CN102349999A - Multifunctional adriamycin precursor medicament as well as preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of new drug preparation, and in particular relates to a multifunctional doxorubicin prodrug, its preparation method and the application of the doxorubicin prodrug in cancer cell targeting, pH stimulation response and inhibition of cancer cell activity. The drug carrier is a complex of ZnO nanoparticles and doxorubicin, ZnO forms O-Zn-O complex particles with doxorubicin as a Zn2+ source, and the mass loading capacity of doxorubicin in the doxorubicin prodrug 4% to 20%. The invention reduces the toxicity to normal cells, improves the targeting property of the drug, and provides a new idea for the exploration of anticancer drugs.

Description

A kind of multifunctional doxorubicin prodrug, preparation method and application thereof

technical field

The invention belongs to the technical field of new drug preparation, and in particular relates to a multifunctional doxorubicin prodrug, its preparation method and the application of the doxorubicin prodrug in cancer cell targeting, pH stimulation response and inhibition of cancer cell activity.

Background technique

Doxorubicin (DOX) is a spectrum anti-tumor antibiotic, it can inhibit the synthesis of RNA and DNA, and its inhibitory effect on RNA is relatively strongest. Tumor cells in each growth cycle have a killing effect. Clinically widely used in breast cancer, lung cancer, ovarian cancer, osteosarcoma, neuroblastoma, bladder tumor, thyroid tumor, rhabdomyosarcoma, Wilms tumor, prostate cancer, soft tissue sarcoma, acute lymphoblastic leukemia, testicular cancer , Choriocarcinoma, gastric cancer, liver cancer, etc. However, like most anti-tumor drugs, it is prone to drug resistance and has poor selectivity to cancer cells. While acting on diseased cells, it will also have toxic and side effects on non-diseased cells. The side effects include nausea, hair loss, A series of toxic and side effects such as leukopenia, local congestion, sensitivity to light, discoloration of urination, etc., have encountered bottlenecks in clinical application. Therefore, the targeted therapy of tumors and the controlled release of drugs are proposed to minimize the damage of drugs to normal cells. Targeting refers to the selective concentration of drugs used for treatment in diseased tissues, so that the damage to normal tissues can be minimized, and controlled release refers to the release of drugs in a specific environment. No release or even zero release.

Modern nanotechnology has been widely used in medical treatment. The targeted positioning of tumor cells after the combination of nanoparticles and drugs, the specific binding of modified nanoparticles to the receptors of targeted cells (ie cancer cells) overexpress malignant tumor cells, can greatly increase the therapeutic effect of drugs, the maximum To reduce the side effects on normal cells, but the current technology focuses more on the selective targeting of cancer cells, while ignoring the release in the pathophysiological area of diseased cells. Semiconductor quantum dots have excellent qualities as biological nanoprobes. In recent years, there have been many reports on quantum dots as probe molecules and as nano drug carriers. O.C.Farokhzad et al. published on NanoLett. (2007, pages 3065-3070) using quantum dots with CdSe/ZnS core structure as A However, the obvious cytotoxicity of cadmium quantum dots limits the application of this system in biomedicine. ZnO has become an ideal biomaterial due to its low price and good biocompatibility. However, the preparation of ZnO nanoparticles has always been a challenging subject. Ying-Song Fu et al reported that ZnO nanoparticles with surface-modified oleic acid molecules have strong blue light emission in J.Am.Chem.Soc. (2007, pages 16029-16033), but the hydrophobicity of the product limits It is applied in the field of biomedicine. Therefore, the preparation of nano-drug systems with good water solubility, biocompatibility, and ability to target and release the lesion is still facing great challenges.

Contents of the invention

The present invention aims at the above-mentioned deficiencies in the prior art, and provides a functional doxorubicin prodrug using ZnO nanoparticles and doxorubicin through covalent bonds, its preparation method, and the prodrug in targeting cancer cells. Cellular, pH-stimuli-responsive, and cancer-cell-inhibiting applications.

