CN113912841A - PH and Redox dual-response diblock amphiphilic polymer prodrug and preparation method thereof - Google Patents
PH and Redox dual-response diblock amphiphilic polymer prodrug and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an amphiphilic block polymer, a pH and Redox dual-response amphiphilic block polymer prodrug and a preparation method thereof. The molecular formula of the two-block amphiphilic polymer is mPEG-b-PAE, and the molecular structural formula is shown as a formula 1. The molecular formula of the pH and Redox dual-response diblock amphiphilic polymer prodrug is mPEG-b-PAE-ss-DOX, and the molecular structural formula is shown as a formula 2. The preparation method comprises the steps of firstly preparing a macromonomer mPEG-AA, preparing an amphiphilic block polymer mPEG-b-PAE with pH responsiveness by a Michael stepwise addition method, and then linking adriamycin to hydroxyl positions of side chains of the mPEG-b-PAE through a disulfide bond sensitive to Redox to obtain a prodrug of the amphiphilic block polymer with pH and Redox dual responses. The prodrug is applied to a system for preparing a nano-micelle medicament, can be self-assembled into a prodrug nano-micelle with a stable structure in an aqueous solution, can quickly and accurately respond to the change of the pH value of the environment and Redox, and can effectively relieve burst release and control the medicament release behavior, thereby effectively killing cancer cells.
Description
Technical Field
The invention belongs to the technical field of polymer synthesis and biomedical materials, and particularly relates to a pH and Redox dual-response diblock amphiphilic polymer prodrug and a preparation method thereof.
Background
Nowadays, with the development of science and technology and the progress of society, the development of drugs for various serious diseases is endless, so that the medical level of people is rapidly improved, but cancer (tumor) is still the killer with the highest fatality rate in the world, and becomes the first cause of death of human diseases, and the morbidity and mortality rate are continuously increased, thus posing a great threat to human health. According to statistics, the number of new malignant tumor cases in China in 2015 is about 392.9 ten thousands, the increase is 12.5 thousands compared with 2014, the growth rate is 3.2%, 7.5 individuals are diagnosed as cancer per minute on average, and about 1810 ten new cancer cases exist globally in 2018, wherein Asia accounts for nearly half, 960 ten thousand cancer death cases account for nearly seven. Cancer has increasingly become a major killer threatening human life and health, particularly in asia.
Malignant tumors have the characteristics of high growth rate, easy metastasis, high recurrence and the like, so that the malignant tumors are difficult to effectively treat by common treatment means. Currently, treatment methods are largely divided into chemotherapy, radiotherapy and surgical treatment, with chemotherapy (chemotherapy) being the most common and effective method in the clinic. The small-molecule chemotherapy drugs widely used in clinic, such as adriamycin, paclitaxel, camptothecin, cyclopamine and the like, are mostly hydrophobic drugs, have poor water solubility, and cannot circulate in blood for a long time stably, so the injection dosage is large, the targeting is not realized, the drugs cannot be enriched at the tumor focus part in a targeted manner, and finally the drugs kill normal tissue cells in a human body while killing tumor cells, so the function loss of normal tissues and organs of the human body is caused, the treatment efficiency is low, the toxic and side effects are large, and the price is high. In short, chemotherapy still faces many challenges as the most clinically important method for treating cancer, and the most important one is how to effectively improve the treatment efficiency (drug bioavailability) while reducing the toxic and side effects to human body.
In order to effectively solve the current defects of chemotherapy and improve the efficiency of chemotherapy in tumor clinical treatment, drug delivery is an effective solution strategy, which is widely researched by a plurality of scholars worldwide. Especially, with the continuous development of nanotechnology and material science in recent decades, the functional nano drug-carrying system based on organic polymer is a hot spot of the current research. The amphiphilic polymer can be self-assembled in an aqueous solution to form nanoparticles, a micromolecule hydrophobic drug is wrapped in a core, good solubilization effect is achieved on the amphiphilic polymer, high drug loading capacity is achieved, good stability is achieved, the system is effectively prevented from being cleared away by a reticuloendothelial system (RES) in the in-vivo circulation process, the drug loading system is enabled to have long blood circulation time, enrichment is achieved on tumor tissues through the effect of Enhanced Permeation and Retention (EPR), bioavailability of the drug is improved, meanwhile, the functional polymer can respond to special microenvironment physiological characteristics (such as low pH value and high GSH concentration) of the tumor tissues (cells), and therefore targeted delivery and controlled release of the drug are achieved. However, the conventional method of preparing nanoparticles by using polymer-embedded anticancer drugs still has some disadvantages, such as high burst release amount of the drugs, partial drug leakage, poor controlled release performance of the drugs, etc.
Therefore, the anticancer drug is connected on the functional amphiphilic polymer through the stimulus-response chemical bond, so that the multifunctional polymer drug-loaded nano system is prepared, the bioavailability of the drug can be effectively improved, the toxic and side effects of the drug are reduced, and the tumor treatment effect is optimized. Therefore, based on the above theories, in order to overcome various disadvantages in chemotherapy, it is urgently needed to develop a functional amphiphilic polymer as a carrier material, which can effectively chemically load and target and deliver an anticancer drug to a tumor focus part, and the prepared drug-loaded system can realize the controlled release of the anticancer drug, thereby realizing the improvement of the treatment effect while reducing the toxic and side effects.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a pH and Redox dual-response diblock amphiphilic polymer prodrug and a preparation method thereof. The specific technical scheme is as follows:
the invention provides an amphiphilic diblock polymer, the molecular formula of which is mPEG-b-PAE, the molecular structural formula of which is shown in formula 1,
wherein x is 10-30.
