CN111135309A - Tilazamine drug carrier with core-shell structure and preparation method and application thereof - Google Patents
Tilazamine drug carrier with core-shell structure and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a tirapazamine drug carrier with a core-shell structure, a preparation method and application thereof, belonging to the technical field of drug preparation. The hyaluronic acid, the dithiodiacetic acid, the tetraamino zinc phthalocyanine and the L-carnitine are connected through chemical action to form a tirapazamine drug carrier with a core-shell structure, wherein the tetraamino zinc phthalocyanine-the L-carnitineThe part is the core, the hyaluronic acid-dithiodiacetic acid part is the shell, and the hyaluronic acid and the L-carnitine are utilized to improve the hydrophobicity of the tetra-amino zinc phthalocyanine so as to form a carrier micelle with hydrophilicity, targeting cell membrane and mitochondrion double-targeting effects. The tirapazamine drug (TPZ) is loaded into the carrier to form a tirapazamine drug-loaded micelle with a core-shell structure, oxygen is consumed through photodynamic therapy to activate the tirapazamine drug (TPZ), so that the cooperative therapy of photodynamic therapy and chemotherapy is realized, the drug resistance of chemotherapy and the resistance of cells to photodynamic therapy are avoided, and the tirapazamine drug-loaded micelle has a good application prospect in preparing antitumor drugs.
Description
Technical Field
The invention belongs to the technical field of medicine preparation, and particularly relates to a tirapazamine medicine carrier with a core-shell structure, and a preparation method and application thereof.
Background
At present, tumors are still one of the major diseases threatening the life and health of human beings, and the photodynamic therapy is taken as a treatment means for treating the tumors, and can utilize a photosensitizer to generate ROS under the excitation of a specific excitation wavelength to kill tumor cells. However, the ROS has short existence time in cells and low action distance range, so that the utilization rate is low, the photosensitizer is delivered to mitochondria through the mitochondrial targeting molecules, and the ROS directly damages the mitochondria, so that the photodynamic force can be effectively enhanced. Meanwhile, the photodynamic action consumes oxygen to cause the hypoxia state in cells, thereby activating the function of the prodrug tirapazamine, enabling the photodynamic action and the chemotherapy to play a role simultaneously and enhancing the killing effect of tumor cells.
Therefore, a need exists for preparing a tirapazamine vector with targeted mitochondria.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a tirapazamine drug carrier with a core-shell structure; the second purpose of the invention is to provide a preparation method of the tirapazamine drug carrier with a core-shell structure; the invention also aims to provide a tirapazamine drug-carrying micelle with a core-shell structure; the fourth purpose of the invention is to provide a preparation method of tirapazamine drug-carrying micelle with a core-shell structure; the fifth purpose of the invention is to provide the application of the tirapazamine drug-carrying micelle with the core-shell structure in preparing anti-tumor drugs.
In order to achieve the purpose, the invention provides the following technical scheme:
1. a tirapazamine drug carrier with a core-shell structure is hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-L-carnitine, wherein the tetraaminozinc phthalocyanine-L-carnitine part is a core and the hyaluronic acid-dithiodiacetic acid part is a shell; the structural formula of the carrier is shown as follows:
2. The preparation method of the drug carrier micelle with the core-shell structure comprises the following steps:
(1) preparation of tetraaminozinc phthalocyanine-2-acetic acid dithiodipyridine: adding 2-acetic acid dithiodipyridine into N, N-dimethylformamide for dissolving, then adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), reacting for 5-30 min under an ice bath condition, removing the ice bath, continuing to react and activate for 30-60 min, then adding tetraaminozinc phthalocyanine, reacting for 24-36 h at room temperature, removing the solvent, and washing with acetone to obtain tetraaminozinc phthalocyanine-2-acetic acid dithiodipyridine;
(2) preparation of tetraamino zinc phthalocyanine-2-acetic acid dithiodipyridine-l-carnitine: dissolving the tetraaminozinc phthalocyanine-2-acetic acid dithiodipyridine prepared in the step (1) in a mixed solvent of DMF and water to form a tetraaminozinc phthalocyanine-2-acetic acid dithiodipyridine solution, dissolving L-carnitine in PBS, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), activating for 1-6 h, adding the solution to the tetraaminozinc phthalocyanine-2-acetic acid dithiodipyridine solution, reacting for 12-48 h, and washing with water to obtain the tetraaminozinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine;
(3) preparing hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-l-carnitine: dissolving hyaluronic acid sulfydryl in PBS, adding N, N-dimethylformamide under an ice bath condition, uniformly mixing, continuously adding the tetraamino zinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine, stirring and reacting at room temperature for 12-48 h after ultrasonic dissolution, removing DMF through dialysis, freeze-drying, repeatedly adding DMF, performing ultrasonic treatment and centrifuging to obtain the hyaluronic acid-dithiodiacetic acid-tetraamino zinc phthalocyanine-L-carnitine.
Preferably, the molar volume ratio of the 2-acetic acid dithiodipyridine, the N, N-dimethylformamide, the 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride, the N-hydroxysuccinimide and the tetraaminozinc phthalocyanine in the step (1) is 0.03-0.06: 5-20: 0.078:0.078:0.156, mol: L: mol: mol.
Further preferably, the preparation method of the 2-acetic acid dithiodipyridine is as follows:
(1) firstly, sufficiently dissolving dithiodipyridine in any one solvent of ethanol, methanol or dichloromethane, and adding glacial acetic acid for oscillation; dropwise adding 3-mercaptopropionic acid, reacting at room temperature for 12-48 h, removing ethanol, and extracting by column chromatography to obtain 2-ethylcarboxyl dipyridyl disulfide, wherein the molar ratio of the dipyridyl disulfide to the 3-mercaptopropionic acid is 2: 0.5-1;
(2) fully dissolving the 2-ethylcarboxyl dithiodipyridine in a mixed solvent formed by dichloromethane and ethanol, performing vacuum extraction to remove acetic acid, and then removing the ethanol, wherein the volume ratio of the dichloromethane to the ethanol is 3: 1-5;
(3) and (3) repeating the operation of the step (2) until the acetic acid is completely removed to obtain the 2-acetic acid dipyridyl disulfide.
