weighed10000-b-PBLG25-b-PPLG20Dissolving in 50mL of dichloroacetic acid, adding 15mL of hydrogen bromide, stirring at 30 ℃ for reaction for 1.5h, settling by diethyl ether, dissolving the settled product in 15mL of DMF, stirring at room temperature overnight, dialyzing, and freeze-drying to obtain a white powder product, namely polyethylene glycol monomethyl ether-b-poly (ethylene glycol mono-methyl ether-b-poly (ethylene glycol) (PEG)) (IV) with a structure of formula IVL-glutamic acid) -b-poly (gamma-propargyl-L-glutamate) triblock copolymer mPEG10000-b-PLGA25-b-PPLG20。
Example 18:
5g of mPEG prepared in example 9 were weighed10000-b-PBLG50-b-PPLG40Dissolving in 50mL of dichloroacetic acid, adding 15mL of hydrogen bromide, stirring at 30 ℃ for reaction for 1.5h, settling by diethyl ether, dissolving the settled product in 15mL of DMF, stirring at room temperature overnight, dialyzing, and freeze-drying to obtain a white powder product, namely polyethylene glycol monomethyl ether-b-poly (ethylene glycol mono-methyl ether-b-poly (ethylene glycol) (PEG)) (IV) with a structure of formula IVL-glutamic acid) -b-poly (gamma-propargyl-L-glutamate) triblock copolymer mPEG10000-b-PLGA50-b-PPLG40。
Example 19:
5g of mPEG prepared in example 10 were weighed10000-b-PBLG100-b-PPLG80Dissolving in 50mL of dichloroacetic acid, adding 15mL of hydrogen bromide, stirring at 30 ℃ for reaction for 1.5h, settling by diethyl ether, dissolving the settled product in 15mL of DMF, stirring at room temperature overnight, dialyzing, and freeze-drying to obtain a white powder product, namely polyethylene glycol monomethyl ether-b-poly (ethylene glycol mono-methyl ether-b-poly (ethylene glycol) (PEG)) (IV) with a structure of formula IVL-glutamic acid) -b-poly (gamma-propargyl-L-glutamate) triblock copolymer mPEG10000-b-PLGA100-b-PPLG80。
Example 20:
0.86g of mPEG prepared in example 11 was weighed2000-b-PLGA25-b-PPLG20And 0.68g of 2-ethyl azide-N, N-diisopropylamine obtained in example 1 were dissolved in 40ml of anhydrous DMSO, nitrogen was bubbled for 20 minutes to remove oxygen, 0.1225g of copper sulfate pentahydrate and 0.214g of sodium ascorbate were weighed into the reaction system, bubbled with nitrogen for 15 minutes to remove oxygen, and then sealed and reacted at 40 ℃ with stirring for 72 hours. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I) with a structure of formula IIL-glutamic acid) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG2000-b-PLGA25-b-PGTA20。
Example 21:
0.76g of the mPEG prepared in example 12 was weighed2000-b-PLGA50-b-PPLG40And 0.68g of 2-ethyl azide-N, N-diisopropylamine obtained in example 1 were dissolved in 40ml of anhydrous DMSO, nitrogen was bubbled for 20 minutes to remove oxygen, 0.1225g of copper sulfate pentahydrate and 0.214g of sodium ascorbate were weighed into the reaction system, bubbled with nitrogen for 15 minutes to remove oxygen, and then sealed and reacted at 40 ℃ with stirring for 72 hours. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I) with a structure of formula IIL-glutamic acid) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG2000-b-PLGA50-b-PGTA40。
Example 22:
0.71g of the mPEG prepared in example 13 was weighed2000-b-PLGA100-b-PPLG80And 0.68g of 2-ethyl azide-N, N-diisopropylamine obtained in example 1 were dissolved in 40ml of anhydrous DMSO, nitrogen was bubbled for 20 minutes to remove oxygen, 0.1225g of copper sulfate pentahydrate and 0.214g of sodium ascorbate were weighed into the reaction system, bubbled with nitrogen for 15 minutes to remove oxygen, and then sealed and reacted at 40 ℃ with stirring for 72 hours. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I) with a structure of formula IIL-glutamic acid) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG2000-b-PLGA100-b-PGTA80。
Example 23:
1.16g of mPEG prepared in example 14 were weighed5000-b-PLGA25-b-PPLG20And 0.68g of 2-ethyl azide-N, N-diisopropylamine obtained in example 1 were dissolved in 40ml of anhydrous DMSO, nitrogen was bubbled for 20 minutes to remove oxygen, 0.1225g of copper sulfate pentahydrate and 0.214g of sodium ascorbate were weighed into the reaction system, bubbled with nitrogen for 15 minutes to remove oxygen, and then sealed and reacted at 40 ℃ with stirring for 72 hours. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I) with a structure of formula IIL-glutamic acid) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG5000-b-PLGA25-b-PGTA20。
For mPEG obtained in the above5000-b-PLGA25-b-PGTA20Detection is carried out, and FIG. 4 shows mPEG prepared in example 23 of the present invention5000-b-PLGA25-b-PGTA20The result shows that mPEG5000-b-PLGA25-b-PGTA20Has a structure of a formula II.
