CN107551275B - Preparation of magnetic nano-drug carrier and method for loading doxorubicin hydrochloride by using magnetic nano-drug carrier - Google Patents
Preparation of magnetic nano-drug carrier and method for loading doxorubicin hydrochloride by using magnetic nano-drug carrier Download PDFInfo
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- MWWSFMDVAYGXBV-RUELKSSGSA-N Doxorubicin hydrochloride Chemical compound Cl.O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 MWWSFMDVAYGXBV-RUELKSSGSA-N 0.000 title claims abstract description 57
- 229960002918 doxorubicin hydrochloride Drugs 0.000 title claims abstract description 57
- 239000003937 drug carrier Substances 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 229920002674 hyaluronan Polymers 0.000 claims abstract description 66
- 229960003160 hyaluronic acid Drugs 0.000 claims abstract description 66
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 64
- 229910021389 graphene Inorganic materials 0.000 claims abstract description 64
- 229920000642 polymer Polymers 0.000 claims abstract description 24
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 45
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 42
- 238000003756 stirring Methods 0.000 claims description 36
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- 238000006243 chemical reaction Methods 0.000 claims description 25
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 24
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- 238000004108 freeze drying Methods 0.000 claims description 19
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 18
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 18
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 13
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- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims description 8
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- 238000002329 infrared spectrum Methods 0.000 description 5
- SZVJSHCCFOBDDC-UHFFFAOYSA-N iron(II,III) oxide Inorganic materials O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 4
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Abstract
The invention relates to a preparation method of a magnetic nano-drug carrier and a method for loading doxorubicin hydrochloride by using the magnetic nano-drug carrier, belonging to the technical field of drug transportation of magnetic nano-materials. The invention mainly solves the technical problems of high toxicity and poor treatment effect in the prior art. The technical scheme of the invention is as follows: a preparation method of a magnetic nano-drug carrier comprises the following steps: 1) preparing cyclodextrin-hyaluronic acid supermolecule polymer; 2) preparing magnetic graphene oxide; 3) preparing the cyclodextrin-hyaluronic acid polymer functionalized magnetic nano-drug carrier. Compared with the prior art, the invention has strong biocompatibility, low toxic and side effects, cancer cell targeting positioning effect and drug controllable release characteristic, and has important application value in the aspect of biological drug carriers.
Description
Technical Field
The invention belongs to the technical field of magnetic nano material drug transportation, and particularly relates to a preparation method of a magnetic nano drug carrier and a method for loading doxorubicin hydrochloride by using the magnetic nano drug carrier.
Background
The traditional antitumor drugs have the defects of systemic distribution, high cytotoxicity and the like. Anthraquinone drugs are used as broad-spectrum antitumor drugs, and drug molecules are embedded into a DNA chain to block the replication of the DNA chain, so that the purpose of resisting tumors is achieved. Doxorubicin hydrochloride (DOX) as a new generation of anthraquinone drugs has lower toxicity and weaker cardiac inhibitory effect than other anthraquinone drugs, but has a certain influence on liver function. In order to achieve safe and efficient treatment effects of DOX, it is important to research a drug carrier with a targeted delivery function to improve the curative effect and safety.
In addition, the hydrophilic group of GO is easily lost by heating to influence the further dispersion and loading of the nanocomposite, while β -cyclodextrin (β -CD) has a ring structure, has a hydrophilic outer cavity and a lipophilic inner cavity, and can form an inclusion compound with many drugs to improve the bioavailability, solubility and stability of drug molecules.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a preparation method of a magnetic nano-drug carrier and a method for loading doxorubicin hydrochloride by using the same, and solves the technical problems of high toxicity and poor treatment effect in the prior art.
The invention is realized by the following technical scheme: a method for preparing a magnetic nano-drug carrier, wherein: the method comprises the following steps:
1) preparation of cyclodextrin-hyaluronic acid supramolecular polymer:
firstly, slowly adding 17-19g of β -cyclodextrin and 11-12g of paratoluensulfonyl chloride into 100-150mL of pyridine solvent in sequence, stirring, reacting for 5-8h at room temperature, removing the pyridine solvent by rotary evaporation, recrystallizing in water, washing and purifying with cold acetone to obtain precipitate mono-6-deoxy-6- (paratoluensulfonyl) - β -cyclodextrin;
then 4-6g of mono-6-deoxy-6- (p-toluenesulfonyl) - β -cyclodextrin and 10-12g of ethylenediamine are dissolved in 20-30mL of N-N-dimethylformamide, and are magnetically stirred and reacted for 18-24 hours at 80 ℃ in a nitrogen environment, after the reaction is finished, the mixture is cooled to room temperature, and then is washed for 3-5 times by cold acetone to remove the residual ethylenediamine, and is dried under vacuum, so that white powder is prepared;
finally, activating 40-42mg hyaluronic acid by 22-24mg 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride and 13-15mg N-hydroxysuccinimide solution, continuously stirring for 2 hours to prepare dispersion liquid, adding white powder into the dispersion liquid, reacting the mixed liquid for 18-24 hours at room temperature, dialyzing and purifying in deionized water, and freeze-drying to prepare the cyclodextrin-hyaluronic acid supramolecular polymer;
