阅读说明：本技术 一种响应型紫杉醇二聚体和光敏剂共组装纳米靶向给药系统及制备方法 (Response type taxol dimer and photosensitizer co-assembled nano targeting drug delivery system and preparation method thereof ) 是由 王占峰 韩海玲 谢志刚 柳时 于 2021-06-11 设计创作，主要内容包括：本发明提供一种响应型紫杉醇二聚体和光敏剂共组装纳米靶向给药系统,载体为具有生物相容性的聚合物,载体担载有前药和光敏剂形成的共组装体,前药为利用偶氮苯键将紫杉醇偶联形成的乏氧响应型紫杉醇二聚体。可以在乏氧的肿瘤微环境中特异性响应释放化疗药物紫杉醇,精准杀伤肿瘤细胞,具有更高的紫杉醇含量,减少了相关辅料的毒副作用,并且可在特定波长的辐照下消耗氧气并发挥光动力治疗效果,促进乏氧响应型紫杉醇二聚体的更多释放,实现化疗和光动力治疗的联合治疗,稳定性良好具有较大的临床转化应用潜能。(The invention provides a response type taxol dimer and photosensitizer co-assembled nano-targeting drug delivery system, wherein a carrier is a polymer with biocompatibility, the carrier carries a co-assembly formed by a prodrug and a photosensitizer, and the prodrug is an oxygen-deficient response type taxol dimer formed by coupling taxol by utilizing an azobenzene bond. The paclitaxel laser-induced hypoxia tumor cell specifically responds and releases chemotherapeutic drug paclitaxel in hypoxic tumor microenvironment, accurately kills tumor cells, has higher paclitaxel content, reduces toxic and side effects of related auxiliary materials, can consume oxygen under irradiation of specific wavelength and exert photodynamic treatment effect, promotes more release of hypoxic response type paclitaxel dimers, realizes combined treatment of chemotherapy and photodynamic treatment, has good stability and has larger clinical transformation application potential.)

1. A response type taxol dimer and photosensitizer co-assembled nano targeting drug delivery system is characterized in that: the carrier of the targeted drug delivery system is a polymer with biocompatibility, a prodrug and a photosensitizer are loaded in the carrier to form a co-assembly, and the prodrug is a hypoxia-responsive paclitaxel dimer formed by coupling paclitaxel by using azobenzene bonds.

2. The responsive paclitaxel dimer and photosensitizer co-assembled nano-targeting drug delivery system of claim 1, wherein: the photosensitizer is selected from but not limited to indocyanine green.

3. The responsive paclitaxel dimer and photosensitizer co-assembled nano-targeting drug delivery system of claim 1, wherein: the biocompatible polymer carrier is selected from any one of polymer poloxamer, poly (ethylene glycol) -polylactic acid and poly (ethylene glycol) -polycaprolactone.

4. The responsive paclitaxel dimer and photosensitizer co-assembled nano-targeting drug delivery system of claim 1, wherein: the hypoxia response type paclitaxel dimer has the following specific structural formula:

。

5. the nano-targeting delivery system of claims 1 and 4, wherein the combination of the responsive paclitaxel dimer and the photosensitizer is characterized in that: the preparation method of the hypoxia-responsive paclitaxel dimer comprises the following steps:

(1) preparing molecules connected by azobenzene bonds;

adding p-nitrobenzyl alcohol into an aqueous solution of sodium hydroxide, slowly adding zinc powder, refluxing for 10 hours under stirring, and filtering to obtain an orange solid which is a compound 1; dissolving Compound 1 in anhydrous Dichloromethane (CH)₂Cl₂) To the solution, cooling the solution to 0 deg.C, adding excess 4-Dimethylaminopyridine (DMAP) and triethylamine, and adding dropwise anhydrous CH of p-nitrophenyl chloroformate₂Cl₂Stirring the solution at room temperature for 2 hours, washing the mixture with saturated ammonium chloride solution after the reaction is completed, separating the organic layer, drying with anhydrous magnesium sulfate, and purifying the crude product by silica gel column chromatography to obtain compound 2 as an orange solid;

