CN111135309B - 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, and a preparation method and application thereof, and belongs to the technical field of drug preparation. The invention relates to a tirapazamine drug carrier with a core-shell structure, which is formed by connecting hyaluronic acid, dithiodiacetic acid, tetra-amino zinc phthalocyanine and L-carnitine through chemical action, wherein the tetra-amino zinc phthalocyanine-L-carnitine part is taken as a core, the hyaluronic acid-dithiodiacetic acid part is taken as a shell, and the hydrophobicity of the tetra-amino zinc phthalocyanine is improved by utilizing the hyaluronic acid and the L-carnitine, so that carrier micelles with hydrophilic, targeted cell membranes and mitochondria double-targeting effects are formed. The tirapazamine drug (TPZ) is loaded into the carrier to form a tirapazamine drug-loaded micelle with a core-shell structure, oxygen is consumed for activating the tirapazamine drug (TPZ) through photodynamic therapy, so that the photodynamic and chemotherapy cooperative therapy is realized, the drug resistance of chemotherapy and the resistance of cells to photodynamic are avoided, and the tirapazamine drug-loaded micelle has 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 major diseases threatening the life health of human beings, and photodynamic therapy is a treatment means for treating tumors, and can utilize photosensitizers to generate ROS under excitation of specific excitation wavelengths so as to kill tumor cells. However, ROS exist in cells for a short time and have a low range of action distances, so that the utilization rate is low, the photosensitizer is delivered to mitochondria through the mitochondrial targeting molecule, the ROS directly damage the mitochondria, and the photodynamic can be effectively enhanced. At the same time, the photodynamic action consumes oxygen to cause a low oxygen state in cells, so that the action of the prodrug tirapazamine is activated, the photodynamic action and the chemotherapy are simultaneously exerted, and the killing effect of tumor cells is enhanced.
It is therefore desirable to prepare a tirapazamine vector with a targeted mitochondria.
Disclosure of Invention
In view of the above, one of the objects 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 a tirapazamine drug carrier with a core-shell structure; the invention aims at providing a tirapazamine drug-loaded micelle with a core-shell structure; the fourth purpose of the invention is to provide a preparation method of a tirapazamine drug-loaded micelle with a core-shell structure; the invention aims at providing an application of a tirapazamine drug-loaded micelle with a core-shell structure in preparation of antitumor drugs.
In order to achieve the above purpose, the present invention provides the following technical solutions:
1. a tirapazamine drug carrier with a core-shell structure, wherein the carrier is hyaluronic acid-dithiodiacetic acid-tetraminozinc phthalocyanine-L-carnitine, a tetraminozinc phthalocyanine-L-carnitine part is a core, and a 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 tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine: adding 2-acetic acid dithiodipyridine into N, N-dimethylformamide for dissolution, then adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), reacting for 5-30 min under the ice bath condition, removing the ice bath, continuing to activate for 30-60 min, then adding tetra-amino zinc phthalocyanine, reacting for 24-36 h under the room temperature condition, removing the solvent, and washing with acetone to obtain tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine;
(2) Preparation of tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine-l-carnitine: dissolving tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine prepared in the step (1) in a mixed solvent of DMF and water to form tetra-amino zinc 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 tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine solution, reacting for 12-48 h, and washing with water to obtain tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine;
(3) Preparation of hyaluronic acid-dithiodiacetic acid-tetra-amino zinc phthalocyanine-l-carnitine: dissolving hyaluronate mercapto in PBS, adding N, N-dimethylformamide under ice bath condition, mixing uniformly, continuously adding tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine, stirring at room temperature for reaction for 12-48 h after ultrasonic dissolution, removing DMF through dialysis, freeze-drying, repeatedly adding DMF for ultrasonic and centrifugal treatment, and obtaining hyaluronate-dithiodiacetic acid-tetra-amino zinc phthalocyanine-L-carnitine.
Preferably, in step (1), the 2-acetic acid dithiodipyridine, N, N-dimethylformamide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride molar volume of N-hydroxysuccinimide and tetra-amino Zinc Phthalocyanine the ratio of the catalyst to the catalyst is 0.03-0.06:5-20:0.078:0.078:0.156, and the mol ratio of the catalyst to the catalyst is L mol.
Further preferably, the preparation method of the 2-acetic acid dithiodipyridine is as follows:
(1) Firstly, fully dissolving dithiodipyridine in any one of ethanol, methanol or dichloromethane, and adding glacial acetic acid for oscillation; then dropwise adding 3-mercaptopropionic acid, reacting for 12-48 hours at room temperature, removing ethanol, and extracting by column chromatography to obtain 2-ethylene carboxyl dithiodipyridine, wherein the molar ratio of the dithiodipyridine to the 3-mercaptopropionic acid is 2:0.5-1;
(2) Fully dissolving the 2-ethylene carboxyl dithiodipyridine in a mixed solvent formed by dichloromethane and ethanol, vacuum extracting to remove acetic acid, and then removing ethanol, wherein the volume ratio of the dichloromethane to the ethanol is 3:1-5;
(3) Repeating the operation of the step (2) until the acetic acid is completely removed, thereby obtaining the 2-acetic acid dithiodipyridine.