Example 24:
0.91g of the mPEG prepared in example 15 was weighed5000-b-PLGA50-b-PPLG40And 0.68g of 2-ethyl azide-N, N-diisopropylamine obtained in example 1 were dissolved in 40ml of anhydrous DMSO, nitrogen was bubbled for 20 minutes to remove oxygen, 0.1225g of copper sulfate pentahydrate and 0.214g of sodium ascorbate were weighed into the reaction system, bubbled with nitrogen for 15 minutes to remove oxygen, and then sealed and reacted at 40 ℃ with stirring for 72 hours. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I) with a structure of formula IIL-glutamic acid) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG5000-b-PLGA50-b-PGTA40。
Example 25:
0.78g of the mPEG prepared in example 16 was weighed5000-b-PLGA100-b-PPLG80And 0.68g of 2-ethyl azide-N, N-diisopropylamine obtained in example 1 were dissolved in 40ml of anhydrous DMSO, nitrogen was bubbled for 20 minutes to remove oxygen, 0.1225g of copper sulfate pentahydrate and 0.214g of sodium ascorbate were weighed into the reaction system, bubbled with nitrogen for 15 minutes to remove oxygen, and then sealed and reacted at 40 ℃ with stirring for 72 hours. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I) with a structure of formula IIL-glutamic acid) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG5000-b-PLGA100-b-PGTA80。
Example 26:
1.66g of mPEG prepared in example 17 were weighed10000-b-PLGA25-b-PPLG20And 0.68g of 2-ethyl azide-N, N-diisopropylamine obtained in example 1 were dissolved in 40ml of anhydrous DMSO, nitrogen was bubbled for 20 minutes to remove oxygen, 0.1225g of copper sulfate pentahydrate and 0.214g of sodium ascorbate were weighed into the reaction system, bubbled with nitrogen for 15 minutes to remove oxygen, and then sealed and reacted at 40 ℃ with stirring for 72 hours. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I) with a structure of formula IIL-glutamic acid) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG10000-b-PLGA25-b-PGTA20。
Example 27:
1.16g of mPEG prepared in example 18 were weighed10000-b-PLGA50-b-PPLG40And 0.68g of 2-ethyl azido-N, N-diisopropylamine prepared in example 1, dissolved in 40ml of anhydrous DMSO, bubbled with nitrogen for 20 minutes to remove oxygen, and 0.1225g of copper sulfate pentahydrate were weighedAnd 0.214g of sodium ascorbate were added to the reaction system, and nitrogen was bubbled for 15 minutes to remove oxygen, followed by sealing and stirring the reaction at 40 ℃ for 72 hours. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I) with a structure of formula IIL-glutamic acid) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG10000-b-PLGA50-b-PGTA40。
Example 28:
0.91g of the mPEG prepared in example 19 was weighed10000-b-PLGA100-b-PPLG80And 0.68g of 2-ethyl azide-N, N-diisopropylamine obtained in example 1 were dissolved in 40ml of anhydrous DMSO, nitrogen was bubbled for 20 minutes to remove oxygen, 0.1225g of copper sulfate pentahydrate and 0.214g of sodium ascorbate were weighed into the reaction system, bubbled with nitrogen for 15 minutes to remove oxygen, and then sealed and reacted at 40 ℃ with stirring for 72 hours. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I) with a structure of formula IIL-glutamic acid) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG10000-b-PLGA100-b-PGTA80。
Example 29:
0.4g of the mPEG prepared in example 20 was weighed2000-b-PLGA25-b-PGTA20Dissolving in 40ml of anhydrous DMSO, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.038g of dopamine hydrochloride, adding to the above solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.028g of NHS, adding to the above mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.046g of EDC hydrochloride, adding to the above mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, sealing and reacting at 25 ℃ for 48h with stirring. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I)L-glutamic acid/gamma-3, 4-dihydroxyphenethyl-L-Glutamine) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG2000-b-PL(GA19CA6)-b-PGTA20。
Example 30:
0.4g of the mPEG prepared in example 20 was weighed2000-b-PLGA25-b-PGTA20Dissolving in 40ml of anhydrous DMSO, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.12g of dopamine hydrochloride, adding to the solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.089g of NHS, adding to the mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.146g of EDC hydrochloride, adding to the mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, sealing, and reacting at 25 ℃ for 48 hours with stirring. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I)LGlutamic acid/gamma-3, 4-dihydroxyphenethyl-L-Glutamine) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG2000-b-PL(GA6CA19)-b-PGTA20。
Example 31:
0.366g of the mPEG prepared in example 21 were weighed out2000-b-PLGA50-b-PGTA40Dissolving in 40ml of anhydrous DMSO, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.038g of dopamine hydrochloride, adding to the above solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.028g of NHS, adding to the above mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.046g of EDC hydrochloride, adding to the above mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, sealing and reacting at 25 ℃ for 48h with stirring. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I)L-glutamic acid/gamma-3, 4-Dihydroxy phenethyl-L-Glutamine) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG2000-b-PL(GA38CA12)-b-PGTA40。
Example 32
0.366g of the mPEG prepared in example 21 were weighed out2000-b-PLGA50-b-PGTA40Dissolving in 40ml of anhydrous DMSO, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.12g of dopamine hydrochloride, adding to the solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.089g of NHS, adding to the mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.146g of EDC hydrochloride, adding to the mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, sealing, and reacting at 25 ℃ for 48 hours with stirring. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I)LGlutamic acid/gamma-3, 4-dihydroxyphenethyl-L-Glutamine) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG2000-b-PL(GA12CA38)-b-PGTA40。
Example 33
0.349g of mPEG prepared in example 22 was weighed2000-b-PLGA100-b-PGTA80Dissolving in 40ml of anhydrous DMSO, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.038g of dopamine hydrochloride, adding to the above solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.028g of NHS, adding to the above mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.046g of EDC hydrochloride, adding to the above mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, sealing and reacting at 25 ℃ for 48h with stirring. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I)L-glutamic acid/gamma-3, 4-dihydroxybenzeneEthyl-L-Glutamine) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG2000-b-PL(GA76CA24)-b-PGTA80。
Example 34:
0.349g of mPEG prepared in example 22 was weighed2000-b-PLGA100-b-PGTA80Dissolving in 40ml of anhydrous DMSO, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.12g of dopamine hydrochloride, adding to the solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.089g of NHS, adding to the mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.146g of EDC hydrochloride, adding to the mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, sealing, and reacting at 25 ℃ for 48 hours with stirring. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I)LGlutamic acid/gamma-3, 4-dihydroxyphenethyl-L-Glutamine) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG2000-b-PL(GA24CA76)-b-PGTA80。
Example 35:
0.5g of the mPEG prepared in example 23 was weighed5000-b-PLGA25-b-PGTA20Dissolving in 40ml of anhydrous DMSO, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.038g of dopamine hydrochloride, adding to the above solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.028g of NHS, adding to the above mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.046g of EDC hydrochloride, adding to the above mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, sealing and reacting at 25 ℃ for 48h with stirring. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I)LGlutamic acid/gamma-3, 4-dihydroxyphenethyl-L-Glutamine) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG5000-b-PL(GA19CA6)-b-PGTA20。
For mPEG obtained in the above5000-b-PL(GA19CA6)-b-PGTA20Detection is carried out, and FIG. 5 shows mPEG prepared in example 35 of the present invention5000-b-PL(GA19CA6)-b-PGTA20The result shows that mPEG5000-b-PL(GA19CA6)-b-PGTA20Has a structure shown in formula I.