The invention provides a preparation method of an amphiphilic diblock polymer mPEG-b-PAE, which comprises the following steps:
(1) under the conditions of inert gas protection and ice bath, dropwise adding Triethylamine (TEA) and acylating agent Acryloyl Chloride (AC) into Dichloromethane (DCM) solution of hydroxyl-terminated polyethylene glycol monomethyl ether (mPEG-OH), reacting for 1-4h, heating to room temperature, reacting for 18-32h, cooling, precipitating and drying to obtain hydroxyl-terminated acrylated macromonomer polyethylene glycol monomethyl ether (mPEG-AA);
(2) dropwise adding an anhydrous chloroform solution of 3-amino-1-propanol (AP) into an anhydrous chloroform solution of a macromonomer mPEG-AA and 1, 4-Butanediol Diacrylate (BD) by adopting a Michael stepwise addition method under the conditions of inert gas protection and no water, reacting for 72-120h at 45-65 ℃, cooling, concentrating, precipitating and drying to obtain the amphiphilic polymer mPEG-b-PAE.
Wherein the molecular structural formula of mPEG-AA is as follows:
the molecular structural formulas of TEA and AC are respectively:
the molecular structural formulas of BD and AP are respectively:
in the technical scheme of the invention, the mole parts of the reactants in the reaction system for generating mPEG-AA in the step (1) are as follows:
1-5 parts of hydroxyl-terminated polyethylene glycol monomethyl ether
5-50 parts of triethylamine
15-25 parts of an acylating agent;
the mole parts of the reactants in the reaction system for generating the mPEG-b-PAE in the step (2) are as follows:
1-5 parts of mPEG-AA
5-50 parts of 1, 4-butanediol diacrylate
6-55 parts of 3-amino-1-propanol.
In the above technical solution of the present invention, the operations of precipitating and drying in step (1) are specifically: adding 8-12 times volume of 0 ℃ n-hexane into the solution for precipitation, and performing vacuum drying on the obtained product mPEG-AA at 25-45 ℃ under 30-40mbar for 24-72 h;
the specific operations of precipitation and drying in the step (2) are as follows: adding 10 times volume of 0 ℃ n-hexane into the solution concentrated by rotary evaporation for precipitation, and performing vacuum drying on the obtained product mPEG-b-PAE at 25-45 ℃ under 30-40mbar for 24-72 h.
The third aspect of the invention provides a pH and Redox dual-response diblock amphiphilic polymer prodrug, the molecular formula of which is mPEG-b-PAE-ss-DOX, the molecular structural formula of which is shown in formula 2,
wherein x is 10-30, and y is 10-30.
Further, the number average molecular weight of the pH and Redox double-response diblock amphiphilic polymer prodrug is 10520-21560 g/mol.
The fourth aspect of the invention provides a preparation method of a pH and Redox double-response diblock amphiphilic polymer prodrug, which comprises the following steps:
1) preparing a diblock amphiphilic polymer mPEG-b-PAE;
2)3,3' -dithiodipropionic acid (DTDP) reacts with acetyl chloride (ACC) to obtain dithiodipropionic anhydride (DTDPA), and the dithiodipropionic anhydride reacts with doxorubicin hydrochloride (DOX-HCl) to obtain functionalized DOX molecules DOX-ss-COOH;
3) adding Dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DMAP) into a solution of DOX-ss-COOH in anhydrous Dichloromethane (DCM), then adding a solution of mPEG-b-PAE in anhydrous dichloromethane, heating to 25-40 ℃, reacting for 36-60h in the absence of light, precipitating, filtering, drying, dialyzing, and freeze-drying to obtain the pH and Redox dual-response amphiphilic polymer prodrug mPEG-b-PAE-ss-DOX.
Wherein, the molecular structural formulas of DCC and DMAP are respectively as follows:
in the technical scheme of the invention, the preparation of the functionalized DOX molecule DOX-ss-COOH comprises the following specific operations: refluxing 3,3' -dithiodipropionic acid and acetyl chloride at 40-70 deg.C for 1-3h, concentrating, adding excessive diethyl ether, and stirring for 1-5h to obtain dithiodipropionic anhydride; dissolving doxorubicin hydrochloride and triethylamine in anhydrous N, N-Dimethylformamide (DMF), keeping out of the sun, stirring for 10min-1h, centrifuging, adding dithiodipropionic anhydride, and reacting at room temperature for 18-32h to obtain the compound;
preferably, the mole fraction of the reactants in the reaction system for forming DOX-ss-COOH is as follows:
15-25 parts of dithiodipropionic anhydride
15-25 parts of doxorubicin hydrochloride
15-25 parts of triethylamine;
preferably, the rotation speed of the centrifugation is 12000-16000 rpm, and the time is 1-3 min;
preferably, the diblock amphiphilic polymer mPEG-b-PAE is prepared by the method;
preferably, the operations of precipitating, filtering and drying in the step 3) are as follows: adding 8-12 times volume of 0 ℃ n-hexane into the solution for precipitation, and performing vacuum drying at 25-45 ℃ under 30-40mbar for 24-72 hours;
preferably, the dialysis in step 3) is specifically operative to: and dissolving the obtained solid in N, N-dimethylformamide, placing the solid in a dialysis bag, dialyzing the solid in N, N-dimethylformamide as a medium for 24-72 hours, then changing the medium into deionized water, and continuously dialyzing the medium for 24-72 hours to obtain mPEG-b-PAE-ss-DOX.
Wherein, the molecular structural formulas of DTDP and ACC are respectively as follows:
the molecular structural formula of DTDPA is as follows:
the molecular structural formula of DOX-HCl is respectively as follows:
in the above technical scheme of the present invention, the mole fraction of the reactants in the reaction system for generating dithiodipropionic anhydride in step 2) is as follows:
1-5 parts of 3,3' -dithiodipropionic acid
5-15 parts of acetyl chloride;
the mole parts of the reactants in the reaction system for generating mPEG-b-PAE-ss-DOX in the step 3) are as follows:
the invention also provides a drug-loaded nano-micelle system, which comprises the pH and Redox dual-response diblock amphiphilic polymer prodrug;
preferably, the preparation method of the drug-loaded nano-micelle system comprises the following steps: dissolving the pH and Redox dual-response diblock amphiphilic polymer prodrug in an organic solvent, stirring for 30min at room temperature, then placing in a dialysis bag, dialyzing for 48h with deionized water, filtering, and freeze-drying to obtain a drug nano-micelle system;
preferably, the organic solvent is dimethyl sulfoxide or dimethylformamide.