Further preferably, the preparation method of the tetra-amino zinc phthalocyanine comprises the following steps:
(1) Preparation of tetranitrozinc phthalocyanine: firstly, mixing urea, 4-nitrophthalimide and ammonium molybdate according to the molar ratio of 0.1665:0.02:0.000147, grinding, heating and stirring to melt, then adding zinc acetate, continuously heating and stirring to melt, reacting at 160 ℃ until no bubbles are generated, cooling, carrying out micro-boiling treatment for 1-5 h by using hydrochloric acid with the concentration of 0.5-1.5 mol/L, filtering and washing the solid to be neutral, continuously carrying out micro-boiling treatment for 2h by using sodium hydroxide solution with the concentration of 1mol/L, filtering and washing the solid to be neutral, and repeatedly washing until a blue-violet solid is obtained, namely tetranitrozinc phthalocyanine, wherein the molar ratio of the 4-nitrophthalimide to the zinc acetate is 4:1;
(2) Preparation of tetra amino zinc phthalocyanine: adding tetranitro zinc phthalocyanine and sodium sulfide nonahydrate into DMF according to the molar ratio of 1:12-30, vacuumizing, filling nitrogen, heating to 60 ℃ under slow stirring, stirring at the constant temperature of 60 ℃ for 1-5 hours, pouring into water, mixing uniformly, filtering, repeatedly washing the solid until the washing liquid is neutral, and freeze-drying the washed solid to obtain the tetramino zinc phthalocyanine.
Preferably, in the step (2), the volume ratio of DMF and water in the mixed solvent is 1:1-1.2, and the mass ratio of the L-carnitine to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide to the tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine is 8-20:22:14:100.
Preferably, in the step (3), the molar volume ratio of the thiol group of hyaluronic acid, PBS, DMF and tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine is 0.5:50:50:0.5, nmol:ml:ml:mmol, the molecular weight of a dialysis bag used in dialysis is 10KD, and the rotating speed in centrifugation is 8000rpm.
3. The medicine carrying micelle of the tirapazamine medicine with the core-shell structure takes the carrier as the carrier, and the tirapazamine medicine 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 micelle: adding the carrier hyaluronic acid-dithiodiacetic acid-tetra-amino zinc phthalocyanine-L-carnitine into a mixed solvent of PBS and THF with the same volume, stirring to fully dissolve the carrier hyaluronic acid-dithiodiacetic acid-tetra-amino zinc phthalocyanine-L-carnitine, and removing the THF by rotary evaporation to form a carrier micelle with a core-shell structure;
(2) Preparation of tirapazamine drug:
firstly, slowly dropwise adding a 50% cyanamide solution into o-nitroaniline at 50 ℃ according to the mol volume ratio of 0.1449:18, heating to 100 ℃ after the dropwise addition is finished, cooling to room temperature after the reaction system turns dark red to separate out orange solid, continuously slowly dropwise adding 12mol/L concentrated hydrochloric acid for 15min, heating to 100 ℃ until layering occurs, slowly dropwise adding 16mol/L NaOH solution for 15min after stirring for 30min, continuously heating to 100 ℃ for reacting until a viscous solid suspension appears, adding water, stirring, cooling to room temperature, recrystallizing to separate out yellow solid, repeatedly washing with water and ethyl acetate, and drying to obtain yellow powder which is 3-amino-1, 2, 4-benzotriazine-1-oxide, wherein the mol volume ratio of the o-nitroaniline, the 50% cyanamide solution, HCl and NaOH is as follows: 0.1449:18:18:18, mol:mL:mol;
then 220mL of glacial acetic acid is added into the 3-amino-1, 2, 4-benzotriazine-1-oxide (4.4 g,0.03 mol), the mixture is stirred until suspension appears, 107mL of 30% hydrogen peroxide is dropwise added after the temperature is raised to 50 ℃, the mixture reacts under the dark condition until the solution is programmed to be light red, the solvent is removed, then the mixture is evaporated to red solid matters, and the red solid matters are recrystallized to obtain red crystal matters, 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 and the 30% hydrogen peroxide is 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 PBS buffer solution, dropwise adding the solution into the carrier micelle prepared in the step (1), stirring overnight after the dropwise adding, rotationally evaporating to remove THF, and dialyzing to remove the unloaded 3-amino-1, 2, 4-benzotriazine-1, 4-dioxide by adopting a dialysis bag with the molecular weight of 30KD to obtain the tirapazamine drug-carrying micelle with a 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: according to the invention, 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 taken as a core, the hyaluronic acid-dithiodiacetic acid part is taken as a shell, the contained tetra-amino zinc phthalocyanine is a nontoxic photosensitizer with better biocompatibility, a large amount of ROS can be generated to cause damage to tumor cells under the excitation of laser with the wavelength of 720nm, and the hyaluronic acid and L-carnitine can solve the application limitation of the tetra-amino zinc phthalocyanine due to stronger hydrophobicity, so that carrier micelles with the particle size of about 100nm are formed, and the carrier micelle has hydrophilic, targeted cell membrane and mitochondrial double-targeting effects. Compared with a pure photosensitizer, the carrier can avoid the risk of being rapidly metabolized out by kidneys in the blood circulation process, can be enriched around tumor cell tissues through an EPR effect, HAs the characteristic of targeting CD44, can enhance the phagocytic effect of tumor cells on the carrier, can realize powerful photodynamic therapy through the photosensitizer targeting mitochondria, can finally load tirapazamine medicine (TPZ) into micelles to form a tirapazamine medicine carrying micelle with a core-shell structure, and can consume oxygen to activate the tirapazamine medicine (TPZ) through photodynamic therapy, 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 carrier HAs good application prospect in preparing the antitumor medicine.
