CN111978553B

CN111978553B - Triple-stimulus responsive interfacial crosslinked polymer micelle and preparation method and application thereof

Info

Publication number: CN111978553B
Application number: CN202010718748.0A
Authority: CN
Inventors: 雷忠利; 郭琼; 杨红
Original assignee: Shaanxi Normal University
Current assignee: Shaanxi Normal University
Priority date: 2020-07-23
Filing date: 2020-07-23
Publication date: 2021-09-14
Anticipated expiration: 2040-07-23
Also published as: CN111978553A

Abstract

The invention discloses a triple stimulus-responsive interfacial crosslinked polymer micelle, a preparation method and an application thereof, wherein the polymer micelle is prepared by taking a triple stimulus-responsive hydrophobic polymer chain segment as a core, taking polymethyl dimethylaminoethyl methacrylate as a shell and taking polyethylene glycol methyl ether methacrylate as a crown and utilizing a crosslinking reaction with N, N' -di (bromoacetyl) cystamine, and the structural formula is as follows:

wherein x is an integer of 20 to 80, y is an integer of 3 to 20, m is an integer of 2 to 8, and n is an integer of 5 to 40. The polymer micelle is simple to prepare and uniform in appearance, the interface crosslinking structure improves the stability of the micelle, the drug loading efficiency is improved, and the drug loading capacity of the polymer micelle is improved due to a large number of oxazole rings contained in the polymer structure. When the micelle is used as a drug delivery carrier, the rapid release of the hydrophobic drug under different conditions can be realized, and the micelle has wide application prospects in the fields of drug delivery, targeted release and the like.

Description

Triple-stimulus responsive interfacial crosslinked polymer micelle and preparation method and application thereof

Technical Field

The invention belongs to the technical field of stimulus-responsive polymer materials, and particularly relates to a light-based, oxidation-based and reduction-based triple stimulus-responsive interfacial crosslinked polymer micelle and a preparation method and application thereof.

Background

The stimulus-responsive polymer material can have a structure or property that can be greatly changed under the action of a small external stimulus (pH, redox, glucose, enzyme, temperature, light, magnetic field, current), and the reaction can be a reversible process or an irreversible process. The polymer has higher drug loading capacity as a drug carrier, increases the solubility of the drug, prolongs the circulation time of the drug in blood, reduces the toxic and side effects of the drug on human bodies, and has high permeability and retention effect to ensure that the drug is accumulated in tumor tissues, thereby obtaining better disease treatment effect. The polymer is easy to prepare and store, and the structure and the appearance are easy to control, so that the polymer can be widely applied to the fields of drug delivery, disease monitoring and diagnosis, gene therapy and the like.

In recent years, many researchers at home and abroad are dedicated to exploring and researching an ideal Drug Delivery System (DDS) with the characteristics of biocompatibility, low toxicity, high drug loading, high stability and the like. In most block polymer systems, the number of stimuli-sensitive groups is limited, and the assembled structure of the drug delivery system cannot be completely destroyed under environmental stimuli, resulting in slow, incomplete drug release. In addition, cancer cells proliferate rapidly, the slow drug release process can cause that good treatment effect is difficult to achieve, the physiological environment in human body is complex, and the single stimulation response can not meet the requirement of drug controlled release. The inherent instability of the polymer micelle can cause the polymer micelle to generate a 'burst release' phenomenon under the condition of blood dilution, the release speed of the drug in the subsequent process becomes very slow due to the diffusion effect of the drug, the drug leakage causes the drug-loaded polymer to have the drug loading content lower than the level of killing cancer cells before being endocytosed by the cancer cells, and the poor therapeutic effect is shown, which is one of the great challenges facing the clinical application.

The environment of cancer cells and tissues has the characteristics of three lower and one higher, namely low pH value, hypoxia, low sugar and higher Glutathione (GSH) concentration, the concentration of Glutathione (GSH) or cysteine in cancer cells is more than several times of that of normal tissue cells, and disulfide bonds are stable under physiological conditions, but are rapidly broken in the presence of high-concentration GSH. To date, the most effective strategy for stabilizing the micelle structure has been to crosslink the micelles to form a crosslinked structure. The redox-stimuli-responsive core-crosslinked Polymer nanomicelles prepared by Liu et al are used for the transport of anticancer drug Doxorubicin (DOX), and the core-crosslinked Polymer micelles are verified to have good stability in an extremely dilute concentration or in an organic solvent (European Polymer Journal, 2016, 83, 230-.

Disclosure of Invention

The invention aims to provide an interface cross-linked polymer micelle which has uniform size, good stability, light, oxidation and reduction triple stimulus response and good biocompatibility, and provides a simple preparation method and application for the polymer micelle.

The structural formula of the light, oxidation and reduction triple stimulus responsive interfacial crosslinked polymer micelle used for solving the technical problems is as follows:

wherein x is an integer of 20-80, y is an integer of 3-20, m is an integer of 2-8, and n is an integer of 5-40; preferably, x is an integer of 20-40, y is an integer of 3-10, m is an integer of 2-4, and n is an integer of 8-20.

The synthetic route and the specific preparation method of the light, oxidation and reduction triple stimulus responsive interfacial crosslinked polymer micelle are as follows:

1. using Dichloromethane (DCM) as a solvent, and reacting 2-propyne-1-alcohol with oxalyl chloride to obtain propynyl oxy oxalyl chloride; and reacting the propynyloxyoxalyl chloride with bis (2-hydroxyethyl) disulfide and Triethylamine (TEA) to obtain the compound of the formula I.