The drug-loaded nano-micelle system is used as a carrier material for drug delivery.
The invention has the beneficial effects that:
(1) in the pH and Redox dual-response diblock amphiphilic polymer prodrug provided by the invention, the PAE material has pH sensitivity and amphipathy, and the disulfide bond in the functionalized DOX drug has Redox sensitivity. The pH and Redox double-response diblock amphiphilic polymer prodrug molecule can be self-assembled into a nano drug-loaded micelle with a stable structure in an aqueous solution, and the nano micelle not only can rapidly and accurately respond to the change of the pH value and Redox of the environment, but also can effectively relieve burst release and control the drug release behavior, thereby effectively killing cancer cells. The hydrophilic PEG of the outer layer of the drug-loaded nano-micelle has the advantages of no toxicity, no immunogenicity, no antigenicity and the like, and can prolong the circulation time of the micelle in blood while increasing the stability of the micelle.
(2) The PAE is hydrophobic at pH 7.4, forms a hydrophobic core of a micelle with hydrophobic anticancer drug molecules, effectively enhances the stability of the micelle core, and better protects the activity of the anticancer drug; in a tumor tissue weak acid (pH5.0-7.0) microenvironment, a tertiary amine matrix in the PAE block is protonated and converted into hydrophilicity, and meanwhile, the PAE block is positively charged, so that a micelle structure is damaged; the pro-drug molecule with positive charge enters tumor cells and is included in an inclusion body and a lysosome with lower pH, PAE is completely protonated and the positive charge is enhanced, so that the PAE escapes from the inclusion body/the lysosome through a proton-sponge effect, the pro-drug molecule enters into cytoplasm, and the high-concentration GSH in the cells can efficiently cause the breakage of disulfide bonds, so that the anti-cancer drug molecules connected to the polymer molecules are released into organelles such as cell nucleus and the like, and the apoptosis of the tumor cells is caused. The change of the pH value of a microenvironment and the concentration of GSH in cells in the in vivo circulation process can be accurately responded, the structure stability of a drug delivery system is maintained, the in vivo circulation time is prolonged, the accumulation amount of the drug delivery system at a focus part is increased, the cell uptake can be effectively improved, the bioavailability of the drug is increased, the toxic and side effects are reduced, and the treatment effect of tumors is optimized.
(3) The pH and Redox dual-response diblock amphiphilic polymer prodrug provided by the invention has a treatment effect and can be independently used as a pharmaceutical preparation. Meanwhile, the pH and Redox dual-response diblock amphiphilic polymer prodrug can also be used as a drug carrier, and other therapeutic drugs are loaded into the nano-micelle of the material disclosed by the invention by other means such as physical embedding, so that the combined therapeutic effect of multiple drugs is realized. The DOX drug and the PAE polymer are connected through chemical bonds, so that the stability is stronger, and the early release of the drug can be effectively reduced. Upon reaching the tumor microenvironment, the disulfide bonds linking the DOX and PAE polymers are cleaved by pH and Redox responses, releasing the drug.
(4) The invention adopts a method of Michael addition one-step generation when synthesizing the diblock amphiphilic polymer mPEG-b-PAE, has low reaction temperature, is simple and convenient compared with other complex synthetic methods, has mild conditions and high yield.
Drawings
FIG. 1 shows mPEG-AA in example 1 of the present invention1H-NMR chart, solvent is d-CDCl3。
FIG. 2 is a diagram of mPEG-b-PAE in example 1 of the present invention1H-NMR chart with the solvent ofd-CDCl3。
FIG. 3 shows DTDPA of example 1 of the present invention1H-NMR chart, solvent is d-DMSO.
FIG. 4 shows DOX-ss-DOX in example 1 of the present invention1H-NMR chart with solvent D2O。
FIG. 5 shows the preparation of mPEG-b-PAE-ss-DOX in example 1 of the present invention1H-NMR chart, solvent is d-CDCl3。
FIG. 6 is a test curve of critical micelle concentration of mPEG-b-PAE-ss-DOX in example 1 of the present invention.
FIG. 7 is a graph showing the relationship between the particle size and pH of mPEG-b-PAE-ss-DOX self-assembled micelle of example 1.
FIG. 8 is a graph showing the relationship between the zeta potential and pH of mPEG-b-PAE-ss-DOX self-assembled micelles obtained in example 1.
FIG. 9 is a UV spectrum of mPEG-b-PAE-ss-DOX of example 1 under different pH and DTT concentrations.
FIG. 10 is a graph showing the in vitro release profile of the polymeric prodrug micelle of application example 1 of the present invention.
FIG. 11(A) is a graph showing the toxicity of the amphiphilic polymers of mPEG-b-PAE in example 1 against 3LL cells.
FIG. 11(B) is a graph of toxicity of mPEG-B-PAE-ss-DOX prodrug nanomicelles and free doxorubicin on 3LL cells after 24h in example 1.
FIG. 11(C) is a graph of toxicity of mPEG-b-PAE-ss-DOX prodrug nanomicelles and free doxorubicin on 3LL cells after 48h in example 1.
Detailed Description
In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer. Example 1: preparation of pH and Redox dual-response diblock amphiphilic polymer prodrug molecule mPEG-b-PAE-ss-DOX
(1) Preparation of end-functionalized macromonomer polyethylene glycol monomethyl ether (mPEG-AA): under the protection of inert gas and in the absence of water, the solvents DCM (20mL), mPEG-OH (100mg, 0.02mmol) and TEA (13.9. mu.L, 0.1mmol) are sequentially added into a round-bottom flask provided with a stirrer, the round-bottom flask is cooled to 0 ℃ by ice, AC (24.27. mu.L, 0.3mmol) is dropwise added under stirring, the round-bottom flask is reacted for 2 hours at 0 ℃, the reaction is continued for 24 hours after the temperature is raised to room temperature, the reacted solution is added into 300mL (corresponding to 10 times of volume of n-hexane at 0 ℃ for precipitation, the solution is filtered, and then the solution is dried in vacuum at 45 ℃ for 48 hours to obtain the macromonomer mPEG-AA after the acidification of the hydroxyl group. The synthetic reaction formula is shown in formula (1), the nuclear magnetism result is shown in figure 1, and the yield is 90%.