2) preparing magnetic graphene oxide:
stripping 180-220mg of graphene oxide in deionized water for 1 hour by ultrasonic treatment to prepare graphene oxide dispersion, and then placing the graphene oxide dispersion in a three-neck flask and heating to 60-80 ℃;
introducing nitrogen into the graphene oxide solution for 10-15 minutes to remove air in the graphene oxide solution, mechanically stirring the graphene oxide solution at the speed of 800r/min in a nitrogen environment, and adding 2.7g of FeCl3·6H2O and 1.0g FeCl2·4H2Dissolving O in the graphene oxide solution to prepare a reaction solution;
then dropwise adding concentrated ammonia water until the pH value of the reaction solution is adjusted to 9-12; stirring the reactant at 80 ℃ for 1-3 hours to obtain a magnetic black precipitate; cooling to room temperature, performing magnetic hysteresis separation, repeatedly washing the black precipitate with deionized water and absolute ethyl alcohol, and freeze-drying for 20-30h to obtain magnetic graphene oxide powder;
3) preparing a cyclodextrin-hyaluronic acid polymer functionalized magnetic nano-drug carrier:
firstly, ultrasonically dispersing the magnetic graphene oxide powder obtained in the step 2) of 180-220mg in 220mL of ethanol of 180-220mL for 30-40 minutes to ensure that the magnetic graphene oxide powder is completely dissolved (the concentration is 1mg/mL), adding 400 mu L of 3-aminopropyltriethoxysilane into the solution under the protection of nitrogen, and mechanically stirring the solution at room temperature for 6-8 hours; after the reaction is finished, removing supernatant through magnetic hysteresis separation, washing the precipitate for more than 3 times by using ethanol and deionized water respectively, and freeze-drying to obtain an aminated magnetic graphene oxide nano compound;
carrying out ultrasonic treatment on 48-52mg of the cyclodextrin-hyaluronic acid supramolecular polymer obtained in the step 1) in deionized water for 1-3 hours, adding 22-24mg of 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride and 13-15mg of N-hydroxysuccinimide after the cyclodextrin-hyaluronic acid supramolecular polymer is completely dissolved, and continuously stirring for 2-4 hours to activate carboxyl of the cyclodextrin-hyaluronic acid supramolecular polymer; adding the aminated magnetic graphene oxide nano-composite into the activated cyclodextrin-hyaluronic acid supermolecule polymer solution, stirring the reactants at room temperature for 24-48 hours, and freeze-drying to obtain the cyclodextrin-hyaluronic acid polymer functionalized magnetic nano-drug carrier.
Further, the reaction process of β -cyclodextrin and p-toluenesulfonyl chloride in step 1) is carried out in an anhydrous state, and the reaction time is 5 hours.
Further, in the step 1), the reaction time is 18 hours under the condition of magnetic stirring at 80 ℃ in a nitrogen environment; the number of washes of the mixture cooled to room temperature in cold acetone was 3; the reaction time of the mixture at room temperature was 24 hours.
Further, placing the graphene oxide dispersion liquid in the step 2) in a three-neck flask, and heating to 80 ℃; the time for introducing nitrogen into the graphene oxide solution is 10 minutes; dropwise adding concentrated ammonia water until the pH value of the reaction liquid is 10; the reaction was stirred at 80 ℃ for 2 hours; the black precipitate was washed repeatedly with deionized water and absolute ethanol and then freeze-dried for 24 h.
Further, in the step 3), the magnetic graphene oxide powder is ultrasonically dispersed in ethanol for 30 minutes; the adding amount of the 3-aminopropyltriethoxysilane is 300 mu L; the mechanical stirring time at room temperature after addition of 3-aminopropyltriethoxysilane was 7 hours.
Further, after adding 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in the step 3), continuously stirring for 2 hours; adding the aminated magnetic graphene oxide nano-composite into the activated cyclodextrin-hyaluronic acid supermolecule polymer solution, and stirring at room temperature for 48 hours.
A method for loading doxorubicin hydrochloride by using a magnetic nano-drug carrier is disclosed, wherein: the method comprises the following steps:
adding 0.5mg of cyclodextrin-hyaluronic acid polymer functionalized magnetic nano-drug carrier into 4mL of doxorubicin hydrochloride solution with the concentration range of 1-300mg/L, and carrying out hysteresis separation after oscillating for 7 hours at normal temperature.
According to the invention, magnetic Fe is introduced on the large specific surface of Graphene Oxide (GO)3O4The Hyaluronic Acid (HA) is a linear polysaccharide with good biocompatibility, biodegradability and nonimmunity, and HAs proved that HA can be combined with receptors excessively expressed on the surface of tumor cells, effectively enhances the combination and internalization capacity of the HA and the tumor cells, rapidly enters the tumor cells, shows huge potential in various biomedical applications, and takes hyaluronic acid as a targeting carrier to endow the drug inclusion compound with a cancer cell targeting and positioning function, can realize the targeting of the drug, reduce the toxic and side effects, and the cyclodextrin-hyaluronic acid polymer (β -CD-HA) is assembled on a Magnetic Graphene Oxide (MGO) nano-sheet to synthesize a cyclodextrin-hyaluronic acid functionalized nano-material (MGO- β -CD-HA) to produce a high-efficiency tumor targeting drug composite material (MGO- β -CD-3838) and transport the CD-3838-HA to a supermolecular drug-binding site.
Compared with the prior art, the invention has strong biocompatibility, low toxic and side effects, cancer cell targeting positioning effect and drug controllable release characteristic, and has important application value in the aspect of biological drug carriers.