2. Tetrahydrofuran (THF) is used as a solvent, and 5- (2-hydroxyethoxy) -2-nitrobenzol reacts with dibromo isobutyryl bromide and TEA under the protection of inert gas to obtain the compound shown in the formula II.

3. And (3) reacting the compound shown in the formula II with sodium azide (NaN3) by using N, N-Dimethylformamide (DMF) as a solvent to obtain a compound shown in the formula III (N3-ONB-N3).

4. The preparation method comprises the steps of taking cuprous bromide (CuBr) as a catalyst, pentamethyl diethylenetriamine (PMDETA) as a ligand and acetonitrile as a solvent, and preparing the compound shown in the formula I and the compound shown in the formula III into the hydrophobic block polymer shown in the formula IV through a click chemical reaction under the protection of inert gas.

5. Carrying out Atom Transfer Radical Polymerization (ATRP) by taking CuBr as a catalyst, PMDETA as a ligand and DMF as a solvent and under the protection of inert gas and taking 2-bromo-2-butynylmethacrylate (alkyne-Br) as an initiator and dimethylaminoethyl methacrylate (DMAEMA) as a monomer to obtain dimethylaminoethyl methacrylate (alkyne-PDMAEMA-Br) with the end group of the formula V as alkynyl.

6. Under the protection of inert gas, taking CuBr as a catalyst, PMDETA as a ligand and DMF as a solvent, taking alkyne-PDMAEMA-Br as a macroinitiator and polyethylene glycol methyl ether methacrylate (PEGMA) as a monomer, and carrying out ATRP reaction to obtain the formula VI, wherein the end group is alkynyl, namely the polymethyl methacrylate dimethylaminoethyl methacrylate-b-polyethylene glycol methyl ether methacrylate (alkyne-PDMAEMA-PPEGMA-Br).

7. CuBr is used as a catalyst, PMDETA is used as a ligand, DMF is used as a solvent, and the amphiphilic block polymer shown as the formula VII is prepared by carrying out click chemical reaction on the hydrophobic block polymer shown as the formula IV and alkyne-PDMAEMA-PPEGMA-Br under the protection of inert gas.

8. Dissolving the amphiphilic block polymer shown in the formula VII in DMF, and forming an uncrosslinked micelle by a dialysis method; adding N, N-di (bromoacetyl) cystamine (BBAC) into the uncrosslinked micelle to carry out crosslinking reaction to obtain the light, oxidation and reduction triple stimulus responsive interfacial crosslinked polymer micelle.

In the step 1, the molar ratio of 2-propyn-1-ol to oxalyl chloride is preferably 1 (1-3), the molar ratio of propynyloxyoxalyl chloride, bis (2-hydroxyethyl) disulfide and TEA is preferably (1-3) to 1 (1-3), and the reaction is preferably carried out at normal temperature for 3-15 hours.

In the step 2, the molar ratio of the 5- (2-hydroxyethoxy) -2-nitrobenzol, the dibromo-isobutyryl bromide and the TEA is preferably 1 (2-4) to (2-4), and the reaction is preferably carried out at normal temperature for 20-30 hours. Wherein the 5- (2-hydroxyethoxy) -2-nitrobenzol is synthesized according to the method disclosed in the literature "Chemical Science,2013,4(6): 2573-.

In the step 3, the compound of formula II and NaN are preferable₃In a molar ratio ofIs 1 (2-4), the reaction temperature is preferably 50-70 ℃ and the reaction time is preferably 20-30 hours.

In the step 4, the molar ratio of the compound of formula I, the compound of formula III, CuBr and PDMETA is preferably 1 (1-1.5) to 0.3-1, and the reaction is preferably carried out at 60-70 ℃ for 10-18 hours.

In the step 5, the molar ratio of alkyne-Br, DMAEMA, CuBr and PMDETA is preferably 1 (20-100) to (0.5-1.5), the reaction temperature is preferably 50-70 ℃ and the reaction time is preferably 5-10 hours.

In the step 6, the molar ratio of alkyne-PDMAEMA-Br, PEGMA, CuBr and PMDETA is preferably 1 (5-20): (0.5-2): 0.5-2), and the reaction temperature is preferably 50-90 ℃ and the reaction time is preferably 5-10 hours.

In the step 7, the mole ratio of the hydrophobic block polymer of formula IV, alkyne-PDMAEMA-PPEGMA-Br, CuBr and PMDETA is preferably 1 (2-6) to (10-30), and the reaction temperature is preferably 50-80 ℃ and the reaction time is preferably 10-20 hours.

In the step 8, the molar ratio of the amphiphilic block Polymer of the formula VII to BBAC is preferably 1: 30-80, the temperature of the crosslinking reaction is preferably 20-50 ℃, and the time is 20-72 hours, wherein the BBAC is synthesized according to the method disclosed in the literature "Polymer Chemistry,2013,4, 1199-.

The invention relates to an application of a triple-stimulus responsive interface cross-linked polymer micelle as a hydrophobic anticancer drug carrier.

Compared with the prior art, the invention has the following advantages:

1. the invention prepares the amphiphilic block polymer with simple structure by a mature synthesis method and widely applied monomers; the polymer micelle with an interface crosslinking structure is prepared by crosslinking molecules BBAC, the micelle has better stability in aqueous solution, and the Critical Micelle Concentration (CMC) is 0.0159 mg/mL.

2. The preparation method of the triple-stimulus responsive interfacial crosslinked polymer micelle is simple, does not have harsh and special reaction conditions, does not need any protection and deprotection steps, and has simple and easy operation.

3. The interface cross-linked polymer micelle has light, oxidation and reduction triple stimulus responsiveness, contains amino and a large number of oxazole rings in the structure, can adsorb certain anti-cancer substances (such as adriamycin), enzyme and the like through static electricity, and has a simple drug loading process.

