CN112675305A

CN112675305A - Amphiphilic molecule self-assembly nano-drug for tumor treatment and preparation method and application thereof

Info

Publication number: CN112675305A
Application number: CN202110090262.1A
Authority: CN
Inventors: 张卫; 刘奔; 赖毓霄
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2021-01-22
Filing date: 2021-01-22
Publication date: 2021-04-20
Anticipated expiration: 2041-01-22
Also published as: CN112675305B; WO2022156424A1

Abstract

The invention relates to an amphiphilic molecule self-assembly nano-drug for treating tumors, a preparation method and application thereof. Particularly discloses an amphiphilic compound which is a conjugate obtained by coupling an organic photo-thermal agent modified by a hydrazine group and an anti-tumor drug containing carbonyl through a hydrazone bond. Further, a nanoparticle is disclosed, said nanoparticle being obtained by self-assembly of the above amphiphilic compound in an aqueous solution. The nanoparticles have an anti-tumor effect, can be enriched and dissociated in a tumor region, and have a combined treatment synergistic function of photothermal treatment and chemotherapy.

Description

Amphiphilic molecule self-assembly nano-drug for tumor treatment and preparation method and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to an amphiphilic molecule self-assembly nano-medicine for treating tumors, a preparation method and application thereof.

Background

Cancer is seriously threatening the life health and safety of human beings, and attacking cancer is one of the hot problems all over the world. Although there is some progress in the treatment and prevention of cancer, the morbidity and mortality of cancer is still in a state of rapid rise, and related reports show that the mortality of lung cancer patients is as high as 18.4%, followed by colorectal cancer (9.2%), gastric cancer and liver cancer are both 8.2%; for the incidence, lung cancer ranks first, accounting for 11.6% of cancer cases, female breast cancer (11.6%), male prostate cancer (7.1%), and colorectal cancer (6.1%), so the development of new therapeutic approaches is of particular importance. The current clinical treatment means for cancer mainly include surgical resection, radiotherapy and chemotherapy. Surgical resection, as the name suggests, is to remove tumor tissue by surgery, but the residual tumor tissue after resection is very likely to cause a series of problems such as secondary recurrence of cancer. At present, the radiotherapy mainly kills tumor cells by X-rays, so that the tumor cells die, but normal body organs and tissues can be damaged to different degrees while the tumor is killed. Chemotherapy is the main means of cancer treatment at present, but the utilization rate of single chemotherapy means drugs is low, and the drug resistance problem is easy to generate, especially for patients with malignant tumors and cancer in advanced stage. Therefore, the combination therapy is adopted in clinic, the anti-tumor effect is improved through the synergistic effect of the medicines, the anti-tumor mechanisms of different medicines are inconsistent, and the generation of drug resistance can be restrained as much as possible. However, the combination therapy does not solve the inherent disadvantages of the chemotherapeutic drugs, for example, most drugs do not have the function of targeting tumor tissues, and act on normal cells while acting on tumor cells, thereby causing side effects.

With the development of nanotechnology, researchers have great potential to apply nanotechnology to tumor therapy, and have generated a great number of nanomaterials with good properties for the delivery of anticancer drugs. The drug system constructed by the nano material can be combined with various ligands and drugs, so that higher targeting and specificity are obtained, the drug resistance mechanism caused by the traditional drugs is overcome, and more importantly, the nano system can be enriched in a tumor area by means of passive or active targeting, the drug concentration in normal tissues is reduced, and the systemic toxicity is reduced.

The carrier-free self-assembly nano-drug system does not need a nano-material as a carrier, and forms the nano-drug by self-assembly of the drug, so that the theoretical drug loading rate is 100 percent, and the method is simpler and more efficient. Therefore, the invention provides the self-assembled nano-particles for treating the tumor and the preparation method thereof, which improve the utilization rate of the drug, reduce the waste of the drug, avoid the problems of increased production cost, difficult metabolism of human bodies and the like caused by introducing the nano-carrier in the manufacturing process and reduce the instability and complexity of a nano-system.