Drawings
FIG. 1 is an infrared spectrum of GO, MGO-APTES and MGO- β -CD-HA;
FIG. 2 is a thermogravimetric analysis of MNPs, MGO and MGO- β -CD-HA;
FIG. 3 is an X-ray diffraction pattern of MGO and MGO- β -CD-HA;
FIG. 4 is a hysteresis plot of the MGO;
FIG. 5 is a fluorescence spectrum of 0.0025mg/mL DOX, DOX + MGO supernatant and DOX + MGO- β -CD-HA;
FIG. 6 is a graphical representation of the effect of pH on the net fluorescence intensity of MGO- β -CD-HA + DOX, where F0: fluorescence intensity of 0.03mg/mL DOX, F: fluorescence intensity of supernatant after DOX is adsorbed by MGO-CD-HA;
FIG. 7 is a graph of MGO- β -CD-HA vs DOX adsorption isotherms;
FIG. 8 is a graph of the cumulative release behavior of MGO- β -CD-HA nanocomposites at different temperatures and different pH values;
FIG. 9 shows MGO- β -CD-HA, MGO (0.4mg/mL) and buffer PBS at different concentrations (0.4 and 0.8mg/mL) over 2W cm-2After irradiation with near infrared light, the temperature rise is plotted as a function of time;
FIG. 10 is a photothermal curve of MGO- β -CD-HA (1.2mg/mL) at different power intensities;
FIG. 11 is a graph showing the release profile of DOX in a timed laser on/off (irradiation/non-irradiation) state after loading DOX on the surface of MGO- β -CD-HA nanomaterial, wherein both are at pH 5.3 and 37 deg.C, the laser irradiation wavelength is 808nm, and the intensity is 2Wcm-2；
FIG. 12 is a diagram showing DOX inclusion bodies incubated with human hepatoma cells BEL-7402 for various periods of time, and observed by a fluorescence inverted microscope;
figure 13 is a graph of cytotoxicity of drug inclusion complexes at different concentrations.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
Example 1:
the preparation method of the magnetic nano-drug carrier in the embodiment comprises the following steps:
1) preparation of cyclodextrin-hyaluronic acid supramolecular polymer (β -CD-HA):
firstly, slowly adding 18g of β -cyclodextrin (β -CD) and 11.06g of p-toluenesulfonyl chloride (TsCl) into 120mL of pyridine solvent in turn, stirring, reacting for 6h at room temperature, removing the pyridine solvent through rotary evaporation, recrystallizing in water, washing and purifying with cold acetone to obtain precipitate mono-6-deoxy-6- (p-toluenesulfonyl) - β -cyclodextrin (M-6-O-Ts- β -CD);
then 5g of mono-6-deoxy-6- (p-toluenesulfonyl) - β -cyclodextrin and 10.55g of Ethylenediamine (EDA) were dissolved in 25mL of N-Dimethylformamide (DMF) and reacted under magnetic stirring at 80 ℃ for 18 hours under a nitrogen atmosphere, after completion of the reaction, the mixture was cooled to room temperature, then washed 3 times with cold acetone to remove the residual ethylenediamine and dried under vacuum to obtain white powder (β -CD-EDA);
finally, activating 40.85mg of Hyaluronic Acid (HA) by 23.23mg of 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 13.8mg of N-hydroxysuccinimide (NHS) solution, continuously stirring for 2 hours to obtain a dispersion, adding white powder into the dispersion, reacting the mixture at room temperature for 24 hours, dialyzing and purifying the mixture in deionized water, and freeze-drying to obtain cyclodextrin-hyaluronic acid supramolecular polymer (β -CD-HA);
2) preparation of Magnetic Graphene Oxide (MGO):
stripping 200mg of Graphene Oxide (GO) in deionized water for 1 hour by ultrasonic treatment to prepare graphene oxide dispersion, and then placing the graphene oxide dispersion in a three-neck flask and heating to 80 ℃;
introducing nitrogen into the graphene oxide solution for 10 minutes to remove air in the graphene oxide solution, mechanically stirring the graphene oxide solution at the speed of 800r/min in a nitrogen environment, and adding 2.7g of FeCl3·6H2O and 1.0g FeCl2·4H2Dissolving O in the graphene oxide solution to obtain a reactionLiquid;
then dropwise adding concentrated ammonia water until the pH value of the reaction solution is adjusted to 10; stirring the reactant at 80 ℃ for 2 hours to obtain a magnetic black precipitate; cooling to room temperature, performing magnetic hysteresis separation, repeatedly washing the black precipitate with deionized water and absolute ethyl alcohol, and freeze-drying for 24h to obtain Magnetic Graphene Oxide (MGO) powder;
3) preparation of cyclodextrin-hyaluronic acid polymer functionalized magnetic nano-drug carrier (MGO- β -CD-HA):
firstly, 200mg of the magnetic graphene oxide powder obtained in the step 2) is ultrasonically dispersed in 200mL of ethanol for 30 minutes to ensure complete dissolution (MGO concentration is 1mg/mL), 300 μ L of 3-Aminopropyltriethoxysilane (APTES) is added under the protection of nitrogen, and mechanical stirring is carried out at room temperature for 7 hours; after the reaction is finished, removing supernatant through magnetic hysteresis separation, washing the precipitate for more than 3 times by using ethanol and deionized water respectively, and freeze-drying to obtain an aminated Magnetic Graphene Oxide (MGO) nano compound;
50mg of the cyclodextrin-hyaluronic acid supramolecular polymer obtained in the step 1) is subjected to ultrasonic treatment in deionized water for 2 hours, after the cyclodextrin-hyaluronic acid supramolecular polymer is completely dissolved, 23.23mg of 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 13.8mg of N-hydroxysuccinimide (NHS) are added, continuous stirring is carried out for 2 hours so as to activate carboxyl of the cyclodextrin-hyaluronic acid supramolecular polymer, an aminated magnetic graphene oxide nano-composite is added into the activated cyclodextrin-hyaluronic acid supramolecular polymer solution, and after a reactant is stirred for 48 hours at room temperature, the reactant is frozen and dried so as to prepare the cyclodextrin-hyaluronic acid polymer functionalized magnetic nano-drug carrier (MGO- β -CD-HA).
