CN111973555A - Anti-tumor and anti-tumor metastasis dual-pH-sensitive polymer-drug conjugate mixed micelle composition - Google Patents

Anti-tumor and anti-tumor metastasis dual-pH-sensitive polymer-drug conjugate mixed micelle composition Download PDF

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CN111973555A
CN111973555A CN201910422001.8A CN201910422001A CN111973555A CN 111973555 A CN111973555 A CN 111973555A CN 201910422001 A CN201910422001 A CN 201910422001A CN 111973555 A CN111973555 A CN 111973555A
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drug
oxazoline
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刘艳
周远航
邹洋
李馨儒
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Peking University
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    • A61P35/04—Antineoplastic agents specific for metastasis

Abstract

The invention provides a preparation method of a double-pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-transfer drug conjugate, a mixed micelle composition and application thereof. The acid-sensitive chemical bond is taken as a connecting arm to couple the poly (2-alkyl-2-oxazoline) -hydrophobic chain with the anti-tumor drug and the anti-transfer drug. The conjugate disclosed by the invention can respond to an acidic environment in a cell to quickly release a drug, and can be self-assembled in an aqueous environment to form a dual pH-sensitive conjugate mixed micelle. The conjugate mixed micelle can avoid the premature release of the drug in systemic circulation, increase the accumulation of the drug in tumor sites and reduce toxic and side effects. In addition, the anti-tumor drug and the anti-metastasis drug in the conjugate mixed micelle have the effects of synergistic anti-tumor growth and anti-metastasis. The conjugate mixed micelle has potential application in the aspects of drug targeted delivery, controlled release, drug toxic and side effect reduction, synergistic antitumor and antitumor metastasis.

Description

Anti-tumor and anti-tumor metastasis dual-pH-sensitive polymer-drug conjugate mixed micelle composition
Technical Field
The invention belongs to the field of pharmaceutical preparations, and relates to a polymer micelle composition for resisting tumor and tumor metastasis, in particular to a polymer-drug conjugate micelle.
Background
Although radiotherapy and chemotherapy can effectively inhibit the growth of some primary tumors, under the existing treatment means, the prognosis of metastatic tumors is still not ideal due to serious toxic and side effects or postoperative recurrence. Tumor metastasis is an important biological feature of malignant tumors, and statistical studies have shown that 90% of patients who die from cancer die from tumor metastasis, not from primary tumors. Tumor metastasis is a continuous process of a series of multi-step, multifactorial interactions between tumor cells, the host and the tumor microenvironment. Due to the complexity of tumor metastasis mechanism, the effect of single drug therapy is often not ideal, and the antitumor drug and the anti-tumor metastasis drug are combined to make the two drugs play a synergistic effect to achieve a better therapeutic effect. For example, patent publication No. CN108309943A prepares nanocrystals for co-delivery of paclitaxel and selemarieside for the purpose of inhibiting tumor cell proliferation and metastasis. Patent publication No. CN105963306B combines metformin with ursolic acid and its derivatives, and has significant inhibitory effect on proliferation, migration, invasion, etc. of tumor cells. However, the low solubility of chemotherapeutic drugs such as paclitaxel, doxorubicin, etc. in water greatly limits their clinical applications. The nano-carrier can obviously improve the solubility of insoluble drugs.
Generally, the nano-drug delivery system has the following advantages: (1) improving the solubility and bioavailability of the drug; (2) protecting the entrapped drug from degradation; (3) increasing the uptake of the drug by the cells; (4) change the tissue distribution and the pharmacokinetic behavior of the drug and increase the drug aggregation at the tumor part; (5) reduce the toxicity of the medicine to normal cells or tissues. However, the release of drug from the carrier is affected by a number of factors. The medicine is released too early before reaching the tumor part to cause toxic and side effects on the whole body, and the medicine concentration in the target area is reduced; too slow release in the target area reduces the efficacy of the drug and may cause drug resistance in tumor cells. Thus, controlled release of the drug by the carrier is critical to drug delivery systems. In recent years, in order to solve the problem of premature release of drugs in systemic circulation, stimulus-responsive drug delivery systems have been extensively studied in which external factors (light, temperature, ultrasound, electrochemical triggers) or the tumor microenvironment (pH, enzymatic activity, redox properties) trigger the release of drugs. Among these stimuli-responsive systems, pH-sensitive polymeric micelles are of interest. The pH of normal tissue and blood is 7.4, while the extracellular pH of tumor tissue is only about 6.7-7.1. In addition, when the nanocarrier is taken up by the cell by endocytosis, it can also enter organelles of different pH values, for example, the pH of endosome is about 5.5-6.0, while the pH of lysosome is only 4.5-5.0. pH sensitive polymer micelles can be designed for these pH gradients to selectively release drugs in low pH tumor tissues or organelles. The pH sensitive polymeric micelles mainly comprise two forms: one is that the micellar material is a polymer with ionizable groups that can accept or donate protons upon a change in the ambient pH. Poly (2-alkyl-2-oxazoline) (PAOz) is a biocompatible polymeric material in which, when used as the hydrophilic shell of a micelle, the amide groups on the PAOz backbone are positively charged by proton incorporation under acidic conditions, and the drug is released as the micelle swells or disintegrates due to charge repulsion between the hydrophilic chains. Utilizing this property of PAOz, the patent of CN109303767A prepared a polymeric micelle composition with antitumor drug resistance and metastasis, however, the effect of synergistic inhibition of tumor metastasis and reduction of side effects in the nanocarrier is not ideal, and the ratio of the two drugs co-entrapped in the micelle is not easy to control. Another pH-sensitive polymer micelle couples a drug on a polymer through an acid-sensitive chemical bond, and after the drug reaches tumor tissues, the chemical bond is broken due to the change of pH, so that the drug is released. Common acid-sensitive bonds are benzoimine bonds, acetal bonds, hydrazone bonds, and the like. For example, CN102475891A discloses a polyethylene glycol-doxorubicin conjugate linked by a benzimine bond and a preparation method thereof, and CN103736101A discloses a polyethylene glycol-polylactic acid-curcumin conjugate linked by a hydrazone bond and a preparation method thereof. However, polyethylene glycol cannot be degraded in vivo, and accumulation occurs in long-term use, which has a potential safety problem. In addition, PEG on the hydrophilic surface of micelles may also hinder cellular uptake and subsequent intracellular processes of the micelle.
In conclusion, the combined use of the antitumor drug and the anti-tumor metastasis drug can play a role in synergistic antitumor and anti-metastasis effects, and enhance the curative effect. The pH-sensitive polymer-drug conjugate can reduce the early release of the drug and increase the accumulation of the drug at the tumor site so as to reduce the toxic and side effects and improve the curative effect. In addition, two or more drugs are coupled with the polymer and prepared into mixed micelles, so that the drug proportion can be conveniently controlled, and the drugs can play a synergistic anti-tumor and anti-metastasis role. Therefore, the development of the pH sensitive polymer-drug conjugate mixed micelle for co-delivering the insoluble antitumor drug and the antitumor metastasis drug to the tumor part has important significance for improving the curative effect and reducing the toxic and side effects.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a double-pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-transfer drug conjugate. The poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-transfer drug conjugate which is combined with the antitumor drug in a covalent mode not only keeps the pharmacological activity of the antitumor drug/anti-transfer drug, but also overcomes the defects of premature drug release and slow drug release at tumor parts or in cells in the systemic circulation, thereby reducing the toxic and side effects of the antitumor drug/anti-transfer drug.
Another object of the present invention is to provide a method for preparing the poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-metastatic drug conjugate.
It is yet another object of the present invention to provide mixed micelle compositions of poly (2-alkyl-2-oxazoline) -hydrophobic chain-antineoplastic/anti-metastatic drug conjugates. The poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-transfer drug conjugate can be self-assembled in water to form a nano mixed micelle which takes the hydrophobic chain-antitumor drug/anti-transfer drug as a hydrophobic inner core and the poly (2-alkyl-2-oxazoline) as a hydrophilic shell, and the particle size of the formed mixed micelle is less than 200 nm. The problem that the proportion of the drugs of the conventional physical polymer micelle carrying multiple drugs is difficult to control is solved, and the aim of combined administration treatment of multiple drugs can be achieved.
The poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-transfer drug conjugate is characterized in that the structural schematic diagram of the conjugate is as follows:
Figure BSA0000183380290000031
the hydrophilic segment of the pH-sensitive amphiphilic block polymer is pH-sensitive poly (2-alkyl-2-oxazoline) (PAOz), and the alkyl in the PAOz is an alkyl containing 1 to 6 carbon atoms, preferably an alkyl containing 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, n-butyl, more preferably methyl, ethyl. The molecular weight of PAOz is 600-20000, preferably 1000-15000, more preferably 2000-8000. The hydrophobic chain segment is selected from polylactic acid, polycaprolactone, polybutanelactone, polypentanolide, polyglycolide, polylactide, cholic acid, vitamin E succinate, phospholipid and phospholipid derivatives. Preferably from polylactic acid, polycaprolactone, polyglycolide, polylactide, vitamin E succinate, phospholipids. More preferably selected from polylactic acid, vitamin E succinate, phospholipids. The molecular weight of the hydrophobic chain segment is 600-10000, preferably 800-8000, and more preferably 1000-6000.
The poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/transfer-resistant drug conjugate is characterized in that the connecting arm is a benzoimine bond, an acetal bond, a hydrazone bond, a hydrazide bond, an oxime bond or a ketal bond. Benzoimine, acetal, and hydrazone bonds are preferable, and benzoimine and acetal bonds are more preferable.
The poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-transfer drug conjugate is characterized in that the antitumor drug is mainly any one of or derivatives of anthracycline, taxanes, cyclosporins, camptothecins and podophyllotoxin antitumor drugs. Preferably doxorubicin, paclitaxel, more preferably doxorubicin. The anti-transfer drug is selected from, but not limited to, honokiol, quercetin, wustatin, atorvastatin, tetrandrine, 6-elemene, kaempferol, psoralen, matrine, ginsenoside Rb, clove, curcumin and the like. Preferably selected from honokiol and curcumin.
The preparation method of the poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-transfer drug conjugate provided by the invention comprises one of the following steps:
(1) dissolving an amino-containing anti-tumor drug and a poly (2-alkyl-2-oxazoline) -hydrophobic chain with aldehyde group at one end in a solvent, and reacting to obtain the poly (2-alkyl-2-oxazoline) -hydrophobic chain-anti-tumor drug/anti-transfer drug conjugate containing benzoimine bond.
(2) Dissolving a poly (2-alkyl-2-oxazoline) -hydrophobic chain with one end being hydroxyl in a solvent, adding a catalyst 1 and an aldehyde group reagent for reaction to obtain the poly (2-alkyl-2-oxazoline) -hydrophobic chain with one end containing aldehyde group. And then the amino-containing anti-tumor drug/anti-transfer drug reacts with the amphiphilic polymer chain to obtain the benzoimine bond-containing poly (2-alkyl-2-oxazoline) -hydrophobic chain-anti-tumor drug/anti-transfer drug conjugate.
(3) The hydroxyl-containing anti-tumor drug/anti-transfer drug is reacted with an amination reagent to obtain the aminated drug. Dissolving a poly (2-alkyl-2-oxazoline) -hydrophobic chain with aldehyde group at one end in a solvent, adding aminated drugs, and reacting to obtain the poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-transfer drug conjugate containing benzoimine bonds.
(4) Dissolving the hydroxyl-containing anti-tumor drug/anti-transfer drug, the catalyst 2 and the poly (2-alkyl-2-oxazoline) -hydrophobic chain with one end of a vinyl ether structure in a solvent, and reacting to obtain the poly (2-alkyl-2-oxazoline) -hydrophobic chain-anti-tumor drug/anti-transfer drug conjugate containing acetal bonds.
(5) Dissolving a poly (2-alkyl-2-oxazoline) -hydrophobic chain with one end of a hydroxyl group in a solvent, adding a catalyst 3 and a carboxylation reagent for reaction, and reacting with ethylene glycol monovinyl ether and the catalyst 3 to ensure that one end of a polymer chain is the vinyl ether. The polymer, the antitumor drug/anti-transfer drug containing hydroxyl and the catalyst 2 are dissolved in a solvent to react to obtain the acetal bond-containing poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-transfer drug conjugate.
(6) Dissolving the carbonyl-containing anti-tumor drug/anti-transfer drug in a solvent, dissolving a poly (2-alkyl-2-oxazoline) -hydrophobic chain with a hydrazide group at one end and a small amount of acetic acid in the solvent, mixing the two solutions, and reacting to obtain the poly (2-alkyl-2-oxazoline) -hydrophobic chain-anti-tumor drug/anti-transfer drug conjugate with a hydrazone bond.
(7) Dissolving the hydroxyl-containing anti-tumor drug/anti-transfer drug in a solvent, adding a catalyst 3 and a carbonylation reagent, and reacting to obtain the carbonylation anti-tumor drug/anti-transfer drug. Dissolving the carbonylated antitumor drug/anti-transfer drug in a solvent, adding a double-bond-containing hydrazide reagent, and reacting to obtain the hydrazone-bond-containing antitumor drug/anti-transfer drug. And co-dissolving the poly (2-alkyl-2-oxazoline) -hydrophobic chain with the end group being sulfydryl and the anti-tumor drug/anti-transfer drug containing a hydrazone bond in a solvent for reaction to obtain the poly (2-alkyl-2-oxazoline) -hydrophobic chain-anti-tumor drug/anti-transfer drug conjugate containing the hydrazone bond.
(8) Dissolving the hydroxyl-containing anti-tumor drug/anti-transfer drug in a solvent, adding a catalyst 3 and a carbonylation reagent, and reacting to obtain the carbonylation anti-tumor drug/anti-transfer drug. Dissolving the carbonyl-containing anti-tumor drug/anti-transfer drug in a solvent, dissolving a poly (2-alkyl-2-oxazoline) -hydrophobic chain with a hydrazide group at one end and a small amount of acetic acid in the solvent, mixing and reacting in a dark place to obtain the poly (2-alkyl-2-oxazoline) -hydrophobic chain-anti-tumor drug/anti-transfer drug conjugate with a hydrazone bond.
(9) Dissolving the hydroxyl-containing anti-tumor drug/anti-transfer drug in a solvent, adding a catalyst 3 and a carbonylation reagent, and reacting to obtain the carbonylation anti-tumor drug/anti-transfer drug. Dissolving the carbonylated antitumor drug/anti-transfer drug in a solvent, adding a double-bond-containing hydrazide reagent, and reacting to obtain the hydrazone-bond-containing antitumor drug/anti-transfer drug. Dissolving the antitumor drug/anti-transfer drug containing hydrazone bond in solvent, adding thioglycolic acid, and reacting to obtain carboxylated antitumor drug/anti-transfer drug. Dissolving carboxylated anti-tumor drugs/anti-transfer drugs in a solvent, adding a catalyst and a poly (2-alkyl-2-oxazoline) -hydrophobic chain with one amino end, and reacting to obtain the poly (2-alkyl-2-oxazoline) -hydrophobic chain-anti-tumor drug/anti-transfer drug conjugate containing hydrazone bonds.
The solvent in the preparation method is selected from dimethyl sulfoxide, N-dimethylformamide, trichloromethane, tetrahydrofuran, dichloromethane, methanol, acetonitrile or a combination thereof.
The catalyst 1 in the preparation method is dicyclohexylcarbodiimide and 4-dimethylaminopyridine; catalyst 2 is p-toluenesulfonic acid; catalyst 3 was 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine.
The hydroformylation reagent in the preparation method is p-formylbenzoic acid.
The amination reagent in the preparation method is ethylenediamine, butanediamine and hexanediamine.
The carboxylation reagent in the preparation method is succinic anhydride, glutaric anhydride and adipic anhydride.
The carbonylation reagent in the preparation method is p-acetylbenzoic acid.
The double bond-containing hydrazide reagent in the preparation method is maleimide butyryl hydrazine hydrochloride.
The feeding molar ratio of the poly (2-alkyl-2-oxazoline) -hydrophobic chain polymer to the antitumor drug/anti-transfer drug in the preparation method is 1: 1-10, preferably 1: 1-6, and more preferably 1: 1-4.
The reaction time in the preparation method is 24-96 h.
According to the invention, a chemical bond sensitive to the acidic microenvironment of tumor cells, such as a benzoimide bond, a hydrazone bond, a hydrazide bond, an oxime bond, a ketal bond or an acetal bond, is introduced between the poly (2-alkyl-2-oxazoline) -hydrophobic chain and the anti-tumor drug/anti-metastatic drug to form the poly (2-alkyl-2-oxazoline) -hydrophobic chain-anti-tumor drug/anti-metastatic drug conjugate, so that the conjugate can respond to the acidic microenvironment in the tumor cells to break the pH sensitive chemical bond and quickly release the anti-tumor drug/anti-metastatic drug. The poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-transfer drug conjugate improves the solubility of the drug, retains the antitumor/anti-transfer activity of the drug, reduces the toxic and side effects of the antitumor drug/anti-transfer drug, and avoids the drug resistance of tumor cells.
In addition, the invention also provides a poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-transfer drug conjugate mixed micelle composition. The pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-transfer drug conjugate has amphipathy, can be self-assembled in an aqueous medium to form a nano mixed micelle which takes the hydrophobic chain-antitumor drug/anti-transfer drug as a hydrophobic core and the poly (2-alkyl-2-oxazoline) as a hydrophilic shell, and the particle size of the formed mixed micelle is less than 200nm, preferably less than 150 nm. The proportion of two or more medicaments in the micelle is easy to control, and the co-delivery of the two or more medicaments can be realized for combined treatment, so that the aims of synergistically enhancing the anti-tumor and anti-metastasis effects are fulfilled. The anti-tumor medicament can be further coated in a physical coating way, so that the medicament loading rate of the anti-tumor medicament and/or the common delivery of different anti-tumor medicaments are improved, and the purpose of synergy is achieved.
The mass ratio of the antitumor drug to the anti-transfer drug in the pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-transfer drug conjugate mixed micelle is 1: 1-25: 1, preferably 1: 1-10: 1, and more preferably 1: 1-5: 1.
In addition, the conjugate mixed micelle composition of the present invention may further comprise pharmaceutically acceptable additives.
The conjugate mixed micelle composition of the present invention can be prepared by methods known in the art, such as dialysis, thin film dispersion, emulsification, solvent evaporation, lyophilization, co-solvent evaporation, infusion, and the like.
The poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-transfer drug conjugate mixed micelle can be prepared into oral, mucosal, injection or external preparations by adopting preparation technology known in the field.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) one of the raw materials used for preparing the poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/transfer-resistant drug conjugate is a poly (2-ethyl-2-oxazoline) -hydrophobic chain which is non-toxic, has good biocompatibility and has biodegradability.