Photothermal therapy is an emerging mode for cancer treatment, most cancer cells have weaker high temperature resistance than normal cells, and irreversible damage to the cancer cells can be caused by high heat, so that the cancer cells are killed. The substance capable of raising temperature by laser irradiation is called as a photo-thermal agent, and the photo-thermal agent is enriched in a tumor area to locally generate high temperature to kill cancer cells so as to achieve the purpose of treating cancer.

Disclosure of Invention

The invention provides a self-assembled nano-medicament for treating tumors and a preparation method thereof.

The invention provides an amphiphilic compound which is a conjugate formed by coupling an organic photo-thermal agent modified by a hydrazine group and an anti-tumor medicine containing carbonyl through a hydrazone bond.

In an embodiment of the invention, the organic photo-thermal agent is selected from organic photo-thermal agents of heptamethine cyanine class, and the organic photo-thermal agent is selected from IR-780, IR-783, IR-808, IR-825, IR-1045, IR-1048, IR-1061 and IR-26.

In an embodiment of the present invention, the carbonyl-containing antitumor drug is selected from any one of doxorubicin, daunorubicin, doxorubicin B, epirubicin, idarubicin, pirarubicin, paclitaxel, docetaxel, formestane, and epothilone.

In an embodiment of the present invention, the hydrazine group in the organic photothermal agent modified with a hydrazine group and the carboxyl group in the carbonyl-containing antitumor agent are coupled by forming a hydrazone bond through a dehydration reaction.

In an embodiment of the invention, a hydrazine group modification refers to the modification with

Modifications are made wherein n is 0, 1, 2, 3, 4.

In an embodiment of the invention, hydrazine group modification is by modification of the amino group

Obtained by reaction with the chlorine group of an organic photothermal agent, having

The organic photo-thermal agent modified by the group, wherein n is 0, 1, 2, 3, 4.

In an embodiment of the present invention, the amphiphilic compound has a structural formula as shown in formula I below:

wherein n is 0, 1, 2, 3, 4;

x is

A is antineoplastic containing carbonyl, and carbon in carbonyl is coupled with nitrogen in hydrazone bond, preferably antineoplastic containing carbonyl is any one of adriamycin, daunorubicin, doxorubicin B, epirubicin, idarubicin, pirarubicin, paclitaxel, docetaxel, formestane and epothilone, structure of A is such as

And the like.

In a preferred embodiment of the present invention, the amphiphilic compound has a structural formula as shown in formula II below

In a second aspect, the present invention provides a nanoparticle obtained by self-assembly of the above amphiphilic compound in an aqueous solution.

In an embodiment of the present invention, the nanoparticle has a particle size of 40 to 200 nm.

In an embodiment of the present invention, the nanoparticle does not include a high molecular polymer.

The third aspect of the invention provides the application of the nanoparticles in preparing antitumor drugs.

The invention provides a double-effect anti-tumor medicament in a fourth aspect, wherein the medicament contains the nanoparticles.

In the embodiment of the invention, the dosage form of the double-effect anti-tumor medicament is injection, oral preparation and parenteral preparation.

In an embodiment of the invention, the tumor is selected from osteosarcoma, skin cancer, bladder cancer, ovarian cancer, breast cancer, gastric cancer, prostate cancer, colon cancer, lung cancer, bone cancer, brain cancer, rectal cancer, esophageal cancer, tongue cancer, kidney cancer, cervical cancer, uterine corpus cancer, endometrial cancer, testicular cancer, urinary cancer, melanoma, astrocytic cancer, meningioma, hodgkin lymphoma, non-hodgkin lymphoma, acute lymphatic leukemia, chronic lymphatic leukemia, acute myeloid leukemia, chronic myeloid leukemia, adult T-cell leukemia lymphoma, hepatocellular carcinoma, bronchial cancer, small cell lung cancer, non-small cell lung cancer, multiple myeloma, basal cell tumor, seminoma, chondrosarcoma, myosarcoma, fibrosarcoma.