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 objects and other advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the specification.
Drawings
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in the following preferred detail with reference to the accompanying drawings, in which:
FIG. 1 is a graph showing the ultraviolet absorption spectra of different substances, wherein A is a change graph of ultraviolet absorption before and after hyaluronic acid-dithiodiacetic acid-tetra-amino zinc phthalocyanine-levocarnitine (HA-S-S-ZnPc-Ic) drug-loaded Tirapazamine (TPZ), B is a graph showing the ultraviolet absorption contrast of hyaluronic acid-dithiodiacetic acid-tetra-amino zinc phthalocyanine-levocarnitine carrier (HA-S-S-ZnPc-Ic) carrier and tetra-amino zinc phthalocyanine-levocarnitine (ZnPc-Ic) formed after disulfide bond rupture, and C is a graph showing the trend of drug release of tirapazamine drug-loaded micelles (HA-S-ZnPc-ic@TPZ) before and after Glutathione (GSH) addition;
FIG. 2 is a graph showing the particle size distribution of a tirapazamine drug-loaded micelle (HA-S-S-ZnPc-ic@TPZ) prepared by the invention;
FIG. 3 is a TEM image of different substances, wherein a is a TEM image of a tirapazamine drug-loaded micelle (HA-S-S-ZnPc-ic@TPZ), and b is a nanoparticle degradation image of the drug-loaded micelle after Glutathione (GSH) is added;
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 1-a 3 are 4T1 cells (mouse breast cancer cells), b 1-b 3 are 4T1 cells after the CD44 receptor HAs been previously blocked with hyaluronic acid, and c 1-c 3 are HUVECs cells (human umbilical vein endothelial cells);
FIG. 5 shows the distribution of FITC-labeled hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-levocarnitine micelles (HA-S-S-ZnPc-ic@FITC) and FITC-labeled hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine (HA-S-S-ZnPc@FITC) in the mitochondria of cells, wherein a1 is the fluorescence profile of HA-S-S-ZnPc-ic@FITC, a2 is the fluorescence profile of a1 and a2, a1 is the superimposed graph of HA-S-S-ZnPc@FITC, a2 is the fluorescence profile of a mitochondrial rhodamine, and b3 is the superimposed graph of b1 and b 2;
FIG. 6 shows cytotoxicity of different substances on cells with and without laser irradiation, wherein a is the toxicity of tirapazamine drug (TPZ), tirapazamine drug carrier (HA-S-S-ZnPc-Ic) and tirapazamine drug-carrying micelle (HA-S-S-ZnPc-lc@TPZ) on 4T1 cells without laser irradiation, and b is a graph of killing effect of different concentrations of tirapazamine drug (TPZ), tirapazamine drug carrier (HA-S-ZnPc-Ic) and tirapazamine drug-carrying micelle (HA-S-S-ZnPc-lc@TPZ) on 4T1 cells with laser irradiation.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention. It should be noted that the illustrations provided in the following embodiments merely illustrate the basic idea of the present invention by way of illustration, and the following embodiments and features in the embodiments may be combined with each other without conflict.
Example 1
The preparation method of the drug carrier hyaluronic acid-dithiodiacetic acid-tetra-amino zinc phthalocyanine-levocarnitine micelle (HA-S-S-ZnPc-Ic) with the core-shell structure comprises the following specific steps:
1. preparation of thiolated hyaluronic acid (HA-SH)
(1) Hyaluronic Acid (HA) (200 mg,0.5 mmol) is dissolved in 40mL PBS buffer solution with pH value of 6.8, after stirring and mixing uniformly, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (287.6 mg,1.5 mmol) and 1-hydroxybenzotriazole (202.7 mg,1.5 mmol) are sequentially added, the mixed solution is subjected to activation reaction for 2h at room temperature, and then cysteamine hydrochloride ((337.8 mg,1.5 mmol) is added into the activated mixed solution, and stirred and reacted for 24h at room temperature 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 ethyl alcohol (to remove unreacted cysteamine hydrochloride, 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride and 1-hydroxybenzotriazole), dissolving in water again after washing, and freeze-drying to finally obtain the product white cotton-shaped thiolated hyaluronic acid (HA-SH).
