CN109320636B - Triple stimulus-responsive core-crosslinked polymer micelle and preparation method and application thereof - Google Patents
Triple stimulus-responsive core-crosslinked polymer micelle and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a triple stimulus responsive core crosslinked polymer micelle, a preparation method and application thereof, wherein the structural formula of the micelle is as follows:
wherein x is an integer of 30 to 70, y is an integer of 20 to 50, x1The value of (b) is 20-50% of x. The invention obtains amphiphilic diblock polymer by Atom Transfer Radical Polymerization (ATRP), modifies the amphiphilic diblock polymer by using azide reaction, and the azide product can self-assemble into a core-shell structure in water, and obtains the nucleus-crosslinked polymer micelle with light, oxidation and reduction triple stimulus responsiveness by click chemistry under the action of a crosslinking agent with redox sensitivity, anhydrous copper sulfate and sodium ascorbate. The triple-stimulus responsive core-crosslinked polymer micelle has good in-vitro stability, can realize efficient hydrophobic drug controlled release behavior under dual stimulus of light and oxidation or reduction, and has good application prospect in the aspects of carrying and controlled release of hydrophobic anticancer drugs.
Description
Technical Field
The invention relates to a light, oxidation and reduction triple stimulus responsive core crosslinked polymer micelle and a preparation method and application thereof.
Background
With the rapid development of nanoscience technology in recent years, more and more novel nanomaterials are used for drug delivery systems. At present, polymer-based nano-carriers mainly comprise drug precursors, vesicles, nano-cages, microgels, polymer micelles and the like. Due to the unique core-shell nano structure of the polymer micelle, when the polymer micelle is used as a drug carrier, the solubility of a slightly soluble drug can be improved, and the drug in the core can be protected from being degraded or enzymolyzed. Because the medicine carrying polymer micelle is small in size, the accumulation amount of the medicine in cancer cells can be improved through enhanced cell permeability and EPR (ethylene propylene rubber) effect, and meanwhile, the hydrophilic shell of the polymer micelle can not cause the increase of vascular resistance and blood viscosity in the blood transportation process, so that the side effect on normal organisms is small. However, in the transportation process of the polymer micelle in vivo, due to the change of conditions such as dilution, ionic strength, pH value and the like, the polymer micelle can have the phenomena of structural damage, premature release of carried drugs, low cancer cell targeting efficiency and the like. Therefore, the stability of the polymer micelle in the physiological environment of a human body is enhanced, the damage of the medicament to other parts of the body in the transportation process is reduced, and the effective utilization rate of the medicament is improved, which is a new direction for research.
In order to enhance the anti-interference capability of the carrier and improve the stability of the polymer micelle, the crosslinking mode is widely adopted so as to be better applied to the biological environment. The crosslinking sites can be divided into three types, namely hydrophobic core crosslinking, hydrophilic shell crosslinking and core-shell interface crosslinking. The shell cross-linked micelle and the core-shell interface cross-linked micelle have good stability, and the cross-linked structure can protect the medicine from being influenced by external environmental factors. However, the crosslinking reaction generally requires preparation under highly diluted conditions to avoid aggregate generation due to crosslinking between micelles, which makes large-scale preparation and application difficult. The core crosslinking is a means of crosslinking an active group carried by a hydrophobic core layer of a polymer micelle by a chemical reaction to form an assembly. The crosslinked micelle inner core layer becomes more compact and can better wrap the drug to reduce leakage. To date, core crosslinking techniques have been extensively and extensively studied. Zhu and the like design and synthesize a biodegradable core-crosslinked polymer micelle with reduction response, wherein polyethylene glycol is a hydrophilic shell layer, polycaprolactone and poly-5, 5-diazide methyl trimethylene carbonate are hydrophobic cores, and a crosslinking agent is propargyl dithio propionate. In addition, hydrophobic fluorescent molecule nile red is used as a probe, and the in-vitro controlled release behavior of the nile red is researched. Lim and the like successfully prepare poly (ethylene oxide) -b-poly (glycidyl methacrylate) amphiphilic block polymers and different cross-linking agents through reversible addition-fragmentation chain transfer polymerization, and the influence of the self-assembly behavior of the poly (ethylene oxide) -b-poly (glycidyl methacrylate) amphiphilic block polymers in water and the different cross-linking agents on the performance of the formed nuclear cross-linked polymer micelle is researched.