The ZnO nano particles prepared by the invention have uniform size, and the ZnO nano particles have good water solubility, high quantum yield and monodisperse through ligand exchange reaction on the quantum dot surface. The invention reduces the toxicity to normal cells, improves the targeting property of the drug, and provides a new idea for the exploration of anticancer drugs.

At present, a large number of studies have proved that the number and activity of folate receptors in most tumor cells far exceeds that of normal cells. Therefore, folate receptors in cancer cells have received extensive attention, and folic acid compounds can be used as targeting molecules for tumor cells. In this patent, after the surface of ZnO quantum dots is modified with functional groups, folic acid molecules are grafted, so that the ZnO nanoparticles have a targeting effect on cancer cells. In addition, the pH value of the normal tissue environment is neutral to slightly alkaline (about 7.4), but the pH value of the surrounding environment of tumor cells is about 6.0, while the pH value of the internal environment of tumor cells, such as lysosomes and endosomes, is about 4.5 ~6.5, so the ZnO nanoparticles can quickly dissolve and release the drug at this acidity value, and exist stably at pH7.4, so the drug carrier can be used to achieve pH-responsive release.

The multifunctional doxorubicin prodrug of the present invention is characterized in that: it is a complex of ZnO nanoparticles and doxorubicin, and Zn ²⁺ on the surface of ZnO nanoparticles is complexed with doxorubicin drug molecules to form a A new prodrug, in which the mass loading of doxorubicin is 4% to 20%, and cancer cell targeting molecules are grafted on the surface of ZnO, which can realize the targeted delivery of drugs to cancer cells .

Multifunctional doxorubicin prodrug of the present invention, its preparation method is as follows:

Step 1: Synthesis of ZnO nanoparticles

Mix zinc acetate and magnesium acetate with a molar ratio of 10.0 to 40.0:0 to 2.0 and dissolve them in solvent 1 at 45°C to 80°C to obtain a solution of zinc acetate and magnesium acetate. The molar ratio of solvent 1 to zinc acetate is 250 to 300: 1~3; in another container, dissolve the alkali in the same temperature and the same type of solvent 1 to prepare an alkali solution with a molar concentration of 0.1~0.5mol/L; put the zinc acetate and magnesium acetate solutions in Under ice-bath conditions, the alkali solution is quickly added dropwise to the zinc acetate and magnesium acetate solutions, the volume ratio of the alkali solution to the zinc acetate and magnesium acetate solutions is 1:1 to 3, and then the mixed solution is stirred for 5 to 10 hours, Discard the supernatant after precipitating with a precipitant to obtain ZnO nanoparticles with a particle size of 3-4 nm;

The solvent 1 is absolute ethanol or n-hexane, the alkali is NaOH or KOH, and the precipitant is n-hexane or acetone.

Step 2: Synthesis of amino-modified ZnO nanoparticles (ZnO-NH ₂ )

Take 100 mg of ZnO nanoparticles obtained in the first step, ultrasonically disperse them in 10-50 mL of solvent 2, then add 5-20 μL of aminosilane coupling agent into the above solution, and stir at 80-120 °C for 1-24 hours to obtain Amino-modified ZnO nanoparticle solution; then place the amino-modified ZnO nanoparticle solution in a centrifuge for separation, discard the centrifugate, wash the precipitate obtained after centrifugation with solvent 3, and then redisperse it in 10mL In water, a clear and transparent amino-modified ZnO nanoparticle solution (ZnO—NH ₂ ) was obtained.

The solvent 2 described in the second step is anhydrous N, N'-dimethylformamide (DMF), toluene or dimethylsulfoxide (DMSO); the aminosilane coupling agent is 3-aminopropyl Amino group-containing silane coupling agents such as triethoxysilane, Y-aminopropyltrimethoxysilane, etc.; the solvent 3 is DMF or absolute ethanol.