(2)PTX₂-preparation of Azo;

dissolving Compound 2 in anhydrous CH₂Cl₂And the solution was cooled to 0 ℃, 2 equivalents of Paclitaxel (PTX) and DMAP were added, and the mixture was stirred overnight, washed with saturated sodium bicarbonate and dried over anhydrous magnesium sulfate, and the product was purified by silica gel column chromatography to give hypoxic responsive paclitaxel dimer, abbreviated as PTX₂-Azo。

6. The nano-targeting drug delivery system with co-assembly of responsive paclitaxel dimer and photosensitizer according to claim 1, characterized in that the preparation method comprises the following steps:

mixing a polymeric carrier, PTX₂-Azo、ICGDissolving in tetrahydrofuran at a mass ratio of 7:1.5:1.5, dropwise adding the mixture into deionized water under stirring, stirring overnight at room temperature in a dark place, volatilizing the organic solvent, centrifuging the mixture to remove unassembled impurities, and dialyzing the supernatant with deionized water for two days to obtain antitumor nanoparticles M @ (PTX)₂-Azo/ICG)。

7. The use of the responsive paclitaxel dimer and photosensitizer co-assembled nano-targeting drug delivery system according to claim 1 for anti-tumor therapy.

8. The use of the responsive paclitaxel dimer and photosensitizer co-assembled nano-targeting drug delivery system according to claim 7, wherein the drug delivery system is used in combination therapy of chemotherapy and photodynamic therapy for the treatment of human cervical cancer.

9. The use of the responsive paclitaxel dimer and photosensitizer co-assembled nano-targeting drug delivery system according to claims 7 and 8, wherein the drug delivery method is injection, oral administration or topical administration.

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a response type taxol dimer and photosensitizer co-assembled nano targeted drug delivery system and a preparation method thereof, and the response type taxol dimer and photosensitizer co-assembled nano targeted drug delivery system is applied to antitumor therapy.

Background

Cancer has been a problem that afflicts human life health. Paclitaxel (PTX), a representative chemotherapeutic drug, has been widely used clinically in the treatment of breast cancer, ovarian cancer, lung cancer, and some head and neck cancers. However, PTX has poor water solubility, and its commercial injectable formulation (Taxol) is prepared by dissolving PTX in ethanol and Cremophor EL (Cremophor EL), which is a nonionic surfactant, has serious side effects and causes pain to patients. Therefore, there is an urgent need to develop new drug delivery systems to achieve better chemotherapeutic effects.

In recent years, self-assembly nanotechnology of dimer prodrug becomes an emerging highly efficient chemotherapeutic drug delivery platform. The dimer prodrug is formed by connecting two drug molecules together through a bridging molecule, and under the in vivo condition, the dimer is depolymerized to release the drug molecules to play a therapeutic role. The PTX molecule is very easy to form needle crystals, and the introduction of the bridging molecule with a flexible chain structure is equivalent to providing a structural defect for the molecule, so that the molecule can rotate more freely, the self-assembly of a dimer is promoted, and the self-assembly can be carried out in water into nano aggregates even under the condition without an auxiliary agent. Compared with PTX, the self-assembly performance of the dimer molecule enables the dimer molecule to have better compatibility with a carrier, thereby realizing ultrahigh loading of the drug and reducing the side effect related to the carrier.

In addition, single chemotherapy is susceptible to drug resistance, and the combination of multiple different treatments can significantly ameliorate this condition. Photodynamic therapy (PDT) is less invasive and less invasive than surrounding normal tissueLess damage has attracted the attention of many researchers. Generally, under light conditions, some have high singlet oxygen (ii) ((iii))¹O₂) The photosensitizer produces Reactive Oxygen Species (ROS), especially¹O₂This ROS can directly kill tumor cells.

Indocyanine green (ICG) is an FDA-approved near-infrared organic dye, commonly used for intraoperative imaging in the clinic. At present, the long wavelength absorption and safety of ICG in the absence of light irradiation have led to its extensive study as a photosensitizer in PDT, finding that it exhibits excellent ROS generation properties at 808nm radiation. However, ICG-based PDT also presents several challenges, including rapid in vivo clearance, aqueous solution instability, and photobleaching characteristics. Assembly of ICG into nanoparticles can overcome these problems to some extent.