(2) The poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-transfer drug conjugate has the advantages of mild preparation conditions, simple method and operation and easy implementation.
(3) The invention introduces biodegradable and nontoxic poly (2-alkyl-2-oxazoline) -hydrophobic chain polymer on hydrophobic anti-tumor drug/anti-transfer drug molecules, so that the polymer has amphipathy, can be self-assembled in an aqueous medium to form a nano mixed micelle, forms the inner core of the mixed micelle relative to the hydrophobic chain-anti-tumor drug/anti-transfer drug, and forms the outer shell of the mixed micelle by highly hydrophilic poly (2-alkyl-2-oxazoline), thereby having the functions of stabilizing the micelle and avoiding reticuloendothelial system phagocytosis and plasma protein adsorption in organisms.
(4) The poly (2-alkyl-2-oxazoline) adopted by the invention has pH sensitivity, so that the poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-transfer drug conjugate has dual pH sensitivity. The poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-transfer drug conjugate is a good macromolecular prodrug, can rapidly release the antitumor drug/anti-transfer drug by breaking a pH sensitive chemical bond in an extracellular matrix and an intracellular acidic microenvironment, is a micelle forming material with pH sensitivity, can rapidly release the drug in a weakly acidic environment in tumor cells, realizes dual pH responsive drug release, reduces premature release of the drug in systemic circulation, and improves accumulation of the drug in tumor tissues.
(5) The poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-transfer drug conjugate disclosed by the invention is self-assembled to form micelles, and the proportion of the combined drugs can be easily controlled according to the requirements of tumor combined treatment.
In another aspect, the invention also relates to the use of pH sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain-anti-tumor drug/anti-metastatic drug conjugate mixed micelles for the treatment of tumors and tumor metastases.
In yet another aspect, the present invention relates to the following:
Scheme 1. a mixed micelle of dual pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-transfer drug conjugate, wherein poly (2-alkyl-2-oxazoline) -hydrophobic chain polymer is coupled with antitumor drug/anti-transfer drug by pH-sensitive chemical bond, and optional pharmaceutical excipients.
Scheme 2. a dual pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain-anti-tumor drug/anti-transfer drug conjugate mixed micelle as described in scheme 1, wherein the structural schematic of the amphiphilic pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain polymer and the anti-tumor drug/anti-transfer drug conjugate is as follows:
Figure BSA0000183380290000061
characterized in that the chemical bond of the linker arm is selected from the group consisting of a benzoimine bond, an acetal bond, a hydrazone bond, a hydrazide bond, an oxime bond, and a ketal bond, preferably a benzoimine bond, an acetal bond, and a hydrazone bond, more preferably a benzoimine bond and an acetal bond.
Scheme 3. a dual pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain-antineoplastic/anti-metastatic drug conjugate mixed micelle as described in scheme 1, wherein the alkyl group in the amphiphilic pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain polymer is an alkyl group containing 1 to 6 carbon atoms, preferably an alkyl group containing 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, n-butyl, more preferably methyl, ethyl. The poly (2-alkyl-2-oxazoline) has a molecular weight of 600 to 20000, preferably 1000 to 15000, more preferably 2000 to 8000.
Scheme 4. the dual pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain-antineoplastic/anti-metastatic drug conjugate mixed micelle of any of scheme 1, wherein the hydrophobic chain segment of the amphiphilic pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain polymer is selected from the group consisting of polylactic acid, polycaprolactone, polybutanolide, polypentanolactone, polyglycolide, polylactide, cholic acid, vitamin E succinate, phospholipids, phospholipid derivatives. Preferably from polylactic acid, polycaprolactone, polyglycolide, polylactide, vitamin E succinate, phospholipids. More preferably from polylactic acid, vitamin E succinate, phospholipids. The molecular weight of the hydrophobic chain segment is 600-10000, preferably 800-8000, and more preferably 1000-6000.
Scheme 5. the conjugate mixed micelle composition according to schemes 1-4, wherein the micelle may further encapsulate an anti-tumor drug by physical entrapment.
Scheme 6. the dual pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain-anti-neoplastic drug/anti-metastatic drug conjugate mixed micelle of scheme 1, wherein the mass ratio of the antineoplastic drug to the anti-metastatic drug is 1: 1-25: 1, preferably 1: 1-10: 1, more preferably 1: 1-5: 1.
Scheme 7. the mixed micelle of dual pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain-anti-neoplastic agent/anti-metastatic agent conjugate as described in any of the schemes 1 to 6, wherein the anti-neoplastic agent is selected from the group consisting of anthracyclines such as doxorubicin, taxanes such as paclitaxel, camptothecins such as 9-nitrocamptothecin, semisynthetic derivatives of podophyllotoxin such as teniposide, and tennini such as solitinib. Preferably selected from paclitaxel, docetaxel and doxorubicin. The anti-tumor metastasis medicine is selected from, but not limited to, honokiol, quercetin, neferine, tetrandrine, paeonol, 6-elemene, kaempferol, daidzein, psoralen, matrine, ginsenoside Rb, curcumin, etc. Preferably selected from honokiol and curcumin.
Scheme 8. a dual pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain-anti-neoplastic drug/anti-metastatic drug conjugate mixed micelle of any of schemes 1 to 7, wherein the average particle size of the micelle is below 200nm, preferably below 150 nm.
Scheme 9 an oral, mucosal, injectable or topical formulation, characterized in that it comprises a micellar composition according to any one of claims 5 to 7.
Scheme 10. use of the dual pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain-anti-tumor drug/anti-metastatic drug conjugate mixed micelle of any of schemes 1-8 for the preparation of a drug for anti-tumor and anti-tumor metastasis.
In order to achieve the aim of the invention, the invention carries out in vitro release test, cytotoxic test and in vivo and in vitro anti-tumor metastasis test of the poly (2-alkyl-2-oxazoline) -hydrophobic chain-anti-tumor drug/anti-metastasis drug conjugate mixed micelle. The in vitro release test is used for explaining the pH sensitivity of the poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-metastatic drug conjugate mixed micelle, the cytotoxicity test is used for explaining the in vitro antitumor activity of the poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-metastatic drug conjugate mixed micelle, and the in vitro and in vivo antitumor transfer test is used for explaining the antitumor transfer effect of the poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-metastatic drug conjugate mixed micelle.
The foregoing and other objects and features of the invention will be apparent from the following description of the invention, which proceeds with reference to fig. 1-24.
FIG. 1 is a drawing showing the preparation of hydroxy-terminated poly (2-ethyl-2-oxazoline) in example 1 1H-NMR spectrum.
FIG. 2 is a GPC chart of poly (2-ethyl-2-oxazoline) terminated with hydroxyl groups in example 1.
FIG. 3 is a drawing showing the preparation of poly (2-ethyl-2-oxazoline) -polylactic acid having a hydroxyl group as a terminal in example 11H-NMR spectrum.
Fig. 4 is a GPC diagram of poly (2-ethyl-2-oxazoline) -polylactic acid terminated with a hydroxyl group in example 1.
FIG. 5 shows the preparation of poly (2-ethyl-2-oxazoline) -polylactic acid having an aldehyde group as a terminal in example 11H-NMR spectrum.
FIG. 6 is a drawing of a poly (2-ethyl-2-oxazoline) -polylactic acid-benzoinimine linkage-doxorubicin conjugate prepared in example 11H-NMR spectrum.
FIG. 7 shows the curcumin starting material and hydroxy-modified curcumin derivative in example 21H-NMR chart.
FIG. 8 is a drawing showing the preparation of poly (2-ethyl-2-oxazoline) -polylactic acid having a carboxyl group as a terminal group in example 21H-NMR spectrum.
FIG. 9 is a drawing of example 2 of poly (2-ethyl-2-oxazoline) -polylactic acid terminated with vinyl ether1H-NMR spectrum.
FIG. 10 is a drawing of the poly (2-ethyl-2-oxazoline) -polylactic acid-acetal linkage-curcumin conjugate of example 21H-NMR spectra, and preparation method of hydroxyl-modified curcumin derivative, poly (2-ethyl-2-oxazoline) -polylactic acid with terminal group of vinyl ether, and poly (2-ethyl-2-oxazoline) -polylactic acid-acetal bond-curcumin conjugate 13C-NMR chart.
Fig. 11 is a distribution diagram of the particle size of the poly (2-ethyl-2-oxazoline) -polylactic acid-benzoinmine bond-doxorubicin conjugate/poly (2-ethyl-2-oxazoline) -polylactic acid-acetal bond-curcumin conjugate mixed micelle of example 12 measured by a dynamic light scattering method.
Fig. 12 is a transmission electron micrograph of poly (2-ethyl-2-oxazoline) -polylactic acid-benzoinmine linkage-doxorubicin conjugate/poly (2-ethyl-2-oxazoline) -polylactic acid-acetal linkage-curcumin conjugate mixed micelles of example 12.
FIG. 13 is a graph of the in vitro drug release of the conjugate mixed micelles and single-drug conjugate micelles of example 12 under different pH conditions.
FIG. 14 is a cytotoxicity plot of the conjugate mixed micelles, single-drug conjugate micelles, and free drug of example 12.
FIG. 15 is a graph showing the results of the effect of the conjugate mixed micelles and single-drug conjugate micelles of example 12 on tumor cell adhesion. In the figure, "ns" indicates p > 0.05 and "×" indicates p < 0.001.
FIG. 16 is a graph of inhibition of tumor cell invasion by the conjugate mixed micelles and single conjugate micelles of example 12. In the figure, "ns" indicates p > 0.05, "+" indicates p < 0.05 and "+" indicates p < 0.01.