A fifth aspect of the present invention provides a method for preparing the above amphiphilic compound, the method comprising the steps of:

1) will be provided with

A group-modified organic photothermal agent;

2) carrying out dehydration reaction on the modified organic photo-thermal agent obtained in the step 1) and carboxyl in the carbonyl-containing anti-tumor drug to form a hydrazone bond for coupling to obtain the amphiphilic compound.

In an embodiment of the invention, the organic photothermal agent in step 1) is selected from the group of heptamethine cyanine type organic photothermal agents selected from the group consisting of IR-780, IR-783, IR-805, IR-808, IR-825, IR-1045, IR-1048, IR-1061, IR-26.

In an embodiment of the present invention, the carbonyl-containing antitumor agent in step 2) is selected from any one of doxorubicin, daunorubicin, doxorubicin B, epirubicin, idarubicin, pirarubicin, paclitaxel, docetaxel, formestane, and epothilone.

In an embodiment of the invention, the reaction conditions in step 1) are under an inert atmosphere

And the organic photo-thermal agent are dispersed in an organic solvent and react to the completion.

In the embodiment of the invention, in the step 2), the reaction condition is inert atmosphere, the modified organic photo-thermal agent obtained in the step 1) and the anti-tumor medicine containing carbonyl are dispersed in an organic solvent together, and a catalyst trifluoroacetic acid is added to react at 50-80 ℃ until the reaction is complete.

In an embodiment of the invention, a purification step is also included in step 2).

In an embodiment of the invention, the compound in step 1)

Is prepared by reacting hydrazine hydrate with

Dispersing in organic solvent, and reacting to obtain the final product

The sixth aspect of the present invention provides a method for preparing the above nanoparticles, wherein the method comprises the following steps:

a) dispersing the amphiphilic compound in an organic solvent to prepare an organic solution;

b) dispersing the organic solution in an aqueous solution, and volatilizing the organic solvent to obtain a nanoparticle aqueous solution.

In an embodiment of the invention, said step b) further comprises the step of mechanically dispersing the aqueous solution.

In an embodiment of the present invention, the organic solvent in step b) is a mixed solvent of one or more organic solvents capable of dissolving the amphiphilic compound and miscible with water.

In a specific embodiment of the present invention, the organic solvent in step b) is a mixed solvent of chloroform and acetone, preferably the volume ratio of chloroform to acetone is 1: 9.

The seventh aspect of the present invention provides a use of the above amphiphilic compound in the preparation of nanoparticles for treating or preventing tumors.

Advantageous effects

1. The carrier-free self-assembly nano-drug system does not need a nano-material as a carrier, and forms nano-drugs by self-assembly of the drugs, so that the theoretical drug loading rate is 100%, the method is simpler and more efficient, the drug utilization rate is improved, and the drug waste is reduced.

2. The nanometer medicine prepared with the photothermal agent and the anticancer medicine containing carbonyl is enriched and dissociated in tumor area and has combined treating function of photothermal treatment and chemotherapy. The experimental results prove that the synergistic effect is generated on the anti-tumor effect.

Drawings

Fig. 1 is a distribution diagram of hydrodynamic diameters of nanoparticles prepared in example 2.

Fig. 2 is a transmission electron micrograph of the nanoparticles prepared in example 2.

FIG. 3 shows the dissociation effect of nanoparticles under different pH conditions.

FIG. 4 is a temperature rise curve of nanoparticles with different concentrations under laser (808nm) irradiation.

FIG. 5 is a graph of cell viability after treatment with different conditions.

FIG. 6 is a mass spectrum of Compound c obtained in example 1.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below, but the present invention is not to be construed as being limited to the implementable range thereof.