4. The triple-stimulus responsive interfacial cross-linked polymer micelle takes polyethylene glycol widely applied to the pharmaceutical industry as the micelle corona, has a phase transition temperature close to the human body temperature (37 ℃), is beneficial to the release of the drug-loaded micelle in cancer cell drugs, and is also beneficial to the increase of the biocompatibility of the drug-loaded micelle.

5. In the controlled release process of the drug, the cumulative release amount of the drug can be significantly improved under the triple stimulation of light, oxidation and reduction, and the drug-loaded micelle can play a role in the microenvironment of the cancer cells in a rapid and targeted manner.

6. The triple stimulus responsive interfacial crosslinked polymer micelle of the present invention has good drug loading capacity (for example, when the dosage is 10% of the micelle mass, the coating rate is 60%).

Drawings

FIG. 1 is a nuclear magnetic hydrogen spectrum of the amphiphilic block polymer of formula VII-1 prepared in example 1 (solvent is CDCl)₃)。

FIG. 2 is an IR spectrum of the dimethylaminoethyl methacrylate (a) with alkynyl group at the end group of formula V-1, the dimethylaminoethyl methacrylate-b-polyethylene glycol methyl ether methacrylate (b) with alkynyl group at the end group of VI-1, the hydrophobic block polymer (c) of formula IV-1 and the amphiphilic block polymer (d) of formula VII-1 prepared in example 1.

FIG. 3 shows the fluorescence spectra (excitation wavelength 340nm) of pyrene in triple stimulus-responsive interfacial cross-linked polymer micelles prepared in example 1 at different concentrations.

FIG. 4 is I of pyrene in FIG. 3₃/I₁And an analytical spectrum of log concentration (logC) of the triple stimulus-responsive interfacially crosslinked polymer micelle prepared in example 1.

FIG. 5 is a UV spectrum of the triple stimulus-responsive interfacially crosslinked polymer micelle prepared in example 1 under different illumination time (micelle concentration is 0.5 mg/mL).

FIG. 6 shows the triple stimulus-responsive interfacially crosslinked polymer micelles prepared in example 1 under different conditions (no stimulus, UV light, 10mM H)₂O₂、10mM GSH、10mM H₂O₂&365nm UV、10mM GSH&365nm UV、、10mM H₂O₂&10mM GSH&365nm UV) of the sample (micelle concentration of 0.2 mg/mL).

FIG. 7 is a DLS curve spectrum (micelle concentration of 0.2mg/mL) of the uncrosslinked micelles and the triple stimulus-responsive interfacially crosslinked polymeric micelles prepared in example 1 under different conditions (no stimulation, 10-fold dilution with DMF).

FIG. 8 shows NR-loaded uncrosslinked micelles prepared in example 2 under different stimulus triggers (no stimulus, UV light, 10mM H₂O₂、10mM GSH、10mM H₂O₂&365nm UV、10mM GSH&365nm UV、10mM H₂O₂&10mM GSH&365nm UV) drug release profile (micelle concentration of 0.2 mg/mL).

FIG. 9 shows NR-loaded triple stimulus-responsive interfacially crosslinked polymer micelles prepared in example 2 under different stimulus triggers (no stimulus, UV light, 10mM H)₂O₂、10mM GSH、10mM H₂O₂&365nm UV、10mM GSH&365nm UV、10mM H₂O₂&10mM GSH&365nm UV) drug release profile (micelle concentration of 0.2 mg/mL).

FIG. 10 is a fluorescence spectrum of NR-loaded uncrosslinked micelles under 365nm UV light irradiation at different times (micelle concentration of 0.2 mg/mL).

FIG. 11 is a fluorescence spectrum of NR-loaded triple stimulus-responsive interfacially crosslinked polymeric micelles under 365nm UV light irradiation at different times (micelle concentration of 0.2 mg/mL).

FIG. 12 shows the presence of NR-loaded uncrosslinked micelles in different H₂O₂Fluorescence spectrum at concentration (micelle concentration 0.2 mg/mL).

FIG. 13 shows that NR-loaded triple-stimulus-responsive interfacial cross-linked polymer micelles differ in H₂O₂Fluorescence at concentrationSpectrophotometers (micelle concentration 0.2 mg/mL).

FIG. 14 is a fluorescence spectrum of NR-loaded non-crosslinked micelles at different GSH concentrations (micelle concentration of 0.2 mg/mL).

FIG. 15 is a fluorescence spectrum of NR-loaded triple stimulus-responsive interfacially crosslinked polymeric micelles at different GSH concentrations (micelle concentration of 0.2 mg/mL).

FIG. 16 shows NR-loaded uncrosslinked micelles at 10mM H₂O₂And fluorescence spectra under 365nm UV light irradiation at different times (micelle concentration 0.2 mg/mL).

FIG. 17 shows NR-loaded triple stimulus-responsive interfacially crosslinked polymer micelles at 10mM H₂O₂And fluorescence spectra under 365nm UV light irradiation at different times (micelle concentration of 0.2 mg/mL).

FIG. 18 is a fluorescence spectrum of NR-loaded uncrosslinked micelles under irradiation with 10mM GSH and UV light at different times of 365nm (micelle concentration of 0.2 mg/mL).

FIG. 19 is a fluorescence spectrum of NR-loaded triple stimulus-responsive interfacially crosslinked polymeric micelles under irradiation with 10mM GSH and 365nm UV light at different times (micelle concentration of 0.2 mg/mL).

FIG. 20 shows NR-loaded uncrosslinked micelles at 10mM H₂O₂&GSH and fluorescence spectra under 365nm UV light at different times (micelle concentration 0.2 mg/mL).