FIG. 17 shows the inhibition of tumor cell migration by the conjugate mixed micelle of example 12. In the figure, "+" indicates p < 0.05, and "+" indicates p < 0.01.
FIG. 18 is a graph of inhibition of tumor cell scratch healing by the conjugate mixed micelles and single conjugate micelles of example 12.
FIG. 19 is a graph of in vivo images of a nude mouse lung metastasis model after administration of the conjugate mixed micelle and single conjugate micelle of example 12 to nude mice.
FIG. 20 is a graph showing the bioluminescence in the lungs of nude mice after administration of the conjugate mixed micelle and single conjugate micelle of example 12 to nude mice.
FIG. 21 is an H & E stained section of the lungs of nude mice after administration of the conjugate mixed micelles and single conjugate micelles of example 12 to nude mice. The white arrows in the figure indicate lung metastasis nodules in nude mice.
FIG. 22 is a graph showing the body weight change of nude mice during administration of the conjugate mixed micelle and the single conjugate micelle of example 12 to the nude mice.
FIG. 23 is a graph comparing the lung coefficients of nude mice after administration of the conjugate mixed micelle and the single conjugate micelle of example 12 to the nude mice.
FIG. 24 is an H & E stained section of the heart and kidney of nude mice after administration of the conjugate mixed micelle and the single conjugate micelle of example 12 to the nude mice. The white arrows in the figure indicate the vacuoles of the cardiomyocytes in nude mice.
Detailed Description
The following examples serve to illustrate the invention in further detail, but in no way limit the scope of the invention.
Example 1 Synthesis of Poly (2-Ethyl-2-oxazoline) -polylactic acid-Benzoimine linkage-Adriamycin conjugate
(1) Preparation of hydroxy-terminated poly (2-ethyl-2-oxazoline) (molecular weight 2200)
Putting the 4A molecular sieve in a muffle furnace, activating at 400 ℃ for 4h, cooling to room temperature in a dryer, and adding a proper amount of 2-ethyl-2-oxazoline (EOz), acetonitrile and methanol to remove water. A250 mL round bottom flask was charged with 20mL EOz (0.2mol) and 1.24g methyl p-toluenesulfonate (6.7mmol) and 60mL anhydrous acetonitrile. Vacuumizing, introducing nitrogen for three times, and reacting at 100 ℃ for 24 hours under the protection of nitrogen. After cooling to room temperature, 60mL of KOH methanol solution (0.38M) was added, and after 4h of reaction under nitrogen protection, the solvent was removed by rotary evaporation, dissolved in 50mL of dichloromethane, filtered, precipitated with cold ether three times, and dried under reduced pressure. White powder was obtained.
(2) Preparation of hydroxy-terminated poly (2-ethyl-2-oxazoline) -polylactic acid (molecular weight 4000)
Precisely 5g (1.23mmol) of the product of (1) were weighed into a 250mL round-bottomed flask, 80mL of toluene was added, azeotropic dehydration was carried out under heating, cooling was carried out to room temperature, and 3.2g of lactide (22mmol) and 16mg of Sn (Oct) were added2Vacuumizing, introducing nitrogen for three times, and reacting at 100 ℃ for 48 hours. After the reaction is finished, cooling and spin-drying the solvent, adding 30mL of dichloromethane for dissolution, precipitating and purifying by 500mL of cold ether for three times, and drying under reduced pressure to obtain light yellow powder.
(3) Preparation of poly (2-ethyl-2-oxazoline) -polylactic acid with end group as aldehyde group
The solvent Tetrahydrofuran (THF) was dehydrated with activated molecular sieves.
4.0g of the product (1mmol) of (2), 742.3mg of p-formylbenzoic acid (5mmol), 1036.7mg of dicyclohexylcarbodiimide (5mmol) and 123.9mg of 4-dimethylaminopyridine (1mmol) were precisely weighed out and dissolved in 100mL of THF to react at ordinary temperature for 24 hours.
Centrifuging reaction solution 6000r/min for 10min, collecting supernatant, filtering with 0.22 μm filter membrane, purifying with cold diethyl ether, and drying under reduced pressure. The solid obtained is dissolved in 10mL dichloromethane, then passes through a 0.22 mu m filter membrane, is purified by cold ether for three times, and is dried under reduced pressure to obtain a white solid product.
(4) Preparation of poly (2-ethyl-2-oxazoline) -polylactic acid-benzoinimine bond-doxorubicin conjugate
358mg of doxorubicin hydrochloride (HCl. DOX) (0.6mmol) is weighed and dissolved in 200mL of anhydrous methanol, 504 μ L of triethylamine (3.6mmol) is added, after 24h of reaction in the dark at room temperature, the solvent is dried by spinning, 150mL of chloroform is added for dissolution, 2.5g of poly (2-ethyl-2-oxazoline) -polylactic acid (0.6mmol) with aldehyde group as the end group in (3) is added, and the reaction is carried out for 24h in the dark at room temperature. After the reaction is finished, the solvent is dried by spinning, the product is dissolved by 30mL of dichloromethane, the solution is centrifuged for 10min at 5000r/min, red supernatant is collected, the red supernatant is filtered by a 0.22 mu m filter membrane, purified for three times by 20 times of cold ether, and dried under reduced pressure to obtain pink product.
FIG. 1 is a preparation of poly (2-ethyl-2-oxazoline) terminated with hydroxyl group1H-NMR spectrum, the chemical shift of each characteristic proton is marked in the figure. FIG. 2 is a Gel Permeation Chromatography (GPC) graph of hydroxy-terminated poly (2-ethyl-2-oxazoline). FIG. 3 is a drawing of poly (2-ethyl-2-oxazoline) -polylactic acid terminated with hydroxyl groups1H-NMR spectrum, the chemical shift of each characteristic proton is marked in the figure. Fig. 4 is a Gel Permeation Chromatography (GPC) graph of poly (2-ethyl-2-oxazoline) -polylactic acid terminated with hydroxyl groups. FIG. 5 is a scheme of poly (2-ethyl-2-oxazoline) -polylactic acid with an aldehyde group as a terminal group1H-NMR spectrum, the chemical shift of each characteristic proton is marked in the figure. FIG. 6 is a poly (2-ethyl-2-oxazoline) -polylactic acid-doxorubicin conjugate1H-NMR spectrum, the chemical shift of each characteristic proton is marked in the figure.
Example 2 preparation of poly (2-ethyl-2-oxazoline) -polylactic acid-acetal linkage-curcumin conjugate
(1) Preparation of hydroxy-modified curcumin derivatives
Curcumin (1g, 2.72mmol) was weighed precisely into a 50mL round-bottom flask, potassium carbonate (0.75g, 5.44mmol), potassium iodide (46mg, 0.272mmol), tribromo-1-propanol (295. mu.L, 3.26mmol) were added, dissolved with 10mL DMF under ultrasound, and reacted at room temperature for 24 h. After completion of the reaction, the reaction in the flask was dissolved with 50mL of ethyl acetate, transferred to a 250mL separatory funnel, extracted three times with an equal volume of 0.1M hydrochloric acid, and extracted three times with distilled water. Standing for 10min, transferring the upper oil phase into a conical flask, and adding appropriate amount of anhydrous sodium sulfate for overnight dewatering. The water-depleted liquid was concentrated, passed through a silica gel column and monitored by thin layer chromatography. The first component eluted first with dichloromethane methanol at 200: 1 was unreacted curcumin. Eluting with eluent of dichloromethane and methanol at ratio of 100: 1 to remove the second component. The ratio of the developing solvent to the dichloromethane to the methanol is 25: 1 when the plate is dotted. Collecting the eluent of the second component, concentrating and spin-drying to obtain Cur-OH.
(2) Preparation of poly (2-ethyl-2-oxazoline) -polylactic acid with carboxyl as end group
DMAP (0.4066g, 3.3334mmol) and succinic anhydride (0.3334g, 3.3334mmol) were weighed out separately and placed in a 20mL beaker, 10mL of DMF was added and activated with stirring at room temperature for 1 hour. 3.52g of the hydroxy-terminated poly (2-ethyl-2-oxazoline) -polylactic acid (1.1mmol) prepared in example 1(2) was weighed and placed in a 50mL round-bottomed flask, 10mL of DMF and 158. mu.L of triethylamine (2.224mmol) were added, and the activated mixture in the beaker was added dropwise to the round-bottomed flask and reacted at room temperature for 24 hours. After the reaction, the reaction solution was transferred to a dialysis bag with a cut-off molecular weight of 1000, dialyzed in deionized water for 24 hours, filtered through a 0.45 μm filter membrane, and lyophilized.
(3) Preparation of poly (2-ethyl-2-oxazoline) -polylactic acid with vinyl ether as end group
The carboxyl-modified poly (2-ethyl-2-oxazoline) -polylactic acid (1.5g, 0.39mmol) synthesized in (2), DMAP (143mg, 1.17mmol) and EDC & HCl (224.3mg, 1.17mmol) were precisely weighed in a 50mL round-bottomed flask, and 15mL of DMF was added and activated for 2 hours in an ice-water bath. Then, 110. mu.L of ethylene glycol monovinyl ether (EGVE) was added to the system, and vacuum-pumping was carried out and nitrogen gas was introduced three times. The reaction was carried out at room temperature for 24 h. After the reaction was completed, the reaction solution was transferred into a dialysis bag with a cut-off molecular weight of 1000 and dialyzed in deionized water for 48 hours. Centrifuging the suspension in dialysis bag at 3000r/min for 10min, filtering the supernatant with 0.45 μm filter membrane, and lyophilizing.