Example 36:
0.5g of the mPEG prepared in example 23 was weighed5000-b-PLGA25-b-PGTA20Dissolving in 40ml of anhydrous DMSO, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.12g of dopamine hydrochloride, adding to the solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.089g of NHS, adding to the mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.146g of EDC hydrochloride, adding to the mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, sealing, and reacting at 25 ℃ for 48 hours with stirring. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I)LGlutamic acid/gamma-3, 4-dihydroxyphenethyl-L-Glutamine) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG5000-b-PL(GA6CA19)-b-PGTA20。
Example 37:
0.416g of the mPEG prepared in example 24 was weighed5000-b-PLGA50-b-PGTA40Dissolving in 40ml anhydrous DMSO, removing oxygen in the reaction system by bubbling nitrogen, weighing 0.038g dopamine hydrochloride, adding into the above solution, removing oxygen in the reaction system by bubbling nitrogen, weighing 0.028g NHS, adding into the above mixed solutionThen, oxygen in the reaction system was removed by bubbling with a nitrogen stream, and 0.046g of EDC hydrochloride was weighed and added to the above mixed solution, and oxygen in the reaction system was removed by bubbling with a nitrogen stream, sealed and reacted with stirring at 25 ℃ for 48 hours. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I)LGlutamic acid/gamma-3, 4-dihydroxyphenethyl-L-Glutamine) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG5000-b-PL(GA38CA12)-b-PGTA40。
Example 38:
0.416g of the mPEG prepared in example 24 was weighed5000-b-PLGA50-b-PGTA40Dissolving in 40ml of anhydrous DMSO, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.12g of dopamine hydrochloride, adding to the solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.089g of NHS, adding to the mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.146g of EDC hydrochloride, adding to the mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, sealing, and reacting at 25 ℃ for 48 hours with stirring. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I)LGlutamic acid/gamma-3, 4-dihydroxyphenethyl-L-Glutamine) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG5000-b-PL(GA12CA38)-b-PGTA40。
Example 39:
0.374g of the mPEG prepared in example 25 was weighed5000-b-PLGA100-b-PGTA80Dissolving in 40ml anhydrous DMSO, removing oxygen in the reaction system by bubbling nitrogen, weighing 0.038g dopamine hydrochloride, adding to the above solution, removing oxygen in the reaction system by bubbling nitrogen, weighing 0.028g NHS, adding to the above mixed solution, and removing the dissolved substance with the aid of the above solutionThe reaction system was purged with nitrogen gas, and 0.046g of EDC hydrochloride was weighed and added to the above mixed solution, and the reaction system was purged with nitrogen gas, sealed and stirred at 25 ℃ for 48 hours. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I)LGlutamic acid/gamma-3, 4-dihydroxyphenethyl-L-Glutamine) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG5000-b-PL(GA76CA24)-b-PGTA80。
Example 40:
0.374g of the mPEG prepared in example 25 was weighed5000-b-PLGA100-b-PGTA80Dissolving in 40ml of anhydrous DMSO, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.12g of dopamine hydrochloride, adding to the solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.089g of NHS, adding to the mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.146g of EDC hydrochloride, adding to the mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, sealing, and reacting at 25 ℃ for 48 hours with stirring. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I)LGlutamic acid/gamma-3, 4-dihydroxyphenethyl-L-Glutamine) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG5000-b-PL(GA24CA76)-b-PGTA80。
Example 41:
0.167g of mPEG prepared in example 26 was weighed10000-b-PLGA25-b-PGTA20Dissolving in 40ml anhydrous DMSO, removing oxygen in the reaction system by bubbling nitrogen gas, weighing 0.038g dopamine hydrochloride, adding into the above solution, removing oxygen in the reaction system by bubbling nitrogen gas, weighing 0.028g NHS, adding into the above mixed solution, and removing nitrogen gas by bubbling nitrogen gasThe reaction system was purged with oxygen by bubbling with a gas stream, and 0.046g of EDC hydrochloride was further weighed and added to the above mixed solution, and the reaction system was purged with oxygen by bubbling with a nitrogen stream, sealed and stirred at 25 ℃ for 48 hours. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I)LGlutamic acid/gamma-3, 4-dihydroxyphenethyl-L-Glutamine) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG10000-b-PL(GA19CA6)-b-PGTA20。
Example 42:
0.167g of mPEG prepared in example 26 was weighed10000-b-PLGA25-b-PGTA20Dissolving in 40ml of anhydrous DMSO, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.12g of dopamine hydrochloride, adding to the solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.089g of NHS, adding to the mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.146g of EDC hydrochloride, adding to the mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, sealing, and reacting at 25 ℃ for 48 hours with stirring. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I)LGlutamic acid/gamma-3, 4-dihydroxyphenethyl-L-Glutamine) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG10000-b-PL(GA6CA19)-b-PGTA20。
Example 43:
0.25g of the mPEG prepared in example 27 was weighed10000-b-PLGA50-b-PGTA40Dissolving in 40ml anhydrous DMSO, removing oxygen in the reaction system by bubbling nitrogen, weighing 0.038g dopamine hydrochloride, adding into the above solution, removing oxygen in the reaction system by bubbling nitrogen, weighing 0.028g NHS, adding into the above mixed solution, adding nitrogen, and removing oxygen in the reaction system by bubbling nitrogenOxygen in the reaction system was removed by bubbling a stream, and 0.046g of EDC hydrochloride was weighed and added to the above mixed solution, and oxygen in the reaction system was removed by bubbling a nitrogen stream, sealed and reacted at 25 ℃ with stirring for 48 hours. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I)LGlutamic acid/gamma-3, 4-dihydroxyphenethyl-L-Glutamine) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG10000-b-PL(GA38CA12)-b-PGTA40。
Example 44:
0.25g of the mPEG prepared in example 27 was weighed10000-b-PLGA50-b-PGTA40Dissolving in 40ml of anhydrous DMSO, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.12g of dopamine hydrochloride, adding to the solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.089g of NHS, adding to the mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.146g of EDC hydrochloride, adding to the mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, sealing, and reacting at 25 ℃ for 48 hours with stirring. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I)LGlutamic acid/gamma-3, 4-dihydroxyphenethyl-L-Glutamine) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG10000-b-PL(GA12CA38)-b-PGTA40。
Example 45:
0.416g of mPEG10000-b-PLGA obtained in example 28 was weighed out100-b-PGTA80Dissolving in 40ml anhydrous DMSO, removing oxygen in the reaction system by bubbling nitrogen, weighing 0.038g dopamine hydrochloride, adding into the above solution, removing oxygen in the reaction system by bubbling nitrogen, weighing 0.028g NHS, adding into the above mixed solution, removing by bubbling nitrogenThe oxygen in the reaction system was removed, and 0.046g of EDC hydrochloride was weighed and added to the above mixed solution, and the oxygen in the reaction system was removed by bubbling a nitrogen stream, sealed and reacted at 25 ℃ for 48 hours with stirring. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I)LGlutamic acid/gamma-3, 4-dihydroxyphenethyl-L-Glutamine) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG10000-b-PL(GA76CA24)-b-PGTA80。
Example 46:
0.416g of the mPEG prepared in example 28 was weighed10000-b-PLGA100-b-PGTA80Dissolving in 40ml of anhydrous DMSO, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.12g of dopamine hydrochloride, adding to the solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.089g of NHS, adding to the mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, weighing 0.146g of EDC hydrochloride, adding to the mixed solution, removing oxygen in the reaction system by bubbling with a nitrogen stream, sealing, and reacting at 25 ℃ for 48 hours with stirring. After the reaction, the reaction product solution is dialyzed and lyophilized. Obtaining a light yellow powder product of polyethylene glycol monomethyl ether-b-poly (I)LGlutamic acid/gamma-3, 4-dihydroxyphenethyl-L-Glutamine) -b-poly (. gamma. -1N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) triblock copolymer mPEG10000-b-PL(GA24CA76)-b-PGTA80。
Comparative examples 1 to 6: preparation of Individual drug Carriers
100mg of the products prepared in example 29, example 30, example 31, example 32, example 33 and example 34 are weighed out separately;
and dissolving 10mg of adriamycin hydrochloride in 5mL of DMSO respectively, adding 40 mu L of triethylamine into the mixed solution, uniformly mixing by vortex oscillation, dropwise adding into stirred water, stirring for 12 hours at room temperature in a dark place, dialyzing the mixed solution, and freeze-drying to obtain a single drug carrier system.
The individual drug carriers prepared in comparative examples 1 to 6 were similar to those of comparative example 7 in anticancer effect as antitumor drugs.
Comparative example 7: preparation of Individual drug Carriers
100mg of mPEG prepared in example 35 were weighed out separately5000-b-PL(GA19CA6)-b-PGTA20And 10mg of adriamycin hydrochloride, dissolving in 5mL of DMSO, adding 40 mu L of triethylamine into the mixed solution, uniformly mixing by vortex oscillation, dropwise adding into stirred water, stirring for 12 hours at room temperature in a dark place, dialyzing the mixed solution, and freeze-drying to obtain a single drug carrier system.