FIG. 21 shows NR-loaded triple stimulus-responsive interfacially crosslinked polymer micelles at 10mM H₂O₂&GSH and fluorescence spectra under 365nm UV light at different times (micelle concentration 0.2 mg/mL).

Detailed Description

The invention will be further described in detail with reference to the following figures and examples, but the scope of the invention is not limited to these examples.

Example 1

1. Oxalyl chloride (18.00mL, 209.76mmol) and anhydrous dichloromethane (DCM, 60.00mL) were added to a 100mL single-neck round-bottom flask, 2-propyn-1-ol (5.00g, 89.28mmol) was added dropwise under ice-bath conditions, and after the addition was complete, the reaction was carried out at room temperature for 3 h. After completion of the reaction, the reaction mixture was distilled under reduced pressure at 98 ℃ to give propynyloxyoxalyl chloride (8.65g, yield 81%) as a colorless transparent oil.

Bis (2-hydroxyethyl) disulfide (1.84g, 11.93mmol), TEA (2.40g, 24.00mmol) and purified THF (10.00mL) were charged into a 100mL single-neck round-bottom flask, and after dropping of oxalyl propiolate (4.00g, 27.30mmol) was added dropwise thereto under ice bath, followed by reaction at ordinary temperature for 12 hours to obtain a colorless transparent liquid, i.e., the compound of formula I (3.87g, yield 87%).

2. Under the protection of nitrogen, 5- (2-hydroxyethoxy) -2-nitrobenzol (2.50g, 11.68mmol), TEA (3.05g, 29.90mmol) and THF (80.00mL) were added to a 250mL three-necked flask, and 2-bromoisobutyryl bromide (6.00g, 26.09mmol) was added dropwise, followed by reaction at room temperature for 24 h. After the reaction is finished, the mixture is purified by a column chromatography separation method, the developing agent is a mixed solution of ethyl acetate and petroleum ether with the volume ratio of 1:4, the solvent is removed by vacuum rotary evaporation at 30 ℃, and the mixture is dried in vacuum at 25 ℃ to obtain a light yellow solid (5.06g, the yield is 85 percent), namely the compound of the formula II.

3. Mixing the compound of formula II (2.30g, 4.50mmol) with NaN₃(1.00g, 15.38mmol) and DMF (30.00mL) were charged to a 100mL single neck round bottom flask and reacted at 60 ℃ for 24 h. After the reaction is finished, adding 50.00mL of ethyl acetate, washing with saturated aqueous solution of ammonium chloride and water for three times respectively, collecting the organic phase, drying for 12h at 25 ℃, separating and purifying by column chromatography, wherein the developing agent is a mixed solution of ethyl acetate and petroleum ether with the volume ratio of 1:3, removing the solvent by a vacuum rotary evaporation method, and drying for 12h at 25 ℃ in vacuum to obtain a light yellow liquid (1.51g, 77 percent), namely the compound of the formula III.

4. Adding the compound of the formula I (0.35g, 0.93mmol), the compound of the formula III (0.43g, 0.98mmol), PMDETA (0.08g, 0.46mmol) and dry acetonitrile (5.00mL) into a 50mL Schlenk tube, continuously freezing and thawing for degassing for 2 times, rapidly adding CuBr (0.06g, 0.42mmol), continuously freezing and thawing for degassing for 1 time, keeping a nitrogen atmosphere, and reacting at 60 ℃ for 12 hours. The compound of formula III (0.05g, 0.11mmol) was added again and the reaction was continued for 4 h. After the reaction was completed, the reaction mixture was quenched with THF (2.00mL), passed through a neutral alumina column, dialyzed against deionized water for three days (dialysis bag: 3.5kD), and freeze-dried to obtain a pale yellow substance (0.68g), which is a hydrophobic block polymer of formula IV-1.

5. Adding alkyne-Br (0.0771g, 0.33mmol), PMDETA (0.0506g, 0.29mmol), DMAEMA (3.27g, 20.80mmol) and dried DMF (5.00mL) into a 50mL Schlenk tube in sequence, carrying out continuous freeze-thaw and degassing for 2 times, rapidly adding CuBr (0.052g, 0.36mmol), carrying out continuous freeze-thaw and degassing for 1 time, keeping the nitrogen atmosphere, and reacting for 6 hours at 60 ℃. After the reaction was completed, the reaction mixture was quenched with THF (2.00mL), passed through a neutral alumina column, and after most of the solvent was removed by rotary evaporation, it was dialyzed against deionized water for three days (dialysis bag: 3.5kD), and lyophilized to obtain a white solid (0.93g), i.e., dimethylaminoethyl methacrylate having an alkynyl group as the terminal group of formula V-1.

6. Sequentially adding poly (dimethylaminoethyl methacrylate) (0.90g, 0.19mmol) with alkynyl as a terminal group in the formula V-1, PMDETA (0.052g, 0.30mmol), PEGMA (0.60g, 2.00mmol) with the number average molecular weight of about 300 and dry DMF (5.00mL) into a 50mL Schlenk tube, continuously freezing and thawing for degassing for 2 times, rapidly adding CuBr (0.053g, 0.37mmol), continuously freezing and thawing for degassing for 1 time, keeping the nitrogen atmosphere, and reacting for 6 hours at 75 ℃. After the reaction was completed, the reaction mixture was quenched with THF (2.00mL), passed through a neutral alumina column, and after most of the solvent was removed by rotary evaporation, dialyzed against deionized water for three days (dialysis bag: 5kD), and lyophilized to obtain a white solid (0.66g), i.e., dimethylaminoethyl methacrylate-b-polyethylene glycol methyl ether methacrylate having an alkynyl group as the terminal group of formula VI-1.