2. Preparation of 2-acetic acid dithiodipyridine
(1) A solution of dithiodipyridine (3.75 g,0.017 mol) was dissolved in an ethanol solvent (99.5%, 10 ml), then 0.4ml of glacial acetic acid was added, 3-mercaptopropionic acid (0.9 g,0.008 mol) was dropwise added after vigorous shaking, and reacted at room temperature for 20 hours,
the reaction can be carried out for 12 to 48 hours by controlling the mol ratio of the dithiodipyridine to the 3-mercaptopropionic acid to be in the range of 2:0.5 to 1, and the effect of the reaction can be obtained;
(2) Then removing ethanol under vacuum condition at 40deg.C, purifying by column chromatography, eluting with mixed solvent of DCM, ethanol and acetic acid (the volume ratio of DCM, ethanol and acetic acid is 15:4:1) to obtain viscous oily substance which is 2-ethoxyl dithiodipyridine;
(3) Dissolving the obtained viscous oily substance (2-ethylene carboxyl dithiodipyridine) in a mixed solvent composed of 2ml of dichloromethane and ethanol (wherein the volume ratio of the dichloromethane to the ethanol is 3:2), vacuumizing to remove unreacted acetic acid, removing the ethanol by rotary evaporation, repeating the operation until the acetic acid is thoroughly removed to obtain the diacetic acid dithiodipyridine,
in addition, the volume ratio of dichloromethane to ethanol in the mixed solvent used for dissolving viscous oily matter (2-ethylene carboxyl dithiodipyridine) can be in the range of 3:1-5.
3. Preparation of tetra-amino Zinc phthalocyanine
(1) Preparation of tetranitrozinc phthalocyanine: firstly, uniformly mixing urea (10 g,0.1665 mol), 4-nitrophthalimide (3.84 g,0.02 mol) and ammonium molybdate (0.05 g, 0.000147mol), grinding by a mortar, putting into a three-mouth bottle of 250ml, loading a thermometer into the three-mouth bottle, heating to 160 ℃ to melt the three-mouth bottle, adding zinc acetate (0.9 g,0.005 mol) into a molten product after all solids are melted, and adding a stirrer for accelerating stirring in the process to uniformly mix the molten product with the zinc acetate; then continuously reacting at a constant temperature of 160 ℃, gradually generating a blue-violet solid from the original brown liquid in the reaction process, and continuously expanding bubbles in the reaction process until no bubbles are generated, so as to indicate the termination of the reaction; finally, cooling the reaction mixture and then carrying out post-treatment (the reaction mixture is firstly subjected to micro-boiling treatment by using 500ml of hydrochloric acid with the concentration of 1mol/L for 1h, filtering, washing the solid to be neutral, then carrying out micro-boiling treatment by using 500ml of sodium hydroxide solution with the concentration of 1mol/L for 2h, filtering the washing value for neutral, and washing the solid to be neutral;
in the process of preparing tetranitro zinc phthalocyanine, the micro-boiling treatment process can be treated with hydrochloric acid with the concentration of 0.5-1.5 mol/L for 1-5 hours, so that tetranitro zinc phthalocyanine can be obtained.
(2) Preparation of tetra amino zinc phthalocyanine:
putting the prepared solid powder tetranitro zinc phthalocyanine (0.758 g,1 mmol) and sodium sulfide nonahydrate (2.88 g,12 mmol) into a three-mouth bottle of 250ml, mounting a condenser tube and a thermometer on the three-mouth bottle, adding 20ml of DMF, vacuumizing, filling nitrogen, heating and slowly magnetically stirring, accelerating the stirring speed when the temperature reaches 60 ℃, keeping the temperature for 1.5h 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 tetramino zinc phthalocyanine;
in the preparation process, excessive sodium sulfide nonahydrate and tetranitro zinc phthalocyanine can be added for reaction, and the tetranitro zinc phthalocyanine and 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 tetra-amino Zinc phthalocyanine-2-acetic acid dithiodipyridine
(1) 2-Acidithiodipyridine (11 mg,0.052 mmol) and 10ml of N, N-Dimethylformamide (DMF) are added into a 25ml single-port volumetric flask, stirred to be uniformly mixed, then 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (15 mg,0.078 mmol) and N-hydroxysuccinimide (9 mg,0.078 mmol) are sequentially added under ice bath condition, the ice bath is removed after reaction for 5min, and the reaction is continued for activation for 30min, tetra-amino zinc phthalocyanine (100 mg,0.156 mmol) is continuously added, the reaction is carried out for 24h under room temperature condition, and the solvent is removed by vacuum rotary evaporation to obtain a crude product;
the process for preparing the crude product may also be carried out under the following conditions: the molar volume ratio of the 2-acetic acid dithiodipyridine to the N, N-dimethylformamide to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide to the tetra-amino zinc phthalocyanine is in the range of 0.03-0.06:5-20:0.078:0.078:0.156 mol: L: 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 tetra-amino zinc phthalocyanine is added, and the properties of the crude product prepared by the reaction are not affected 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, to finally obtain pure tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine.
