CN115501348A - Nano-drug carrier with anticancer activity and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biomedicine, and in particular relates to a nano drug carrier with anticancer activity and its preparation method and application. Specifically, it is a method and technology for using glucose oxidase (GOx) to modify metal-organic framework (MOFs) materials. The modified MOFs materials have good dispersion and long-term stability in water, and the modified MOFs can be used as drug carriers. One or more other drugs or biomolecules. The invention loads nano-silver particles on the surface of MOFs modified by GOx, realizes uniform dispersion and loading of silver nanoparticles in MOFs, and has excellent near-infrared light absorption and photothermal conversion performance after the combination of the two. The nanomedicine has excellent starvation and photothermal synergistic therapeutic properties in both cell and animal experiments, which can significantly reduce the survival rate of cancer cells and improve the therapeutic effect of cancer.

Description

A nano drug carrier with anticancer activity and its preparation method and application

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to a nano drug carrier with anticancer activity, its preparation method and application.

Background technique

As a major disease that affects the health of the body, cancer has brought great distress to patients themselves and their families. Cancer treatment has always been the focus of technical research and development in the field of medicine. The most commonly used and most conventional treatment methods in clinical practice are still surgical treatment, chemotherapy and radiotherapy, etc., but the above-mentioned treatments will have a relatively serious negative impact on the patient's body, there will be more side effects, and may even have a negative effect on the patient's body. Serious damage to the patient's body. In order to reduce the toxic and side effects of conventional therapy on the patient's body, the field of pharmaceutical development has been researching new treatments, such as photodynamic therapy, sonodynamic therapy, photothermal conversion therapy and chemodynamic therapy, which have been proven to have greater therapeutic effects. The prospect is applied to the treatment of cancer. However, these "new" treatments all need to rely on a suitable drug delivery system to deliver the drug to the lesion. Therefore, the research and development of a mature drug delivery system has become the key to the promotion and application of the above-mentioned therapies in clinical cancer treatment.

Metal-organic frameworks (MOFs), as a crystalline porous material composed of metal ions/clusters and organic ligands, have the characteristics of high porosity, adjustable pore size, and huge specific surface area. , biosensing, drug delivery and other aspects have good application prospects. The large specific surface area of MOFs can adsorb and deliver small molecule drugs such as chemotherapy drugs, photosensitizers, fluorescent dyes, etc., and can also be used to immobilize or encapsulate macromolecular drugs such as biological enzymes, which can realize chemotherapy, photodynamic therapy and biomedical imaging. Combined treatment of multidimensional states. However, as biomaterials, MOFs are easy to agglomerate in water, making them large in size and difficult to be taken up by cells, and their instability in water will further lead to their skeleton collapse and decomposition during drug delivery. Or there is an early release of the load. The biological stability and dispersibility of MOFs have limited their application in nano-drug delivery. Therefore, the research and development of new functional modification methods has become an urgent problem to be solved.

Glucose oxidase (GOx), as an endogenous biological enzyme, can effectively consume glucose in cells, thereby inhibiting cell growth and activity. Delivering GOx into cancer cells can achieve starvation therapy. This therapy has no toxic side effects, The advantages of remarkable anticancer effect. In addition, the surface of GOx has many hydrophilic groups, which has good dispersibility in water, and has broad research prospects in the field of anticancer.

Based on previous investigations, there is still a lack of public reports and technical research on the use of GOx to modify MOFs and simultaneously achieve starvation therapy, and there is no research method and method for simultaneous modification of MOFs with GOx and nano-metal particles for starvation/photothermal synergistic therapy of cancer. technology.

Contents of the invention

Aiming at the problems existing in the clinical application of existing innovative therapies, the present invention realizes the construction of a multifunctional system for cancer starvation therapy and drug delivery through the modification of metal-organic frameworks (MOFs) by glucose oxidase (GOx); the MOFs modified by GOx The loading of nano-silver particles realizes the construction of a multi-dimensional therapeutic system for cancer starvation therapy, photothermal synergistic therapy, and drug delivery. Through the functional modification of MOFs, not only the long-term stability and water dispersibility of MOFs materials in vivo can be significantly improved, but also the release controllability of MOFs materials can be achieved, so as to realize the controllable release of drugs and achieve targeted The multidimensional treatment of cancer reduces the toxic and side effects during treatment.

In order to realize the above-mentioned purpose of the invention, the specific technical scheme of the present invention is as follows:

In one aspect, the present invention provides a nano-drug carrier with anticancer activity, the drug carrier is a metal-organic framework MOFs nano-drug carrier modified with glucose oxidase GOx, represented by MOFs@GOx.