Further preferably, the preparation method of the tetraaminozinc phthalocyanine is as follows:
(1) preparation of tetranitrozinc phthalocyanine: mixing urea, 4-nitrophthalimide and ammonium molybdate according to a molar ratio of 0.1665:0.02:0.000147, grinding, heating and stirring to melt the mixture, adding zinc acetate, continuing heating and stirring to melt the mixture, reacting at 160 ℃ until no bubbles are generated, cooling, then carrying out micro-boiling treatment on the mixture for 1-5 hours by using hydrochloric acid with the concentration of 0.5-1.5 mol/L, filtering and washing the solid to be neutral, continuing micro-boiling treatment on the mixture for 2 hours by using a sodium hydroxide solution with the concentration of 1mol/L, filtering and washing the mixture to be neutral, and repeatedly washing until a blue-purple solid is obtained, namely tetranitrozinc phthalocyanine, wherein the molar ratio of the 4-nitrophthalimide to the zinc acetate is 4: 1;
(2) preparation of tetraaminozinc phthalocyanine: adding the tetranitro zinc phthalocyanine and sodium sulfide nonahydrate into DMF according to a molar ratio of 1: 12-30, vacuumizing, filling nitrogen, heating to 60 ℃ under slow stirring, stirring at a constant temperature of 60 ℃ for 1-5 h at a high speed, pouring into water, mixing uniformly, performing suction filtration, repeatedly washing the solid until the washing liquid is neutral, and freeze-drying the washed solid to obtain the tetraamino zinc phthalocyanine.
Preferably, the volume ratio of DMF to water in the mixed solvent in the step (2) is 1: 1-1.2, and the mass ratio of L-carnitine, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and tetraaminozinc phthalocyanine-2-acetic acid dithiodipyridine is 8-20: 22:14: 100.
Preferably, the molar volume ratio of the hyaluronic acid sulfhydryl, the PBS, the DMF and the tetraaminozinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine in the step (3) is 0.5:50:50:0.5, nmol: ml: ml: mmol, the molecular weight of the dialysis bag adopted during dialysis is 10KD, and the rotation speed during centrifugation is 8000 rpm.
3. The tirapazamine drug-carrying micelle of the core-shell structure takes the carrier as a carrier, and the tirapazamine drug is 3-amino-1, 2, 4-benzotriazine-1, 4-dioxide.
4. The preparation method of the drug-loaded micelle comprises the following steps:
(1) preparing carrier micelles: adding the carrier hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-L-carnitine into a mixed solvent of PBS and THF (tetrahydrofuran), stirring to fully dissolve the carrier hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-L-carnitine, and removing THF (tetrahydrofuran) by rotary evaporation to form a carrier micelle with a core-shell structure;
(2) preparing a tirapazamine medicament:
firstly, slowly dripping a cyanamide solution with the concentration of 50% into o-nitroaniline with the temperature of 50 ℃ according to the molar volume ratio of 0.1449:18, heating to 100 ℃ after finishing dripping, cooling to room temperature after a reaction system turns to dark red to precipitate an orange solid, continuously and slowly dripping 12mol/L concentrated hydrochloric acid for 15min, heating to 100 ℃ until layering occurs, stirring for 30min, slowly dripping 16mol/L NaOH solution for 15min, continuously heating to 100 ℃ for reaction until a sticky solid suspension occurs, adding water, stirring, cooling to room temperature, recrystallizing to precipitate a yellow solid, repeatedly washing with water and ethyl acetate in sequence, and drying to obtain yellow powder, namely the synthesis of 3-amino-1, 2, 4-benzotriazine-1-oxide, wherein the molar volume ratio of the o-nitroaniline, the 50% cyanamide solution, the HCl and the NaOH is as follows: 0.1449:18:18:18, mol: mL: mol;
then, adding 220mL of glacial acetic acid into the 3-amino-1, 2, 4-benzotriazine-1-oxide (4.4g,0.03mol), stirring until a suspension appears, heating to 50 ℃, then dropwise adding 107mL of 30% hydrogen peroxide, reacting under the condition of keeping out of the sun until the solution is programmed to be light red, removing the solvent, evaporating to obtain a red solid substance, and recrystallizing to obtain a red crystal substance, namely the tirapazamine drug 3-amino-1, 2, 4-benzotriazine-1, 4-dioxide, wherein the molar volume ratio of the 3-amino-1, 2, 4-benzotriazine-1-oxide to the glacial acetic acid to the 30% hydrogen peroxide is 0.03:220:107, and the molar volume ratio is mL: mL;
(3) fully dissolving the 3-amino-1, 2, 4-benzotriazine-1, 4-dioxide prepared in the step (2) in a PBS (phosphate buffer solution), dropwise adding the solution into the carrier micelle prepared in the step (1), stirring overnight after dropwise adding, removing THF (tetrahydrofuran) by rotary evaporation, and dialyzing by using a dialysis bag with the molecular weight of 30KD to remove unloaded 3-amino-1, 2, 4-benzotriazine-1, 4-dioxide to obtain the tirapamine drug-loaded micelle with the core-shell structure, wherein the mass ratio of the carrier to the 3-amino-1, 2, 4-benzotriazine-1, 4-dioxide is as follows: 5-10: 1.