A method for loading doxorubicin hydrochloride (DOX) on a magnetic nano-drug carrier comprises the following steps:
adding 0.5mg of cyclodextrin-hyaluronic acid polymer functionalized magnetic nano-drug carrier (MGO- β -CD-HA) into 4mL of doxorubicin hydrochloride solution with the concentration range of 1-300mg/L, oscillating for 7h at normal temperature, and performing magnetic hysteresis separation.
Example 2:
the preparation method of the magnetic nano-drug carrier in the embodiment comprises the following steps:
1) preparation of cyclodextrin-hyaluronic acid supramolecular polymer (β -CD-HA):
firstly, slowly adding β -cyclodextrin (β -CD) and 11g of paratoluensulfonyl chloride (TsCl) into 100mL of pyridine solvent in turn, stirring, reacting for 5 hours at room temperature, removing the pyridine solvent by rotary evaporation, recrystallizing in water, washing and purifying by cold acetone to obtain precipitate mono-6-deoxy-6- (paratoluenesulfonyl) - β -cyclodextrin (M-6-O-Ts- β -CD);
then 4g of mono-6-deoxy-6- (p-toluenesulfonyl) - β -cyclodextrin and 10g of Ethylenediamine (EDA) were dissolved in 20ml of N-Dimethylformamide (DMF) and reacted under magnetic stirring at 80 ℃ for 18 hours under a nitrogen atmosphere, after completion of the reaction, the mixture was cooled to room temperature, then washed 3 times with cold acetone to remove the residual ethylenediamine and dried under vacuum to obtain a white powder (β -CD-EDA);
finally, activating 40mg of Hyaluronic Acid (HA) by 22mg of 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 13mg of N-hydroxysuccinimide (NHS) solution, continuously stirring for 2 hours to obtain a dispersion, adding white powder (β -CD-EDA) into the dispersion, allowing the mixture to react at room temperature for 18 hours, dialyzing and purifying the mixture in deionized water, and freeze-drying to obtain cyclodextrin-hyaluronic acid supramolecular polymer (β -CD-HA);
2) preparation of Magnetic Graphene Oxide (MGO):
stripping 180mg of Graphene Oxide (GO) in deionized water for 1 hour by ultrasonic treatment to prepare graphene oxide dispersion, and then placing the graphene oxide dispersion in a three-neck flask and heating to 60 ℃;
introducing nitrogen into the graphene oxide solution for 10 minutes to remove air in the graphene oxide solution, mechanically stirring the graphene oxide solution at the speed of 800r/min in a nitrogen environment, and adding 2.7g of FeCl3·6H2O and 1.0g FeCl2·4H2Dissolving O in the graphene oxide solution to prepare a reaction solution;
then dropwise adding concentrated ammonia water until the pH value of the reaction solution is adjusted to 9; stirring the reactant at 80 ℃ for 1 hour to obtain a magnetic black precipitate; cooling to room temperature, performing magnetic hysteresis separation, repeatedly washing the black precipitate with deionized water and absolute ethyl alcohol, and freeze-drying for 20h to obtain Magnetic Graphene Oxide (MGO) powder;
3) preparation of cyclodextrin-hyaluronic acid polymer functionalized magnetic nano-drug carrier (MGO- β -CD-HA):
firstly, ultrasonically dispersing 180mg of the Magnetic Graphene Oxide (MGO) powder obtained in the step 2) in 180mL of ethanol for 30 minutes to ensure complete dissolution (MGO concentration is 1mg/mL), adding 200 μ L of 3-Aminopropyltriethoxysilane (APTES) under the protection of nitrogen, and mechanically stirring at room temperature for 6 hours; after the reaction is finished, removing supernatant through magnetic hysteresis separation, washing the precipitate for more than 3 times by using ethanol and deionized water respectively, and freeze-drying to obtain an aminated Magnetic Graphene Oxide (MGO) nano compound;
ultrasonically treating 48mg of cyclodextrin-hyaluronic acid supramolecular polymer (β -CD-HA) obtained in the step 1) in deionized water for 1 hour, adding 22mg of 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 13mg of N-hydroxysuccinimide (NHS) after the cyclodextrin-hyaluronic acid supramolecular polymer is completely dissolved, continuously stirring for 2 hours to activate carboxyl of the cyclodextrin-hyaluronic acid supramolecular polymer (β -CD-HA), adding an aminated Magnetic Graphene Oxide (MGO) nanocomposite into the activated cyclodextrin-hyaluronic acid supramolecular polymer (β -CD-HA) solution, stirring reactants at room temperature for 24 hours, and freeze-drying to obtain the cyclodextrin-hyaluronic acid polymer functionalized magnetic nano-drug carrier (MGO- β -CD-HA).
A method for loading doxorubicin hydrochloride (DOX) on a magnetic nano-drug carrier comprises the following steps:
adding 0.5mg of cyclodextrin-hyaluronic acid polymer functionalized magnetic nano-drug carrier (MGO- β -CD-HA) into 4mL of doxorubicin hydrochloride solution with the concentration range of 1-300mg/L, oscillating for 7h at normal temperature, and performing magnetic hysteresis separation.