In the present invention, the structural formula of IR-780 is

The structural formula of the IR-783 is

The structural formula of the IR-808 is

The structural formula of IR-825 is

The structural formula of the IR-1045 is

The structural formula of the IR-1048 is

The structural formula of the IR-1061 is

The structural formula of the IR-26 is

In some embodiments of the invention, the amphiphilic compound is selected from

The amphiphilic compound coupled by the hydrazone bond is prepared by using the photo-thermal agent after chemical modification and the anticancer drug containing carbonyl, and the photo-thermal agent IR-780, the drug doxorubicin hydrochloride and the coupling agent methyl thioglycolate are taken as examples.

1) Compound (I)a

The synthesis of (2): under nitrogen atmosphere, methyl thioglycolate and hydrazine hydrate are dispersed in methanol, stirred at room temperature, the obtained reaction liquid is dried in a spinning mode, and the colorless transparent oily liquid, namely the compound, is obtained after chromatography and column chromatography.

2) Compound b

The synthesis of (2): and dispersing the compound a and IR-780 in chloroform under a nitrogen environment, stirring at room temperature, spin-drying the obtained reaction liquid, adding dichloromethane to dissolve, washing an organic phase with water, drying the organic phase, and evaporating to dryness to obtain a compound b.

3) Compound c

The synthesis of (2): and dispersing the compound b and doxorubicin hydrochloride in methanol under a nitrogen environment, dropwise adding a catalytic amount of trifluoroacetic acid, stirring at 60 ℃, spin-drying the obtained reaction liquid, adding dichloromethane to dissolve, washing an organic phase with water, drying the organic phase, concentrating, and performing chromatography on a column to obtain a dark green solid, namely a compound c.

EXAMPLE 1 preparation of amphiphilic Compounds

The synthetic scheme is as follows:

1) synthesis of Compound a: methyl thioglycolate (0.24g,2mmol) and hydrazine hydrate (0.24g,4.8mmol) were dispersed in 5mL of methanol under nitrogen, stirred at room temperature for 12h, and the resulting reaction solution was spun dry and chromatographed on a column to give 0.15g of a colorless transparent oily liquid, i.e., compound a, yield: 62.5 percent.

2) Synthesis of Compound b: under a nitrogen atmosphere, dispersing the compound a (0.0288g,0.24mmol) and IR-780(0.08g,0.12mmol) in 20mL of chloroform, stirring at room temperature for 12h, spin-drying the obtained reaction solution, adding dichloromethane to dissolve, washing the organic phase with water, drying the organic phase, and evaporating to dryness to obtain the compound b.

3) Synthesis of Compound c: dispersing compound b (0.04mmol) and doxorubicin hydrochloride (0.0348g,0.06mmol) in 20mL of methanol under a nitrogen atmosphere, dropwise adding a catalytic amount of trifluoroacetic acid, stirring at 60 ℃ for 48h, spin-drying the obtained reaction solution, adding dichloromethane to dissolve, washing the organic phase with water, drying the organic phase, concentrating, and performing chromatography to obtain 0.012g of a dark green solid, namely compound c, yield: 23.5 percent. The mass spectrometric detection molecular weight is 1148.56, and the mass spectrogram is shown in FIG. 6.

Example 2 preparation of nanoparticles

Dissolving 5mg of the compound c in 500 mu L of mixed solvent (50 mu L of chloroform +450 mu L of acetone), slowly dropping the mixture into 5mL of vigorously stirred deionized water, and volatilizing the organic solvent to obtain the nanoparticle aqueous solution of the compound c. The hydrodynamic diameter of the nanoparticles of compound c prepared in this example is shown in fig. 1, and the transmission electron micrograph of the nanoparticles of compound c prepared in this example is shown in fig. 2.

Example 3 dissociation experiments to simulate tumor microenvironment

The in vitro simulation of the tumor microenvironment was performed, the nanoparticle aqueous solution of the compound c was placed in a dialysis bag (cut-off 2000), and the dialysis bag was immersed in the aqueous solution at pH 5.0 and 7.5, samples were taken at intervals (12, 24, 48, 60, 72, 84, 96, 108, and 120 hours), and the degree of dissociation of the nanoparticles was calculated by measuring the ultraviolet absorbance of the precipitated doxorubicin, as shown in fig. 3, which demonstrates that the nanoparticles of the compound c have a good dissociation effect under acidic conditions.