(2) Synthesizing a pH sensitive amphiphilic diblock polymer, namely polyethylene glycol monomethyl ether-poly beta amino ester (mPEG-b-PAE): adding solvents of anhydrous chloroform (20mL), macromonomer mPEG-AA (100mg, 0.02mmol) and BD (19.8mg, 0.1mmol) into an eggplant-shaped reaction bottle in sequence under the protection of inert gas and in the absence of water by adopting a Michael stepwise addition method to obtain a clear solution, dropwise adding AP (9.01mg, 0.12mmol) under stirring, reacting for 96 hours at 60 ℃, precipitating, filtering and drying to obtain an amphiphilic diblock polymer mPEG-b-PAE with pH responsiveness. The synthetic reaction formula is shown in formula (2), the nuclear magnetic result is shown in figure 2, and the yield is 82%.
(3) Preparing a functionalized drug molecule DOX-ss-COOH: DTDP (1g, 4.75mmol) and ACC (3.312g, 42.19mmol) were added sequentially to the round bottom flask, heated to 65 deg.C under reflux for 2h, added excess ether (50mL), stirred for an additional 3h, filtered, and dried to give DTDPA with nuclear magnetism as shown in FIG. 3, 55% yield. Adding DOX-HCl (15mg, 0.026mmol) and TEA (5 mu L, 0.036mmol) in anhydrous DMF (5mL), placing in a round-bottomed flask with a stirrer, keeping out of the light, stirring for 30min, centrifuging, adding DTDPA (5.4mg, 0.028mmol) in anhydrous DMF (5mL), reacting at room temperature for 24h in the absence of the light, adding the reacted solution to 300mL (corresponding to 10 times of the volume) of n-hexane at 0 ℃, precipitating, filtering, and drying in vacuum at 45 ℃ for 48h to obtain the functionalized DOX molecule DOX-ss-COOH. The synthetic reaction formula is shown in formula (3), the nuclear magnetic result is shown in figure 4, and the yield is 60%.
(4) Preparation of pH and Redox dual-responsive diblock amphiphilic polymeric prodrugs: DOX-ss-COOH (6.2mg, 0.01mmol), DCC (20.6mg, 0.1mmol), DMAP (12.2mg, 0.1mmol) and solvent anhydrous DCM (10mL) were added sequentially under anhydrous conditions to a round bottom flask equipped with a stirrer, stirring for 30min, adding anhydrous DCM (20mL) solution of mPEG-b-PAE (10.52mg, 0.001mmol), heating to 35 deg.C, reacting for 48h in the dark, adding the reacted solution into 300mL (10 times of volume) of 0 deg.C n-hexane for precipitation, filtering, then dried under vacuum at 45 ℃ for 48h, the solid was redissolved in DMF (10mL), placed in dialysis bags (MWCO1500Da), dialyzing in a DMF medium for 24h, then changing the DMF medium into deionized water, continuing to dialyze for 48h, and freeze-drying the obtained solution to obtain the pH and Redox double-response amphiphilic polymer prodrug mPEG-b-PAE-ss-DOX. The synthetic reaction formula is shown in formula (4), the nuclear magnetic result is shown in figure 5, and the yield is 75%.
Example 2: preparation of pH and Redox dual-response diblock amphiphilic polymer prodrug molecule mPEG-b-PAE-ss-DOX
(1) Preparation of end-functionalized macromonomer polyethylene glycol monomethyl ether (mPEG-AA): under the protection of inert gas and in the absence of water, sequentially adding solvents DCM (20mL), mPEG-OH (100mg, 0.02mmol) and TEA (139 uL, 1mmol) into a round-bottomed flask provided with a stirrer, cooling to 0 ℃ with ice, dropwise adding AC (40.45 uL, 0.5mmol) under stirring, reacting for 2h at 0 ℃, heating to room temperature, continuing to react for 24h, adding the reacted solution into 300mL (corresponding to 10 times of volume of n-hexane at 0 ℃ for precipitation, filtering, and then drying in vacuum at 45 ℃ for 48h to obtain the hydroxyl terminated acryloyl acidified macromonomer mPEG-AA. The yield was 85%.
(2) Synthesizing a pH sensitive diblock amphiphilic polymer mPEG-b-PAE: adding solvents of anhydrous chloroform (20mL), macromonomer mPEG-AA (100mg, 0.02mmol) and BD (198mg, 1mmol) into an eggplant-shaped reaction bottle in sequence under the conditions of inert gas protection and no water by adopting a Michael stepwise addition method to obtain a clear solution, dropwise adding AP (90.1mg, 1.2mmol) under stirring, reacting for 96h at 60 ℃, precipitating, filtering and drying to obtain an amphiphilic block polymer mPEG-b-PAE with pH responsiveness. The yield was 70%.
(3) Preparing a functionalized drug molecule DOX-ss-COOH: DTDP (1g, 4.75mmol) and ACC (5.6g, 71.25mmol) were added sequentially to a round bottom flask, heated to 65 ℃ under reflux for 2h, added excess ether (50mL), stirred for an additional 3h, filtered and dried to give DTDPA in 50% yield. Adding DOX-HCl (15mg, 0.026mmol) and TEA (6.14 mu L, 0.043mmol) into anhydrous DMF (5mL), putting into a round-bottomed flask with a stirrer, keeping out of the light, stirring for 30min, centrifuging, adding a solution (5mL) of DTDPA (12.54mg, 0.065mmol) in anhydrous DMF, reacting for 24h at room temperature keeping out of the light, adding the reacted solution into 300mL (corresponding to 10 times of volume of n-hexane at 0 ℃ for precipitation, filtering, and drying in vacuum for 48h at 45 ℃ to obtain the functionalized DOX molecule DOX-ss-COOH. The yield was 57%.