Example 3:
the preparation method of the magnetic nano-drug carrier in the embodiment comprises the following steps:
1) preparation of cyclodextrin-hyaluronic acid supramolecular polymer (β -CD-HA):
firstly, slowly adding β -cyclodextrin (β -CD) and 12g of p-toluenesulfonyl chloride (TsCl) into 150mL of pyridine solvent in turn, stirring, reacting for 8 hours at room temperature, removing the pyridine solvent by rotary evaporation, recrystallizing in water, washing and purifying with cold acetone to obtain precipitate mono-6-deoxy-6- (p-toluenesulfonyl) - β -cyclodextrin (M-6-O-Ts- β -CD);
then 6g of mono-6-deoxy-6- (p-toluenesulfonyl) - β -cyclodextrin (M-6-O-Ts- β -CD) and 12g of Ethylenediamine (EDA) were dissolved in 30mL of N-N-Dimethylformamide (DMF) and reacted under magnetic stirring at 80 ℃ for 24 hours under a nitrogen atmosphere, after completion of the reaction, the mixture was cooled to room temperature, and then washed 5 times with cold acetone to remove the residual Ethylenediamine (EDA) and dried under vacuum to obtain white powder (β -CD-EDA);
finally, activating 42mg of Hyaluronic Acid (HA) by 24mg of 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 15mg of N-hydroxysuccinimide (NHS) solution, continuously stirring for 2 hours to obtain a dispersion, adding white powder (β -CD-EDA) into the dispersion, reacting the mixture at room temperature for 24 hours, dialyzing and purifying the mixture in deionized water, and freeze-drying the mixture to obtain the cyclodextrin-hyaluronic acid supramolecular polymer (β -CD-HA);
2) preparation of Magnetic Graphene Oxide (MGO):
stripping 220mg of Graphene Oxide (GO) in deionized water for 1 hour by ultrasonic treatment to prepare graphene oxide dispersion, and then placing the graphene oxide dispersion in a three-neck flask and heating to 80 ℃;
introducing nitrogen into the graphene oxide solution for 15 minutes to remove air in the graphene oxide solution, mechanically stirring the graphene oxide solution at the speed of 800r/min in a nitrogen environment, and adding 2.7g of FeCl3·6H2O and 1.0g FeCl2·4H2Dissolving O in the graphene oxide solution to prepare a reaction solution;
then dropwise adding concentrated ammonia water until the pH value of the reaction solution is adjusted to 12; stirring the reactant at 80 ℃ for 3 hours to obtain a magnetic black precipitate; cooling to room temperature, performing magnetic hysteresis separation, repeatedly washing the black precipitate with deionized water and absolute ethyl alcohol, and freeze-drying for 30h to obtain Magnetic Graphene Oxide (MGO) powder;
3) preparation of cyclodextrin-hyaluronic acid polymer functionalized magnetic nano-drug carrier (MGO- β -CD-HA):
firstly, 220mg of the Magnetic Graphene Oxide (MGO) powder obtained in the step 2) is ultrasonically dispersed in 220mL of ethanol for 40 minutes to ensure complete dissolution (MGO concentration is 1mg/mL), 400 μ L of 3-Aminopropyltriethoxysilane (APTES) is added under the protection of nitrogen, and mechanical stirring is carried out at room temperature for 8 hours; after the reaction is finished, removing supernatant through magnetic hysteresis separation, washing the precipitate for more than 3 times by using ethanol and deionized water respectively, and freeze-drying to obtain an aminated Magnetic Graphene Oxide (MGO) nano compound;
carrying out ultrasonic treatment on 52mg of cyclodextrin-hyaluronic acid supramolecular polymer (β -CD-HA) obtained in the step 1) in deionized water for 3 hours, adding 24mg of 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 15mg of N-hydroxysuccinimide (NHS) after the cyclodextrin-hyaluronic acid supramolecular polymer is completely dissolved, continuously stirring for 4 hours to activate carboxyl of the cyclodextrin-hyaluronic acid supramolecular polymer (β -CD-HA), adding an aminated Magnetic Graphene Oxide (MGO) nanocomposite into the activated cyclodextrin-hyaluronic acid supramolecular polymer (β -CD-HA) solution, stirring reactants at room temperature for 48 hours, and carrying out freeze drying to obtain the cyclodextrin-hyaluronic acid polymer functionalized magnetic nano drug carrier (MGO- β -CD-HA).
A method for loading doxorubicin hydrochloride (DOX) on a magnetic nano-drug carrier comprises the following steps:
adding 0.5mg of cyclodextrin-hyaluronic acid polymer functionalized magnetic nano-drug carrier (MGO- β -CD-HA) into 4mL of doxorubicin hydrochloride solution with the concentration range of 1-300mg/L, oscillating for 7h at normal temperature, and performing magnetic hysteresis separation.
After the adsorption performance is researched, the cyclodextrin-hyaluronic acid polymer functionalized magnetic nano-drug carrier (MGO- β -CD-HA) is eluted by absolute ethyl alcohol, ultrasonically oscillated for 10min, subjected to magnetic hysteresis separation again, washed by secondary water and absolute ethyl alcohol for three times respectively, and dried in vacuum to obtain a magnetic nano-complex subjected to dissociative adsorption, and the magnetic nano-complex can be recycled.