As is known to all, the microenvironment in cancer cells is very complex, so that the polymer micelle is an ideal drug carrier, and not only needs to have good blood stability, but also needs to have a stimulation response function to meet clinical application. Common stimuli-responsive polymeric micelles include oxidative, reductive, temperature, pH, salt ion, electromagnetic field, light, glucose, and other stimuli-response. The reductive stimulation responsive polymer micelle is gradually a hotspot in the research field of drug carriers due to the excellent characteristics of rapid drug release, small toxic and side effects, biodegradability and the like. For example, Wu et al synthesized a novel reduction-responsive core crosslinked polymer micelle based on chitosan, crosslinked by 3, 3-dithiodipropionic acid, and used it as a nanocarrier to carry anticancer drugs. However, studies have shown that single stimuli-responsive core-crosslinked polymeric micelles tend to have certain limitations in application.
So far, there are few reports on the research on designing and synthesizing a core-crosslinked polymer micelle having multiple stimulus responses simultaneously for delivery of anticancer drugs. Therefore, in order to cope with the complicated environment of cancer cells, more excellent drug delivery systems need to be developed.
Disclosure of Invention
The invention aims to solve the technical problem of providing a triple stimulus-responsive core crosslinked polymer micelle with light, oxidation and reduction stimulus responses, and a preparation method and application thereof.
The structural formula of the triple stimulus-responsive core-crosslinked polymer micelle adopted by the invention is shown as follows:
wherein x is an integer of 30 to 70, y is an integer of 20 to 50, x1The value of (b) is 20-50% of x.
The synthetic route and the specific preparation method of the triple stimulus-responsive core-crosslinked polymer micelle are as follows:
1. under the protection of inert gas, carrying out Atom Transfer Radical Polymerization (ATRP) reaction on o-nitrobenzyl methacrylate (NBM) and Glycidyl Methacrylate (GMA) under the initiation of methoxy polyethylene glycol-bromine (mPEG-Br) by using cuprous bromide (CuBr) as a catalyst, Pentamethyl Diethylenetriamine (PDMETA) as a ligand and N, N-Dimethylformamide (DMF) as a solvent to obtain an amphiphilic diblock polymer (mPEG-b-P (GMA-co-NBM)) shown in formula I.
2. The amphiphilic diblock polymer obtained in the step 1 and sodium azide (NaN)3) Ammonium chloride (NH)4Cl) is dissolved in DMF, and the azide reaction is carried out under the stirring condition to obtain the azide modified amphiphilic diblock polymer (mPEG-b-P (GMA-N) shown as the formula II3-co-NBM))。
3. Taking Dichloromethane (DCM) as a solvent, and reacting 3-butyn-1-ol with oxalyl chloride at normal temperature to obtain butynyl oxalyl chloride; reacting butynyl oxyoxalyl chloride with bis (2-hydroxyethyl) disulfide and Triethylamine (TEA) at normal temperature to obtain a Cross-linking agent (Cross-linker) shown in a formula III;
4. dissolving the azide-modified amphiphilic diblock polymer obtained in the step 2 and the cross-linking agent obtained in the step 3 in Tetrahydrofuran (THF), forming micelles by using a dialysis method, and then adding copper sulfate pentahydrate (CuSO)4·5H2O) and sodium ascorbate are subjected to click chemical reaction under the protection of nitrogen to obtain the light, oxidation and reduction triple stimulus responsive core crosslinked polymer micelle shown in the formula IV.
In the step 1, the molar ratio of NBM to GMA, mPEG-Br, CuBr and PDMETA is preferably 50-70: 30-50: 1: 1-2, the ATRP reaction temperature is preferably 50-70 ℃, and the time is preferably 18-24 hours.
In the step 2, mPEG-b-P (GMA-co-NBM) and NaN are preferred3、NH4The mol ratio of Cl is 1: 1-6, and the preferable temperature of the nitridization reaction is 25-60℃,The time is 24-32 hours.
In the step 3, the molar ratio of 3-butyn-1-ol to oxalyl chloride is preferably 1: 1-3, and the molar ratio of bis (2-hydroxyethyl) disulfide to TEA and butynyl oxalyl chloride is preferably 1: 1-3.
In the above step 4, mPEG-b-P (GMA-N) is preferred3-co-NBM) with Cross-linker, CuSO4·5H2The molar ratio of O to sodium ascorbate is 1-2: 1: 1-5, the temperature of the click chemical reaction is preferably 25-40 ℃, and the time is preferably 48-72 hours.