5. Preparation of tetra-amino Zinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine
L-carnitine (24 mg) was dissolved in 2ml of PBS solution, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC, 22mg,0.117 mol) N-hydroxysuccinimide (NHS, 14mg,0.117 mol) was added, activated for two hours, and then added to a mixture containing 100mg of tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine (the solvent of the mixture was DMF and water mixed in equal volumes), and after 24 hours of reaction, EDC, NHS and unreacted L-carnitine were washed off with water to give tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine;
the volume ratio of DMF and water in the solvent in the mixed solution in the reaction process can be 1:1-1.2, and the mass ratio of L-carnitine, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine can be 8-20:22:14:100, and the tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine can be obtained by the reaction.
6. Preparation of drug Carrier hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-L-carnitine micelle (HA-S-S-ZnPc-Ic) with core-shell structure
Wherein n is normally distributed and the intermediate value is 20.
(1) Thiolated hyaluronic acid (200 mg,0.5 mmol) was added to 50ml of PBS solution, and 50ml of N, N-Dimethylformamide (DMF) was poured under ice-bath conditions and stirred well;
(2) Then tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine (437 mg,0.5 mmol) is added, and the mixture is fully dissolved by ultrasonic treatment, and stirred and reacted for 24 hours at room temperature;
(3) Removing DMF from the reacted product by dialysis (dialysis bag with molecular weight of 10 KD), and freeze-drying;
(4) Adding the freeze-dried product into 50ml of N, N-Dimethylformamide (DMF), dissolving unreacted tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine in the DMF by ultrasonic, centrifuging at 8000rpm, collecting a solid part, repeating the dissolving ultrasonic until the unreacted tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine is removed completely, and obtaining pure hyaluronic acid-dithiodiacetic acid-tetra-amino zinc phthalocyanine-L-carnitine;
(5) 10mg of hyaluronic acid-dithiodiacetic acid-tetra-amino zinc phthalocyanine-L-carnitine is added into 5ml of PBS solution and 5ml of THF, stirred for 10min, fully dissolved, and the THF is slowly evaporated by a rotary evaporator to form a drug carrier hyaluronic acid-dithiodiacetic acid-tetra-amino zinc 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. preparation of the 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 (20 g,0.1449 mol) into a 250mL three-neck flask, heating to 50 ℃, stirring, slowly dropwise adding 18mL of a 50% cyanamide solution, continuously heating to 100 ℃ after dropwise adding, 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 for 15min, heating to 100 ℃ after dripping, continuously stirring for 30min, slowly dripping 16mol/L NaOH solution for 15min, heating to 100 ℃ after dripping, maintaining 100 ℃, and continuously reacting for 12h;
(3) After the reaction is finished, suspending sticky solid appears, adding 100-200 ml of water, stirring, cooling to room temperature, precipitating yellow solid, washing with water and ethyl acetate for 3 times in sequence, and finally drying to obtain yellow powder, namely 3-amino-1, 2, 4-benzotriazine-1-oxidation;
(4) 3-amino-1, 2, 4-benzotriazine-1-oxide (4.4 g,0.03 mol) is added into a 500mL three-necked flask, 220mL glacial acetic acid is added, suspension is stirred, 30% hydrogen peroxide solution (107 mL) is added dropwise after the temperature is raised to 50 ℃, reaction is carried out for 10 hours under the dark condition, the solution turns light red, the solvent is removed by vacuum rotary evaporation, the red solid substance is obtained after evaporation, and then absolute ethyl alcohol is used for recrystallization, so that the red crystal is the tirapazamine drug 3-amino-1, 2, 4-benzotriazine-1, 4-dioxide (TPZ).
2. Preparation of core-shell Structure tirapazamine drug-loaded micelle (HA-S-S-ZnPc-ic@TPZ)
(1) 10mg of hyaluronic acid-dithiodiacetic acid-tetra-amino zinc phthalocyanine-L-carnitine (HA-S-S-ZnPc-Ic) is added into 5ml of PBS solution and 5ml of THF, and stirred for 10min to be fully dissolved to form micelle;
(2) Then slowly dropwise adding PBS buffer solution (1 mg/ml) of Tirapazamine (TPZ), stirring overnight after the dropwise adding, slowly evaporating to remove THF by a rotary evaporator, gradually reducing THF in the process, and finally forming micelle of a core shell due to the hydrophilic effect of tetra-amino zinc phthalocyanine-L-carnitine and hyaluronic acid, wherein part of tirapazamine is wrapped in the center of the core shell to form hyaluronic acid-dithiodiacetic acid-tetra-amino zinc phthalocyanine-L-carnitine micelle (HA-S-S-ZnPc-ic@TPZ) carrying medicine, removing the carried tirapazamine by a dialysis method (MW=30KD dialysis bag), dialyzing for three days, freeze-drying the micelle, and observing particles of about 200 nanometers by a transmission electron microscope (shown in figure 2).