(4) Preparation of poly (2-ethyl-2-oxazoline) -polylactic acid-acetal bond-curcumin conjugate
Weighing 7.1mg of p-toluenesulfonic acid into a 2mL volumetric flask, adding anhydrous DMF to dissolve and fixing the volume. The product (1.5g, 0.441mmol) in (3), the product (375.7mg, 0.882mmol) in (1) and 1g of activated 4A molecular sieve powder were weighed out into a 50mL round-bottomed flask, then p-toluenesulfonic acid solution (236. mu.L, 0.0004mmol) was added, vacuum was applied through N2 three times, and the reaction was carried out at 50 ℃ for 4 days in the absence of light. After the reaction was completed, the brown reaction solution was centrifuged and passed through a 0.22 μm organic filter, and DMF was removed by rotary evaporation using a diaphragm pump to obtain a brown transparent gummy solid. 10mL of dichloromethane was added to dissolve, and the solution was filtered through a 0.22 μm organic filter and precipitated with 200mL of cold ether for 3 times. Drying under reduced pressure for 3h to obtain orange solid.
FIG. 7 shows curcumin and hydroxy-modified curcumin derivatives1H-NMR spectrum, the chemical shift of each characteristic proton is marked in the figure. FIG. 8 is a diagram showing the end groups beingProcess for preparing carboxy poly (2-ethyl-2-oxazoline) -polylactic acid1H-NMR spectrum, the chemical shift of each characteristic proton is marked in the figure. FIG. 9 is a preparation of poly (2-ethyl-2-oxazoline) -polylactic acid with vinyl ether end group1H-NMR spectrum, the chemical shift of each characteristic proton is marked in the figure. FIG. 10 is of poly (2-ethyl-2-oxazoline) -polylactic acid-curcumin conjugate 1H-NMR spectra and preparation of curcumin derivatives modified by hydroxyl, poly (2-ethyl-2-oxazoline) -polylactic acid with vinyl ether as end group, and poly (2-ethyl-2-oxazoline) -polylactic acid-curcumin conjugates13C NMR comparison chart.
Example 3 preparation of poly (2-ethyl-2-oxazoline) -polylactic acid-acetal linkage-honokiol conjugate
(1) Preparation of hydroxy-modified honokiol derivatives
Honokiol (723.5mg, 2.72mmol) was weighed precisely into a 50mL round-bottomed flask, added with potassium carbonate (0.75g, 5.44mmol), potassium iodide (46mg, 0.272mmol), tribromo-1-propanol (295. mu.L, 3.26mmol), dissolved with 10mL DMF under ultrasound, and reacted at normal temperature for 24 h. After completion of the reaction, the reaction in the flask was dissolved with 50mL of ethyl acetate, transferred to a 250mL separatory funnel, extracted three times with an equal volume of 0.1M hydrochloric acid, and extracted three times with distilled water. Standing for 10min, transferring the upper oil phase into a conical flask, and adding appropriate amount of anhydrous sodium sulfate for overnight dewatering. Concentrating the liquid after water removal, and purifying the product by a silica gel column.
(2) Preparation of poly (2-ethyl-2-oxazoline) -polylactic acid-acetal bond-honokiol conjugate
Poly (2-ethyl-2-oxazoline) -polylactic acid (1.5g, 0.441mmol) with vinyl ether as the end group in example 2, hydroxy-modified honokiol (285.7mg, 0.882mmol) and 1g of activated 4A molecular sieve powder are precisely weighed into a 50mL round-bottom flask, then the p-toluenesulfonic acid solution (236. mu.L, 0.0004mmol) in example 2 is added, vacuum pumping is carried out, nitrogen is introduced for three times, and reaction is carried out for 4 days at 50 ℃ in the dark. After the reaction was completed, the reaction solution was centrifuged and passed through a 0.22 μm organic filter, and DMF was removed by rotary evaporation using a diaphragm pump to obtain a transparent colloidal solid. 10mL of dichloromethane was added to dissolve, and the solution was filtered through a 0.22 μm organic filter and precipitated with 200mL of cold ether for 3 times. Drying for 3h under reduced pressure to obtain the product.
Example 4 preparation of poly (2-ethyl-2-oxazoline) -polylactic acid-acetal linkage-paclitaxel conjugate
The poly (2-ethyl-2-oxazoline) -polylactic acid (1.5g, 0.441mmol) with the terminal group of vinyl ether in example 2, paclitaxel (752.3mg, 0.882mmol) and 1g of activated 4A molecular sieve powder are precisely weighed into a 50mL round-bottomed flask, then the p-toluenesulfonic acid solution (236. mu.L, 0.0004mmol) in example 2 is added, vacuum is pumped in for three times, and reaction is carried out at 50 ℃ for 4 days in the dark. After the reaction was completed, the reaction solution was centrifuged and passed through a 0.22 μm organic filter, and DMF was removed by rotary evaporation using a diaphragm pump to obtain a transparent colloidal solid. 10mL of dichloromethane was added to dissolve, and the solution was filtered through a 0.22 μm organic filter and precipitated with 200mL of cold ether for 3 times. Drying for 3h under reduced pressure to obtain the product.
Example 5 preparation of Poly (2-ethyl-2-oxazoline) -polylactic acid-hydrazone linkage-Doxorubicin
(1) Preparation of poly (2-ethyl-2-oxazoline) -polylactic acid with end group of methyl ester group
Poly (2-ethyl-2-oxazoline) -polylactic acid (20.2g, 9.5mmol) with carboxyl end group in example 2 is dissolved in toluene, water is removed by a water separator, after removing the toluene by evaporation under reduced pressure, the solution is redissolved by 70mL of anhydrous methanol, under the condition of a salt-ice bath (0-5 ℃), thionyl chloride (11.6mL, 0.156mmol) is slowly dropped, then a few drops of dimethylformamide are added, and after stirring is continued for 2h under the condition of the salt-ice bath, heating reflux is carried out overnight. Concentrating the solution, recrystallizing with anhydrous ether, evaporating under reduced pressure to remove solvent, and vacuum drying to obtain poly (2-ethyl-2-oxazoline) -polylactic acid with end group of methyl ester group.
(3) Preparation of poly (2-ethyl-2-oxazoline) -polylactic acid with terminal group of hydrazide group
The product of step (1) (11.2g, 0.4mmol) was dissolved in 50mL of methanol and slowly added dropwise to a hydrazine hydrate-methanol (8mmol) solution and stirred at room temperature for 24 h. Evaporating under reduced pressure to remove solvent, redissolving with deionized water, dialyzing with dialysis bag with molecular weight cutoff of 1000 in weak acid solution for 24 hr, and lyophilizing to obtain the product.
(4) Preparation of poly (2-ethyl-2-oxazoline) -polylactic acid-hydrazone bond-adriamycin conjugate
The product of step (3) (0.74g, 0.28mmol), doxorubicin hydrochloride (0.33g, 0.57mmol) and acetic acid (274. mu.L, 0.005mmol) were dissolved in anhydrous dimethylsulfoxide and stirred at room temperature away from light for 24 h. Recrystallizing with anhydrous ether to remove most of dimethyl sulfoxide and adriamycin, evaporating under reduced pressure to remove solvent, re-dissolving with phosphate buffer solution with pH of 7.8, separating and purifying with G25 Sephadex column, and lyophilizing to obtain the final product.
Example 6 preparation of Poly (2-ethyl-2-oxazoline) -polylactic acid-hydrazone linkage-curcumin conjugate
(1) Preparation of end-formyl curcumin
Separately, levulinic acid (0.1893g, 1.63mmol), 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (0.3123g, 1.63mmol) and 4-methylaminopyridine (0.1986g, 1.629mmol) were dissolved in 30mL of dichloromethane and activated with stirring at room temperature for 1 hour. Dissolving curcumin (0.6g, 1.63mmol) in 40mL of dichloromethane, dissolving with ultrasound, slowly adding activated liquid dropwise into curcumin solution, vacuumizing, introducing nitrogen, and reacting at room temperature in dark place for 24 h. Spin-drying the solvent, extracting the product with ethyl acetate, distilled water and a small amount of salt solution for 5 times, discarding the water phase, drying the organic phase with anhydrous sodium sulfate to remove water, and separating the pure product by silica gel column chromatography.
(2) Preparation of poly (2-ethyl-2-oxazoline) -polylactic acid-hydrazone bond-curcumin conjugate
The synthesized product (0.4g, 0.87mmol) in (1) and 1.5g of poly (2-ethyl-2-oxazoline) -polylactic acid with terminal hydrazide group prepared in example 7(3) are weighed into a 100mL round-bottom flask, and added with 20mL of dimethylformamide for dissolution, and reacted for 48h under the condition of room temperature and light shielding. After the reaction is finished, putting the reaction solution into a buffer solution with the pH value of 7.0 for dialysis for 24 hours, then centrifuging the liquid in the dialysis bag at 12000rpm for 5min, taking the supernatant, and freeze-drying to obtain the product.