5. The drug-loaded micelle is applied to the preparation of antitumor drugs.
The invention has the beneficial effects that: hyaluronic acid, dithiodiacetic acid, tetra-amino zinc phthalocyanine and L-carnitine are connected through chemical action to form a tirapazamine drug carrier with a core-shell structure, wherein the tetra-amino zinc phthalocyanine-L-carnitine part is a core, the hyaluronic acid-dithiodiacetic acid part is a shell, the tetra-amino zinc phthalocyanine contained in the tirapazamine drug carrier is a photosensitizer with good non-toxic biocompatibility, a large amount of ROS can be generated under excitation of laser with the wavelength of 720nm to damage tumor cells, the application limitation of the tetra-amino zinc phthalocyanine due to strong hydrophobicity can be solved by the hyaluronic acid and the L-carnitine, a carrier micelle with the particle size of about 100nm is formed, and the tirapazamine drug carrier has hydrophilic, targeting cell membrane and mitochondrion double-targeting effects. Compared with a simple photosensitizer, the carrier disclosed by the invention can avoid the risk of being rapidly metabolized by the kidney in the blood circulation process, and can be enriched to the periphery of tumor cell tissues through an EPR (ethylene propylene rubber) effect, in addition, the Hyaluronic Acid (HA) HAs the characteristic of targeting CD44, the phagocytic effect of tumor cells on the carrier can be enhanced, then, the powerful photodynamic therapy can be realized through the photosensitizer targeting mitochondria, finally, the tirapazamine drug (TPZ) is loaded in the micelle to form a tirapazamine drug-loaded micelle with a core-shell structure, oxygen is consumed to activate the tirapazamine drug (TPZ) through the photodynamic therapy, the synergistic therapy of photodynamic and chemotherapy is realized, the drug resistance of chemotherapy and the resistance of cells to the photodynamic are avoided, and the carrier HAs a good application prospect in preparing antitumor drugs.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objectives and other advantages of the invention may be realized and attained by the means of the instrumentalities and combinations particularly pointed out hereinafter.
Drawings
For the purposes of promoting a better understanding of the objects, aspects and advantages of the invention, reference will now be made to the following detailed description taken in conjunction with the accompanying drawings in which:
FIG. 1 is a diagram showing ultraviolet absorption spectra of different substances, wherein A is a diagram showing ultraviolet absorption changes before and after hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-L-carnitine (HA-S-S-ZnPc-Ic) carrying Tirapazamine (TPZ), B is a diagram showing ultraviolet absorption contrast between a hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-L-carnitine carrier (HA-S-S-ZnPc-Ic) and a tetraaminozinc phthalocyanine-L-carnitine (ZnPc-Ic) formed after disulfide bond rupture, and C is a diagram showing drug release tendency of tirapazamine drug-carrying micelles (HA-S-S-ZnPc-Ic @ TPZ) before and after Glutathione (GSH) addition;
FIG. 2 is a particle size distribution diagram of a tirapazamine drug-loaded micelle (HA-S-S-ZnPc-Ic @ TPZ) prepared by the invention;
FIG. 3 is TEM images of different substances, wherein a is TEM image of tirapazamine drug-loaded micelle (HA-S-S-ZnPc-Ic @ TPZ), and b is nanoparticle degradation image of drug-loaded micelle after Glutathione (GSH) is added;
FIG. 4 shows the FITC labeled hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-L-carnitine micelles (HA-S-S-ZnPc-Ic @ FITC) phagocytosed by different cells, wherein a 1-a 3 are 4T1 cells (mouse breast cancer cells), b 1-b 3 are 4T1 cells after CD44 receptor is blocked by hyaluronic acid in advance, and c 1-c 3 are HUVECs (human umbilical vein endothelial cells);
FIG. 5 is the distribution of FITC-labeled hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-L-carnitine micelles (HA-S-S-ZnPc-Ic @ FITC) and FITC-labeled hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine (HA-S-S-ZnPc @ FITC) in cell mitochondria, wherein a1 is the fluorescence distribution of HA-S-S-ZnPc-Ic @ FITC, a2 is the rhodamine staining map for mitochondria, a3 is the overlay of a1 and a2, b1 is the fluorescence distribution of HA-S-S-ZnPc @ FITC, a2 is the rhodamine staining map for mitochondria, b3 is the overlay of b1 and b 2;
FIG. 6 is a graph showing the toxicity of different substances to cells under laser irradiation and without laser irradiation, wherein a is the toxicity of tirapazamine drug (TPZ), a tirapazamine drug carrier (HA-S-S-ZnPc-Ic) and a tirapazamine drug-loaded micelle (HA-S-S-ZnPc-lc @ TPZ) to 4T1 cells under the condition without laser irradiation, and b is the killing effect of tirapazamine drug (TPZ), a tirapazamine drug carrier (HA-S-S-ZnPc-Ic) and a tirapazamine drug-loaded micelle (HA-S-S-ZnPc-lc @ TPZ) with different concentrations to 4T1 cells under the laser irradiation.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention. It should be noted that the drawings provided in the following embodiments are only for illustrating the basic idea of the present invention in a schematic way, and the features in the following embodiments and examples may be combined with each other without conflict.
Example 1
The preparation method of the drug carrier hyaluronic acid-dithiodiacetic acid-tetraamino zinc phthalocyanine-levocarnitine micelle (HA-S-S-ZnPc-Ic) with the core-shell structure comprises the following steps:
1. preparation of thiolated hyaluronic acid (HA-SH)
(1) Dissolving Hyaluronic Acid (HA) (200mg, 0.5mmol) in 40mL PBS buffer solution with pH value of 6.8, stirring to mix uniformly, then adding 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride (287.6mg,1.5mmol) and 1-hydroxybenzotriazole (202.7mg,1.5mmol) in sequence, carrying out activation reaction on the mixed solution at room temperature for 2h, then adding cysteamine hydrochloride ((337.8mg,1.5mmol) into the activated mixed solution, and stirring at room temperature for 24h to obtain a reaction product solution;
(2) pouring 40ml of the reaction product solution into 200ml of ethanol solvent to generate flocculent white precipitate, centrifuging for 10min at the rotating speed of 10000rpm to obtain white precipitate, repeatedly washing with absolute ethanol (to remove unreacted cysteamine hydrochloride, 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride and 1-hydroxybenzotriazole), dissolving in water again after washing is finished, and freeze-drying to finally obtain white cotton-like thiolated hyaluronic acid (HA-SH).