7. Adding the hydrophobic block polymer (0.10g, 0.012mmol) of the formula IV-1, the poly (dimethylaminoethyl methacrylate) -b-polyethylene glycol methyl ether methacrylate (0.21g, 0.036mmol) with the end group of the formula VI-1 being alkynyl, PMDETA (0.04g, 0.23mmol) and DMF (5.00mL) into a 50mL Schlenk tube, continuously freezing and thawing for degassing for 2 times, quickly adding CuBr (0.03g, 0.21mmol), continuously freezing and thawing for degassing for 1 time, keeping the nitrogen atmosphere, and reacting at 60 ℃ for 13 h. The mixture was diluted with THF (2.00mL), passed through a neutral alumina column, dialyzed against deionized water for three days (dialysis bag: 10kD), and freeze-dried to give a milky sponge (0.24g), an amphiphilic block polymer of formula VII-1.

8. The amphiphilic block polymer of formula VII-1 (40.00mg, 0.002mmol) was dissolved in THF (10.00mL) and micelles were formed by dialysis to give uncrosslinked micelles. The uncrosslinked micelle (20.00mL) was taken out into a 100mL round-bottomed flask, a THF solution (2.00mL, 0.11mmol) of 25.00mg/mLBBAC was added dropwise, stirred at 30 ℃ for 24 hours, and then dialyzed against deionized water for three days (dialysis bag: 10kD) to obtain a triple stimuli-responsive interfacially crosslinked polymer micelle.

And characterizing the obtained sample by adopting a nuclear magnetic resonance spectrometer, an infrared spectrometer, a laser particle analyzer, a fluorescence spectrophotometer, a laser light scattering gel chromatograph and an ultraviolet spectrophotometer, wherein the result is shown in the figure 1-7. As can be seen from FIG. 1, the chemical shift of the triazole ring occurs at 8.22ppm, while the three peaks at 6.91ppm, 6.94ppm and 7.06ppm belong to the aromatic proton hydrogen in the compound of formula III and the two peaks at 5.45ppm and 4.30ppm belong to the two-CH groups in the compound of formula I₂Chemical shift peaks of-4.03 ppm and 2.26ppm assigned to DMAEMA and 3.36ppm assigned to PEGMA, indicating that click chemistry has occurred, indicating that the amphiphilic block polymer has been successfully synthesized. 2117cm, as can be seen in FIG. 2^-1Characteristic absorption peaks of azide and azole rings appear at 1585cm^-1The characteristic peaks of benzene rings appear, and the combination of the characteristic peaks indicates that the amphiphilic block copolymer has been successfully synthesized. As can be seen from FIGS. 3 and 4, the CMC value of the triple stimulus-responsive interfacially crosslinked polymer micelle was 0.0159 mg/mL. The number average molecular weight of the poly (dimethylaminoethyl methacrylate) with the end group of the formula V-1 being alkynyl is 4.85kD and the number average molecular weight of the poly (dimethylaminoethyl methacrylate) -b-polyethylene glycol methyl ether methacrylate with the end group of the formula VI-1 being alkynyl is 1.124, the number average molecular weight of the poly (dimethylaminoethyl methacrylate) -b-polyethylene glycol methyl ether methacrylate with the end group of the formula VI-1 being alkynyl is 5.799kD and the number average molecular weight of the Poly (PDI) is 1.071, the number average molecular weight of the amphiphilic block polymer of the formula VII-1 is 19.779kD and the number average molecular weight of the Poly (PDI) is 1.094, the molecular weight and the molecular weight distribution of the polymer are confirmed through GPC measurement results, and the hydrophobic part in the polymer has 10 polymerization units.

As can be seen from FIG. 5, the characteristic absorption peak of o-nitrobenzol near 265nm decreases with the increase of the 365nm UV light irradiation time in the triple stimulus-responsive interfacial crosslinked polymer micelle solution, which indicates thatThe heavy stimulus responsive interfacial crosslinked polymer micelle has light stimulus responsiveness. As can be seen from FIG. 6, the skin was irradiated with 365nm UV light for 30min and H, respectively, without applying stimulus₂O₂Action 10h, GSH action 10h and 10mM GSH&H₂O₂After the reaction is carried out for 10 hours, the average particle sizes of the polymer micelles are respectively 120nm, 145nm, 130nm and 160 nm; h₂O₂Action 10h, GSH action 10h and 10mM GSH&H₂O₂After the reaction is carried out for 10 hours, the UV light is irradiated for 30min, the particle sizes of the micelles are respectively changed into 140nm, 140nm and 250nm, and the particle size change and the particle size dispersion degree (the width of a DLS curve peak) can clearly prove that the interface cross-linked polymer micelle synthesized by the embodiment has light, oxidation and reduction stimulation responsiveness.

As can be seen from FIG. 7, the average particle size of the uncrosslinked micelle was 160nm, PDI was 0.115, and after 10-fold dilution with THF, the average particle size of the micelle became 320 nm; the average particle size of the triple stimulus-responsive interfacial crosslinked polymer micelle is 120nm, PDI is 0.145, and after 10 times of THF dilution, the average particle size of the micelle is 240 nm. The grain size of the interface crosslinked micelle is smaller than that of the non-crosslinked micelle, because the chain segment of the micelle is more compact due to crosslinking, after 10 times of THF (tetrahydrofuran) dilution, the grain size change of the interface crosslinked micelle is obviously smaller than that of the non-crosslinked micelle, because the crosslinked structure can stabilize the micelle structure, the micelle structure is not easy to damage under the dilution condition. The data show that the triple stimulus-responsive interfacial crosslinked polymer micelle synthesized by the embodiment has better structural stability.