Example 7 preparation of poly (2-ethyl-2-oxazoline) -polylactic acid-benzoinimine bond-doxorubicin conjugate/poly (2-ethyl-2-oxazoline) -polylactic acid-acetal bond-honokiol conjugate mixed micelle
The poly (2-ethyl-2-oxazoline) -polylactic acid-benzoinmimine bond-doxorubicin conjugate of example 1 and the poly (2-ethyl-2-oxazoline) -polylactic acid-acetal bond-honokiol conjugate of example 3 (total mass: 40mg) were precisely weighed in a 50mL heart-shaped flask, and after 20mL of anhydrous methanol was added to fully dissolve the conjugate, the conjugate was slowly evaporated by rotation in a water bath at 25 ℃ to form a uniform and transparent film on the wall of the glass flask. And then adding 20mL of 60 ℃ deionized water into the heart bottle, carrying out vortex oscillation for 5min, filtering with a 0.22 mu m filter membrane to obtain a micelle solution, and carrying out freeze drying to obtain the white loose micelle freeze-dried powder. The whole process is carried out in a dark place.
Example 8 preparation of poly (2-ethyl-2-oxazoline) -polylactic acid-acetal linkage-paclitaxel conjugate/poly (2-ethyl-2-oxazoline) -polylactic acid-acetal linkage-honokiol conjugate mixed micelle
The poly (2-ethyl-2-oxazoline) -polylactic acid-acetal bond-paclitaxel conjugate in example 4 and the poly (2-ethyl-2-oxazoline) -polylactic acid-acetal bond-honokiol conjugate in example 3 (total mass: 40mg) are precisely weighed in a 50mL heart-shaped flask, 20mL of anhydrous methanol is added to fully dissolve the conjugate, and then the mixture is slowly rotated and evaporated in a water bath at 25 ℃ to form a uniform and transparent film on the wall of the glass flask. And then adding 20mL of 60 ℃ deionized water into the heart-shaped flask, carrying out vortex oscillation for 5min, filtering with a 0.22 mu m filter membrane to obtain a micelle solution, and carrying out freeze drying to obtain micelle freeze-dried powder.
Example 9 preparation of Poly (2-ethyl-2-oxazoline) -polylactic acid-acetal linkage-paclitaxel conjugate/Poly (2-ethyl-2-oxazoline) -polylactic acid-hydrazone linkage-curcumin conjugate Mixed micelle
The poly (2-ethyl-2-oxazoline) -polylactic acid-acetal bond-paclitaxel conjugate in example 4 and the poly (2-ethyl-2-oxazoline) -polylactic acid-hydrazone bond-curcumin conjugate in example 6 (total mass: 40mg) were precisely weighed in a 50mL heart-shaped flask, and after 20mL of anhydrous methanol was added to fully dissolve the conjugate, the mixture was slowly rotated and evaporated in a water bath at 25 ℃ to form a uniform and transparent film on the wall of the glass flask. And then adding 20mL of 60 ℃ deionized water into the heart-shaped flask, carrying out vortex oscillation for 5min, filtering with a 0.22 mu m filter membrane to obtain a micelle solution, and carrying out freeze drying to obtain micelle freeze-dried powder. The whole process is carried out in a dark place.
Example 10 verapamil-entrapped poly (2-ethyl-2-oxazoline) -polylactic acid-doxorubicin conjugate/poly (2-ethyl-2-oxazoline) -polylactic acid-curcumin conjugate mixed micelle
4mg of verapamil, 40mg of the total mass of the poly (2-ethyl-2-oxazoline) -polylactic acid-benzoinimine bond-doxorubicin conjugate of example 1 and the poly (2-ethyl-2-oxazoline) -polylactic acid-acetal bond-curcumin conjugate of example 2 were placed in a 50mL flask, 20mL of anhydrous methanol was added to dissolve them sufficiently, and then slowly rotary evaporation was performed in a water bath at 25 ℃ to form a uniform and transparent film on the wall of the flask. And then adding 20mL of 60 ℃ deionized water into the heart-shaped flask, carrying out vortex oscillation for 5min, filtering with a 0.22 mu m filter membrane to obtain a micelle solution, and carrying out freeze drying to obtain micelle freeze-dried powder.
Example 11 honokiol-entrapped poly (2-ethyl-2-oxazoline) -polylactic acid-doxorubicin conjugate/poly (2-ethyl-2-oxazoline) -polylactic acid-curcumin conjugate mixed micelle
4mg of honokiol, 40mg of the total mass of the poly (2-ethyl-2-oxazoline) -polylactic acid-benzoinmine bond-doxorubicin conjugate in example 1 and the poly (2-ethyl-2-oxazoline) -polylactic acid-acetal bond-curcumin conjugate in example 2 were placed in a 50mL heart-shaped flask, 20mL of anhydrous methanol was added to dissolve the conjugate sufficiently, and then the mixture was slowly rotated and evaporated in a water bath at 25 ℃ to form a uniform and transparent film on the wall of the glass flask. And then adding 20mL of 60 ℃ deionized water into the heart-shaped flask, carrying out vortex oscillation for 5min, filtering with a 0.22 mu m filter membrane to obtain a micelle solution, and carrying out freeze drying to obtain micelle freeze-dried powder. The whole process is carried out in a dark place.
Example 12 preparation of poly (2-ethyl-2-oxazoline) -polylactic acid-benzoinmine bond-doxorubicin conjugate/poly (2-ethyl-2-oxazoline) -polylactic acid-acetal bond-curcumin conjugate mixed micelle
The poly (2-ethyl-2-oxazoline) -polylactic acid-adriamycin conjugate in example 1 and the poly (2-ethyl-2-oxazoline) -polylactic acid-curcumin conjugate (total mass: 40mg) in example 2 are precisely weighed in a 50mL heart-shaped flask, 20mL of anhydrous methanol is added to fully dissolve the conjugate, and then the mixture is slowly rotated and evaporated in a water bath at 25 ℃ to form a uniform and transparent film on the wall of the glass flask. And then adding 20mL of 60 ℃ deionized water into the heart-shaped bottle, carrying out vortex oscillation for 5min, filtering with a 0.22 mu m filter membrane to obtain a micelle solution, and carrying out freeze drying to obtain orange loose micelle freeze-dried powder for later use. The whole process is carried out in a dark place.
In order to set single-drug conjugate micelle controls in subsequent experiments, poly (2-ethyl-2-oxazoline) -polylactic acid-benzoinmine bond-doxorubicin conjugate micelles and poly (2-ethyl-2-oxazoline) -polylactic acid-acetal bond-curcumin conjugate micelles were prepared as described above.
The dynamic light scattering measures the particle size and distribution of the micelles, and the results are shown in FIG. 11. As can be seen, the conjugate mixed micelle prepared in example 3 has an average particle size of about 105nm, a polydispersity index of 0.15, and a uniform particle size distribution. The appearance of the micelle measured by a transmission electron microscope is shown in figure 12, and the micelle is spherical and has uniform size distribution.
Test example 1 in vitro release test of poly (2-ethyl-2-oxazoline) -polylactic acid-benzoinmine bond-doxorubicin conjugate/poly (2-ethyl-2-oxazoline) -polylactic acid-acetal bond-curcumin conjugate mixed micelles
To evaluate the pH sensitivity of the conjugate mixed micelles of the invention, the in vitro release of the conjugate mixed micelles of example 12 at different pH was examined as follows.
1mL of the micelle solution of example 12 of the present invention with a concentration of 2mg/mL was put in a dialysis bag with activated molecular weight cut-off of 8000, both ends of which were fastened with a thin wire, and then placed in 30mL of acetate buffer (pH5.0, 10mM) or PBS (pH7.4, 10mM) and shaken in a water bath at 37 ℃ and 100 rpm. At each preset time point, 1mL of the released sample was withdrawn while being supplemented with 1mL of fresh medium, in triplicate. The content of adriamycin and curcumin were measured by fluorescence spectrophotometry and HPLC, respectively, and the cumulative percentage release of the drug was calculated. To further understand the release characteristics of the conjugate mixed micelles, the release behavior of the conjugate micelles of example 1 and the conjugate micelles of example 2 were measured under the same conditions for comparison. The results are shown in FIG. 13.
As can be seen from fig. 13, the poly (2-ethyl-2-oxazoline) -polylactic acid-benzoinmimine bond-doxorubicin conjugate/poly (2-ethyl-2-oxazoline) -polylactic acid-acetal bond-curcumin conjugate mixed micelle of example 12 of the present invention has pH sensitivity to release of doxorubicin and curcumin. The drug release of the mixed micelle in the medium of pH5.0 is more than that of the mixed micelle in the medium of pH7.4. In addition, at ph7.4, the release of the poly (2-ethyl-2-oxazoline) -polylactic acid-benzoinmine linkage-doxorubicin conjugate micelle for 10h was greater than 70%, while the release of doxorubicin for 96h of the mixed micelle was less than 30%. Therefore, the conjugate mixed micelle of the embodiment 12 of the invention can reduce the release of the adriamycin in systemic circulation and reduce the toxic and side effects thereof.
Test example 2 cytotoxicity test of mixed micelles of poly (2-ethyl-2-oxazoline) -polylactic acid-benzoinmine bond-doxorubicin conjugate/poly (2-ethyl-2-oxazoline) -polylactic acid-acetal bond-curcumin conjugate
To evaluate the inhibition of tumor cell growth by the conjugate mixed micelles of the invention, the conjugate mixed micelles of example 12 of the invention were subjected to cytotoxicity test as follows, with free doxorubicin and the conjugate micelles of example 1 as controls.