(4) Preparation of pH and Redox dual-responsive diblock amphiphilic polymeric prodrugs: DOX-ss-COOH (12.4mg, 0.02mmol), DCC (154.5mg, 0.75mmol), DMAP (24.4mg, 0.2mmol) and solvent anhydrous DCM (10mL) were added sequentially under anhydrous conditions to a round bottom flask equipped with a stirrer, stirring for 30min, adding anhydrous DCM (20mL) solution of mPEG-b-PAE (10.52mg, 0.001mmol), heating to 35 deg.C, reacting for 48h in the dark, adding the reacted solution into 300mL (10 times of volume) of 0 deg.C n-hexane for precipitation, filtering, then dried under vacuum at 45 ℃ for 48h, the solid was redissolved in DMF (10mL), placed in dialysis bags (MWCO1500Da), dialyzing in a DMF medium for 24h, then changing the DMF medium into deionized water, continuing to dialyze for 48h, and freeze-drying the obtained solution to obtain the pH and Redox double-response amphiphilic polymer prodrug mPEG-b-PAE-ss-DOX. The yield was 72%.
Example 3: preparation of pH and Redox dual-response diblock amphiphilic polymer prodrug molecule mPEG-b-PAE-ss-DOX
(1) Preparation of end-functionalized macromonomer polyethylene glycol monomethyl ether (mPEG-AA): under the protection of inert gas and in the absence of water, the solvents DCM (20mL), mPEG-OH (100mg, 0.02mmol) and TEA (27.8. mu.L, 0.2mmol) are sequentially added into a round-bottom flask provided with a stirrer, the round-bottom flask is cooled to 0 ℃ by ice, AC (8.09. mu.L, 0.1mmol) is dropwise added under stirring, the round-bottom flask is reacted for 2 hours at 0 ℃, the reaction is continued for 24 hours after the temperature is raised to room temperature, the reacted solution is added into 300mL (corresponding to 10 times of volume of 0 ℃ n-hexane for precipitation, filtered and then dried in vacuum at 45 ℃ for 48 hours, and the hydroxyl-terminated acrylic acid-containing macromonomer mPEG-AA is obtained. The yield was 80%.
(2) Synthesizing a pH sensitive diblock amphiphilic polymer mPEG-b-PAE: adding solvents of anhydrous chloroform (20mL), macromonomer mPEG-AA (100mg, 0.02mmol) and BD (39.6mg, 0.2mmol) into an eggplant-shaped reaction bottle in sequence under the protection of inert gas and in the absence of water by adopting a Michael stepwise addition method to obtain a clear solution, dropwise adding AP (45.05mg, 0.6mmol) under stirring, reacting for 96 hours at 60 ℃, precipitating, filtering and drying to obtain an amphiphilic diblock polymer mPEG-b-PAE with pH responsiveness. The yield was 51%.
(3) Preparing a functionalized drug molecule DOX-ss-COOH: DTDP (1g, 4.75mmol) and ACC (1.86g, 23.75mmol) were added sequentially to a round bottom flask, heated to 65 ℃ under reflux for 2h, added excess ether (50mL), stirred for an additional 3h, filtered and dried to give DTDPA in 48% yield. Adding DOX-HCl (15mg, 0.026mmol) and TEA (3.61 mu L, 0.026mmol) into anhydrous DMF (5mL), putting into a round-bottomed flask with a stirrer, keeping out of the light, stirring for 30min, centrifuging, adding a DTDPA (10.03mg, 0.052mmol) solution (5mL) of anhydrous DMF, reacting for 24h at room temperature in the absence of the light, adding the reacted solution into 300mL (corresponding to 10 times of volume of n-hexane at 0 ℃ for precipitation, filtering, and drying in vacuum at 45 ℃ for 48h to obtain the functionalized DOX molecule DOX-ss-COOH. The yield was 60%.
(4) Preparation of pH and Redox dual-responsive diblock amphiphilic polymeric prodrugs: DOX-ss-COOH (1.24mg, 0.002mmol), DCC (15.45mg, 0.075mmol), DMAP (2.44mg, 0.02mmol) and solvent anhydrous DCM (10mL) were added sequentially under anhydrous conditions to a round bottom flask equipped with a stirrer, stirring for 30min, adding anhydrous DCM (20mL) solution of mPEG-b-PAE (10.52mg, 0.001mmol), heating to 35 deg.C, reacting for 48h in the dark, adding the reacted solution into 300mL (10 times of volume) of 0 deg.C n-hexane for precipitation, filtering, then dried under vacuum at 45 ℃ for 48h, the solid was redissolved in DMF (10mL), placed in dialysis bags (MWCO1500Da), dialyzing in a DMF medium for 24h, then changing the DMF medium into deionized water, continuing to dialyze for 48h, and freeze-drying the obtained solution to obtain the pH and Redox double-response amphiphilic polymer prodrug mPEG-b-PAE-ss-DOX. The yield was 68%.
Example 4: preparation of pH and Redox dual-response diblock amphiphilic polymer prodrug molecule mPEG-b-PAE-ss-DOX
(1) Preparation of end-functionalized macromonomer polyethylene glycol monomethyl ether (mPEG-AA): under the protection of inert gas and in the absence of water, the solvents DCM (20mL), mPEG-OH (100mg, 0.02mmol) and TEA (20.85. mu.L, 0.15mmol) are sequentially added into a round-bottom flask provided with a stirrer, the round-bottom flask is cooled to 0 ℃ by ice, AC (6.47. mu.L, 0.08mmol) is dropwise added under stirring, the round-bottom flask is reacted for 2 hours at 0 ℃, the reaction solution is continuously reacted for 24 hours after being heated to room temperature, the reacted solution is added into 300mL (corresponding to 10 times of volume of n-hexane at 0 ℃ for precipitation, filtered and then dried in vacuum at 45 ℃ for 48 hours, and the macromonomer mPEG-AA after the acidification of the hydroxyl group is obtained. The yield was 82%.