The four amino zinc phthalocyanine-left-handed carnitine (ZnPc-Ic) and the four amino zinc phthalocyanine-left-handed carnitine (HA-S-S-ZnPc-Ic) connected with hyaluronic acid are dissolved in DMSO, and the ultraviolet absorption condition is tested, and the result is shown in figure 1, wherein A is a change chart of ultraviolet absorption before and after the medicine-carrying Tirapazamine (TPZ) of hyaluronic acid-dithiodiacetic acid-four amino zinc phthalocyanine-left-handed carnitine carrier (HA-S-S-ZnPc-Ic) and the ultraviolet absorption spectrogram of the four amino zinc phthalocyanine-left-handed carnitine (ZnPc-Ic) formed after disulfide bond rupture of the carrier, and C is a chart of medicine release trend of the medicine-carrying medicine micelle (HA-S-S-ZnPc-Pc-Pc@TPZ) of tirapazamine before and after the medicine-carrying tirapazamine (HA-S-ZnPc@TPZ) is added into glutathione (H). Since tetra-amino zinc phthalocyanine (ZnPc) itself is not soluble in water due to strong hydrophobicity, and thus is aggregated and precipitated in water, HAs no ultraviolet absorption spectrum, but HAs absorption peak in water after the tetra-amino zinc phthalocyanine (ZnPc) is connected with Hyaluronic Acid (HA), and the peak spectrum is widened, the case of ultraviolet absorption peak in B shows that Hyaluronic Acid (HA) and l-carnitine can be successfully connected to tetra-amino zinc phthalocyanine by disulfide bond to form hyaluronic acid-dithiodiacetic acid-tetra-amino zinc phthalocyanine-l-carnitine carrier (HA-S-ZnPc-Ic) by the method of the present invention; according to the change peak spectrogram of hyaluronic acid-dithiodiacetic acid-tetra-amino zinc phthalocyanine-L-carnitine (HA-S-S-ZnPc-Ic) before and after carrying the medicine Tirapazamine (TPZ) shown in the figure 1A, the synthetic carrier can be seen to successfully carry the tirapazamine medicine (TPZ), while the medicine release speed of the tirapazamine medicine carrying micelle (HA-S-S-ZnPc-ic@TPZ) prepared by the invention is slow, and the medicine release speed of the tirapazamine medicine carrying micelle is obviously increased after Glutathione (GSH) is added.
The particle size distribution of the core-shell structured tirapazamine drug carrier micelle (HA-S-S-ZnPc-ic@TPZ) prepared in example 1 is shown in fig. 2, and the TEM diagram of the drug carrier micelle (HA-S-S-ZnPc-ic@TPZ) formed by hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-levocarnitine and tirapazamine drug is shown in fig. 3 a, from which it can be seen that the drug carrier micelle (HA-S-S-ZnPc-ic@TPZ) is a spherical micelle of core-shell structure, and the map of the substance obtained by adding Glutathione (GSH) into the drug carrier micelle is shown in fig. 3 b, which shows that the added Glutathione (GSH) can decompose the HA-S-S-ZnPc-ic@TPZ core-shell micelle, thereby allowing the entrapped drug molecules to 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 the CD44 receptor HAs been previously blocked with hyaluronic acid, 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-ZnPc-ic@fitc) in different cells, and a3, b3 and c3 are superposition diagrams of nuclear staining and fluorescence distribution of the drug-loaded micelles. The phagocytic amount a & gtb & gtc shows that the drug-loaded micelle prepared by the invention HAs the effect of targeting tumor cells of the CD44 receptor, and the targeting effect is only from Hyaluronic Acid (HA) in the carrier, so that the hyaluronic acid of the micelle cannot target the tumor cells after the CDD4 receptor is pre-blocked by the hyaluronic acid in b, and the phagocytic amount is reduced; whereas HUVECs cells in c contain very little CDD4 receptor, resulting in micelles with no targeting to them and little fluorescent phagocytosis.
FIG. 5 shows the distribution of FITC-labeled hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine-levocarnitine micelles (HA-S-S-ZnPc-ic@FITC) and FITC-labeled hyaluronic acid-dithiodiacetic acid-tetraaminozinc phthalocyanine (HA-S-S-ZnPc@FITC) in the mitochondria of cells, wherein a1 is the fluorescence profile of HA-S-S-ZnPc-ic@FITC, a2 is the fluorescence profile of the dye of the mitochondria with rhodamine, a3 is the superimposed pattern of a1 and a2, b1 is the fluorescence profile of the dye of the HA-S-S-ZnPc@FITC, a2 is the superimposed pattern of the dye of the mitochondria with rhodamine, and a3 is the superimposed pattern of b1 and b 2. From the distribution situation in fig. 5, it can be seen that the shell ZnPc in the HA-S-ZnPc-Ic micelle can be targeted to mitochondria after being connected with Ic with the targeting function, so that the formed good book HAs more content distributed in mitochondria, while the pure HA-S-ZnPc does not have the targeting mitochondrial function, and the distribution amount in mitochondria is less.
FIG. 6 shows the cytotoxicity of different substances on cells with and without laser irradiation, where a is the toxicity of tirapazamine drug (TPZ), tetra-amino zinc phthalocyanine (ZnPc) and the corresponding carriers (HA-S-S-ZnPc-Ic) and drug-loaded micelles (HA-S-S-ZnPc-lc@TPZ) on cells without laser irradiation, and b is a graph of the killing effect of different concentrations of tirapazamine drug (TPZ), tetra-amino zinc phthalocyanine (ZnPc) and the corresponding carriers (HA-S-S-ZnPc-Ic) and drug-loaded micelles (HA-S-ZnPc-lc@TPZ) on 4T1 cells with laser irradiation. As can be seen from fig. 6, under the irradiation of laser, the drug-loaded micelle of HA-S-ZnPc-ic@tpz, which combines photodynamic therapy and chemotherapy, HAs the most obvious effect of killing cells.