The particle size of the individual drug carriers was measured by dynamic light scattering, the measurement results are shown in fig. 6; FIG. 6 is a graph of dynamic light scattering of nanoparticles assembled from individual drug carriers prepared in comparative example 7 of the present invention in an aqueous phase; the antitumor activity of the nanoparticles was examined by cytotoxicity assay, and fig. 12 is MTT assay of B16F10 cells. Fig. 12 shows cytotoxicity experiments after the drug-carrier-only nanoparticles prepared in comparative example 7, the protein-carrier-only nanoparticles prepared in comparative example 25, and the drug/protein-carrier nanoparticles prepared in example 53 were incubated with B16F10 tumor cells for 72h, and the experimental results show that the drug-carrier-only nanoparticles prepared in comparative example 7 can reduce the survival rate of cancer cells to 20 ± 3%.
Comparative examples 8 to 18: preparation of Individual drug Carriers
100mg of the products prepared in example 36, example 37, example 38, example 39, example 40, example 41, example 42, example 43, example 44, example 45 and example 46 are each weighed out;
dissolving 10mg of adriamycin hydrochloride in 5mL of DMSO respectively, adding 40 mu L of triethylamine into the mixed solution, uniformly mixing by vortex oscillation, dropwise adding into stirred water, stirring for 12h at room temperature in a dark place, dialyzing the mixed solution, and freeze-drying to obtain a single drug carrier system.
The individual drug carriers prepared in comparative examples 8 to 18 were similar to those of comparative example 7 in anticancer effect as antitumor drugs.
Comparative examples 19 to 24: preparation of Individual protein Carriers
100mg of each of the products obtained in example 29, example 30, example 31, example 32, example 33 and example 34 was weighed, dissolved in 5mL of DMSO, vortexed, shaken and mixed, added dropwise to stirred water, stirred at room temperature in the dark for 12 hours, and the mixture was dialyzed and lyophilized to obtain an empty vector system. Respectively weighing 20mg of the prepared empty carrier, respectively dissolving in 10ml of PBS, further weighing 6.6mg of phenylboronic acid modified ribonuclease A prepared according to reference 3, respectively adding into the mixed solution, stirring at room temperature in a dark place for 2 hours, and performing ultrafiltration purification to obtain a single protein carrier system.
The individual protein carriers prepared in comparative examples 19 to 24 were similar to those of comparative example 25 in anticancer effect as antitumor agents.
Comparative example 25: preparation of Individual protein Carriers
100mg of mPEG prepared in example 35 were weighed out separately5000-b-PL(GA19CA6)-b-PGTA20Dissolving the mixture in 5mL of DMSO, uniformly mixing by vortex oscillation, dropwise adding the mixture into stirred water, stirring for 12 hours at room temperature in a dark place, dialyzing and freeze-drying the mixed solution to obtain an unloaded system. Weighing 20mg of the empty carrier prepared above, dissolving in 10ml of PBS, weighing 6.6mg of phenylboronic acid modified ribonuclease A prepared according to reference 3, adding to the mixed solution, stirring at room temperature in the dark for 2 hours, and purifying by ultrafiltration to obtain a single protein carrier system.
The particle size of the individual protein carriers was measured by dynamic light scattering, and the measurement results are shown in fig. 7; FIG. 7 is a dynamic light scattering diagram of nanoparticles assembled by individual protein carriers prepared in comparative example 25 of the present invention in an aqueous phase; the antitumor activity of the nanoparticles was examined by cytotoxicity assay, and fig. 12 is MTT assay of B16F10 cells. Fig. 12 shows cytotoxicity experiments of the drug-carrier-only nanoparticles prepared in comparative example 7, the protein-carrier-only nanoparticles prepared in comparative example 25, and the drug/protein-carrier nanoparticles prepared in example 53 after being incubated with B16F10 tumor cells for 72h, wherein the experimental results show that the protein-carrier-only nanoparticles prepared in comparative example 25 can reduce the survival rate of cancer cells to 46 ± 4%.
Comparative example 26 to comparative example 36: preparation of Individual protein Carriers
100mg of the products prepared in the examples 36 to 46 are respectively weighed, dissolved in 5mL of DMSO, vortexed, uniformly mixed, dropwise added into stirred water, stirred at room temperature in a dark place for 12 hours, and dialyzed and freeze-dried to obtain an unloaded system. Weighing 20mg of the empty carrier prepared above, dissolving in 10ml of PBS, weighing 6.6mg of phenylboronic acid modified ribonuclease A prepared according to reference 3, respectively adding to the above mixed solution, stirring at room temperature in the dark for 2 hours, and purifying by ultrafiltration to obtain a single protein carrier system.
The individual protein carriers prepared in the above comparative examples have anticancer effects similar to those of comparative example 25 as antitumor agents.