(2) Synthesizing a pH sensitive diblock amphiphilic polymer mPEG-b-PAE: adding solvents of anhydrous chloroform (20mL), macromonomer mPEG-AA (100mg, 0.02mmol) and BD (3.96mg, 0.02mmol) into an eggplant-shaped reaction bottle in sequence under the protection of inert gas and in the absence of water by adopting a Michael stepwise addition method to obtain a clear solution, dropwise adding AP (1.8mg, 0.024mmol) under stirring, reacting for 96 hours at 60 ℃, precipitating, filtering and drying to obtain an amphiphilic diblock polymer mPEG-b-PAE with pH responsiveness. The yield was 50%.
(3) Preparing a functionalized drug molecule DOX-ss-COOH: DTDP (1g, 4.75mmol) and ACC (0.37g, 4.75mmol) were added sequentially to a round bottom flask, heated to 65 ℃ under reflux for 2h, added excess ether (50mL), stirred for an additional 3h, filtered and dried to give DTDPA in 30% yield. Adding DOX-HCl (15mg, 0.026mmol) and TEA (2.17 uL, 0.016mmol) in anhydrous DMF (5mL), putting into a round-bottomed flask with a stirrer, keeping out of the light, stirring for 30min, centrifuging, adding a solution (5mL) of DTDPA (5.79mg, 0.03mmol) in anhydrous DMF, reacting at room temperature for 24h in the absence of the light, adding the reacted solution into 300mL (corresponding to 10 times of the volume) of n-hexane at 0 ℃, precipitating, filtering, and drying at 45 ℃ for 48h in vacuum to obtain the functionalized DOX molecule DOX-ss-COOH. The yield was 66%.
(4) Preparation of pH and Redox dual-responsive diblock amphiphilic polymeric prodrugs: DOX-ss-COOH (9.3mg, 0.015mmol), DCC (15.45mg, 0.075mmol), DMAP (18.3mg, 0.15mmol) and the solvent anhydrous DCM (10mL) were added sequentially under anhydrous conditions to a round bottom flask equipped with a stirrer, stirring for 30min, adding anhydrous DCM (20mL) solution of mPEG-b-PAE (10.52mg, 0.001mmol), heating to 35 deg.C, reacting for 48h in the dark, adding the reacted solution into 300mL (10 times of volume) of 0 deg.C n-hexane for precipitation, filtering, then dried under vacuum at 45 ℃ for 48h, the solid was redissolved in DMF (10mL), placed in dialysis bags (MWCO1500Da), dialyzing in a DMF medium for 24h, then changing the DMF medium into deionized water, continuing to dialyze for 48h, and freeze-drying the obtained solution to obtain the pH and Redox double-response amphiphilic polymer prodrug mPEG-b-PAE-ss-DOX. The yield was 73%.
Example 5: critical micelle concentration CMC value of pH and Redox dual-responsive amphiphilic diblock polymer prodrugs
The pH and critical micelle concentration of the double-responsive Redox amphiphilic diblock amphiphilic polymer prodrug mPEG-b-PAE-ss-DOX prepared in example 1 were tested by a fluorescence probe method.
(1) Preparation of pyrene solution: dissolving pyrene with acetone to obtain a solution with a concentration of 12 × 10-5M pyrene solution.
(2) Preparation of sample solution: weighing 5mg of mPEG-b-PAE-ss-DOX, dissolving in 10mL of acetone, quickly adding the solution into 50mL of deionized water, stirring for 24h to volatilize acetone to obtain a polymer mother solution with the concentration of 0.1mg/mL, and diluting to a series of concentrations (the concentration range is 0.0001-0.1 mg/mL). And (3) taking 20 10mL volumetric flasks, respectively adding 0.1mL of the pyrene solution prepared in the step (1), respectively adding the copolymer solutions with different concentrations to a constant volume, and shaking up to obtain a sample solution. The concentration of pyrene in the sample solution was 12X 10-7M。
(3) Fluorescence spectrum test: and scanning the fluorescence excitation spectrum of the sample solution at 300-350 nm by taking 373nm as an emission wavelength. Taking the intensity ratio (I) of 338nm and 335nm338/I335) The logarithm of the polymer concentration is plotted, and as shown in FIG. 6, the abscissa corresponding to the break point of the curve is lg (CMC). The critical micelle concentration of the mPEG-b-PAE-ss-DOX prepared in example 1 was found to be 2.4 mg/L.
Example 6: preparation of pH and Redox dual-response diblock amphiphilic polymer prodrug nano micelle
The dialysis method is adopted to prepare the prodrug nano micelle system, and the specific method is as follows: 100mg of mPEG-b-PAE-ss-DOX prepared in example 1 is weighed and dissolved in 10mLDMSO, stirred for 30min, and transferred into a dialysis bag (MWCO 3500Da) for dialysis after the polymer prodrug is completely dissolved, deionized water (pH 7.4) is replaced every 2h, and deionized water (pH 7.4) is replaced every 6h after 12h, and the total dialysis time is 48 h. And after the dialysis is finished, filtering the dialysate by using a 0.45-micrometer filter membrane, and freeze-drying the filtrate to obtain red powdery solid which is the prodrug nano micelle.
The particle size, distribution and zeta potential of the prodrug micelles were determined by Dynamic Light Scattering (DLS). The particle size Dh of the prodrug micelle was 115nm, PDI was 0.133, and the zeta potential was 0.7 mV.