As shown in FIG. 1, curve a represents the infrared spectrum of GO, 3424cm-1Strong broad peak corresponding to-OH stretching vibration, 1737cm-1Nearby peaks correspond to carboxyl-C ═ O-O at the GO surface, 1250cm-1And 1052cm-1The nearby peaks correspond to the stretching vibration of the epoxy group and the alkoxy group respectively, and 1650cm-1The peak of (a) corresponds to stretching vibration of aromatic C ═ C; curve b represents the infrared spectrum of MGO, where 584cm-1The characteristic peaks in the vicinity correspond to tensile vibrations of Fe-O bonds, despite the bare Fe3O4Shifted compared to the peak, but still accounting for Fe3O4Successfully bound to GO surface; curve c represents the IR spectrum of MGO-APTES, 1565cm-1And 1184cm-1The nearby peaks correspond to bending vibration of N-H and stretching vibration of C-N, respectively, and curve d shows the infrared spectrum of MGO- β -CD-HA, in which the MGO nanoparticles still have characteristic absorption peak of β -CD after being further modified by β -CD (1200--1) And at 1157cm-1The spectrum peak of the position corresponds to the coupled C-O-C stretching vibration and O-H in-plane bending vibration, and is 1040cm-1And 1079cm-1The characteristic peaks at (A) correspond to coupled C-O, C-C stretching vibrations, and O-H bending vibrations, indicating that β -CD-HA HAs successfully complexed onto the surface of the MGO.
In FIG. 2, curve a is the thermogravimetric curve of naked MNPs, curve b is the thermogravimetric curve of MGO, curve c is the thermogravimetric curve of MGO- β -CD-HA, as shown in the figure, MNPs show the first weight loss at the temperature lower than 125 ℃ in curve a, which can be attributed to the volatilization of adsorbed water in the sample and the loss of surface-OH, and then a weak weight gain process at the temperature of 150 ℃ and 250 ℃ is caused by the oxidation of MNPs in the process of heating weight loss, so that the weight loss rate in the whole temperature range is about 3.5 percent, and curve b shows the weight loss rate at the low temperature (low temperature)<First weight loss at 100 deg.C, which can be attributed to loss of residual solvent and water, followed by weight loss in the range of 120-3O4And similarly, curve c shows two weight losses, the weight loss in the range of about 180-The total mass loss in the reactor was about 36.5%, calculated to be about 200mg g-1Again, β -CD-HA was successfully modified to the surface of the MGO.
FIG. 3a is an X-ray diffraction pattern of MGO, wherein the diffraction peak at 2 θ ═ 11.8 corresponds to the (001) crystal plane of GO, and the diffraction peaks at 2 θ positions 30.31, 35.58, 43.19, 53.58, 57.18, and 62.87 correspond to Fe3O4The results of the diffraction planes of face-centered cubic (220), (311), (400), (422), (511) and (440) are consistent with those of hematite in JCPDS (No.85-1436) standard card, indicating that MNPs are modified to the GO surface and that the synthesized MGO HAs a cubic spinel structure, FIG. 3b is the diffraction pattern of MGO- β -CD-HA, which is compared with that of MGO- β -CD-HA, which is probably because the absorption of β -CD-HA leads to a decrease in the relative content of MGO in the composite material, and furthermore, the particle size of MGO- β -CD-HA is estimated to be 14.9512nm by Scherrer's formula.
D＝0.94λ/Bcosθ (1)
Taken together, the XRD patterns of MGO and MGO- β -CD-HA demonstrate that the modifications of GO and β -CD-HA do not cause Fe3O4A change in configuration.
Fig. 4 is a hysteresis loop diagram of MGO, and it can be seen from the diagram that when the external magnetic field is 0, the magnetic induction of MGO is 0, and the curve passes through the origin, which conforms to the concept of superparamagnetic property, which indicates that MGO is superparamagnetic and can be used for targeted drug delivery, according to the magnetic moment experiment result: the magnetization value of MGO was 32.46 emu/g.
In FIG. 5, curve a is a fluorescence spectrum of DOX of 0.0025mg/mL, curve b is a spectrum of DOX + MGO supernatant, and curve c is a spectrum of DOX + MGO- β -CD-HA, it can be seen from the figure that different magnetic nanomaterials MGO and MGO- β -CD-HA have a certain load effect on drug DOX, the calculated net fluorescence intensity of MGO- β -CD-HA on DOX is reduced by 99%, and MGO on DOX is reduced by 80%, which shows that MGO- β -CD-HA HAs a better drug loading effect than MGO, and can be attributed to a larger specific surface and a hydrophobic cavity of MGO- β -CD-HA, so that the MGO- β -CD-HA HAs excellent load capacity.
FIG. 6 is a graph showing the effect of pH on net fluorescence intensity of MGO- β -CD-HA + DOX after interaction of MGO- β -CD-HA with 0.03mg/mL DOXThe drug loading experiment is a dynamic equilibrium process, lower or higher pH can lead to high protonation of DOX in acid or alkaline environment, leading to partial dissociation of hydrogen bonds and poor loading capacity, while at pH7.0, the-OH, -COOH and-OH, -NH in the drug of MGO- β -CD-HA2Multiple strong hydrogen bonding interactions between the two favour the association of MGO- β -CD-HA with the drug, resulting in a rapid increase in loading.
As shown in FIG. 7, which is an adsorption isotherm of MGO- β -CD-HA on DOX, it can be seen that the loading capacity of MGO- β -CD-HA on DOX increases with increasing initial DOX concentration (where the initial DOX concentration is 0.5mg/mL) after removing the effect of DOX alone, whereas the adsorbed amount reaches 450mg g-1The maximum adsorption amount of MGO- β -CD-HA to DOX was found to be 485.43mg g by Langmuir isothermal adsorption curve fitting-1。
FIG. 8 is a graph showing the cumulative release behavior of GO- β -CD-HA nanocomposites at different temperatures and different pH values, at pH 7.4, DOX is released from MGO- β -CD-HA nanocomposite at a very slow rate and only less than 15% of the total drug is released after 10 hours, whereas at acidic conditions (pH 5.3), DOX is released from MGO- β -CD-HA at a rapid rate of increase and gradually decreases within 5-10 hours, and at pH 5.3 within 8 hours before estimation, the maximum cumulative release of DOX is about 50% due to the presence of acidic lysosomes within tumor cells, and it can be seen that higher temperature drug release indicates that MGO- β -CD-HA/DOX is a temperature sensitive carrier, therefore, water-dispersed MGO- β -CD-HA serves as an ideal delivery carrier and can then deliver DOX to target sites for targeted cancer delivery by endocytosis.