The SRB method is adopted. After digestion of MDA-MB-231 cells fused to 90%, they were counted, seeded at a density of 5X 104 cells/mL into 96-well plates at 200. mu.L per well, and an equal amount of PBS solution was added to the peripheral wells. And (3) placing the 96-well plate in an incubator for 24h, removing the culture solution, adding the solution to be detected with different volumes into each well, setting 6 parallel wells for each concentration, and taking a cell-free PBS group as a blank group and a DMEM complete culture solution group as a control group. The culture was continued for 24h, the test solution was removed, washed 3 times with PBS, 200. mu.L of 10% trichloroacetic acid solution was added to each well, and the mixture was allowed to stand and fix at 4 ℃ for 1 h. The trichloroacetic acid solution was removed, washed 5 times with deionized water, dried at 40 deg.C, 100. mu.L of 1% acetic acid solution of 0.4% SRB was added to each well, and allowed to stand and stain at room temperature for 30 min. The SRB dye was removed, rinsed 5 times with 1% acetic acid and dried at 40 ℃. 150 μ L of 10mM Tris buffer was added to each well, and the wells were shaken at 37 ℃ for 30min, and the absorbance at 540nm was measured with a microplate reader to calculate the relative survival rate of the cells and the IC50 value. The results are shown in FIG. 14.
As can be seen from FIG. 14, the IC50 values of free doxorubicin, the conjugate micelle of the present invention in example 1, and the conjugate mixed micelle of example 12 were (0.26. + -. 0.11). mu.g/mL, (0.32. + -. 0.07). mu.g/mL, and (0.13. + -. 0.04). mu.g/mL, respectively, indicating that the conjugate of the present invention retains the antitumor activity of the drug, and the conjugate mixed micelle has a synergistic antitumor effect of the two drugs.
Test example 3 in vitro anti-tumor metastasis effect of poly (2-ethyl-2-oxazoline) -polylactic acid-benzoinmine bond-doxorubicin conjugate/poly (2-ethyl-2-oxazoline) -polylactic acid-acetal bond-curcumin conjugate mixed micelle
In order to evaluate the anti-tumor metastasis effect of the conjugate mixed micelle of the present invention, an in vitro anti-tumor metastasis test including measurement of cell adhesion rate, tumor cell invasion rate, tumor cell mobility rate and a scratch test was performed on the conjugate mixed micelle of example 12 of the present invention as follows.
Measurement of cell adhesion rate: taking MDA-MB-231 cells in logarithmic growth phase, re-suspending with complete culture medium after digestion, and adjusting density to 3.0 × 105one/mL, in a volume of 2mL per well, was seeded into six well plates. After 24h, the cells were about 80% -90% of the bottom of the wells. After washing with PBS for 2 times, 2mL of the single-drug conjugate micelles of the invention of example 1 and example 2 and the conjugate mixed micelle solution of example 12 were added, and incubated at 37 ℃ for 48 hours in serum-free DMEM as a control, the cells in each well were carefully rinsed 3 times with PBS, and after 200. mu.L of pancreatin was added to each well, 2mL of complete medium was added, and the cells were stopped and transferred to a centrifuge tube. Centrifuging at 1000r/min for 3min, discarding supernatant, adding serum-free DMEM, resuspending and diluting to 3.0 × 10 5one/mL. 100 μ L of cell suspension was added to a 96-well plate plated with MDA-MB-231 cell monolayer (or Matrigel, or HUVEC cell monolayer), each cell suspension was plated with 6 duplicate wells, and another set of serum-free DMEM was added as a control and adhered at 37 ℃ for 1 h. Then, the supernatant was aspirated off, washed carefully with PBS for three times to wash away non-adherent cells, 100. mu.L of 4% paraformaldehyde was added to each well and fixed at room temperature for 30min, washed with PBS for 2 times, and then 100. mu.L of 0.1% crystal violet solution was added and stained at room temperature in the dark for 30 min. The excess dye was washed with PBS and dried, and after photographing under a fluorescence inverted microscope, 150. mu.L of 33% acetic acid was added to each well, followed by shaking and decoloring at 37 ℃ for 1 hour, and the OD at 570nm was measured. According to the following formulaAnd (3) calculating the adhesion rate:
Figure BSA0000183380290000161
ODtestthe OD value, OD, of the cell adhesion well of each administration groupcontrolOD value, OD, of adhesion well for MDA-MB-231 cells incubated with DMEMblankOD of the wells with no adherent cells added.
The result of measuring the tumor cell adhesion rate is shown in fig. 15, compared with the control group, the homogeneous adhesion of MDA-MB-231 cells in the conjugate mixed micelle administration group of the invention example 12 did not change significantly, the adhesion with Matrigel did not change significantly, and the adhesion rate with HUVEC cell monolayer was decreased, which indicates that the conjugate mixed micelle of the invention example 12 can inhibit the adhesion with the vascular wall during the tumor cell metastasis process.
Determination of cell invasion rate: serum-free DMEM at 4 ℃ was aspirated through a pre-cooled pipette tip and the dispensed Matrigel was diluted to 0.53 mg/mL. 100 μ L of the suspension was added slowly to the upper chamber of the Transwell plate to avoid the formation of bubbles. This was then incubated at 37 ℃ for 2h to gel. After gelling, the supernatant was carefully aspirated, and 100 μ L of each micellar solution was added to the upper chamber of a Transwell plate, and wells with serum-free DMEM were used as controls. Subsequently, MDA-MB-231 cells in logarithmic growth phase were taken, digested, centrifuged, resuspended in serum-free DMEM and diluted to 5X 105one/mL, 100. mu.L of the single-drug conjugate micelles of examples 1 and 2 of the present invention and the conjugate mixed micelle solution of example 12 of the present invention were added to the upper chamber of the Transwell plate in a volume of 100. mu.L per well and gently shaken. The lower layer was carefully added 500. mu.L of complete medium to avoid air bubbles between the upper and lower chambers. Adding 5% CO at 37 deg.C2And (5) culturing for 48 hours in an incubator. After the incubation was completed, the Transwell plate was removed, the chamber was washed 3 times with PBS, uninfected cells in the upper chamber were wiped off with a wet cotton swab, and 600. mu.L of 4% paraformaldehyde was added to the lower side of the membrane of the chamber and fixed at room temperature for 30 min. Then washed with PBS 3 times, and then 600. mu.L of 0.1% crystal violet solution is added for dyeing for 30min at room temperature in the dark. After the dyeing is finished, excess dye is washed away by PBS and dried, and then fluorescence is carried out Photographs were taken under an inverted microscope. After photographing, adding 600 mu L of 33% acetic acid into the lower layer of each hole to immerse the lower layer of the small chamber, oscillating and decoloring for 1h at 37 ℃, taking acetic acid decoloring solution into a 96-hole plate, adding 150 mu L of acetic acid decoloring solution into each hole, and measuring the OD value under 570nm in an enzyme labeling instrument. The cell invasion rate was calculated according to the following formula:
Figure BSA0000183380290000162
ODtest、ODcontrolrespectively representing the OD value of each experimental group and the OD value, OD, of the blank control groupblankRepresenting the background value used to subtract the blanks.
The results of measuring the tumor cell invasion rate are shown in fig. 16, and the results show that the conjugate mixed micelle of the invention in the embodiment 12 can effectively inhibit the invasion of the tumor cells, and the inhibition effect is enhanced with the increase of the administration concentration. In addition, the inhibition rate of the conjugate mixed micelle of example 12 is greater than the sum of the inhibition rates of the two single-drug conjugates of example 1 and example 2, i.e., the two drugs in the conjugate mixed micelle of the invention have synergistic anti-tumor cell invasion effect.
Determination of cell migration: MDA-MB-231 cells in logarithmic growth phase are taken, digested, centrifuged, resuspended in serum-free DMEM and diluted to 5X 105Each well was seeded in 100. mu.L volume per well on a Transwell plate, and 100. mu.L of the single-drug conjugate micelles of examples 1 and 2 and the conjugate mixed micelle solution of example 12 of the present invention were added thereto, and the mixture was gently shaken. The lower layer was carefully added 500. mu.L of complete medium to avoid air bubbles between the upper and lower chambers. Adding 5% CO at 37 deg.C 2And (5) culturing for 48 hours in an incubator. After the incubation was completed, the Transwell plate was removed, the chamber was washed 3 times with PBS, the non-migrated cells in the upper chamber were wiped off with a wetted cotton swab, and 600. mu.L of 4% paraformaldehyde was added to the lower side of the membrane of the chamber and fixed at room temperature for 30 min. Then washed with PBS 3 times, and then 600. mu.L of 0.1% crystal violet solution is added for dyeing for 30min at room temperature in the dark. After the staining was finished, excess dye was washed off with PBS and dried, and photographed under a fluorescent inverted microscope. After the photographing is finished, adding the lower layer of each holeImmersing the lower layer of the small chamber with 600 mu L of 33% acetic acid, shaking and decoloring at 37 ℃ for 1h, taking acetic acid decoloring solution, adding 150 mu L of acetic acid decoloring solution into a 96-well plate, and measuring the OD value at 570nm in a microplate reader. And tumor cell migration was calculated according to the following formula:
Figure BSA0000183380290000171
ODtest、ODcontrolrespectively representing the OD value of each experimental group and the OD value, OD, of the blank control groupblankRepresenting the background value used to subtract the blanks.
The measurement result of the tumor cell migration rate is shown in fig. 17, and the result shows that the conjugate mixed micelle in the embodiment 12 of the invention can significantly inhibit the migration of the tumor cells, and the inhibition effect is enhanced with the increase of the administration concentration. In addition, the inhibition rate of the conjugate mixed micelle of example 12 is greater than the sum of the inhibition rates of the two single-drug conjugate micelles of examples 1 and 2, i.e., the two drugs in the conjugate mixed micelle of example 12 of the present invention have a synergistic antitumor cell migration effect.