In conclusion, the invention connects hyaluronic acid, dithiodiacetic acid, tetra-amino zinc phthalocyanine and L-carnitine through chemical action to form a tirapazamine drug carrier with a core-shell structure, wherein tetra-amino zinc phthalocyanine-L-carnitine part is taken as a core, the hyaluronic acid-dithiodiacetic acid part is taken as a shell, the contained tetra-amino zinc phthalocyanine is a photosensitizer with better nontoxic biocompatibility, a large amount of ROS can be generated to cause damage to tumor cells under the excitation of laser with the wavelength of 720nm, and the hyaluronic acid and L-carnitine can solve the application limitation of tetra-amino zinc phthalocyanine due to stronger hydrophobicity, so that carrier micelle with the particle size of about 100nm is formed, and the carrier micelle has hydrophilic, targeted cell membrane and mitochondrial double-targeting effect. Compared with a pure photosensitizer, the carrier can avoid the risk of being rapidly metabolized out by kidneys in the blood circulation process, can be enriched around tumor cell tissues through an EPR effect, HAs the characteristic of targeting CD44, can enhance the phagocytic effect of tumor cells on the carrier, can realize powerful photodynamic therapy through the photosensitizer targeting mitochondria, can finally load tirapazamine medicine (TPZ) into micelles to form a tirapazamine medicine carrying micelle with a core-shell structure, and can consume oxygen to activate the tirapazamine medicine (TPZ) through photodynamic therapy, 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 carrier HAs good application prospect in preparing the antitumor medicine.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the present invention, which is intended to be covered by the claims of the present invention.
Claims (10)
1. A tirapazamine drug carrier with a core-shell structure, which is characterized in that the carrier is hyaluronic acid-dithiodiacetic acid-tetra-amino zinc phthalocyanine-L-carnitine; the carrier is of a core-shell structure, wherein a tetra-amino zinc phthalocyanine-L-carnitine part is a core, and a hyaluronic acid-dithiodiacetic acid part is a shell;
the structural formula of the carrier is shown as follows:
2. The preparation method of the tirapazamine drug carrier with the core-shell structure as claimed in claim 1, which is characterized by comprising the following steps:
(1) Preparation of tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine: adding 2-acetic acid dithiodipyridine into N, N-dimethylformamide for dissolution, then adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for reaction for 5-30 min under the ice bath condition, removing the ice bath, continuing to activate for 30-60 min, then adding tetra-amino zinc phthalocyanine, reacting for 24-36 h under the room temperature condition, removing the solvent, and then washing with acetone to obtain tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine;
(2) Preparation of tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine-l-carnitine: dissolving tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine prepared in the step (1) in a mixed solvent of DMF and water to form tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine solution, dissolving levo-carnitine in PBS, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, activating for 1-6 h, adding the tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine solution, reacting for 12-48 h, and washing with water to obtain tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine-levo-carnitine;
(3) Preparation of hyaluronic acid-dithiodiacetic acid-tetra-amino zinc phthalocyanine-l-carnitine: dissolving hyaluronate mercapto in PBS, adding N, N-dimethylformamide under ice bath condition, mixing uniformly, continuously adding tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine-L-carnitine, stirring at room temperature for reaction for 12-48 h after ultrasonic dissolution, removing DMF through dialysis, freeze-drying, repeatedly adding DMF for ultrasonic and centrifugal treatment, and obtaining hyaluronate-dithiodiacetic acid-tetra-amino zinc phthalocyanine-L-carnitine.
3. The process according to claim 2, wherein in the step (1), the 2-acetic acid dithiodipyridine is N, N-dimethylformamide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride molar volume of N-hydroxysuccinimide and tetra-amino Zinc Phthalocyanine the ratio of the catalyst to the catalyst is 0.03-0.06:5-20:0.078:0.078:0.156, and the mol ratio of the catalyst to the catalyst is L mol.
4. A process according to claim 3, wherein the process for the preparation of 2-acetic acid dithiodipyridine is as follows:
(1) Firstly, fully dissolving dithiodipyridine in any one of ethanol, methanol or dichloromethane, and adding glacial acetic acid for oscillation; then dropwise adding 3-mercaptopropionic acid, reacting for 12-48 hours at room temperature, removing ethanol, and extracting by column chromatography to obtain 2-ethylene carboxyl dithiodipyridine, wherein the molar ratio of the dithiodipyridine to the 3-mercaptopropionic acid is 2:0.5-1;
(2) Fully dissolving the 2-ethylene carboxyl dithiodipyridine in a mixed solvent formed by dichloromethane and ethanol, vacuum extracting to remove acetic acid, and then removing ethanol, wherein the volume ratio of the dichloromethane to the ethanol is 3:1-5;
(3) Repeating the operation of the step (2) until the acetic acid is completely removed, thereby obtaining the 2-acetic acid dithiodipyridine.