Examples 47 to 52: preparation of drug/protein carriers
20mg of each of the drug carriers prepared in comparative examples 1,2,3, 4,5 and 6 was dissolved in 10ml of PBS, and 6.6mg of phenylboronic acid-modified ribonuclease A prepared according to reference 3 was added to the above mixed solution, stirred at room temperature in the dark for 2 hours, and purified by ultrafiltration to obtain a protein/drug co-delivery carrier.
The anti-cancer effect of the drug/protein carrier prepared in the above example is similar to that of example 53 when it is used as an anti-tumor drug.
Example 53: preparation of drug/protein carriers
20mg of each of the individual drug carriers prepared in comparative example 7 was weighed and dissolved in 10ml of PBS, and 6.6mg of phenylboronic acid-modified ribonuclease A prepared according to reference 3 was added to the above mixed solution, stirred at room temperature in the dark for 2 hours, and purified by ultrafiltration to obtain a protein/drug co-delivery carrier.
The particle size of the drug/protein carrier was measured by dynamic light scattering, and the measurement results are shown in fig. 8; FIG. 8 is a dynamic light scattering diagram of nanoparticles assembled by drug/protein carriers prepared in example 53 of the present invention in aqueous phase; the Transmission Electron Microscope (TEM) results are shown in fig. 9, and fig. 9 is a transmission electron microscope image of the nanoparticles assembled by the drug/protein carrier prepared in example 53 of the present invention in the aqueous phase; the results of the drug release behavior in response to acidic pH are shown in FIG. 10, and FIG. 10 is the cumulative drug release results of the drug/protein carrier prepared in example 53 according to the present invention under different pH conditions. The results of the acidic pH-responsive protein release behavior are shown in fig. 11, and fig. 11 is the results of the cumulative protein release of the drug/protein carrier prepared in example 53 according to the present invention under different pH conditions. The antitumor activity of the nanoparticles was examined by cytotoxicity assay, and fig. 12 is MTT assay of B16F10 cells. Fig. 12 shows cytotoxicity experiments of the drug-carrier-only nanoparticles prepared in comparative example 7, the protein-carrier-only nanoparticles prepared in comparative example 25, and the drug/protein-carrier nanoparticles prepared in example 53 after being incubated with B16F10 tumor cells for 72h, wherein the experimental results show that the drug/protein-carrier-only nanoparticles prepared in example 53 can reduce the survival rate of cancer cells to 15 ± 2%. The result-based drug synergistic effect analysis shows that the cytotoxicity of the drug/protein carrier system is significantly higher than that of a single drug carrier system and a single protein carrier system, and the drug/protein carrier system has a synergistic anti-tumor effect.
Examples 54 to 64: preparation of drug/protein carriers
20mg of each of the drug carriers prepared in comparative examples 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18 was dissolved in 10ml of PBS, and 6.6mg of phenylboronic acid-modified ribonuclease A prepared according to reference 3 was added to the above mixed solution, stirred at room temperature in the dark for 2 hours, and purified by ultrafiltration to obtain a protein/drug co-delivery carrier.
The anti-cancer effects of the drugs/protein carriers prepared in examples 54 to 64 as anti-tumor drugs were similar to those of example 53.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A triblock amphiphilic copolymer having the structure of formula I:
wherein x is more than or equal to 10 and less than or equal to 500; y is more than or equal to 5 and less than or equal to 100; z is more than or equal to 5 and less than or equal to 100; m is more than or equal to 5 and less than or equal to 100.
2. Triblock amphiphilic copolymer according to claim 1, characterized in that it is in particular:
mPEG2000-b-PL(GA19CA6)-b-PGTA20、mPEG2000-b-PL(GA6CA19)-b-PGTA20、mPEG2000-b-PL(GA38CA12)-b-PGTA40、mPEG2000-b-PL(GA12CA38)-b-PGTA40、mPEG2000-b-PL(GA76CA24)-b-PGTA80、mPEG2000-b-PL(GA24CA76)-b-PGTA80、mPEG5000-b-PL(GA19CA6)-b-PGTA20、mPEG5000-b-PL(GA6CA19)-b-PGTA20、mPEG5000-b-PL(GA38CA12)-b-PGTA40、mPEG5000-b-PL(GA12CA38)-b-PGTA40、mPEG5000-b-PL(GA76CA24)-b-PGTA80、mPEG5000-b-PL(GA24CA76)-b-PGTA80、mPEG10000-b-PL(GA19CA6)-b-PGTA20、mPEG10000-b-PL(GA6CA19)-b-PGTA20、mPEG10000-b-PL(GA38CA12)-b-PGTA40、mPEG10000-b-PL(GA12CA38)-b-PGTA40、mPEG10000-b-PL(GA76CA24)-b-PGTA80or mPEG10000-b-PL(GA24CA76)-b-PGTA80；
Wherein m is methoxy; PL is poly; GA is L-glutamic acid; CA is gamma-3, 4-dihydroxyphenethyl-L-glutamine.