FIG. 9 shows MGO- β -CD-HA, MGO (0.4mg/mL) and buffer PBS at different concentrations (0.4 and 0.8mg/mL) over 2W cm-2After irradiation with near infrared light, the temperature rise is plotted as a function of time; wherein curve a is the near-infrared laser irradiation relaxationTemperature as a function of time when PBS was washed, curve b as a function of temperature as a function of time when MGO was irradiated with near infrared laser, and c and d as a function of time when MGO- β -CD-HA was irradiated with near infrared laser at a concentration of 0.4mg/mL and 0.8mg/mL, respectively, it can be seen that the temperature at a concentration of 0.8mg/mL of MGO- β -CD-HA was higher than that at 0.4mg/mL and increased by 19 ℃ and 17 ℃ respectively under the same irradiation with near infrared laser, thereby giving a higher photothermal conversion efficiency of MGO- β -CD-HA at 0.8 mg/mL.
In FIG. 10, curves a, b and c show that MGO- β -CD-HA (1.2mg/mL) HAs laser intensities of 1.5, 2.0 and 2.5W/cm, respectively-2The photothermal curve shows that the photothermal conversion efficiency of MGO- β -CD-HA is gradually improved with the increase of the laser intensity under the irradiation of the near-infrared laser.
In summary, the concentration dependence and laser power intensity dependence of MGO- β -CD-HA are illustrated in FIGS. 9 and 10, respectively.
FIG. 11 is a release curve of DOX in a timed laser on/off (irradiation/non-irradiation) state after loading DOX on the surface of MGO- β -CD-HA nanomaterial, wherein curve a is the release curve of DOX in the timed laser off (non-irradiation) state, curve b is the release curve of DOX in the timed laser on (irradiation) state, both of which have a laser irradiation wavelength of 808nm and an intensity of 2Wcm at 37 deg.C and pH of 5.3-2(ii) a As can be seen, the amount of drug released by the timed NIR laser irradiation is significantly higher than that without irradiation, indicating that NIR laser irradiation is beneficial for drug release.
In conclusion, the MGO- β -CD-HA nanocomposite can realize dual stimulation of reactive drug release by pH and near infrared laser, and the composite is an excellent drug transport carrier.
FIG. 12 is a cell map observed by a fluorescence inverted microscope after the DOX inclusion compound and the human hepatoma cell BEL-7402 are incubated for different times, wherein a, b, c and d are the distributions in the cell observed by the fluorescence inverted microscope when the DOX inclusion compound and the human hepatoma cell BEL-7402 are incubated for 0.5h, 1h, 2h and 4h, respectively.
FIG. 13 is a graph of cytotoxicity of drug inclusion complex at different concentrations, where column a (no NIR irradiation) and column b (NIR irradiation) are graphs of relative cellular activities of MGO- β -CD-HA at different concentrations of the complex, and column c (no NIR irradiation) and column d (NIR irradiation) are graphs of relative cellular activities of MGO- β -CD-HA + DOX at different concentrations of the complex.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the concept and the protection scope of the present invention, and those skilled in the art should make various modifications and improvements to the technical solution of the present invention without departing from the design concept of the present invention.
Claims (6)
1. A preparation method of a magnetic nano-drug carrier is characterized by comprising the following steps: the method comprises the following steps:
1) preparation of cyclodextrin-hyaluronic acid supramolecular polymer:
firstly, slowly adding 17-19g of β -cyclodextrin and 11-12g of paratoluensulfonyl chloride into 100-150mL of pyridine solvent in sequence, stirring, reacting for 5-8h at room temperature, removing the pyridine solvent by rotary evaporation, recrystallizing in water, washing and purifying with cold acetone to obtain precipitate mono-6-deoxy-6- (paratoluensulfonyl) - β -cyclodextrin;
then 4-6g of mono-6-deoxy-6- (p-toluenesulfonyl) - β -cyclodextrin and 10-12g of ethylenediamine are dissolved in 20-30mL of N-N-dimethylformamide, and are magnetically stirred and reacted for 18-24 hours at 80 ℃ in a nitrogen environment, after the reaction is finished, the mixture is cooled to room temperature, and then is washed for 3-5 times by cold acetone to remove the residual ethylenediamine, and is dried under vacuum, so that white powder is prepared;
finally, activating 40-42mg hyaluronic acid by 22-24mg 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride and 13-15mg N-hydroxysuccinimide solution, continuously stirring for 2 hours to prepare dispersion liquid, adding white powder into the dispersion liquid, reacting the mixed liquid for 18-24 hours at room temperature, dialyzing and purifying in deionized water, and freeze-drying to prepare the cyclodextrin-hyaluronic acid supramolecular polymer;
2) preparing magnetic graphene oxide:
stripping 180-220mg of graphene oxide in deionized water for 1 hour by ultrasonic treatment to prepare graphene oxide dispersion, and then placing the graphene oxide dispersion in a three-neck flask and heating to 60-80 ℃;
introducing nitrogen into the graphene oxide solution for 10-15 minutes to remove air in the graphene oxide solution, mechanically stirring the graphene oxide solution at the speed of 800r/min in a nitrogen environment, and adding 2.7g of FeCl3·6H2O and 1.0g FeCl2·4H2Dissolving O in the graphene oxide solution to prepare a reaction solution;