In tumor tissue, the balance between oxygen supply and consumption is broken. On the one hand, abnormal neovascularization and poor blood flow can lead to inappropriate diffusion of oxygen in the tumor area, and on the other hand, tumor cells proliferate faster than normal cells, require more energy and oxygen to be consumed, and can also cause the tumor to become hypoxic, forming a hypoxic tumor microenvironment. Hypoxic tumor microenvironment has a great influence on tumor proliferation and drug treatment. For example, by regulating multiple genes, hypoxia promotes tumor proliferation and invasive metastasis; furthermore, in photodynamic therapy, ROS production is closely related to oxygen, and hypoxic tumor microenvironment can also limit photodynamic therapy efficacy.

However, hypoxic tumor microenvironments, both challenging and opportunistic, can construct hypoxic responsive prodrugs based on the hypoxic properties of tumors.

Disclosure of Invention

In view of the urgent need of a new paclitaxel formulation in clinic at present, the invention aims to provide a response type paclitaxel dimer and photosensitizer co-assembled nano targeting drug delivery system, which realizes the combined application of chemotherapy and photodynamic therapy and achieves better anti-tumor effect. Wherein the hypoxia-responsive paclitaxel dimer prodrug is hypoxicThe reduction occurs in the tumor microenvironment to release the active paclitaxel, thereby exerting the chemotherapy effect; at the same time, photosensitizer ICG production¹O₂Further killing tumor cells, oxygen is consumed in the process, the hypoxic degree of the tumor microenvironment is further increased, and the release of the dimer prodrug is promoted.

The purpose of the invention is realized by the following technical scheme:

a response type taxol dimer and photosensitizer co-assembled nano targeting drug delivery system is characterized in that a carrier of the targeting drug delivery system is a biocompatible polymer, a prodrug and a photosensitizer are carried in the carrier to form a co-assembly, and the prodrug is a hypoxia response type taxol dimer formed by coupling taxol through an azobenzene bond.

As a more preferred technical scheme of the invention, the structural formula of the hypoxia-responsive paclitaxel dimer is as follows:

as a more preferable technical scheme, the photosensitizer is ICG, and the ICG can be assembled into a stable nano material with PTX.

As a more preferred embodiment of the present invention, the biocompatible polymer carrier is selected from any one of the polymers poloxamer (e.g. F-127), poly (ethylene glycol) -polylactic acid, and poly (ethylene glycol) -polycaprolactone.

As a more preferred technical scheme of the invention, the preparation method of the hypoxia-responsive paclitaxel dimer comprises the following steps:

(1) preparing molecules connected by azobenzene bonds;

adding p-nitrobenzyl alcohol into an aqueous solution of sodium hydroxide, slowly adding zinc powder, refluxing for 10 hours under stirring, and filtering to obtain an orange solid which is a compound 1;

dissolving Compound 1 in anhydrous Dichloromethane (CH)₂Cl₂) And the solution is cooled to 0 ℃ and an excess of 4-dimethylaminopyridine (D) is addedMAP) and triethylamine, anhydrous CH of p-nitrophenyl chloroformate was added dropwise₂Cl₂Stirring the solution at room temperature for 2 hours, washing the mixture with saturated ammonium chloride solution after the reaction is completed, separating the organic layer, drying with anhydrous magnesium sulfate, and purifying the crude product by silica gel column chromatography to obtain compound 2 as an orange solid;

(2)PTX₂-preparation of Azo;

dissolving Compound 2 in anhydrous CH₂Cl₂And the solution was cooled to 0 ℃, 2 equivalents of PTX and DMAP were added, and the mixture was stirred overnight, washed with saturated sodium bicarbonate and dried over anhydrous magnesium sulfate, and the resulting product was purified by silica gel column chromatography to be an hypoxia-responsive paclitaxel dimer PTX₂-Azo。

The preparation method of the paclitaxel dimer and photosensitizer co-assembled nano targeted drug delivery system comprises the following steps:

mixing the polymer carrier (M), the compound PTX₂Dissolving Azo and ICG in tetrahydrofuran at a mass ratio of 7:1.5:1.5 to obtain a mixture, dropwise adding the mixture into deionized water under stirring, stirring overnight at room temperature in the absence of light, volatilizing the organic solvent, centrifuging the mixture to remove unassembled impurities, and dialyzing the supernatant against deionized water for two days to obtain anti-tumor nanoparticles M @ (PTX)₂-Azo/ICG)。

Still another object of the present invention is that the administration mode of the responsive paclitaxel dimer and photosensitizer co-assembled nano-targeting drug delivery system can be injection, oral administration or topical administration.