The NBM described above was synthesized according to RSC Advances,2015,5, 65847-; mPEG-Br was synthesized according to RSC Advances,2016,6, 88444-.
The application of the triple-stimulus responsive core-crosslinked polymer micelle as a hydrophobic drug carrier comprises the following specific steps: in the step 4, the azide-modified amphiphilic diblock polymer, the cross-linking agent and the drug are dissolved in tetrahydrofuran, micelles are formed by a dialysis method, then copper sulfate pentahydrate and sodium ascorbate are added, and click chemical reaction is carried out under the protection of nitrogen, so that the triple stimulus-responsive nuclear cross-linked polymer drug-loaded micelle is obtained.
The invention has the following advantages:
1. the light, oxidation and reduction triple-stimulation core crosslinked polymer micelle is spherical and has a small particle size distribution range.
2. The light, oxidation and reduction triple-stimulus nuclear cross-linked polymer micelle comprises a light-sensitive hydrophobic nucleus taking o-nitrobenzyl alcohol as a side group, and is beneficial to embedding of hydrophobic molecules; a polyethylene glycol hydrophilic shell with good biocompatibility, so that the polyethylene glycol hydrophilic shell can better adapt to the in-vivo environment; the core cross-linked structure contains peroxyoxalate and disulfide bonds, so that the oxidation and reduction stimuli responsiveness is endowed, and the stability of the micelle is improved, and meanwhile, the controlled release of the drug can be efficiently realized.
3. In the process of controlling the release of the drug, the drug can be released in a certain amount by single stimulation trigger, but the drug can be better controlled to release under the common trigger of multiple stimulations.
4. The synthesis method of the triple-stimulus responsive core-crosslinked polymer micelle is simple, has no special requirements on environment, and is simple to operate if the amphiphilic diblock polymer is synthesized into one-step ATRP polymerization reaction; the cross-linking process of the micelle is click chemical reaction, the reaction condition is mild, and the purification can be realized through dialysis.
Drawings
FIG. 1 is a nuclear magnetic hydrogen spectrum of the azido-modified amphiphilic diblock polymer prepared in example 1, with deuterated chloroform (CDCl) as the solvent3)。
FIG. 2 is a nuclear magnetic hydrogen spectrum of the cross-linking agent prepared in example 1, with deuterated chloroform (CDCl) as the solvent3)。
FIG. 3 is an infrared spectrum of the amphiphilic diblock polymer (a) and the azide-modified amphiphilic diblock polymer (b) prepared in example 1 and the triple stimuli-responsive core-crosslinked polymer micelle (c).
FIG. 4 shows the fluorescence spectra (excitation wavelength 340nm) of pyrene in various concentrations of azide-modified amphiphilic diblock polymer solutions prepared in example 1.
FIG. 5 is the I of pyrene in FIG. 43/I1And analysis spectrum of azide-modified amphiphilic diblock polymer log concentration (logC).
FIG. 6 is a UV spectrum of the triple stimuli-responsive core-crosslinked polymer micelle prepared in example 1 at different illumination times (micelle concentration of 0.2 mg/mL).
FIG. 7 is a Dynamic Light Scattering (DLS) curve spectrum (micelle concentration of 0.2mg/mL) of the triple stimulus-responsive core-crosslinked polymer micelle prepared in example 1 under different conditions (no stimulus, UV (365nm UV) light irradiation, DMF dilution).
FIG. 8 is a DLS profile (micelle concentration of 0.2mg/mL) of the triple stimulus-responsive core-crosslinked polymer micelle prepared in example 1 under different conditions (oxidizing substance, oxidizing substance and UV light).
FIG. 9 is a DLS profile (micelle concentration of 0.2mg/mL) of the triple stimuli-responsive core-crosslinked polymer micelle prepared in example 1 under different conditions (reducing substance, reducing substance and UV light).
Fig. 10 is a drug release profile of the triple stimulus-responsive core cross-linked polymer drug-loaded micelle prepared in example 3 under different stimulus triggers (no stimulus, UV light).
Fig. 11 is a drug release profile of the triple stimulus-responsive core cross-linked polymer drug-loaded micelle prepared in example 3 under different stimulus multiple triggers (oxidizing substance, oxidizing substance and UV light).