2. Preparation of 2-acetic acid dithiodipyridine
(1) Dissolving dithiodipyridine (3.75g, 0.017mol) solution in ethanol solvent (99.5%, 10ml), adding 0.4ml glacial acetic acid, dropping 3-mercaptopropionic acid (0.9g,0.008mol) dropwise after strong shaking, reacting at room temperature for 20h,
controlling the molar ratio of the dithiodipyridine to the 3-mercaptopropionic acid within the range of 2: 0.5-1, and reacting for 12-48 h to obtain the effect of the reaction;
(2) then removing ethanol under the vacuum condition of 40 ℃, purifying the obtained product by column chromatography, and using a mixed solvent of DCM, ethanol and acetic acid (the volume ratio of DCM, ethanol and acetic acid is 15:4:1) as an eluent to obtain a viscous oily substance, namely 2-ethylcarboxyl dithiodipyridine;
(3) dissolving the obtained viscous oily matter (2-ethylcarboxyl dipyridyl disulfide) in 2ml of mixed solvent composed of dichloromethane and ethanol (wherein the volume ratio of dichloromethane to ethanol is 3:2), vacuumizing to remove unreacted acetic acid, removing ethanol by rotary evaporation, repeating the above operations until acetic acid is completely removed to obtain diacetic acid dipyridyl disulfide,
in addition, the volume ratio of dichloromethane to ethanol in the mixed solvent for dissolving the viscous oily substance (2-ethylcarboxyl dithiodipyridine) can be within the range of 3: 1-5.
3. Preparation of Tetraaminozinc Phthalocyanine
(1) Preparation of tetranitrozinc phthalocyanine: firstly, uniformly mixing urea (10g, 0.1665mol), 4-nitrophthalimide (3.84g, 0.02 mol) and ammonium molybdate (0.05g, 0.000147mol), grinding the mixture by a mortar, putting the ground mixture into a 250ml three-necked bottle, putting the three-necked bottle into a thermometer, heating the three-necked bottle to 160 ℃ to melt the mixture, adding zinc acetate (0.9g, 0.005mol) into a molten product after all solids are melted, and adding a stirrer to accelerate stirring in the process to uniformly mix the molten product and the zinc acetate; then continuing to react at a constant temperature of 160 ℃, gradually generating a bluish-purple solid from the original brown liquid in the reaction process, and indicating that the reaction is terminated when bubbles are continuously generated and no longer generated in the reaction process; finally, cooling the reaction mixture and then carrying out post-treatment (firstly, carrying out micro-boiling treatment on the reaction mixture for 1h by using 500ml of hydrochloric acid with the concentration of 1mol/L, filtering, washing the solid to be neutral, then carrying out micro-boiling treatment for 2h by using 500ml of sodium hydroxide solution with the concentration of 1mol/L, filtering with the washing value being neutral, washing the solid to be neutral, repeating the micro-boiling treatment last time) to obtain a blue-violet solid product, namely the tetranitro zinc phthalocyanine;
in the process of preparing the tetranitro zinc phthalocyanine, the slightly boiling treatment process can be carried out for 1 to 5 hours by using hydrochloric acid with the concentration of 0.5 to 1.5mol/L, and the tetranitro zinc phthalocyanine can also be obtained.
(2) Preparation of tetraaminozinc phthalocyanine:
putting the prepared solid powder of tetranitro zinc phthalocyanine (0.758g, 1mmol) and sodium sulfide nonahydrate (2.88g, 12mmol) into a 250ml three-mouth bottle, installing a condenser tube and a thermometer on the three-mouth bottle, adding 20ml of DMF, vacuumizing, filling nitrogen, then starting heating and slowly stirring by magnetic force, accelerating the stirring speed when the temperature reaches 60 ℃, keeping the temperature for 1.5 hours to obtain a crude product, pouring the crude product into 200ml of water, carrying out suction filtration, repeatedly washing until the filtered water solution is neutral, and freeze-drying to obtain a dark green solid product, namely the tetraamino zinc phthalocyanine;
in the preparation process, excessive sodium sulfide nonahydrate and tetranitro zinc phthalocyanine can be added to react, and the tetranitro zinc phthalocyanine and the sodium sulfide nonahydrate react for 1-5 hours according to the molar ratio of 1: 12-30 to obtain the tetraamino zinc phthalocyanine.
4. Preparation of TetraaminoZinc phthalocyanine-2-acetic acid dithiodipyridine
(1) Adding 2-acetic acid dithiodipyridine (11mg,0.052mmol) and 10ml of N, N-Dimethylformamide (DMF) into a 25ml single-mouth volumetric flask, stirring to mix uniformly, then sequentially adding 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride (15mg,0.078mmol) and N-hydroxysuccinimide (9mg,0.078mmol) under an ice bath condition, removing the ice bath after reacting for 5min, continuing to react and activate for 30min, continuing to add tetraaminozinc phthalocyanine (100mg,0.156mmol), reacting for 24h under a room temperature condition, and removing the solvent by vacuum rotary evaporation to obtain a crude product;
the process for the preparation of the crude product can also be carried out under the following conditions: the molar volume ratio of 2-acetic acid dithiodipyridine, N-dimethylformamide, 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride, N-hydroxysuccinimide and tetraaminozinc phthalocyanine is 0.03-0.06: 5-20: 0.078:0.078:0.156, the mol: L: mol: mol: mol, the ice bath reaction time is 5-30 min, the reaction activation time is 30-60 min, the continuous reaction time is 24-36 h after the tetraaminozinc phthalocyanine is added, and the properties of the crude product prepared by the reaction are not influenced under the conditions.