Step 3: Synthesis of surface-modified ZnO nanoparticles (ZnO-FA)

Dissolve 0.5-2 mg of folic acid in 0.5-5 mL of DMSO or DMF, then take 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with the same mass as folic acid and add Add the folic acid-containing solution above, stir or sonicate for 10-60 minutes to obtain a carboxyl-activated folic acid solution; then add the carboxyl-activated folic acid solution to the clear and transparent solution described in the second step, and continue stirring at room temperature for 3 For ~6 hours, make the activated carboxyl group in folic acid complex with the amino-modified ZnO nanoparticles (ZnO-NH ₂ ) through an amide bond to obtain ZnO nanoparticles with surface-modified folic acid molecules, and add water to make up to 10 mL;

Step 4: Synthesis of functional doxorubicin prodrug

Mix the solution of the surface-modified ZnO nanoparticles of folic acid obtained in the third step with a concentration of 1 mg/mL doxorubicin solution, the volume ratio of the two is 1-2: 1-3, and stir for 2-24 hours, the Zn atoms on the ZnO surface Form a complex (O-Zn-O) with doxorubicin, centrifuge, and dry at 25°C to 60°C to obtain ZnO nanoparticle powder combined with doxorubicin molecules, that is, a functional doxorubicin prodrug. The loading amount of doxorubicin in the doxorubicin prodrug is 4%-20% by mass fraction.

Prepare multiple sets of standard concentrations of doxorubicin solutions in aqueous solution, respectively test the UV-visible spectra of multiple sets of standard concentrations of doxorubicin solutions, and perform linear fitting to obtain the standard equation of doxorubicin solution concentration and UV-visible spectra Centrifugal separation of ZnO powder and centrifugation solution (not participating in complexing doxorubicin solution) of complexed doxorubicin, after constant volume, test its ultraviolet-visible spectrum, by standard equation, obtain the concentration of the doxorubicin solution that does not participate in complexed concentration, and then calculate the loading amount of doxorubicin on the ZnO nanoparticles.

Description of drawings

Fig. 1: the wide-angle XRD powder diffraction pattern of the sample prepared in embodiment 2;

Curve a is the wide-angle XRD powder diffraction pattern of ZnO- _NH2 , and curve b is the wide-angle XRD powder diffraction pattern of ZnO- _NH2 surface-modified folic acid, from which it can be seen that the ZnO nanoparticles combined with folic acid still maintain their original crystals structure.

Fig. 2: the transmission electron micrograph (different magnifications) of the ZnO nanoparticle of the monodisperse, size uniform 3～4nm surface modification folic acid prepared in embodiment 2; Can find out that ZnO nanoparticle presents under the high resolution transmission electron microscope Clearly latticed.

Fig. 3: the ZnO-NH that embodiment ₂ prepares EDS spectrogram of quantum dot;

The peak of silicon appears at 1.7 keV in the spectrum, which can prove that 3-aminopropyltriethoxysilane (APTES) is successfully coated on the surface of ZnO;

Fig. 4: the ZnO-NH that embodiment ₂ prepares Quantum dot (c), folic acid (b), the ZnO nanoparticle (a) of surface modification folic acid The infrared spectrogram;

Among them, 1115cm ^-1 and 1030cm ^-1 are silicon-oxygen bond vibrations, 3446cm ^-1 and 1640cm ^-1 are stretching vibrations of nitrogen-hydrogen groups and bending vibrations of amino groups, 2936cm ^-1 and 2880cm ^-1 are propyl groups in APTES The CH stretching vibration peak of the modified folic acid ZnO nanoparticles is due to the disappearance of the 3446 cm ^-1 peak of the amino group and the appearance of the peak at 2578 cm ^-1 and the appearance of the oxygen-hydrogen vibration peak of the carboxyl group at 3110 cm ^-1 , all of which indicate that folic acid Molecules were successfully modified on the particle surface.

Figure 5: ZnO nanoparticles with surface-modified folic acid prepared in Example 2 (a), surface-modified folic acid and ZnO nanoparticles complexed with doxorubicin molecules (b) were added to HeLa 60 cervical cancer cells at different doses, The histogram of the MTT test (cytotoxicity test) results of the independent doxorubicin drug (c);

It shows that the surface-modified ZnO nanoparticles of folic acid show obvious cytotoxicity to cancer cells at 50 μg/mL, indicating that the surface-modified ZnO nanoparticles of folic acid have obvious cancer cell toxicity at this concentration, and can be used as adjuvant cancer therapy; The ZnO nanoparticles modified with folic acid and complexed with doxorubicin, when the concentration was 25 μg/mL, the survival rate of HeLa cells was 28.5%, and the prodrug system showed a good inhibitory effect on cancer cells.