3. A process for the preparation of a triblock amphiphilic copolymer according to any one of claims 1 to 2, comprising the steps of:
in the presence of a solvent and a catalyst, polyethylene glycol monomethyl ether-b-poly (L-glutamic acid) -b-poly (gamma-1Carrying out amidation reaction on the N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) block copolymer and dopamine with a structure of a formula III to obtain a triblock amphiphilic copolymer with a structure of a formula I;
4. the method according to claim 3, wherein the temperature of the amidation reaction is 15 to 80 ℃; the amidation reaction time is 12-96 h.
5. The method as claimed in claim 3, wherein the polyethylene glycol monomethyl ether-b-poly (L-glutamic acid) -b-poly (γ -1The mol ratio of the N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) block copolymer to the dopamine with the structure of the formula III is 1: 1 to 500.
6. The method as claimed in claim 3, wherein the polyethylene glycol monomethyl ether-b-poly (L-glutamic acid) -b-poly (γ -1The N- (N, N-diisopropylethylamine) -1,2, 3-triazole-4-methyl-L-glutamate) block copolymer is prepared from a polyethylene glycol monomethyl ether-b-poly (L-glutamic acid) -b-poly (gamma-propargyl-L-glutamate) block copolymer with a structure shown in a formula IV and 2-ethyl azide-N, N-diisopropylamine with a structure shown in a formula V through a click chemistry reaction:
7. the method as claimed in claim 6, wherein the polyethylene glycol monomethyl ether-b-poly (L-glutamic acid) -b-poly (gamma-propargyl-L-glutamate) block copolymer with the structure of formula IV is prepared from polyethylene glycol monomethyl ether-b-poly (gamma-benzyl-L-glutamate) -b-poly (gamma-propargyl-L-glutamate) with the structure of formula VI by benzyl deprotection reaction:
in the formula VI, x is more than or equal to 10 and less than or equal to 500; y is more than or equal to 5 and less than or equal to 100; z is more than or equal to 5 and less than or equal to 100.
8. The method of claim 7, wherein the methoxypolyethylene glycol-b-poly (γ -benzyl-L-glutamate) -b-poly (γ -propargyl-L-glutamate) having the structure of formula vi is prepared by:
under the initiation of end-aminated polyethylene glycol monomethyl ether, sequentially carrying out ring-opening polymerization in gamma-benzyl-glutamate-N-carboxyl cyclic internal anhydride and gamma-propargyl-glutamate-N-carboxyl cyclic internal anhydride in a solvent, and sealing the end amino by using acetic anhydride to obtain polyethylene glycol monomethyl ether-b-poly (gamma-benzyl-L-glutamate) -b-poly (gamma-propargyl-L-glutamate) with the structure of formula VI.
9. A drug/protein co-delivery vehicle comprising a hydrophobic chemotherapeutic drug, a phenylboronic acid-modified protein drug, and a carrier;
the carrier is a triblock amphiphilic copolymer with a structure shown in a formula I as claimed in any one of claims 1 to 8;
the molar ratio of the carrier to the hydrophobic chemotherapeutic drug is 1: 0.1 to 500; the molar ratio of the carrier to the phenylboronic acid modified protein drug is 1: 0.1 to 500.
10. A method of preparing the drug/protein co-delivery vehicle of claim 9, comprising the steps of:
mixing a hydrophobic chemotherapeutic drug, a triblock amphiphilic copolymer with a structure shown in a formula I and a solvent, dripping the mixture into deionized water, stirring the mixture for reaction, and dialyzing and freeze-drying the mixture to obtain an independent drug carrier;
dissolving the phenylboronic acid modified protein drug and the single drug carrier in a neutral phosphate buffer solution, stirring for reaction, and performing ultrafiltration purification to obtain a drug/protein co-delivery carrier;
the molar ratio of the carrier to the hydrophobic chemotherapeutic drug is 1: 0.1 to 500; the molar ratio of the carrier to the phenylboronic acid modified protein drug is 1: 0.1 to 500.
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