(2) The crude product was washed three times with acetone to remove unreacted 2-acetic acid dithiodipyridine, 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride and N-hydroxysuccinimide, and pure tetraaminozinc phthalocyanine-2-acetic acid dithiodipyridine was finally obtained.
5. Preparation of TetraaminoZinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine
Dissolving L-carnitine (24mg) in 2ml of PBS solution, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC, 22mg, 0.117mol) N-hydroxysuccinimide (NHS, 14mg,0.117mol), activating for two hours, adding the mixture into a mixed solution containing 100mg of tetraaminozinc phthalocyanine-2-acetic acid dithiodipyridine (the solvent of the mixed solution is DMF and water which are mixed in equal volume), reacting for 24 hours, and washing the EDC, the NHS and unreacted L-carnitine with water to obtain the tetraaminozinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine;
in the reaction process, the volume ratio of DMF to water in the solvent in the mixed solution can be 1: 1-1.2, the mass ratio of L-carnitine, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and tetraaminozinc phthalocyanine-2-acetic acid dipyridyl disulfide can also be 8-20: 22:14:100, and the tetraaminozinc phthalocyanine-2-acetic acid dipyridyl disulfide-L-carnitine can be obtained through reaction.
6. Preparation of drug carrier hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-levocarnitine micelle (HA-S-S-ZnPc-Ic) with core-shell structure
Where n is normally distributed with a median value of 20.
(1) Adding thiolated hyaluronic acid (200mg, 0.5mmol) into 50ml of PBS solution, pouring 50ml of N, N-Dimethylformamide (DMF) under the ice bath condition, and uniformly stirring;
(2) then adding tetraamino zinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine (437mg,0.5mmol), performing ultrasonic treatment to fully dissolve the tetraamino zinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine, and stirring and reacting for 24 hours at room temperature;
(3) removing DMF from the reacted product by dialysis (dialysis bag with molecular weight of 10KD is adopted in dialysis), and freeze-drying;
(4) adding the freeze-dried product into 50ml of N, N-Dimethylformamide (DMF), ultrasonically dissolving unreacted tetraamino zinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine in the DMF, centrifuging at the rotating speed of 8000rpm, collecting solid parts, and repeating the dissolving ultrasound until the unreacted tetraamino zinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine is completely removed to obtain pure hyaluronic acid-dithiodiacetic acid-tetraamino zinc phthalocyanine-L-carnitine;
(5) adding 10mg of hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-L-carnitine into 5ml of PBS solution and 5ml of THF, stirring for 10min, fully dissolving, and slowly evaporating to remove THF by using a rotary evaporator to form a medicament carrier hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-L-carnitine micelle (HA-S-S-ZnPc-Ic) with a core-shell structure.
Example 2
The preparation method of the tirapazamine drug-loaded micelle (HA-S-S-ZnPc-Ic @ TPZ) with the core-shell structure comprises the following steps:
1. preparing a tirapazamine drug 3-amino-1, 2, 4-benzotriazine-1, 4-dioxide:
(1) preparation of 3-amino-1, 2, 4-benzotriazine-1-oxide: adding o-nitroaniline (20g,0.1449mol) into a 250mL three-neck flask, heating to 50 ℃, stirring, slowly dropwise adding 18mL of cyanamide solution with the concentration of 50%, continuing heating to 100 ℃ after dropwise adding is finished, heating until the color is dark red, and cooling to room temperature to precipitate orange solid;
(2) continuously and slowly dripping 12mol/L concentrated hydrochloric acid into a three-mouth bottle, wherein the continuous dripping time is 15min, heating to 100 ℃ after the dripping is finished, generating a layered state, continuously stirring for 30min, slowly dripping 16mol/L NaOH solution, the continuous time is also 15min, heating to 100 ℃ after the dripping is finished, keeping the temperature at 100 ℃, and continuously reacting for 12 h;
(3) after the reaction is finished, sticky solid suspension appears, 100-200 ml of water is added, stirring is carried out, cooling is carried out to room temperature, yellow solid is separated out, water and ethyl acetate are sequentially used for washing for 3 times, and finally, drying is carried out to obtain yellow powder, namely 3-amino-1, 2, 4-benzotriazine-1-oxide;
(4) adding 3-amino-1, 2, 4-benzotriazine-1-oxide (4.4g,0.03mol) into a 500mL three-necked bottle, adding 220mL glacial acetic acid, stirring to obtain a suspension, heating to 50 ℃, dropwise adding 30% hydrogen peroxide solution (107mL), reacting for 10 hours under the condition of keeping out of the sun, changing the solution into light red, performing vacuum rotary evaporation to remove the solvent, evaporating to obtain a red solid substance, and then recrystallizing with absolute ethyl alcohol to finally obtain a red crystal, namely the tirapazamine drug 3-amino-1, 2, 4-benzotriazine-1, 4-dioxide (TPZ).
2. Preparation of tirapazamine drug-loaded micelle with core-shell structure (HA-S-S-ZnPc-Ic @ TPZ)
(1) Adding 10mg of hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-L-carnitine (HA-S-S-ZnPc-Ic) into 5ml of PBS solution and 5ml of THF, and stirring for 10min to fully dissolve the hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-L-carnitine to form micelles;
(2) then slowly dropping PBS buffer solution (1mg/ml) of Tirapazamine (TPZ), stirring overnight after the dropping is finished, and then slowly evaporating THF by using a rotary evaporator, wherein in the process, as the THF is gradually reduced, core-shell micelles are finally formed due to the hydrophilic action of the tetraaminozinc phthalocyanine-L-carnitine and hyaluronic acid, part of the tirapazamine is wrapped in the center of the core-shell to form medicament-carrying hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-L-carnitine micelles (HA-S-S-ZnPc-Ic @ TPZ), the tirapazamine which is not loaded is removed by a dialysis method (MW & gt, 30KD dialysis bag), and the micelles are freeze-dried after three days of dialysis, and are observed to be particles of about 200 nanometers by a transmission electron microscope (as shown in figure 2).