Detailed ways

The embodiments of the present invention are described in detail below: the present embodiment is implemented under the premise of the technical solution of the present invention, and detailed implementation and specific operation process are provided, but the protection scope of the present invention is not limited to the following implementation example.

Example 1:

1) Dissolve 2.0mmol (440mg) zinc acetate in 0.51mol (30mL) absolute ethanol at 50°C; in another flask, dissolve NaOH in absolute ethanol at 40°C to make a concentration of 0.5mol/L Alkali solution, 0.5mol/L, 17mL NaOH absolute ethanol solution was quickly added dropwise to the absolute ethanol solution of zinc acetate under ice bath conditions, the mixture was stirred for 8 hours, and n-hexane was used as precipitant to precipitate out ZnO quantum dots, then discard the supernatant to obtain ZnO quantum dots;

2) Take 100 mg of ZnO nanoparticles separated in step 1), and disperse them in 10 mL of DMF by ultrasound, then add 3-aminopropyltriethoxysilane (5 μL) to the above solution, and stir the reaction at 120 °C for 3 Hours, the obtained amino modified ZnO nanoparticle solution was placed in a centrifuge for separation, the centrifuged liquid was discarded, the precipitate obtained after centrifugation was washed with DMF, and then redispersed in 10mL water to obtain a clear and transparent amino Modified ZnO nanoparticle solution (ZnO-NH ₂ );

3) 0.5 mg of folic acid was dissolved in 0.5 mL of DMSO, and then 0.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added to the above solution containing folic acid, Stir for 30 minutes to obtain carboxyl-activated folic acid DMSO solution. Then the carboxyl-activated folic acid solution is added to the clear and transparent amino-modified ZnO nanoparticle solution obtained in the step 2), and the stirring is continued for 3 hours to obtain the ZnO nanoparticle solution of surface-modified folic acid molecules; 10mL.

4) Mix the surface-modified folic acid ZnO nanoparticles solution (5 mL) obtained in step 3) with an aqueous solution of doxorubicin (10 mL, 1 mg/mL) and stir for 24 hours, and the surface-modified folic acid ZnO nanoparticles are complexed with doxorubicin , and then centrifuged to separate the surface-modified folic acid ZnO nanoparticle powder complexed with doxorubicin from the uncomplexed doxorubicin solution. Dilute the separated solution of uncomplexed doxorubicin to 10 mL with water, and test its ultraviolet-visible spectrum.

Prepare the doxorubicin solution of the standard concentration of different concentrations in aqueous solution, test the ultraviolet-visible spectrum of solution, take the absorption peak at 481nm place as reference point, carry out linear fitting, obtain the aqueous solution standard equation of doxorubicin (A=0.0242+ 0.018C, R ² =0.9998, A: the absorbance value of the solution, C: the concentration of doxorubicin).

Finally, the UV spectrum of the isolated solution of doxorubicin not complexed with ZnO was compared with the standard equation of doxorubicin in aqueous solution, and it was calculated that the loading amount of doxorubicin on ZnO nanoparticles was 19.8% by mass fraction.

Example 2:

The zinc acetate in step 1) of Example 1 was replaced with a mixture of 2.0 mmol of zinc acetate and 0.2 mmol of magnesium acetate, and other conditions were unchanged, and ZnO nanoparticles similar to those of Example 1 were obtained.

Example 3:

The absolute ethanol in step 1) of Example 1 was replaced by n-hexane, the precipitating agent was replaced by acetone, and other conditions remained unchanged, and ZnO nanoparticles similar to those in Example 1 were obtained.

Example 4:

The 0.5 mol/L NaOH in step 1) in Example 1 was replaced with 0.1 mol/L KOH, and other conditions were unchanged, to obtain ZnO nanoparticles similar to Example 1.

Example 5:

Replace the 3-aminopropyltriethoxysilane (5 μL) in step 2) in Example 1 with 3-aminopropyltriethoxysilane (20 μL), and keep the other conditions unchanged to obtain a mixture similar to that in Example 1. of ZnO nanoparticles.