Fig. 12 is a drug release profile of the triple stimulus-responsive core cross-linked polymer drug-loaded micelle prepared in example 3 under different stimulus-multiple triggers (reducing agent, reducing agent and UV light).
Fig. 13 is a fluorescence spectrum of the triple stimuli-responsive core-crosslinked polymer drug-loaded micelle prepared in example 3 under different irradiation times of UV light.
FIG. 14 shows the three stimuli-responsive core-crosslinked polymer drug-loaded micelles prepared in example 3 in different hydrogen peroxide (H)2O2) Fluorescence spectra at concentration.
Fig. 15 is a fluorescence spectrum of the triple stimuli-responsive core-crosslinked polymer drug-loaded micelle prepared in example 3 at different concentrations of Dithiothreitol (DTT).
FIG. 16 shows that the triple stimulus responsive core cross-linked polymer drug-loaded micelle prepared in example 3 is 10mmol/L H2O2And fluorescence spectra at different times of UV light irradiation.
FIG. 17 shows fluorescence spectra of the triple stimulus-responsive core-crosslinked polymer drug-loaded micelle prepared in example 3 under different UV irradiation times and at 10mmol/L DTT.
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. Dissolving mPEG-Br (0.2g, 0.1mmol), NBM (1.55g, 7mmol), GMA (0.426g, 3mmol) and PMDETA (0.0173g, 0.1mmol) in 4mL DMF, continuously freezing and thawing and degassing for 2 times, adding CuBr (0.0143g, 0.1mmol), continuously freezing and thawing and degassing for 1 time, reacting for 24 times at 70 ℃ under a nitrogen atmosphere, diluting with THF after the reaction is finished, passing through a neutral alumina column, performing rotary evaporation to remove most of solvent, dialyzing in deionized water for 3 days by using a dialysis bag with molecular weight cutoff of 8kDa, and freeze-drying to obtain a white solid, namely the amphiphilic diblock polymer shown in formula I-1.
2. An amphiphilic diblock polymer of formula I-1 (0.4g, 0.86mmol) and NaN3(0.25g，3.87mmol)、NH4And dissolving Cl (0.21g and 3.87mmol) in 20mL of DMF, reacting for 24 hours at the temperature of 50 ℃ under stirring, filtering out insoluble salt by using a Buchner funnel after the reaction is finished, concentrating the filtrate, dialyzing for 3 days in deionized water by using a dialysis bag with the molecular weight cutoff of 8kDa, and freeze-drying to obtain a white solid, namely the azide-modified amphiphilic diblock polymer shown in the formula II-1.
3. 3-butyn-1-ol (4.2g, 60mmol) is dissolved in 20mL DCM, then oxalyl chloride (14.8g, 120mmol) is slowly added dropwise, reaction is carried out for 1 hour under the condition of stirring at normal temperature after the dropwise addition is finished, and reduced pressure distillation is carried out at 115 ℃ to obtain the butynyl oxalyl chloride. Bis (2-hydroxyethyl) disulfide (0.154g, 1mmol) and TEA (0.24g,2.4mmol) were dissolved in 10mL DCM, followed by the slow dropwise addition of butynyloxyoxalyl chloride (0.38g,2.4mmol) and the reaction was carried out at room temperature for 1 hour with stirring to give a crosslinking agent represented by formula III-1.
4. The azide-modified amphiphilic diblock polymer represented by the formula II-1 (30mg, 0.06mmol) and the crosslinking agent represented by the formula III-1 (17mg, 0.04mmol) were dissolved in 20mL of THF to form micelles by dialysis, and then CuSO was added4·5H2O (30mg, 0.12mmol) and sodium ascorbate (24mg, 0.12mmol) were reacted at 25 ℃ for 48 hours, and unreacted crosslinks were dialyzed out using an 8kDa dialysis bagThe agent is used for obtaining the light, oxidation and reduction triple stimulus responsive core crosslinked polymer micelle shown in the formula IV-1.