Example 4 photothermal temperature raising ability test

Nanoparticles of Compound c were prepared into aqueous solutions of different concentrations (5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, 40. mu.g/mL) and then subjected to a laser (2W/cm) at 808nm²) The temperature rise curve of the measured solution with time under irradiation of (1) is shown in fig. 4, and the nanoparticles of the compound c are proved to have good temperature rise capability.

Example 5 results of antitumor experiments

Nanoparticles (IR-780-DOX) of Compound c, IR-780, doxorubicin hydrochloride and the cell culture solution were prepared separatelySolutions at concentrations of 1. mu.g/mL, 2.5. mu.g/mL, 5. mu.g/mL were co-cultured with 143B cells (osteosarcoma cells) for 3 hours, and a part of the co-cultured compositions in the IR-780-DOX group and IR-780 group was irradiated with 808nm laser (0.8W/cm)²) After 5min of irradiation, another part of the co-culture compositions in the IR-780-DOX group and IR-780 group and the doxorubicin group were incubated for 24h without irradiation, and the cell activity test was performed using the MTT method as shown in FIG. 5. Wherein IR-780 is a group in which IR-780 is co-cultured with tumor cells and not subjected to laser irradiation, IR-780/Lasser 5min is a group in which IR-780 is co-cultured with tumor cells and subjected to laser irradiation, Free IR-780-DOX is a group in which nanoparticles of Compound c (IR-780-DOX) are co-cultured with tumor cells and not subjected to laser irradiation, IR-780-DOX/Lasser 5min is a group in which IR-780-DOX is co-cultured with tumor cells and subjected to laser irradiation, Free DOX is doxorubicin and tumor cells and not subjected to laser irradiation. The experimental results confirmed that four groups except IR-780 had different degrees of tumor-inhibiting effects with increasing concentrations. Wherein, the adriamycin group and the IR-780-DOX/Lasser 5min group have better effect than the Free IR-780-DOX group and the IR-780/Lasser 5min group. However, if the comparison under the condition of considering the amount concentration of the substance shows that the molecular weight of IR-780-DOX is much higher than that of adriamycin, the survival rate of adriamycin under the condition of the same amount concentration of the substance is about two times that of IR-780/Lasser for 5 min. The compound c nano-particles are proved to have potential application in the aspect of treating tumors, and the compound c combines two treatment modes, realizes that a nano-particle structure can be formed without a carrier, is released under a specific pH condition, realizes the aim of passive targeting, and realizes a synergistic effect by the two effects.

Claims

1. An amphiphilic compound is characterized in that the amphiphilic compound is a conjugate formed by coupling an organic photo-thermal agent modified by a hydrazine group and an anti-tumor drug containing carbonyl through a hydrazone bond;

the organic photo-thermal agent is selected from heptamethine cyanine organic photo-thermal agents.

2. The amphiphilic compound of claim 1, wherein the organic photothermal agent is selected from the group consisting of IR-780, IR-783, IR-805, IR-808, IR-825, IR-1045, IR-1048, IR-1061, IR-26;

the antineoplastic medicine containing carbonyl is selected from any one of adriamycin, daunorubicin, aclarubicin B, epirubicin, idarubicin, pirarubicin, paclitaxel, docetaxel, formestane and epothilone.

3. The amphiphilic compound of claim 1, wherein the hydrazine group in the hydrazine group-modified organic photothermal agent and the carboxyl group in the carbonyl group-containing antitumor agent are coupled by forming a hydrazone bond through a dehydration reaction;

the hydrazine group is modified by reacting

A group-modified organic photothermal agent.

4. The amphiphilic compound of claim 1, wherein the amphiphilic compound has a structural formula as shown in formula I below:

wherein n is 0, 1, 2, 3, 4;

x is

A is antineoplastic containing carbonyl, and carbon in the carbonyl is coupled with nitrogen in a hydrazone bond, preferably, the antineoplastic containing carbonyl is any one of adriamycin, daunorubicin, doxorubicin B, epirubicin, idarubicin, pirarubicin, paclitaxel, docetaxel, formestane and epothilone.