Example 2

1. This procedure was the same as in example 1, step 1, to give the compound of formula I.

2. This procedure was the same as in example 1, step 2, to give the compound of formula II.

3. This procedure was the same as in example 1, step 3, to give the compound of formula III.

4. Adding the compound of the formula I (0.23g, 0.61mmol), the compound of the formula III (0.27g, 0.62mmol), PMDETA (0.08g, 0.46mmol) and dry acetonitrile (5.00mL) into a 50mL Schlenk tube, continuously freezing and thawing for degassing for 2 times, rapidly adding CuBr (0.06g, 0.42mmol), continuously freezing and thawing for degassing for 1 time, keeping a nitrogen atmosphere, and reacting at 60 ℃ for 12 hours. The compound of formula III (0.05g, 0.11mmol) was added again and the reaction was continued for 4 h. After the reaction was completed, the reaction mixture was quenched with THF (2.00mL), passed through a neutral alumina column, dialyzed against deionized water for three days (dialysis bag: 3.5kD), and freeze-dried to obtain a pale yellow substance (0.65g), i.e., a hydrophobic block polymer of the formula IV-2.

5. The alkyne-Br (0.0784g, 0.34mmol), PMDETA (0.0506g, 0.29mmol), DMAEMA (3.27g, 20.80mmol) and dried DMF (5.00mL) were added to a 50mL Schlenk tube in sequence, after 2 times of continuous freeze-thaw degassing, CuBr (0.055g, 0.38mmol) was added rapidly, and after 1 time of continuous freeze-thaw degassing, the reaction was carried out at 65 ℃ for 7h while maintaining a nitrogen atmosphere. After the reaction was completed, the reaction mixture was quenched with THF (2.00mL), passed through a neutral alumina column, and after most of the solvent was removed by rotary evaporation, it was dialyzed against deionized water for three days (dialysis bag: 3.5kD), and lyophilized to obtain a white solid (0.95g), i.e., dimethylaminoethyl methacrylate having an alkynyl group as the terminal group of formula V-2.

6. Sequentially adding poly (dimethylaminoethyl methacrylate) (0.92g, 0.20mmol) with alkynyl as a terminal group in the formula V-2, PMDETA (0.053g, 0.30mmol), PEGMA (0.69g, 2.30mmol) with the number average molecular weight of about 300 and dry DMF (5.00mL) into a 50mL Schlenk tube, continuously freezing and thawing for degassing for 2 times, rapidly adding CuBr (0.053g, 0.37mmol), continuously freezing and thawing for degassing for 1 time, keeping the nitrogen atmosphere, and reacting for 8 hours at 65 ℃. After the reaction was completed, the reaction mixture was quenched with THF (2.00mL), passed through a neutral alumina column, and after most of the solvent was removed by rotary evaporation, dialyzed against deionized water for three days (dialysis bag: 5kD), and lyophilized to obtain a white solid (0.65g), i.e., dimethylaminoethyl methacrylate-b-polyethylene glycol methyl ether methacrylate having an alkynyl group as the terminal group of formula VI-2.

7. Adding the hydrophobic block polymer (0.10g, 0.014mmol) of the formula IV-2, the poly (dimethylaminoethyl methacrylate) -b-polyethylene glycol methyl ether methacrylate (0.25g, 0.043mmol) with the end group of the formula V-2 being alkynyl, PMDETA (0.03g, 0.19mmol) and DMF (5.00mL) into a 50mL Schlenk tube, continuously freezing and thawing and degassing for 2 times, quickly adding CuBr (0.02g, 0.14mmol), continuously freezing and thawing and degassing for 1 time, keeping the nitrogen atmosphere, and reacting at 70 ℃ for 15 h. The resulting mixture was purified by dialysis through a neutral alumina column (dialysis bag: 10kD) after dilution with THF (2.00mL), dialyzed for three days, and freeze-dried to give a milky sponge (0.25g), i.e., an amphiphilic block polymer of the formula VII-2.

8. The amphiphilic block polymer of formula VII-2 (37.31mg, 0.002mmol) was dissolved in DMF (10.00mL) and micelles were formed by dialysis to give uncrosslinked micelles. Non-crosslinked micelles (20.00mL) were taken out into a 100mL round-bottomed flask, a THF solution of 25.00mg/mL BBAC (2.00mL, 0.11mmol) was added dropwise, stirred at 30 ℃ for 24 hours, and then dialyzed against deionized water for three days (dialysis bag: 10kD) to obtain triple stimulus-responsive interfacially crosslinked polymeric micelles.

Example 3

Application of triple stimulus-responsive interfacial crosslinked polymer micelle of example 1 as anticancer drug carrier

The amphiphilic block polymer of formula VII-1 (40.00mg), Nile Red (NR) (4mg) were added to DMF (10.00mL), stirred for 1h, then added dropwise to 30.00mL of deionized water and dialyzed for three days (dialysis bag: 10kD) with water change every 6 h. After the completion of dialysis, an NR-loaded non-crosslinked micelle (NCL micelle) was obtained.

The NR-loaded non-crosslinked micelle (20.00mL) was taken out and added dropwise to a THF solution (2.00mL) of 25.00mg/mL BBAC in a 100mL round-bottomed flask, and stirred at 30 ℃ for 24 hours, after the reaction was completed, dialyzed with deionized water for three days (dialysis bag: 10kD), and water was changed every 6 hours. And obtaining the NR loaded triple stimulus responsive interface cross-linked polymer micelle (ICL micelle for short) after the dialysis is finished.