Embodiment 6:

The 3-aminopropyltriethoxysilane in step 2) in Example 1 was replaced with Y-aminopropyltrimethoxysilane, and other conditions remained unchanged, and ZnO nanoparticles similar to those in Example 1 were obtained.

Embodiment 7:

The 0.5 mg folic acid in step 3) in Example 1 was replaced with 2 mg folic acid, the DMSO was replaced with DMF, and other conditions remained unchanged, and ZnO nanoparticles similar to those in Example 1 were obtained.

Embodiment 8:

The doxorubicin solution (10mL, 1mg/mL) in step 4) in Example 1 was replaced with doxorubicin solution (2mL, 1mg/mL), stirred for 2 hours, the loading capacity of ZnO nanoparticles to doxorubicin 4% for mass fraction.

Test the invention

In vitro release of the present invention at different pH values

The ZnO nanoparticle powder loaded with doxorubicin drug obtained in Example 1 was respectively dispersed in 5mL, pH=7.4 phosphate buffer and 5mL, pH=5.0 acetate buffer, respectively packed in dialysis bags (molecular weight 3000), the dialysis bags were put into 20mL beakers containing the same buffer as the solution in the dialysis bags, and released at 37°C for 3 hours. Regularly draw a certain amount of solution outside the dialysis bag, test the UV-Vis spectrum, and add corresponding new buffer solution.

In pH=7.4 phosphate buffer solution and pH=5.0 acetic acid buffer solution, prepare the doxorubicin solution of standard concentration respectively, and test the ultraviolet-visible (UV-Vis) spectrum of solution respectively, carry out linear fitting, obtain doxorubicin respectively Standard equation in pH=7.4 phosphate buffer and pH=5.0 acetate buffer. (Phosphate buffer (pH=7.4): A=0.0271+0.017C, R ² =0.9996; acetate buffer (pH=5.0): A=0.0431+0.016C, R ² =0.9996)

The UV-Vis spectra measured after release in different buffer solutions were compared with the standard equations of corresponding buffer solutions. The release amount of doxorubicin drug determined by calculation is respectively, the release amount in the phosphate buffer solution of pH 7.4 is 0%, and the release amount in the acetate buffer solution of pH 5.0 is 100%, indicating that the prodrug is in It can be effectively released under weak acid conditions and exert its medicinal effect, but it can exist stably in normal body fluids, and has potential applications in the selective treatment of cancer cells.

The cultivation of HeLa cells (human cervical cancer cell line, purchased by Shanghai Institute of Cell Biology, Chinese Academy of Sciences) The culture of HeLa cells is that the mass fraction is 10% (v/v) fetal bovine serum DMEM sugar medium, and adding green chain Mycin double antibody solution (concentrations are 100U mL and 100 μg mL ^-1 ), the culture environment is CO ₂ , the humidity mass fraction is 5%, and cultured in a 37°C incubator for 24 hours.

Cytotoxicity Test (MTT Test)

The cultured HeLa cells were inoculated in a 96-well plate, 100 uL per well, the number of cells was 1*104/well, and the inoculation was done for 24 hours before use. Remove the medium, add ZnO nanoparticles (a) of surface-modified folic acid in different doses (the materials are all obtained in Example 2, and the dosage is shown in Figure 5), and ZnO nanoparticles (a) of surface-modified folic acid and complexed with doxorubicin molecules (b), doxorubicin alone (c), each concentration has three parallel wells, the system was incubated at 37°C, with a mass fraction of 5% CO ₂ and 95% humidity for 48 hours, and the culture was completed for 4 hours Add 10 μL of MTT (5 mg/mL, tetramethylazolazolium blue) solution before, and after 4 hours, the amber dehydrogenase MTT in the mitochondria of the surviving cells is reduced to insoluble blue crystal formazan, and 100 μL of the solution (containing 10 %SDS of 10mM hydrochloric acid), incubated overnight in a 37°C incubator, detected the absorbance at 570nm, and calculated the cell viability, the results are shown in Figure 5.