Dissolving tetraamino zinc phthalocyanine-L-carnitine (ZnPc-Ic) and tetraamino zinc phthalocyanine-L-carnitine (HA-S-S-ZnPc-Ic) connected with hyaluronic acid in DMSO, testing the ultraviolet absorption condition, and the result is shown in figure 1, wherein A is the ultraviolet absorption change chart before and after hyaluronic acid-dithiodiacetic acid-tetraamino zinc phthalocyanine-L-carnitine (HA-S-S-ZnPc-Ic) carrying Tirapazamine (TPZ), B is the ultraviolet absorption spectrum chart of hyaluronic acid-dithiodiacetic acid-tetraamino zinc phthalocyanine-L-carnitine carrier (HA-S-S-ZnPc-Ic) and tetraamino zinc phthalocyanine-L-carnitine (ZnPc-Ic) formed after carrier disulfide bond is broken, and C is the ultraviolet absorption spectrum of tirapazamine drug carrying micelle (HA-S-S-ZnPc-IC-TPZ) added with glutathione Trend plots of drug release before and after (GSH). Because the tetraaminozinc phthalocyanine (ZnPc) per se is insoluble in water due to strong hydrophobicity, so that the tetraaminozinc phthalocyanine (ZnPc) is aggregated and precipitated in water and does not have an ultraviolet absorption spectrum, and the tetraaminozinc phthalocyanine (ZnPc) HAs an absorption peak in water after being connected with Hyaluronic Acid (HA), and the peak spectrum is widened, the method of the invention can successfully connect Hyaluronic Acid (HA) and levocarnitine to the tetraaminozinc phthalocyanine through disulfide bonds to form a hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-levocarnitine carrier (HA-S-S-ZnPc-Ic acid); through the change peak spectrogram before and after hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-levocarnitine (HA-S-S-ZnPc-Ic) carrying Tirapazamine (TPZ) shown in the graph A in figure 1, it can be seen that the synthesized carrier of the invention can successfully carry the tirapazamine medicament (TPZ), and C in the graph 1 can be seen that the medicament release rate of the tirapazamine medicament carrying micelle (HA-S-S-ZnPc-Ic @ TPZ) prepared by the invention is slow, and the medicament release rate is obviously increased after Glutathione (GSH) is added.
The particle size distribution of the tirapazamine drug carrier micelle (HA-S-S-ZnPc-Ic @ TPZ) with the core-shell structure prepared in example 1 is shown in FIG. 2, a TEM image of a drug-loaded micelle (HA-S-S-ZnPc-Ic @ TPZ) formed by hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-L-carnitine and tirapazamine is shown as a in figure 3, the drug-loaded micelle (HA-S-S-ZnPc-Ic @ TPZ) is a spherical micelle with a core-shell structure, a map of a substance obtained by adding Glutathione (GSH) into the drug-loaded micelle is shown as b in figure 3, and the substance is shown as a map, so that the HA-S-S-ZnPc-Ic @ TPZ core-shell micelle can be decomposed by adding Glutathione (GSH), and the encapsulated drug molecules can be released.
FIG. 4 shows the phagocytosis of FITC-labeled hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-L-carnitine micelles (HA-S-S-ZnPc-Ic @ FITC) by different cells, wherein a is 4T1 cells (mouse breast cancer cells), b is 4T1 cells after blocking CD44 receptor with hyaluronic acid in advance, and c is HUVECs cells (human umbilical vein endothelial cells). In FIG. 4, a1, b1 and c1 are nuclear staining conditions, a2, b2 and c2 are fluorescence distribution conditions of drug-loaded micelles (HA-S-S-ZnPc-Ic @ FITC) in different cells, and a3, b3 and c3 are superposed images of the nuclear staining conditions and the fluorescence distribution of the drug-loaded micelles. According to the phagocytosis amount a > b > c, the medicine-carrying micelle prepared by the invention HAs the effect of targeting tumor cells of CD44 receptors, and the targeting effect is only from Hyaluronic Acid (HA) in a carrier, so that the hyaluronic acid of the micelle cannot target the tumor cells after the CDD4 receptors are closed by the hyaluronic acid in b in advance, and the phagocytosis amount is reduced; and in c, HUVECs contain few CDD4 receptors, so that micelles have no targeting effect and have little fluorescence phagocytosis.
FIG. 5 shows the distribution of FITC-labeled hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-L-carnitine micelles (HA-S-S-ZnPc-Ic @ FITC) and FITC-labeled hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine (HA-S-S-ZnPc @ FITC) in cell mitochondria, wherein a1 is the fluorescence distribution of HA-S-S-ZnPc-Ic @ FITC, a2 is the rhodamine staining map of mitochondria, a3 is the overlay of a1 and a2, b1 is the fluorescence distribution of HA-S-S-ZnPc @ FITC, a2 is the rhodamine staining map of mitochondria, and a3 is the overlay of b1 and b 2. From the distribution in fig. 5, it can be seen that the HA-S-ZnPc-Ic micelle with the targeting Ic linked to the shell ZnPc can target the micelle to the mitochondria, resulting in a high distribution content of the formed good book in the mitochondria, while the HA-S-ZnPc alone HAs no mitochondrial targeting function and a low distribution content in the mitochondria.