A fluorescence spectrophotometer is adopted to detect the drug release curve of the polymer micelle under single and multiple stimulations (the excitation wavelength is 560nm, the micelle concentration is 0.2mg/mL), and the result is shown in figures 8-9. As can be seen from FIGS. 8 and 9, the NCL micelles released about 75% of the encapsulated amount at 3h NR, and ICL micelles released about 75% of the encapsulated amountIt takes nearly 10h to release 75% of the NR encapsulation. Under the condition of no stimulation, the release amount of the ICL micelle is slightly smaller than that of the NCL micelle, and the drug encapsulation capacity of the ICL micelle is considered to be superior to that of the NCL micelle. The 365nm UV light is applied for 30min, the release amount of NCL micelle and ICL micelle is 29% and 13%, respectively, and the 365nm UV light destroys the polymer structure, but the crosslinking structure can slow down the release rate of NR molecules. 10mM H₂O₂After 15 hours of action, the release amounts of NCL micelle and ICL micelle are 36% and 14% respectively; the results of applying 365nm UV light for 30min under the same conditions showed that the release of NR was greater under the double stimulation condition and that the release of NR was significantly greater for the NCL micelles than for the ICL micelles under the single stimulation condition, since the crosslinked structure in the ICL micelles prevented the release of NR, as compared to the single stimulation condition, in which the release of NR was 41% and 28%, respectively. After 10 hours of action of 10mM GSH, the release amounts of NCL micelle and ICL micelle are respectively 38% and 20%; under the same condition, 365nm UV light irradiation is increased for 30min, the release amount of NCL micelle and ICL micelle is respectively 53% and 51%, and the result shows that compared with single stimulation, under the condition of double stimulation, the release amount of NR is larger, the release amount of NR of the NCL micelle is slightly larger than that of the ICL micelle, the release rate of NR can be prevented by a cross-linked structure, but the influence on the release amount of the drug is limited, and GSH can destroy disulfide bonds in a polymer chain segment and a cross-linked chain segment, so that the cross-linked structure of the micelle is destroyed. 10mM H₂O₂After the GSH is acted for 10 hours, the release amounts of NCL micelle and ICL micelle are 55% and 53% respectively; under the same condition, 365nm UV light irradiation is increased for 30min, the release amount of NCL micelle and ICL micelle is 77% and 74%, and compared with double stimulation and triple stimulation, the release amount of NR is larger. Under the triple stimulation condition, the release amounts of the NCL micelle and the ICL micelle are basically the same, and the cross-linked structure has almost no influence on the release amount of the polymer. The ICL micelle release rate was significantly slower than the NCL micelle, indicating that the ICL micelle structure stability was superior to the NCL micelle.

The data show that compared with single stimulation, the release amount of NR is larger under multiple stimulation, and the multiple stimulation condition can destroy the stability of the polymer micelle structure to a greater extent and trigger the release of NR more effectively. Under the same single stimulation condition, the release amount of NR of the ICL micelle is obviously lower than that of the NCL micelle; under the multiple stimulation condition, the release amount of the NCL micelle is basically consistent with that of the ICL micelle, and is slightly lower than that of the NCL micelle. The interface crosslinking improves the stability of the polymer micelle structure while ensuring the drug release performance.

The drug release test triggering the NCL micelle and the ICL micelle under different stimulation conditions is further characterized by directly changing the environment of the drug-loaded micelle, such as changing the illumination time and controlling the addition of an oxidation or reduction substance, and the result is shown in the figure 10-21. As can be seen from FIGS. 10-11, the release of the drug can be triggered significantly by increasing the 365nm UV light irradiation time; as can be seen from FIGS. 12 to 13, H is increased₂O₂Concentration, which can effectively trigger the release of the drug; as can be seen from fig. 14-15, increasing the GSH concentration can trigger the release of the drug-loaded micelle drug; as can be seen from FIGS. 16 to 17, in a certain range of H₂O₂Under the concentration, 365nm UV light is applied, and the release amount of the drug is increased along with the increase of the illumination time; as can be seen from fig. 18 to 19, under a certain GSH concentration, 365nm UV light is applied, and the release amount of the drug is continuously increased along with the increase of the illumination time; as can be seen from FIGS. 20 to 21, at a certain concentration H₂O₂&When GSH is adopted, the 365nm UV illumination time is prolonged, the drug release amount is continuously increased, and the drug-loaded micelle drug can be triggered to be released more efficiently and rapidly. Overall, the drug release from ICL micelles was smaller than ICL micelles, indicating that the crosslinked structure contributes to the controlled release of the drug loaded micelle.

Claims

1. A triple stimulus-responsive interfacially crosslinked polymeric micelle, wherein the interfacially crosslinked polymer has the following structural formula:

in the formula, x is an integer of 20-80, y is an integer of 3-20, m is an integer of 2-8, and n is an integer of 5-40.

2. The triple stimulus responsive interfacially crosslinked polymeric micelle of claim 1, wherein: the value of x is an integer of 20-40, the value of y is an integer of 3-10, the value of m is an integer of 2-4, and the value of n is an integer of 8-20.