The invention also aims to provide a response type taxol dimer and photosensitizer co-assembled nano-targeting drug delivery system applied to anti-tumor treatment, in particular to chemotherapy/photodynamic combined treatment of human cervical carcinoma.

The beneficial effects are as follows:

m @ (PTX) provided by the invention₂The particle diameter of the-Azo/ICG) nanoparticle is about 110nm, and the nanoparticle can be greatly enriched at a tumor tissue, specifically responds to release a medicament in a tumor microenvironment, accurately kills tumor cells and inhibits tumorsGrowing and reducing damage to normal tissues and organs; the mass content of the drug is 50-80%, the drug loading is high, the usage amount of the carrier is reduced, and the toxic and side effects related to the carrier are reduced; the ICG is carried, oxygen is consumed under the irradiation of specific wavelength, a large amount of ROS are generated, the photodynamic therapy effect is exerted, the hypoxic condition of a tumor microenvironment is further aggravated due to the consumption of the oxygen, the hypoxic response type taxol dimer prodrug is promoted to release more active taxol, the chemotherapy effect is enhanced, and the combined therapy of chemotherapy and photodynamic therapy is realized.

To sum up: the anti-tumor nano particles have the advantages of good stability, high drug loading, long blood circulation time and larger clinical transformation application potential.

Drawings

FIG. 1 is the hypoxia responsive paclitaxel dimer PTX₂-synthetic roadmaps for Azo;

FIG. 2 shows an antitumor nanoparticle [email protected] (PTX) obtained by using polymer F-127 as a carrier₂-Azo/ICG) particle size diagram;

FIG. 3 is the anti-tumor nanoparticle [email protected] (PTX)₂-transmission electron micrograph of Azo/ICG);

FIG. 4 shows the killing effect of the nanoparticles on tumor cells under normoxic conditions and without light;

FIG. 5 shows the laser irradiation (0.8W/cm) at 808nm under the atmospheric condition²5 minutes), the killing effect of the nanoparticles on tumor cells;

FIG. 6 shows the killing effect of the nanoparticles on tumor cells under hypoxic conditions and without light;

FIG. 7 shows the laser irradiation (0.8W/cm) at 808nm under the hypoxic condition²5 minutes), the killing effect of the nanoparticles on tumor cells.

Detailed Description

The invention is further illustrated by the following examples and figures.

It should be noted that the following examples are illustrative and are intended to provide further explanation of the invention and not to limit the scope of the claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

The invention provides a response type taxol dimer and photosensitizer co-assembled nano targeting drug delivery system, wherein a carrier of the targeting drug delivery system is a polymer with biocompatibility, a prodrug and a photosensitizer are carried in the carrier to form a co-assembly, and the prodrug is a hypoxia response type taxol dimer formed by coupling taxol by utilizing an azobenzene bond.

Example 1

As shown in fig. 1, the preparation method of the hypoxia-responsive paclitaxel dimer according to the present invention comprises the following steps:

(1) 6.0g of p-nitrobenzyl alcohol (39mmol) was added to 70mL of aqueous sodium hydroxide (5.7M), and 5.0g of zinc powder (75mmol) was slowly added to give a mixture. The mixture was refluxed for another 10 hours with stirring, and then the solid was collected by filtration. The solid was then suspended in hot methanol until the components were completely dissolved to give a methanol solution. Finally, the resulting methanol solution was slowly cooled and dried to give an orange solid as compound 1 in 50% yield.