Example 7: pH and Redox response behavior research of pH and Redox dual-response diblock amphiphilic polymer prodrug nano-micelle
(1) 20mg of the prodrug micelles prepared in example 6 were dissolved in PBS buffer solution at pH 7.4, 6.8 and 6.0, respectively, and after incubation for 4h, the particle size, distribution and zeta potential of the prodrug nanomicelles in PBS buffer solution at different pH values were measured by Dynamic Light Scattering (DLS), as shown in FIGS. 7 and 8. As the pH value is reduced from 7.4 to 6.8, the micelle size and the zeta potential are gradually increased, the particle size is mainly increased due to protonation of tertiary amino groups in a polymer main chain segment, the hydrophobic blocks are gradually changed into hydrophilic blocks, the inner cores are expanded, so that the polymer micelle particles are swelled and increased, the zeta potential is increased mainly due to the fact that the tertiary amino groups absorb one hydrogen ion, so that the positive charge is increased, and the potential is increased; as the pH is lowered from 6.8 to 6.0, the prodrug nanomicelle disintegrates and the zeta potential continues to increase due to the complete protonation of the tertiary amino groups in the PAE block.
(2) 20mg of the prodrug micelles prepared in example 6 were dissolved in PBS buffer solutions of pH 7.4 and 6.5 containing different DTT concentrations (0, 10mM) of dithiothreitol, incubated for 4h, centrifuged (3000rpm, 30min), and the UV-vis spectrum of the supernatant was measured by UV spectrometer, as shown in FIG. 9. No characteristic peak of DOX was found at pH 7.4; when the pH value is 6.5, no characteristic DOX peak is still found, and the main reason is that the DOX molecules are connected to the side chain of the polymer through chemical bonds, and at a low pH value, the chemical bonds are kept stable and no DOX molecules are released; when DTT (10mM) is present in the solution, the characteristic peak of DOX molecules is clearly visible at 480nm, due to the cleavage of the disulfide bonds linking the DOX molecules and the polymer by DTT, thus releasing free DOX molecules.
Example 8: in-vitro release behavior research of pH and Redox dual-response diblock amphiphilic polymer prodrug nano-micelle
10mg of the mPEG-b-PAE-ss-DOX prodrug nanomicelles prepared in example 6 were weighed out separately and dispersed in 3mL PBS buffer at different pH values (pH 7.4, 6.0) and DTT concentrations (0, 10 mM). The solution is placed in a dialysis bag (MWCO: 3500Da), transferred into 47mL buffer solution with the same pH value, placed in a drug dissolution instrument, and subjected to in vitro release at 37 ℃ and the rotating speed of 120 rpm. Samples were taken periodically at 1mL for UV analysis, and supplemented with 1mL of fresh buffer. The concentrations of DOX in the release solution at different times were measured by UV spectrophotometry, and an in vitro release curve was plotted, as shown in FIG. 10. As can be seen from FIG. 10, in the normal tissue environment (pH 7.4, which simulates the normal human body environment), the release rate of DOX is very slow, the 24h cumulative release amount is not more than 18%, while when DTT (10mM) is added, the release rate of the drug is obviously accelerated, and the 24h cumulative release amount is close to 80%. Under the condition of reducing the pH value (pH 6.0 and simulating the environment of tumor tissues), the release speed of DOX is not obviously changed compared with that at the pH 7.4, the 24h cumulative release amount is not more than 20 percent, and after DTT (10mM) is added, the release speed of DOX is obviously accelerated, and the 24h cumulative release amount reaches nearly 100 percent. The main reason is that as the pH is lowered, the tertiary amino group in the polymer is continuously protonated, which finally leads to the disintegration of the prodrug micelle, and at the same time, DTT can effectively cause the breakage of the disulfide bond, so that the DOX molecule is released from the polymer to the medium. With the reduction of the pH value and the increase of the DTT concentration, the release rate and the accumulated release amount of the drug from the drug-loaded micelle are gradually increased, so that the controlled release of the drug is realized.
Example 9: PH and Redox dual-response diblock amphiphilic polymer prodrug nano-micelle and cytotoxicity evaluation of polymer thereof
Cytotoxicity evaluation was performed using mPEG-b-PAE-ss-DOX prodrug micelle prepared in example 6 and polymer obtained in example 1 (2). 3LL cells (purchased from ATCC) were packed at 1X 104The cells were plated on a 96-well plate, 200. mu.L of the culture medium was added thereto, and the cells were cultured for 24 hours. A concentration of free Doxorubicin (DOX), polymer and prodrug micelles were added to the well plate and the culture medium was refreshed. Each concentration was replicated in parallel for 3. The well plate was placed in an ovipositor at 5% CO2 and 37 ℃ for 24h and 48h, respectively. Medium in the well plate was replaced with 180. mu.L of fresh medium and 20. mu.L of MTT solution, continued oviposition for 4h, and well plate medium was replaced with 200. mu.L of LDMSO. The well plate is placed in a shaker at 37 ℃ and oscillated for 15min, then the absorbance of each well at 480nm is measured by a microplate reader, the survival rate of the cells is calculated, and the cytotoxicity is evaluated.
FIG. 11(A) is a cytotoxicity plot of mPEG-b-PAE polymers. As can be seen from the figure, the cell survival rate is still maintained at a higher level with the increase of the polymer concentration, and the cell survival rate is still above 90% when the polymer concentration is 400 mug/mL, so that the toxicity of the polymer material to the cells is lower, which indicates that the material has almost no toxic or side effect. Fig. 11(B and C) are cytotoxicity plots of free doxorubicin and polymeric prodrug micelles after 24h and 48h, respectively. The graph shows that the cell survival rate is reduced obviously along with the increase of time and the concentration of the drug-loaded micelle, and particularly at 48h, the cell survival rate approaches 50% when the concentration of the drug-loaded micelle is 1 mu g/mL.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. An amphiphilic diblock polymer is characterized in that the molecular formula of the amphiphilic diblock polymer is mPEG-b-PAE, the molecular structural formula is shown as a formula 1,
wherein x is 10-30.