The inventor adopts a nuclear magnetic resonance spectrometer, an infrared spectrometer, a laser particle analyzer and a fluorescence spectrophotometer to characterize the sample obtained in the embodiment 1, and the results are shown in figures 1-9. As can be seen from FIG. 1, the chemical position at 3.64ppm is the characteristic peak of the initiator methoxypolyethylene glycol-bromine; chemical shifts of 7.60-7.44ppm and 8.11ppm can be assigned to the o-nitrophenyl methacrylate moiety; after opening of the oxirane ring of the polyglycidyl methacrylate block, CH-O (f'), CH are present at chemical shifts of 4.04ppm and 3.39ppm2-O (e') and CH2-N3(g') indicating that the azide-modified amphiphilic diblock polymer was successfully synthesized. From FIG. 2, it can be observed that the chemical shift of 1.98ppm is the characteristic peak of the triple bond group on the crosslinking agent, and the chemical shifts at 2.59ppm, 4.49ppm, 4.34ppm and 2.87ppm can be assigned to triple bond ortho methylene hydrogen, ester bond ortho methylene hydrogen and disulfide bond ortho methylene hydrogen, respectively, thereby demonstrating the successful synthesis of the crosslinking agent. By comparing FIG. 3a with FIG. 3b, it can be seen that the infrared spectrum is 2109cm-1The absorption peak of the azido functional group appears, thereby further illustrating that the azido modified amphiphilic diblock polymer is successfully synthesized. 2109cm in the IR spectrum is observed in FIG. 3c-1The absorption peak of the azido functional group is obviously reduced because the click chemical reaction occurs between the cross-linking agent with the triple bond modified end and the amphiphilic diblock polymer modified by the azide, which proves that the triple stimulus-responsive nuclear cross-linked polymer micelle is successfully prepared. According to the detection of gel permeation chromatography, the number average molecular weight of the amphiphilic diblock polymer shown in the formula I-1 is 9.2k, the molecular weight distribution index (PDI) is 1.38, the number average molecular weight of the azide-modified amphiphilic diblock polymer shown in the formula II-1 is 10.1k, the PDI is 1.27, and the crosslinking degree of the triple-stimulus-responsive core crosslinked polymer micelle is 56% through nuclear magnetic data calculation. As can be seen from FIGS. 4 and 5, the Critical Micelle Concentration (CMC) value of the amphiphilic diblock polymer was 25 mg/L. As can be seen from 6, the triple stimulus-responsive core-crosslinked polymer micelle solution has a molecular weight of 365nmThe characteristic absorption peak value of the o-nitrobenzol near 265nm is reduced by increasing the irradiation time of the UV light, which indicates that the triple stimuli-responsive core-crosslinked polymer micelle has the photostimulation responsiveness. As can be seen from FIGS. 7, 8 and 9, the average particle sizes of the triple stimulus-responsive core-crosslinked polymer micelles were 184nm, 203nm and 338nm, respectively, in the absence of stimulus, UV irradiation for 30 minutes and 10-fold dilution of DMF, and the triple stimulus-responsive core-crosslinked polymer micelle solution was added with H2O26 hours or addition of H2O2After 6 hours of UV irradiation for 30 minutes, the micelle particle size became 2560nm and 5804nm, respectively, whereas after 6 hours of DTT addition or 6 hours of DTT addition, the micelle particle size became 1495nm and 3749nm, respectively, after UV irradiation for 30 minutes. According to the change of the particle size and the particle size dispersion degree (the width of DLS curve peak) under different stimuli, the synthesized triple stimulus responsive core crosslinked polymer micelle has good stability and has light, oxidation and reduction triple stimulus responsiveness.
Example 2
1. Dissolving mPEG-Br (0.4g, 0.2mmol), NBM (2.21g, 10mmol), GMA (1.42g, 10mmol) and PMDETA (0.0346g, 0.2mmol) in 4mL DMF, continuously freezing and thawing and degassing for 2 times, adding CuBr (0.0286g, 0.2mmol), continuously freezing and thawing and degassing for 1 time, reacting for 24 times at 70 ℃ under a nitrogen atmosphere, diluting with THF after the reaction is finished, passing through a neutral alumina column, performing rotary evaporation to remove most of solvent, dialyzing in deionized water for 3 days by using a dialysis bag with molecular weight cutoff of 8KDa, and freeze-drying to obtain a white solid, namely the amphiphilic diblock polymer shown in formula I-2.
2. An amphiphilic diblock polymer of formula I-2 (0.4g, 1.05mmol) and NaN3(0.34g，5.25mmol)、NH4Dissolving Cl (0.28g, 5.25mmol) in 20mL DMF, reacting at 50 deg.C under stirring for 24 hr, filtering out insoluble salt with Buchner funnel, concentrating the filtrate, dialyzing with dialysis bag with cut-off molecular weight of 8kD in deionized waterAnd after 3 days, freezing and drying, obtaining white solid, namely the azide-modified amphiphilic diblock polymer shown in the formula II-2.