then dropwise adding concentrated ammonia water until the pH value of the reaction solution is adjusted to 9-12; stirring the reactant at 80 ℃ for 1-3 hours to obtain a magnetic black precipitate; cooling to room temperature, performing magnetic hysteresis separation, repeatedly washing the black precipitate with deionized water and absolute ethyl alcohol, and freeze-drying for 20-30h to obtain magnetic graphene oxide powder;
3) preparing a cyclodextrin-hyaluronic acid polymer functionalized magnetic nano-drug carrier:
firstly, ultrasonically dispersing the magnetic graphene oxide powder obtained in the step 2) of 180-220mg in 220mL of ethanol of 180-220mL for 30-40 minutes to ensure complete dissolution, adding 400 mu L of 3-aminopropyltriethoxysilane into the solution under the protection of nitrogen, and mechanically stirring the solution at room temperature for 6-8 hours; after the reaction is finished, removing supernatant through magnetic hysteresis separation, washing the precipitate for more than 3 times by using ethanol and deionized water respectively, and freeze-drying to obtain an aminated magnetic graphene oxide nano compound;
carrying out ultrasonic treatment on 48-52mg of the cyclodextrin-hyaluronic acid supramolecular polymer obtained in the step 1) in deionized water for 1-3 hours, adding 22-24mg of 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride and 13-15mg of N-hydroxysuccinimide after the cyclodextrin-hyaluronic acid supramolecular polymer is completely dissolved, and continuously stirring for 2-4 hours to activate carboxyl of the cyclodextrin-hyaluronic acid supramolecular polymer; adding the aminated magnetic graphene oxide nano-composite into the activated cyclodextrin-hyaluronic acid supermolecule polymer solution, stirring the reactants at room temperature for 24-48 hours, and freeze-drying to prepare the cyclodextrin-hyaluronic acid polymer functionalized magnetic nano-drug carrier;
4) the cyclodextrin-hyaluronic acid polymer functionalized magnetic nano-drug carrier loads doxorubicin hydrochloride:
adding 0.5mg of the cyclodextrin-hyaluronic acid polymer functionalized magnetic nano-drug carrier prepared in the step 3) into 4mL of doxorubicin hydrochloride solution with the concentration range of 1-300mg/L, and carrying out magnetic hysteresis separation after oscillating for 7 hours at normal temperature.
2. The method for preparing the magnetic nano-drug carrier according to claim 1, wherein the reaction process of β -cyclodextrin and p-toluenesulfonyl chloride in step 1) is carried out in an anhydrous state and the reaction time is 5 hours.
3. The method for preparing a magnetic nano-drug carrier according to claim 1, characterized in that: the reaction time of the step 1) is 18 hours under the condition of magnetic stirring at 80 ℃ in a nitrogen environment; the number of washes of the mixture cooled to room temperature in cold acetone was 3; the reaction time of the mixture at room temperature was 24 hours.
4. The method for preparing a magnetic nano-drug carrier according to claim 1, characterized in that: placing the graphene oxide dispersion liquid in the step 2) in a three-neck flask, and heating to 80 ℃; the time for introducing nitrogen into the graphene oxide solution is 10 minutes; dropwise adding concentrated ammonia water until the pH value of the reaction liquid is 10; the reaction was stirred at 80 ℃ for 2 hours; the black precipitate was washed repeatedly with deionized water and absolute ethanol and then freeze-dried for 24 h.
5. The method for preparing a magnetic nano-drug carrier according to claim 1, characterized in that: ultrasonically dispersing the magnetic graphene oxide powder in the ethanol for 30 minutes in the step 3); the adding amount of the 3-aminopropyltriethoxysilane is 300 mu L; the mechanical stirring time at room temperature after addition of 3-aminopropyltriethoxysilane was 7 hours.
6. The method for preparing a magnetic nano-drug carrier according to claim 1, characterized in that: adding 1-ethyl (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in the step 3), and continuously stirring for 2 hours; adding the aminated magnetic graphene oxide nano-composite into the activated cyclodextrin-hyaluronic acid supermolecule polymer solution, and stirring at room temperature for 48 hours.
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| BRPI0804172A2 (en) * | 2008-07-15 | 2010-07-06 | Pereira Lopes Jose Emilio Fehr | chemical compounds formed from nanocapsulations and element complexation |
| CN102258788B (en) * | 2011-06-13 | 2012-11-07 | 南开大学 | Targeted transmission assembly of adriamycin anticancer medicine and preparation method thereof |
| CN103030140B (en) * | 2012-12-21 | 2014-07-30 | 郑州大学 | Oxidized graphene modified by hyaluronic acid and preparation method and application of medicinal composition of oxidized graphene modified by hyaluronic acid |
| CN104436199A (en) * | 2014-08-16 | 2015-03-25 | 金陵科技学院 | Preparation method of porous ferroferric oxide composite nanometre microspheres efficiently loaded with pharmorubicin |
| CN104689338B (en) * | 2015-03-10 | 2018-01-05 | 中山大学 | Preparation method and application of the acid-sensitive prodrug of tumor-targeting with magnetic nano-particle conjugate |
| CN104840977B (en) * | 2015-03-23 | 2017-12-12 | 济南大学 | A kind of preparation method of magnetic fluorescence composite Nano pharmaceutical carrier |
| CN106913579A (en) * | 2017-03-01 | 2017-07-04 | 山西大学 | Hyaluronic acid targeting pH sensitive medicaments carriers and its preparation method and application |
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