Scratch test: taking MDA-MB-231 cells in logarithmic growth phase, re-suspending with complete culture medium after digestion, and adjusting density to 3.0 × 105one/mL, in a volume of 2mL per well, was seeded into six well plates. After 24h, the cells were about 80% -90% of the bottom of the wells. A200-mu L gun head is used for marking a straight line mark at the bottom of a six-hole plate, so that the gun head is perpendicular to the bottom of the hole as much as possible. The cells were washed 3 times with PBS and the scraped cells were removed. 2mL of single-drug conjugate micelles, the conjugate mixed micelle solution of the invention example 12, were added to each well in sequence, with serum-free DMEM as a control. Adding 5% CO at 37 deg.C2Culturing in an incubator. And taking out the 6-hole plate after 0h, 24h and 48h respectively, observing the scratch gaps of each group of cells under an inverted microscope, and simultaneously taking the scratched cell light mirror pictures. The width of the scratches between cells was observed.
Results of the scratch test as shown in fig. 18, the conjugate mixed micelle administration group of example 12 was scratched more widely and clearly than the control group. In addition, the effect of the conjugate mixed micelle of example 12 of the present invention on inhibiting tumor cell scratch healing is stronger than that of the single-drug conjugate micelle of example 1 and example 2 at the same administration concentration, and the inhibition effect is concentration-dependent.
Test example 4 anti-tumor metastasis effect in vivo of poly (2-ethyl-2-oxazoline) -polylactic acid-benzoinmine bond-doxorubicin conjugate/poly (2-ethyl-2-oxazoline) -polylactic acid-acetal bond-curcumin conjugate mixed micelle
In order to evaluate the in vivo anti-tumor metastasis effect of the polymeric micelle of the present invention, a bioluminescence imaging experiment was performed on the conjugate mixed micelle of example 12 of the present invention as follows. And physiological saline, single-drug conjugate micelle and physical double-drug-loaded micelle are used as controls.
Establishing a tumor lung metastasis nude mouse model: MDA-MB-231-GFP-luc cells were cultured to a log growth phase, washed three times with sterile PBS, trypsinized and centrifuged, the supernatant discarded, the cells resuspended in serum-free medium, and the cell concentration adjusted to 6X 106After one/mL, 200. mu.L of the cell suspension was injected into nude mice via tail vein.
Immediately after the nude mice were inoculated with MDA-MB-231-GFP-luc cells in the tail vein, the nude mice were randomly divided into five groups of three mice each. The tail vein injection of each micelle solution is carried out 3, 6, 9 and 12 days after inoculation, the dose of the adriamycin is 4mg/kg, and the dose of the curcumin is 20 mg/kg. D-fluorescein potassium salt in PBS (15mg/mL) was injected intraperitoneally at day 14 at a dose of 150 mg/kg. After 10min of injection, the nude mice were anesthetized, bioluminescent imaging was performed, the incidence of tumor metastasis was observed and the bioluminescence intensity was semi-quantitatively analyzed. The results are shown in FIG. 19. Immediately after the live body imaging, the lungs of the nude mice were removed, and after the nude mice were washed with physiological saline, ex vivo luminescence imaging was performed, and the intensity of bioluminescent signals was semi-quantitatively analyzed, as shown in fig. 20. After lung luminescence imaging, groups of nude mice lungs were immersed in 4% paraformaldehyde for 24H for fixation, then paraffin-embedded and H & E stained sections were made, observed with an inverted microscope and photographed. The results are shown in FIG. 21.
On the other hand, in order to evaluate the safety of the formulation, the body weight change of nude mice was recorded after each administration, and the results are shown in fig. 22. After lung luminescence imaging, each lung was weighed and lung coefficients calculated, the results are shown in fig. 23. After the nude mice were sacrificed, the heart and kidney of one nude mouse per group were randomly taken, washed with physiological saline, immersed in 4% paraformaldehyde for fixation for 24 hours, then paraffin-embedded and prepared into H & E stained sections, observed with an inverted microscope and photographed. The results are shown in FIG. 24.
From the therapeutic effect, after the conjugate mixed micelle and the physical double drug-loaded micelle of the invention in example 12 are treated, the intensity of the systemic luminescence (fig. 19) and the pulmonary luminescence (fig. 20) of the nude mice are significantly lower than those of the control group, which indicates that the two double drug-loaded micelles can inhibit the proliferation and the metastasis of tumor cells to the lung of the nude mice. In addition, lung H & E stained sections (fig. 21) show that conjugate mixed micelles have a stronger effect in inhibiting tumor nodule formation than physical drug-loaded micelles.
From the viewpoint of safety, the body weight of nude mice decreased more slowly than the doxorubicin conjugate micelle, the physical dual drug-loaded micelle group, during the conjugate mixed micelle treatment of example 12 of the present invention (fig. 22). The lung coefficient of nude mice in conjugate mixed micelle group of example 12 of the present invention was also closer to normal than other administration groups (fig. 23). In addition, the cardiotoxicity of the conjugate mixed micelle group of example 12 of the present invention was lower than that of the doxorubicin single-drug conjugate micelle and the physical double drug-loaded micelle of example 1 (FIG. 24)
In summary, the poly (2-ethyl-2-oxazoline) -polylactic acid-benzoinmimine bond-doxorubicin conjugate/poly (2-ethyl-2-oxazoline) -polylactic acid-acetal bond-curcumin conjugate mixed micelle of example 12 of the present invention is a combined drug delivery preparation with high safety and effective inhibition of tumor cell growth and metastasis, and has a good application prospect.
While the invention has been described with respect to the specific embodiments described above, it will be recognized that various modifications and changes may be made by those skilled in the art, which also fall within the scope of the invention as defined by the claims.

Claims (10)

1. A mixed micelle of double pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-transfer drug conjugate, wherein the poly (2-alkyl-2-oxazoline) -hydrophobic chain polymer is coupled with the antitumor drug/anti-transfer drug by a pH-sensitive chemical bond, and optional pharmaceutical excipients.
2. The dual pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain-anti-neoplastic agent/anti-metastatic agent conjugate mixed micelle of claim 1, wherein the amphiphilic pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain polymer and the anti-neoplastic agent/anti-metastatic agent conjugate have the following structural schematic:
Figure FSA0000183380280000011
Characterized in that the chemical bond of the linker arm is selected from the group consisting of a benzoimine bond, an acetal bond, a hydrazone bond, a hydrazide bond, an oxime bond, and a ketal bond, preferably a benzoimine bond, an acetal bond, and a hydrazone bond, more preferably a benzoimine bond and an acetal bond.
3. The dual pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain-antineoplastic/anti-metastatic drug conjugate mixed micelle of claim 1, wherein the alkyl group in the amphiphilic pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain polymer is an alkyl group containing 1 to 6 carbon atoms, preferably an alkyl group containing 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, n-butyl, more preferably methyl, ethyl. The poly (2-alkyl-2-oxazoline) has a molecular weight of 600 to 20000, preferably 1000 to 15000, more preferably 2000 to 8000.
4. The dual pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain-antineoplastic/anti-metastatic drug conjugate mixed micelle of any of claim 1, wherein the hydrophobic chain segment of the amphiphilic pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain polymer is selected from the group consisting of polylactic acid, polycaprolactone, polybutanolide, polypentanolide, polyglycolide, polylactide, cholic acid, vitamin E succinate, phospholipids, and phospholipid derivatives. Preferably from polylactic acid, polycaprolactone, polyglycolide, polylactide, vitamin E succinate, phospholipids. More preferably from polylactic acid, vitamin E succinate, phospholipids. The molecular weight of the hydrophobic chain segment is 600-10000, preferably 800-8000, and more preferably 1000-6000.
5. The dual pH-sensitive conjugate mixed micelle composition of claims 1-4, wherein the micelle further comprises an anti-tumor drug entrapped in a physical entrapment.
6. The dual pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain-anti-neoplastic agent/anti-metastatic agent conjugate mixed micelle of claim 1, wherein the mass ratio of the antineoplastic agent to the anti-metastatic agent is 1: 1 to 25: 1, preferably 1: 1 to 10: 1, more preferably 1: 1 to 5: 1.
7. The mixed micelle of the dual pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain-antitumor drug/anti-metastatic drug conjugate as claimed in any one of claims 1 to 6, wherein the antitumor drug is selected from the group consisting of anthracyclines such as doxorubicin, taxanes such as paclitaxel, camptothecins such as 9-nitrocamptothecin, semisynthetic derivatives of podophyllotoxin such as teniposide, and tinib such as solitinib. Preferably selected from paclitaxel, docetaxel and adriamycin. The anti-tumor metastasis medicine is selected from, but not limited to, honokiol, quercetin, neferine, tetrandrine, paeonol, 6-elemene, kaempferol, daidzein, psoralen, matrine, ginsenoside Rb, curcumin, etc. Preferably selected from honokiol and curcumin.
8. The dual pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain-antineoplastic/anti-metastatic drug conjugate mixed micelle of any one of claims 1-7, wherein the average particle size of the micelle is below 200nm, preferably below 150 nm.
9. An oral, mucosal, injectable or topical formulation, characterized in that it comprises a micellar composition according to any one of claims 5 to 7.
10. Use of the dual pH-sensitive poly (2-alkyl-2-oxazoline) -hydrophobic chain-antineoplastic/anti-metastatic drug conjugate mixed micelle of any one of claims 1 to 8 for the preparation of a drug for the anti-tumor and anti-tumor metastasis.
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