3. 3-butyn-1-ol (2.1g, 30mmol) is dissolved in 10mL DCM, then oxalyl chloride (9.3g, 75mmol) is slowly dropped, reaction is carried out for 1 hour under the condition of stirring at normal temperature after dropping, and reduced pressure distillation is carried out at 115 ℃ to obtain butynyl oxalyl chloride. Bis (2-hydroxyethyl) disulfide (0.154g, 1mmol) and TEA (0.3g, 3mmol) were dissolved in 5mL DCM, followed by dropwise addition of butynyloxyoxalyl chloride (0.48g,3mmol) slowly and reaction under stirring at room temperature for 1 hour to give a crosslinking agent represented by formula III-1.
4. The azide-modified amphiphilic diblock polymer represented by the formula II-2 (26mg, 0.06mmol) and the crosslinking agent represented by the formula III-1 (13mg, 0.03mmol) were dissolved in 20mL of THF to form micelles by dialysis, and then CuSO was added4·5H2O (30mg, 0.12mmol) and sodium ascorbate (24mg, 0.12mmol) were reacted at 25 ℃ for 48 hours, and unreacted crosslinking agent was dialyzed out by a dialysis bag of 8kD to obtain a photo-, oxidation-, and reduction-triple stimulus-responsive core-crosslinked polymer micelle represented by the formula IV-2.
The inventors characterized the sample obtained in example 2 by using a nuclear magnetic resonance spectrometer and gel permeation chromatography to obtain a polymer of formula I-2 having a number average molecular weight of 8.4k and a PDI of 1.28, and a polymer of formula II-2 having a number average molecular weight of 9.4k and a PDI of 1.16, and the degree of crosslinking of the triplet-stimuli-responsive core-crosslinked polymer micelle was 63% as calculated from nuclear magnetic data.
Example 3
Application of triple stimuli-responsive core-crosslinked polymer micelle prepared in example 1 as hydrophobic drug carrier
Dissolving azide-modified amphiphilic diblock polymer (30mg, 0.06mmol) shown as formula II-1, nile red (1.5mg, 0.005) and cross-linking agent (17mg, 0.04mmol) shown as formula III-1 in 20mL THF, forming micelle by dialysis, and adding CuSO4·5H2O (30mg, 0.12mmol) and sodium ascorbate (24mg, 0.12mmol) at 25 deg.C for 48 hr, dialyzing out unreacted cross-linking agent with 8kDa dialysis bag to obtain light, oxidation and reduction triple stimulus responseSex core cross-linked polymer drug-loaded micelle.
The release curve of the triple-stimulus responsive core-crosslinked polymer drug-loaded micelle under single stimulus and multiple stimuli is detected by a fluorescence spectrometer (the excitation wavelength is 560nm, the micelle concentration is 0.2mg/mL), and the result is shown in figures 10-12. As can be seen from fig. 10, under the condition of no stimulation or single stimulation of UV light, the cumulative release amounts of the drugs of the drug-loaded micelle for 400 minutes are 5.6% and 50.15% in sequence, and it can be seen that only part of the drugs can be released under the condition of light. As can be seen from FIG. 11, in the case of addition of H2O2Or by addition of H2O2After 6 hours, under the stimulation of UV illumination for 30 minutes, the cumulative release amount of the drug-loaded micelle for 400 minutes is 53.28 percent and 76.74 percent in sequence, which fully proves that under the trigger of dual stimulation of light and oxidation, the cumulative release amount of the drug is obviously higher than that of single stimulation. As can be seen from fig. 12, the cumulative release amount of the drug in 400 minutes is 56.99% and 73.88% respectively after the drug-loaded micelle is irradiated by ultraviolet light for 30 minutes after the drug-loaded micelle is added with DTT6 hours or 6 hours after the drug-loaded micelle is added with DTT, i.e. the cumulative release amount of the drug is higher than the single effect of three stimuli under the dual-stimulus trigger.