Claims (5)

1. multi-functional adriablastina prodrug, it is characterized in that: this pharmaceutical carrier is the complex of ZnO nanoparticle and amycin, and ZnO is as Zn ²⁺Source and amycin form O-Zn-O complexation particle, the quality loading 4%～20% of amycin in adriablastina prodrug.

2. the method for preparing of the described a kind of multi-functional adriablastina prodrug of claim 1, its step is following:

(1) synthetic ZnO nanoparticle

Be 10.0～40.0: 0～2.0 zinc acetate with mol ratio and be dissolved in 45 ℃～80 ℃ solvent 1 after magnesium acetate mixes, obtain zinc acetate and magnesium acetate solution, solvent 1 is 250～300: 1～3 with the mol ratio of zinc acetate; In another container, alkali is dissolved in the solvent 1 of uniform temp, identical type, be made into the aqueous slkali that molar concentration is 0.1～0.5mol/L; Zinc acetate and magnesium acetate solution are placed under the condition of ice bath; Aqueous slkali is added drop-wise in zinc acetate and the magnesium acetate solution rapidly again; The volume ratio of aqueous slkali and zinc acetate and magnesium acetate solution is 1: 1～3; Then mixed liquor was stirred 5～10 hours; With precipitant post precipitation abandoning supernatant; Obtain the ZnO nanoparticle, its particle diameter is 3～4nm;

Described solvent 1 is dehydrated alcohol or normal hexane, and described alkali is NaOH or KOH, and described precipitant is normal hexane or acetone;

(2) the ZnO nanoparticle of synthesizing amino modification

Get the ZnO nanoparticle 100mg that step (1) obtains; Ultra-sonic dispersion is in 10～50mL solvent 2; Then 5～20 μ L amino silicane coupling agents are added in the above-mentioned solution, under 80～120 ℃ of temperature, stirred 1～24 hour, obtain through amido modified ZnO nano-particle solution; To place centrifuge to separate through amido modified ZnO nano-particle solution then; Discard centrifugal liquid; The deposition that obtains after centrifugal is washed through solvent 3, then it is dispersed in the 10mL water again, obtain the amido modified ZnO nano-particle solution of clear;

Solvent 2 is anhydrous N, N '-dimethyl formamide DMF, toluene or dimethyl sulfoxide DMSO; Described amino silicane coupling agent is 3-aminopropyl triethoxysilane or Y-aminopropyl trimethoxysilane; Described solvent 3 is DMF or dehydrated alcohol;

(3) synthetic surface is modified the ZnO nanoparticle of folic acid

The folic acid of 0.5～2mg is dissolved among the DMSO or DMF of 0.5～5mL; Get then with quality such as folic acid 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride join in the above-mentioned solution that contains folic acid; Stir or ultrasonic 10～60 minutes, obtain the folic acid solution of activated carboxylic; Folic acid solution with activated carboxylic joins in the solution of the clear described in second step again; Continue under the room temperature to stir 3～6 hours; Make activatory carboxyl in the folic acid and amido modified ZnO nanoparticle through the amido link complexing; Obtain the ZnO nanoparticle of finishing folic acid, add water and be settled to 10mL;

(4) complex functionality property adriablastina prodrug

Is that 1mg/mL amycin solution mixes with the solution of the ZnO nanoparticle of the finishing folic acid of step (3) gained with concentration, and the two volume ratio is 1～2: 1～3, stirs 2～24 hours, and ZnO is as Zn ²⁺Source and amycin form the O-Zn-O complexation, through centrifugal, and 25 ℃～60 ℃ dryings, the ZnO nanoparticle powder of amycin that obtained complexation, promptly functional adriablastina prodrug, the amycin loading is a mass fraction 4%～20% in adriablastina prodrug.

3. the application of the described a kind of multi-functional adriablastina prodrug of claim 1 aspect target cancer cell.

4. the application of the described a kind of multi-functional adriablastina prodrug of claim 1 aspect the pH stimuli responsive.

5. the application of the described a kind of multi-functional adriablastina prodrug of claim 1 aspect the anticancer activity.

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