3. A method for preparing the triple stimulus-responsive interfacially crosslinked polymeric micelle of claim 1, characterized in that it comprises the steps of:

(1) using dichloromethane as a solvent, and reacting 2-propyne-1-alcohol with oxalyl chloride to obtain propynyl oxyoxalyl chloride; reacting the propinyl oxy oxalyl chloride with bis (2-hydroxyethyl) disulfide and triethylamine to obtain a compound shown in the formula I;

(2) reacting 5- (2-hydroxyethoxy) -2-nitrobenzol with dibromo isobutyryl bromide and triethylamine by taking tetrahydrofuran as a solvent under the protection of inert gas to obtain a compound shown in a formula II;

(3) reacting a compound shown in a formula II with sodium azide by using N, N-dimethylformamide as a solvent to obtain a compound shown in a formula III;

(4) preparing a hydrophobic block polymer shown in a formula IV by using cuprous bromide as a catalyst, pentamethyldiethylenetriamine as a ligand and acetonitrile as a solvent through a click chemical reaction of a compound shown in a formula I and a compound shown in a formula III under the protection of inert gas;

(5) carrying out atom transfer radical polymerization reaction by taking cuprous bromide as a catalyst, pentamethyl diethylenetriamine as a ligand and N, N-dimethylformamide as a solvent and taking 2-bromo-2-methylpropionate butyne as an initiator and dimethylaminoethyl methacrylate as a monomer under the protection of inert gas to obtain dimethylaminoethyl methacrylate with alkynyl as a terminal group in a formula V;

(6) under the protection of inert gas, taking cuprous bromide as a catalyst, taking pentamethyl diethylenetriamine as a ligand, taking N, N-dimethylformamide as a solvent, taking dimethylaminoethyl methacrylate with an alkynyl end group shown in a formula V as a macromolecular initiator and taking polyethylene glycol methyl ether methacrylate as a monomer, and carrying out atom transfer radical polymerization reaction to obtain dimethylaminoethyl methacrylate-b-polyethylene glycol methyl ether methacrylate with an alkynyl end group shown in a formula VI;

(7) carrying out click chemical reaction on a hydrophobic block polymer shown in a formula IV and polymethyl methacrylate-b-polyethylene glycol methyl ether methacrylate with alkynyl as a terminal group shown in a formula VI under the protection of inert gas by using cuprous bromide as a catalyst, pentamethyl diethylenetriamine as a ligand and N, N-dimethylformamide as a solvent to prepare an amphiphilic block polymer shown in a formula VII;

(8) dissolving the amphiphilic block polymer shown in the formula VII in N, N-dimethylformamide, and forming an uncrosslinked micelle by a dialysis method; and adding N, N-di (bromoacetyl) cystamine into the uncrosslinked micelle to carry out crosslinking reaction to obtain the triple-stimulus responsive interfacial crosslinked polymer micelle.

4. The method for preparing a triple stimulus-responsive interfacially crosslinked polymeric micelle according to claim 3, characterized in that: in the step (1), the molar ratio of the 2-propyn-1-ol to the oxalyl chloride is 1 (1-3), the molar ratio of the propynyl oxyoxalyl chloride to the bis (2-hydroxyethyl) disulfide to the triethylamine is (1-3) to 1 (1-3), and the reaction is carried out at normal temperature for 3-15 hours.

5. The method for preparing a triple stimulus-responsive interfacially crosslinked polymeric micelle according to claim 3, characterized in that: in the step (2), the molar ratio of the 5- (2-hydroxyethoxy) -2-nitrobenzol, the dibromo-isobutyryl bromide and the triethylamine is 1 (2-4) to (2-4), and the reaction is carried out at normal temperature for 20-30 hours; in the step (3), the molar ratio of the compound shown in the formula II to the sodium azide is 1 (2-4), the reaction temperature is 50-70 ℃, and the reaction time is 20-30 hours.

6. The method for preparing a triple stimulus-responsive interfacially crosslinked polymeric micelle according to claim 3, characterized in that: in the step (4), the molar ratio of the compound of formula I, the compound of formula III, cuprous bromide and pentamethyldiethylenetriamine is 1 (1-1.5): 0.3-1, the reaction temperature is 60-70 ℃, and the reaction time is 10-18 hours.

7. The method for preparing a triple stimulus-responsive interfacially crosslinked polymeric micelle according to claim 3, characterized in that: in the step (5), the molar ratio of the butynyl 2-bromo-2-methylpropionate, the dimethylaminoethyl methacrylate, the cuprous bromide and the pentamethyl diethylenetriamine is 1 (20-100) (0.5-1.5), the reaction temperature is 50-70 ℃, and the reaction time is 5-10 hours; in the step (6), the molar ratio of the poly (dimethylaminoethyl methacrylate) with alkynyl as the end group V, the polyethylene glycol methyl ether methacrylate, the cuprous bromide and the pentamethyl diethylenetriamine is 1 (5-20): (0.5-2): 0.5-2), the reaction temperature is 50-90 ℃, and the reaction time is 5-10 hours.

8. The method for preparing a triple stimulus-responsive interfacially crosslinked polymeric micelle according to claim 3, characterized in that: in the step (7), the molar ratio of the hydrophobic block polymer shown in the formula IV, the poly (dimethylaminoethyl methacrylate) -b-polyethylene glycol methyl ether methacrylate with alkynyl as the terminal group shown in the formula VI, cuprous bromide and pentamethyldiethylenetriamine is 1 (2-6) to (10-30), the reaction temperature is 50-80 ℃, and the reaction time is 10-20 hours.

9. The method for preparing a triple stimulus-responsive interfacially crosslinked polymeric micelle according to claim 3, characterized in that: in the step (8), the molar ratio of the amphiphilic block polymer shown in the formula VII to the N, N' -di (bromoacetyl) cystamine is 1: 30-80, the temperature of the crosslinking reaction is 20-50 ℃, and the time is 20-72 hours, so that the light, oxidation and reduction triple stimulus-responsive interfacial crosslinked polymer micelle is obtained.

10. Use of the triple stimulus-responsive interfacially crosslinked polymeric micelle of claim 1 as a hydrophobic anticancer drug carrier.