The inventor further realizes different single stimulation trigger drug release tests by changing the environment of the drug-loaded micelle, such as changing the illumination time and controlling the addition of the oxidation or reduction substances, and the results are shown in fig. 13-17. As can be seen, the fluorescence intensity of the drug-loaded micelle does not change significantly with the extension of UV light exposure time (see fig. 13), because the core cross-linked structure can prevent the micelle from being damaged by light exposure; by increasing H2O2The concentration is effective to trigger the release of drug-loaded micelles (see fig. 14); the release of the hydrophobic drug in the drug-loaded micelle can also be triggered by increasing the concentration of DTT (see FIG. 15), and increased by increasing the UV exposure time after 6 hours of reaction with 10mmol of DTT added (see FIG. 16), and by adding 10mmol of H2O2Prolonged exposure to UV light after 6 hours of reaction also increases the amount of hydrophobic drug released.
Claims (10)
1. A triple stimuli-responsive core-crosslinked polymeric micelle characterized in that the micelle has the following structural formula:
wherein x is an integer of 30 to 70, y is an integer of 20 to 50, x1The value of (b) is 20-50% of x.
2. A method for preparing the triple stimuli-responsive core-crosslinked polymeric micelle according to claim 1, which comprises the steps of:
(1) carrying out atom transfer radical polymerization reaction on o-nitrobenzyl methacrylate and glycidyl methacrylate under the initiation of methoxy polyethylene glycol-bromine 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 obtain an amphiphilic diblock polymer shown in a formula I;
(2) dissolving the amphiphilic diblock polymer obtained in the step (1), sodium azide and ammonium chloride in N, N-dimethylformamide, and carrying out an azide reaction under a stirring condition to obtain an azide-modified amphiphilic diblock polymer shown in a formula II;
(3) taking dichloromethane as a solvent, and reacting 3-butyne-1-ol with oxalyl chloride at normal temperature to obtain butynyloxy oxalyl chloride; reacting butynyl oxyoxalyl chloride with bis (2-hydroxyethyl) disulfide and triethylamine at normal temperature to obtain a cross-linking agent shown in a formula III;
(4) and (3) dissolving the azide-modified amphiphilic diblock polymer obtained in the step (2) and the cross-linking agent obtained in the step (3) in tetrahydrofuran, forming micelles by using a dialysis method, adding copper sulfate pentahydrate and sodium ascorbate, and carrying out click chemical reaction under the protection of nitrogen to obtain the triple stimulus-responsive nuclear cross-linked polymer micelle.
3. The method for preparing a triple stimuli-responsive core-crosslinked polymeric micelle according to claim 2, characterized in that: in the step (1), the molar ratio of the o-nitrobenzyl methacrylate to the glycidyl methacrylate, the methoxypolyethylene glycol-bromine, the cuprous bromide and the pentamethyldiethylenetriamine is 50-70: 30-50: 1: 1-2.
4. The method for preparing a triple stimuli-responsive core-crosslinked polymeric micelle according to claim 2 or 3, characterized in that: in the step (1), the temperature of the atom transfer radical polymerization reaction is 50-70 ℃ and the time is 18-24 hours.
5. The method for preparing a triple stimuli-responsive core-crosslinked polymeric micelle according to claim 2, characterized in that: in the step (2), the molar ratio of the amphiphilic diblock polymer to the sodium azide to the ammonium chloride is 1: 1-6.
6. The method for preparing a triple stimuli-responsive core-crosslinked polymeric micelle according to claim 2, characterized in that: in the step (2), the temperature of the azide reaction is 25-60 ℃ and the time is 24-32 hours.
7. The method for preparing a triple stimuli-responsive core-crosslinked polymeric micelle according to claim 2, characterized in that: in the step (3), the molar ratio of the 3-butyn-1-ol to the oxalyl chloride is 1: 1-3, and the molar ratio of the bis (2-hydroxyethyl) disulfide to the triethylamine and butynyl oxalyl chloride is 1: 1-3.
8. The method for preparing a triple stimuli-responsive core-crosslinked polymeric micelle according to claim 2, characterized in that: in the step (4), the azide-modified amphiphilic diblock polymer, the crosslinking agent, the copper sulfate pentahydrate and the sodium ascorbate are in a molar ratio of 1-2: 1: 1-5.
9. The method for preparing a triple stimuli-responsive core-crosslinked polymeric micelle according to claim 2, characterized in that: in the step (4), the temperature of the click chemistry reaction is 25-40 ℃ and the time is 48-72 hours.
10. Use of the triple stimuli-responsive core-crosslinked polymeric micelle of claim 1 as a hydrophobic drug carrier.
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