(2) 24.2mg (0.1mmol) of Compound 1 are dissolved in anhydrous CH₂Cl₂And the solution was cooled to 0 ℃ and excess DMAP and 34.7. mu.L triethylamine (0.24mmol) were added. 34.7. mu.L (0.24mmol) of anhydrous CH of p-nitrophenyl chloroformate was then added dropwise₂Cl₂The solution was stirred at room temperature for 2 hours to give a mixture, and the degree of reaction was monitored by thin layer chromatography. The mixture was washed with saturated ammonium chloride solution and the organic layer was separated. Finally, dried over anhydrous magnesium sulfate and evaporated under reduced pressure, the crude product was purified by silica gel column chromatography to give compound 2 as an orange solid.

(3) 40mg (0.07mmol) of Compound 2 was dissolved in 10mL of anhydrous dichloromethane, and the solution was cooled to0 ℃ is used. 66.8mg of PTX (2 equiv.) and 9.6mg of DMAP were added and the mixture was stirred overnight. Then, the reaction mixture was washed with saturated sodium hydrogencarbonate and dried over anhydrous magnesium sulfate. The product was purified by silica gel column chromatography to give 45.8mg of hypoxia responsive paclitaxel dimer PTX₂-Azo, yield 80%.

Example 2 the antitumor nanoparticles M @ (PTX)₂-Azo/ICG) the preparation method is as follows:

7mg of F-127, 1.5mg of PTX₂-Azo, 1.5mg of ICG in 4mL of tetrahydrofuran, then the mixture is added dropwise to 10mL of deionized water with stirring and stirred overnight at room temperature in the dark. After evaporation of the organic solvent, the mixture was centrifuged at 3500r/min for 5 minutes to remove unassembled material. Finally, the supernatant was dialyzed against deionized water for two days (MWCO: 3.5kDa) to give anti-tumor [email protected] (PTX)₂-Azo/ICG) nanoparticles.

As shown in FIG. 2, [email protected] (PTX) was obtained by dynamic light scattering₂-Azo/ICG) nanoparticles having a particle size of about 110nm and a zeta potential of negative potential (-18.3 ± 2.1 mV).

Determination of [email protected] (PTX) by Transmission Electron microscopy₂The apparent form of Azo/ICG) is shown in FIG. 3, [email protected] (PTX)₂the-Azo/ICG) antitumor nanoparticles have complete spherical morphology and relatively uniform size.

Example 3, [email protected] (PTX)₂And (4) -Azo/ICG) nanoparticle in vitro killing ability of tumor cells.

[email protected] (PTX) was assessed by tetramethylazodicarbonyl blue (MTT) analysis₂-Azo/ICG) cytotoxicity of nanoparticles on human cervical carcinoma cells HeLa. After resuspending the tumor cells, they were seeded in 96-well plates and incubated overnight. Then, with 100. mu.L of [email protected] (PTX) at various concentrations₂Azo/ICG) in place of the original medium. In the normal atmosphere (21% O)₂) Or hypoxia (1% O)₂) After 6 hours of incubation under the conditions of (1), the cells were placed in the dark or irradiated with 808nm laser for 1 minute (0.8W/cm)²). After another 48 hours of incubation under normoxic or hypoxic conditions, the viability of the tumor cells was assessed using a BioTek ELx808 microplate reader.

At normal oxygenUnder the condition without illumination, the killing effect of the nanoparticles on tumor cells is shown in figure 4; under the condition of normal oxygen and 808nm laser irradiation (0.8W/cm)²5 minutes), the killing effect of the nanoparticles on tumor cells is shown in fig. 5; under hypoxic conditions and without light, the killing effect of the nanoparticles on tumor cells is shown in fig. 6; under the condition of hypoxia and laser irradiation of 808nm (0.8W/cm)²5 minutes), the killing effect of the nanoparticles on tumor cells is shown in fig. 7.

According to the above, it can be seen that: under the hypoxic condition, the tumor cells are killed optimally by specific wavelengths, which shows that the hypoxic condition of a tumor microenvironment is further aggravated due to the consumption of oxygen, promotes the hypoxic-responsive paclitaxel dimer prodrug to release more active paclitaxel, enhances the effect of chemotherapy, and realizes the combined treatment of chemotherapy and photodynamic therapy.