FIG. 6 shows the toxicity of different substances to cells under laser irradiation and without laser irradiation, wherein a is the toxicity of tirapazamine drug (TPZ), tetraaminozinc phthalocyanine (ZnPc) and corresponding carrier (HA-S-S-ZnPc-Ic) and drug-loaded micelle (HA-S-S-ZnPc-lc @ TPZ) to cells under laser irradiation, and b is the killing effect graph of different concentrations of tirapazamine drug (TPZ), tetraaminozinc phthalocyanine (ZnPc) and corresponding carrier (HA-S-ZnPc-Ic) and drug-loaded micelle (HA-S-S-ZnPc-lc @ TPZ) to 4T1 cells under laser irradiation. As can be seen from FIG. 6, under the laser irradiation, the drug-loaded micelle of HA-S-S-ZnPc-Ic @ TPZ combined with photodynamic and chemotherapy HAs the most obvious effect on killing cells.
In conclusion, hyaluronic acid, dithiodiacetic acid, tetraamino zinc phthalocyanine and L-carnitine are connected through chemical action to form a tirapazamine drug carrier with a core-shell structure, wherein the tetraamino zinc phthalocyanine-L-carnitine part is a core, the hyaluronic acid-dithiodiacetic acid part is a shell, the contained tetraamino zinc phthalocyanine is a photosensitizer with good non-toxic biocompatibility, a large amount of ROS can be generated under excitation of laser with the wavelength of 720nm to damage tumor cells, the application limitation of the tetraamino zinc phthalocyanine due to strong hydrophobicity can be solved by the hyaluronic acid and the L-carnitine, a carrier micelle with the particle size of about 100nm is formed, and the tirapazamine drug carrier has hydrophilic, cell membrane targeting and mitochondrion double targeting effects. Compared with a simple photosensitizer, the carrier disclosed by the invention can avoid the risk of being rapidly metabolized by the kidney in the blood circulation process, and can be enriched to the periphery of tumor cell tissues through an EPR (ethylene propylene rubber) effect, in addition, the Hyaluronic Acid (HA) HAs the characteristic of targeting CD44, the phagocytic effect of tumor cells on the carrier can be enhanced, then, the powerful photodynamic therapy can be realized through the photosensitizer targeting mitochondria, finally, the tirapazamine drug (TPZ) is loaded in the micelle to form a tirapazamine drug-loaded micelle with a core-shell structure, oxygen is consumed to activate the tirapazamine drug (TPZ) through the photodynamic therapy, the synergistic therapy of photodynamic and chemotherapy is realized, the drug resistance of chemotherapy and the resistance of cells to the photodynamic are avoided, and the carrier HAs a good application prospect in preparing antitumor drugs.
Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.
Claims (10)
1. The tirapazamine drug carrier with the core-shell structure is characterized in that the carrier is hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-L-carnitine; the carrier is of a core-shell structure, wherein the tetraamino zinc phthalocyanine-L-carnitine part is a core, and the hyaluronic acid-dithiodiacetic acid part is a shell.
2. The method for preparing the drug carrier micelle with the core-shell structure according to claim 1, wherein the method comprises the following steps:
(1) preparation of tetraaminozinc phthalocyanine-2-acetic acid dithiodipyridine: adding 2-acetic acid dithiodipyridine into N, N-dimethylformamide for dissolving, then adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, reacting for 5-30 min under an ice bath condition, removing the ice bath, continuing to react and activate for 30-60 min, then adding tetraaminozinc phthalocyanine, reacting for 24-36 h at room temperature, removing the solvent, and washing with acetone to obtain tetraaminozinc phthalocyanine-2-acetic acid dithiodipyridine;
(2) preparation of tetraamino zinc phthalocyanine-2-acetic acid dithiodipyridine-l-carnitine: dissolving the tetraaminozinc phthalocyanine-2-acetic acid dithiodipyridine prepared in the step (1) in a mixed solvent of DMF and water to form a tetraaminozinc phthalocyanine-2-acetic acid dithiodipyridine solution, dissolving L-carnitine in PBS, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, activating for 1-6 h, adding the activated tetraaminozinc phthalocyanine-2-acetic acid dithiodipyridine solution, reacting for 12-48 h, and washing with water to obtain tetraaminozinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine;
(3) preparing hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-l-carnitine: dissolving hyaluronic acid sulfydryl in PBS, adding N, N-dimethylformamide under an ice bath condition, uniformly mixing, continuously adding the tetraamino zinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine, stirring and reacting at room temperature for 12-48 h after ultrasonic dissolution, removing DMF through dialysis, freeze-drying, repeatedly adding DMF, performing ultrasonic treatment and centrifuging to obtain the hyaluronic acid-dithiodiacetic acid-tetraamino zinc phthalocyanine-L-carnitine.
3. The method according to claim 2, wherein the molar volume ratio of 2-acetoxydipyridyl disulfide, N-dimethylformamide, 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride, N-hydroxysuccinimide and tetraaminozinc phthalocyanine in step (1) is 0.03 to 0.06:5 to 20:0.078:0.078:0.156, mol: L: mol: mol.
4. The method according to claim 3, wherein said 2-acetic acid dithiodipyridine is prepared as follows:
(1) firstly, sufficiently dissolving dithiodipyridine in any one solvent of ethanol, methanol or dichloromethane, and adding glacial acetic acid for oscillation; dropwise adding 3-mercaptopropionic acid, reacting at room temperature for 12-48 h, removing ethanol, and extracting by column chromatography to obtain 2-ethylcarboxyl dipyridyl disulfide, wherein the molar ratio of the dipyridyl disulfide to the 3-mercaptopropionic acid is 2: 0.5-1;
(2) fully dissolving the 2-ethylcarboxyl dithiodipyridine in a mixed solvent formed by dichloromethane and ethanol, performing vacuum extraction to remove acetic acid, and then removing the ethanol, wherein the volume ratio of the dichloromethane to the ethanol is 3: 1-5;
(3) and (3) repeating the operation of the step (2) until the acetic acid is completely removed to obtain the 2-acetic acid dipyridyl disulfide.