2. A preparation method of an amphiphilic diblock polymer mPEG-b-PAE is characterized by comprising the following steps:
(1) under the conditions of inert gas protection and ice bath, dropwise adding triethylamine and acylating agent acryloyl chloride into a dichloromethane solution of hydroxyl-terminated polyethylene glycol monomethyl ether, reacting for 1-4h, heating to room temperature, reacting for 18-32h, cooling, precipitating and drying to obtain a hydroxyl-terminated acrylated macromonomer mPEG-AA;
(2) dropwise adding an anhydrous chloroform solution of 3-amino-1-propanol into an anhydrous chloroform solution of a macromonomer mPEG-AA and 1, 4-butanediol diacrylate under the conditions of inert gas protection and no water by adopting a Michael stepwise addition method, reacting for 72-120h at 45-65 ℃, cooling, concentrating, precipitating and drying to obtain the amphiphilic polymer mPEG-b-PAE.
3. The preparation method according to claim 2, wherein the mole parts of the reactants in the reaction system for generating mPEG-AA in the step (1) are as follows:
1-5 parts of hydroxyl-terminated polyethylene glycol monomethyl ether
5-50 parts of triethylamine
15-25 parts of an acylating agent;
the mole parts of the reactants in the reaction system for generating the mPEG-b-PAE in the step (2) are as follows:
1-5 parts of mPEG-AA
5-50 parts of 1, 4-butanediol diacrylate
6-55 parts of 3-amino-1-propanol;
preferably, the precipitation and drying in step (1) are specifically performed by: adding 8-12 times volume of 0 ℃ n-hexane into the solution for precipitation, and performing vacuum drying on the obtained product mPEG-AA at 25-45 ℃ under 30-40mbar for 24-72 h;
preferably, the operation of precipitating and drying in step (2) is as follows: adding 10 times volume of 0 ℃ n-hexane into the concentrated solution for precipitation, and performing vacuum drying on the obtained product mPEG-b-PAE for 24-72h at 25-45 ℃ and 30-40 mbar.
4. A pH and Redox dual-response diblock amphiphilic polymer prodrug is characterized in that the molecular formula of the prodrug is mPEG-b-PAE-ss-DOX, the molecular structural formula is shown as a formula 2,
wherein x is 10-30, and y is 10-30.
5. The pH and Redox dual-responsive amphiphilic polymer prodrug of claim 4, wherein the pH and Redox dual-responsive amphiphilic polymer prodrug has a number average molecular weight of 10520 to 21560 g/mol.
6. A preparation method of a pH and Redox double-response diblock amphiphilic polymer prodrug is characterized by comprising the following steps:
1) preparing a diblock amphiphilic polymer mPEG-b-PAE;
2)3,3' -dithiodipropionic acid reacts with acetyl chloride to obtain dithiodipropionic anhydride, and the dithiodipropionic anhydride reacts with doxorubicin hydrochloride to obtain a functionalized DOX molecule DOX-ss-COOH;
3) adding dicyclohexylcarbodiimide and 4-dimethylaminopyridine into an anhydrous dichloromethane solution of DOX-ss-COOH, then adding an anhydrous dichloromethane solution of mPEG-b-PAE, heating to 25-40 ℃, reacting for 36-60h in the dark, precipitating, filtering, drying, dialyzing, and freeze-drying to obtain the amphiphilic polymer prodrug mPEG-b-PAE-ss-DOX with double responses of pH and Redox.
7. The process according to claim 6, characterized in that the functionalized DOX molecule DOX-ss-COOH is prepared by: refluxing 3,3' -dithiodipropionic acid and acetyl chloride at 40-70 deg.C for 1-3h, concentrating, adding excessive diethyl ether, and stirring for 1-5h to obtain dithiodipropionic anhydride; dissolving doxorubicin hydrochloride and triethylamine in anhydrous N, N-dimethylformamide, keeping out of the sun, stirring for 10min-1h, centrifuging, then adding dithiodipropionic anhydride, and reacting at room temperature for 18-32h to obtain the compound;
preferably, the mole fraction of the reactants in the reaction system for forming DOX-ss-COOH is as follows:
15-25 parts of dithiodipropionic anhydride
15-25 parts of doxorubicin hydrochloride
15-25 parts of triethylamine;
preferably, the rotation speed of the centrifugation is 12000-16000 rpm, and the time is 1-3 min;
preferably, the amphiphilic diblock polymer mPEG-b-PAE is prepared using the process of claim 2;
preferably, the operations of precipitating, filtering and drying in the step 3) are as follows: adding 8-12 times volume of 0 ℃ n-hexane into the solution for precipitation, and performing vacuum drying at 25-45 ℃ under 30-40mbar for 24-72 hours;
preferably, the dialysis in step 3) is specifically operative to: and dissolving the obtained solid in N, N-dimethylformamide, placing the solid in a dialysis bag, dialyzing the solid in N, N-dimethylformamide as a medium for 24-72 hours, then changing the medium into deionized water, and continuously dialyzing the medium for 24-72 hours to obtain mPEG-b-PAE-ss-DOX.
8. The method according to claim 6, wherein the molar fractions of the reactants in the reaction system for producing dithiodipropionic anhydride in step 2) are as follows:
1-5 parts of 3,3' -dithiodipropionic acid
5-15 parts of acetyl chloride;
the mole parts of the reactants in the reaction system for generating mPEG-b-PAE-ss-DOX in the step 3) are as follows:
9. a drug-loaded nanomicelle system comprising a pH and Redox dual-responsive amphiphilic diblock amphiphilic polymer prodrug according to claim 4;
preferably, the preparation method of the drug-loaded nano-micelle system comprises the following steps: dissolving the pH and Redox dual-response diblock amphiphilic polymer prodrug in an organic solvent, stirring for 30min at room temperature, then placing in a dialysis bag, dialyzing for 48h with deionized water, filtering, and freeze-drying to obtain a drug nano-micelle system;
preferably, the organic solvent is dimethyl sulfoxide or dimethylformamide.
10. The drug-loaded nanomicelle system of claim 9 for use as a carrier material for drug delivery.
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