5. A nanoparticle, wherein the nanoparticle is obtained by self-assembly of the amphiphilic compound of any one of claims 1 to 4 in an aqueous solution;

preferably, the particle size of the nanoparticles is 40-200 nm.

6. Use of the nanoparticle according to claim 5 for the preparation of an antitumor drug;

preferably, the tumor is selected from osteosarcoma, skin cancer, bladder cancer, ovarian cancer, breast cancer, stomach cancer, prostate cancer, colon cancer, lung cancer, bone cancer, brain cancer, rectal cancer, esophageal cancer, tongue cancer, kidney cancer, cervical cancer, endometrial cancer, testicular cancer, urinary cancer, melanoma, astrocytic cancer, meningioma, hodgkin's lymphoma, non-hodgkin's lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, adult T-cell leukemia lymphoma, hepatocellular carcinoma, bronchial cancer, small cell lung cancer, non-small cell lung cancer, multiple myeloma, basal cell tumor, seminoma, chondrosarcoma, myosarcoma, fibrosarcoma.

7. A dual-effect anti-tumor drug, which is characterized by comprising the nanoparticles as claimed in claim 5;

preferably, the dosage form of the double-effect anti-tumor medicament is injection, oral preparation and parenteral preparation.

8. Process for the preparation of an amphiphilic compound according to any one of claims 1 to 4, characterized in that it comprises the following steps:

1) reacting a compound with a hydrazine group with an organic photo-thermal agent to obtain the hydrazine group modified organic photo-thermal agent;

2) carrying out dehydration reaction on the modified organic photo-thermal agent obtained in the step 1) and carboxyl in the carbonyl-containing anti-tumor drug to form a hydrazone bond for coupling to obtain an amphiphilic compound;

the organic photo-thermal agent in the step 1) is selected from organic photo-thermal agents of heptamethine cyanine;

preferably, the organic photothermal agent is selected from the group consisting of IR-780, IR-783, IR-805, IR-808, IR-825, IR-1045, IR-1048, IR-1061, IR-26;

preferably, the antitumor drug containing carbonyl in step 2) is selected from any one of doxorubicin, daunorubicin, doxorubicin B, epirubicin, idarubicin, pirarubicin, paclitaxel, docetaxel, formestane and epothilone;

preferably, step 1) is to compound having a hydrazine group

Obtained by reaction with a chlorine group of an organic photo-thermal agent having a structure such as

The hydrazine group modified organic photo-thermal agent is shown, and more preferably, the reaction condition of the step 1) is to carry out reaction under inert atmosphere

Dispersing the mixture and an organic photo-thermal agent in an organic solvent together, and reacting to be complete;

preferably, the reaction condition in the step 2) is inert atmosphere, the modified organic photo-thermal agent obtained in the step 1) and the anti-tumor medicine containing carbonyl are dispersed in an organic solvent together, and a catalyst trifluoroacetic acid is added to react at 50-80 ℃ until the reaction is complete.

9. The method of claim 5, wherein the method comprises the steps of:

a) dispersing the amphiphilic compound of any one of claims 1-5 in an organic solvent to make an organic solution;

b) dispersing the organic solution in an aqueous solution, and volatilizing an organic solvent to obtain a nanoparticle aqueous solution;

preferably, the step b) further comprises the step of mechanically dispersing the aqueous solution;

preferably, the organic solvent in step b) is a mixed solvent of one or more organic solvents capable of dissolving the amphiphilic compound and being miscible with water.

10. Use of an amphiphilic compound according to any one of claims 1-5 for the preparation of nanoparticles for the treatment or prevention of tumors;

preferably, the particle size of the nano-particles is 40-200 nm;

preferably, the nanoparticles do not contain high molecular weight polymers.