5. The method according to claim 3, wherein the tetraaminozinc phthalocyanine is prepared by the following method:
(1) preparation of tetranitrozinc phthalocyanine: mixing urea, 4-nitrophthalimide and ammonium molybdate according to a molar ratio of 0.1665:0.02:0.000147, grinding, heating and stirring to melt the mixture, adding zinc acetate, continuing heating and stirring to melt the mixture, reacting at 160 ℃ until no bubbles are generated, cooling, then carrying out micro-boiling treatment on the mixture for 1-5 hours by using hydrochloric acid with the concentration of 0.5-1.5 mol/L, filtering and washing the solid to be neutral, continuing micro-boiling treatment on the mixture for 2 hours by using a sodium hydroxide solution with the concentration of 1mol/L, filtering and washing the mixture to be neutral, and repeatedly washing until a blue-purple solid is obtained, namely tetranitrozinc phthalocyanine, wherein the molar ratio of the 4-nitrophthalimide to the zinc acetate is 4: 1;
(2) preparation of tetraaminozinc phthalocyanine: adding the tetranitro zinc phthalocyanine and sodium sulfide nonahydrate into DMF according to a molar ratio of 1: 12-30, vacuumizing, filling nitrogen, heating to 60 ℃ under slow stirring, stirring at a constant temperature of 60 ℃ for 1-5 h at a high speed, pouring into water, mixing uniformly, performing suction filtration, repeatedly washing the solid until the washing liquid is neutral, and freeze-drying the washed solid to obtain the tetraamino zinc phthalocyanine.
6. The preparation method according to claim 2, wherein the volume ratio of DMF to water in the mixed solvent in the step (2) is 1:1 to 1.2, and the mass ratio of L-carnitine, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and tetraaminozinc phthalocyanine-2-acetic acid dithiodipyridine is 8 to 20:22:14: 100.
7. The method according to claim 2, wherein the molar volume ratio of the thiol group of hyaluronic acid, PBS, DMF and tetraaminozinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine in step (3) is 0.5:50:50:0.5, nmol: ml: ml: mmol, the molecular weight of the dialysis bag used in the dialysis is 10KD, and the rotation speed in the centrifugation is 8000 rpm.
8. The tirapazamine drug-loaded micelle with a core-shell structure is characterized in that the carrier of claim 1 is used as a carrier in the drug-loaded micelle, and the tirapazamine drug is 3-amino-1, 2, 4-benzotriazine-1, 4-dioxide.
9. The preparation method of the drug-loaded micelle of claim 8, which comprises the following steps:
(1) preparing carrier micelles: adding the carrier hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-L-carnitine into a mixed solvent of PBS and THF (tetrahydrofuran), stirring to fully dissolve the carrier hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-L-carnitine, and removing THF (tetrahydrofuran) by rotary evaporation to form a carrier micelle with a core-shell structure;
(2) preparing a tirapazamine medicament:
firstly, slowly dripping a cyanamide solution with the concentration of 50% into o-nitroaniline with the molar volume ratio of 0.1449:18, heating to 100 ℃ after finishing dripping, cooling to room temperature to precipitate orange solid after the reaction system turns to dark red, continuously and slowly dripping 12mol/L concentrated hydrochloric acid for 15min, heating to 100 ℃ until layering occurs, stirring for 30min, slowly dripping 16mol/L NaOH solution for 15min, continuously heating to 100 ℃ for reaction until viscous solid suspended substances occur, adding water, stirring, cooling to room temperature, recrystallizing to precipitate yellow solid, repeatedly washing with water and ethyl acetate in sequence, and drying to obtain yellow powder, namely the synthesis of 3-amino-1, 2, 4-benzotriazine-1-oxide, wherein the molar volume ratio of the o-nitroaniline, the 50% cyanamide solution, the HCl and the NaOH is 0.1449:18:18:18, mol is mL and mol is;
then, adding glacial acetic acid into the 3-amino-1, 2, 4-benzotriazine-1-oxide, stirring until a suspension appears, heating to 50 ℃, dropwise adding 30% hydrogen peroxide, reacting under the condition of keeping out of the sun until the solution is programmed to be light red, removing the solvent, evaporating to obtain a red solid substance, and recrystallizing to obtain a red crystal substance, namely the tirapazamine drug 3-amino-1, 2, 4-benzotriazine-1, 4-dioxide, wherein the molar volume ratio of the 3-amino-1, 2, 4-benzotriazine-1-oxide, the glacial acetic acid to the 30% hydrogen peroxide is as follows: 0.03:220:107, mol: mL: mL;
(3) fully dissolving the 3-amino-1, 2, 4-benzotriazine-1, 4-dioxide prepared in the step (2) in a PBS (phosphate buffer solution), dropwise adding the solution into the carrier micelle prepared in the step (1), stirring overnight after dropwise adding, removing THF (tetrahydrofuran) by rotary evaporation, and dialyzing by using a dialysis bag with the molecular weight of 30KD to remove unloaded 3-amino-1, 2, 4-benzotriazine-1, 4-dioxide to obtain the tirapazamine drug-loaded micelle with the core-shell structure, wherein the mass ratio of the carrier to the 3-amino-1, 2, 4-benzotriazine-1, 4-dioxide is 5-10: 1.
10. The use of the drug-loaded micelle of claim 8 in the preparation of an anti-tumor drug.
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| CN113460986A (en) * | 2021-07-26 | 2021-10-01 | 山东大学 | Method for preparing core-shell structure hydroxyapatite microspheres by one-step method and application thereof |
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| CN113460986A (en) * | 2021-07-26 | 2021-10-01 | 山东大学 | Method for preparing core-shell structure hydroxyapatite microspheres by one-step method and application thereof |
| CN113460986B (en) * | 2021-07-26 | 2023-07-28 | 山东大学 | Method for preparing hydroxyapatite microsphere with core-shell structure by one-step method and application thereof |
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