5. A process according to claim 3, characterized in that the process for the preparation of tetra-amino zinc phthalocyanine is as follows:
(1) Preparation of tetranitrozinc phthalocyanine: firstly, mixing urea, 4-nitrophthalimide and ammonium molybdate according to the molar ratio of 0.1665:0.02:0.000147, grinding, heating and stirring to melt, then adding zinc acetate, continuously heating and stirring to melt, reacting at 160 ℃ until no bubbles are generated, cooling, carrying out micro-boiling treatment for 1-5 h by using hydrochloric acid with the concentration of 0.5-1.5 mol/L, filtering and washing the solid to be neutral, continuously carrying out micro-boiling treatment for 2h by using sodium hydroxide solution with the concentration of 1mol/L, filtering and washing the solid to be neutral, and repeatedly washing until a blue-violet solid is obtained, namely tetranitrozinc phthalocyanine, wherein the molar ratio of the 4-nitrophthalimide to the zinc acetate is 4:1;
(2) Preparation of tetra amino zinc phthalocyanine: adding tetranitro zinc phthalocyanine and sodium sulfide nonahydrate into DMF according to the molar ratio of 1:12-30, vacuumizing, filling nitrogen, heating to 60 ℃ under slow stirring, stirring at the constant temperature of 60 ℃ for 1-5 hours, pouring into water, mixing uniformly, filtering, repeatedly washing the solid until the washing liquid is neutral, and freeze-drying the washed solid to obtain the tetramino zinc phthalocyanine.
6. The preparation method according to claim 2, wherein the volume ratio of DMF and 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 tetra-amino zinc phthalocyanine-2-acetic acid dithiodipyridine is 8-20:22:14:100.
7. The method according to claim 2, wherein the molar volume ratio of the thiol group of hyaluronic acid, PBS, DMF and tetra-amino zinc 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 used in the dialysis is 10KD, and the rotational speed in the centrifugation is 8000rpm.
8. A tirapazamine drug-loaded micelle with a core-shell structure, which is characterized in that the carrier in claim 1 is used as a carrier, and the tirapazamine drug is 3-amino-1, 2, 4-benzotriazine-1, 4-dioxide.
9. The method for preparing the drug-loaded micelle as claimed in claim 8, wherein the method comprises the following steps:
(1) Preparing carrier micelle: adding the carrier hyaluronic acid-dithiodiacetic acid-tetra-amino zinc phthalocyanine-L-carnitine into a mixed solvent of PBS and THF with the same volume, stirring to fully dissolve the carrier hyaluronic acid-dithiodiacetic acid-tetra-amino zinc phthalocyanine-L-carnitine, and removing the THF by rotary evaporation to form a carrier micelle with a core-shell structure;
(2) Preparation of tirapazamine drug:
firstly, slowly dropwise adding a melamine solution with the concentration of 50% into o-nitroaniline with the molar volume ratio of 0.1449:18, heating to 100 ℃ after the dropwise adding is finished, cooling to room temperature after the reaction system turns dark red to precipitate orange solid, continuously slowly dropwise adding concentrated hydrochloric acid with the concentration of 12mol/L for 15min, heating to 100 ℃ until layering occurs, slowly dropwise adding NaOH solution with the concentration of 16mol/L for 15min after stirring for 30min, continuously heating to 100 ℃ for reaction until a viscous solid suspension appears, adding water, stirring, cooling to room temperature, recrystallizing to separate out yellow solid, repeatedly washing with water and ethyl acetate in sequence, and drying to obtain yellow powder, namely 3-amino-1, 2, 4-benzotriazine-1-oxide, wherein the molar volume ratio of o-nitroaniline to 50% cyanamide solution to HCl to NaOH is 0.1449:18:18:18, and mol:mL:mol:mol;
then adding glacial acetic acid into the 3-amino-1, 2, 4-benzotriazine-1-oxide, stirring until suspension appears, dropwise adding 30% hydrogen peroxide after heating to 50 ℃, reacting under the dark condition until the solution is programmed to be light red, removing the solvent, evaporating until the solution is red solid matters, and recrystallizing to obtain red crystal matters, 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 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 PBS buffer solution, dropwise adding the solution into the carrier micelle prepared in the step (1), stirring overnight after the dropwise adding, rotationally evaporating to remove THF, and dialyzing to remove the unloaded 3-amino-1, 2, 4-benzotriazine-1, 4-dioxide by adopting a dialysis bag with the molecular weight of 30KD to obtain the tirapazamine drug-carrying micelle with a 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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| CN104587485A (en) * | 2014-12-24 | 2015-05-06 | 中国科学院近代物理研究所 | Preparation method of tirapazamine-gold nanoparticle compound and application of tirapazamine-gold nanoparticle compound |
| CN109771659A (en) * | 2019-02-25 | 2019-05-21 | 天津大学 | A kind of weary oxygen responsive nano pharmaceutical carrier and its preparation method and application |
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