Further, the MOFs are selected from UiO-66, MOF-808, MOF-5, HKUST-1, MIL-125, NH ₂ -MIL-125; wherein UiO-66 is formed by reacting terephthalic acid with zirconium oxychloride The prepared metal-organic framework, MOF-808 is a metal-organic framework prepared by the reaction of trimesic acid and zirconium tetrachloride, MOF-5 is a metal-organic framework prepared by the reaction of terephthalic acid and zinc nitrate, HKUST -1 is a metal-organic framework prepared by the reaction of trimesic acid and copper nitrate; MIL-125 is a metal-organic framework prepared by the reaction of terephthalic acid and tetrabutyl titanate; NH ₂ -MIL-125 is a Metal-organic frameworks prepared by the reaction of 2-aminoterephthalic acid and tetrabutyl titanate.

Further, the MOFs@GOx is selected from UiO-66@GOx, MOF-808@GOx, MOF-5@GOx, HKUST-1@GOx, MIL-125@GOx, NH ₂ -MIL-125@GOx.

Further, the particle size of the nano-drug carrier with anticancer activity is 10 nm to 500 nm; preferably, the particle size is 10 to 100 nm.

In another aspect, the present invention provides a method for preparing the nano drug carrier, the method comprising the steps of:

Glucose oxidase is mixed with MOFs materials, stirred and reacted to obtain glucose oxidase-modified MOFs, namely MOFs@GOx.

Further, the MOFs described in the preparation method are selected from UiO-66, MOF-808, MOF-5, HKUST-1, MIL-125, NH ₂ -MIL-125.

Further, in the mixing process described in the preparation method, the specific operation is to place the MOFs material in the aqueous solution of glucose oxidase, and then ultrasonicate, the ultrasonic power is 200W-400W; the ultrasonic power is preferably 300W; in this ultrasonic power range The MOFs can be uniformly dispersed in the GOx solution, which facilitates the uniform modification of GOx to the surface of MOFs.

Further, in the stirring reaction described in the preparation method, the stirring rate is 15 rad to 30 rad, preferably 20 rad; within this stirring rate range, MOFs and GOx can fully react, and the modification effect is ideal.

Further, for the stirring reaction described in the preparation method, the stirring time is 40 min to 90 min; preferably 60 min; within this stirring time interval, the MOFs and GOx can fully react, and the modification effect is ideal.

Further, the stirring reaction described in the preparation method is carried out at room temperature; preferably 20-30°C.

Further, the MOFs@GOx described in the preparation method is selected from UiO-66@GOx, MOF-808@GOx, MOF-5@GOx, HKUST-1@GOx, MIL-125@GOx, NH ₂ -MIL-125 @GOx.

Further, the preparation method of the UiO-66@GOx nano drug carrier includes:

Step 1: Dissolve 1,4-phthalic acid and zirconium oxychloride octahydrate separately and then mix them. Add glacial acetic acid to the mixture, and then carry out hydrothermal synthesis reaction in a reaction kettle to obtain UiO-66.

Step 2: Put UiO-66 in the glucose oxidase aqueous solution, then sonicate, and then stir the reaction to obtain glucose oxidase-modified UiO-66 (that is, UiO-66@GOx).

Further, the mixing process in step 1 is that 1,4-phthalic acid is ultrasonically dissolved in N,N-dimethylformamide (DMF), zirconium oxychloride octahydrate is mechanically stirred and dissolved in DMF, and the latter two well mixed.

Further, glacial acetic acid was added to the mixed liquid described in step 1 and then ultrasonically treated for 1-2 min. Through this step, the particle size of the synthesized UiO-66 could be adjusted in the subsequent hydrothermal reaction.

Further, the reaction temperature of the hydrothermal method described in step 1 is 90°C to 120°C; preferably 90°C;

Further, the reaction time of the hydrothermal method described in step 1 is 10-24 hours; preferably 18 hours.

Further, after the hydrothermal reaction described in step 1 is completed, the post-treatment process includes: centrifuging the reaction product at a speed of 11,000 rpm, and centrifuging for 10 minutes; then performing solvent exchange with DMF and methanol for 3 times; Dry in an oven, the drying temperature is 60°C, and the drying time is 12h.

Further, the ultrasonic power described in step 2 is 200W～400W; preferably 300W.

Further, the stirring rate of the stirring reaction described in step 2 is 15rad-30rad; preferably 20rad.

Further, the stirring time of the stirring reaction described in step 2 is 40 min to 90 min; preferably 60 min.

Further, the reaction temperature of the stirring reaction described in step 2 is room temperature; preferably 20-30°C.

In another aspect, the present invention provides an application of the nano drug carrier MOFs@GOx in the preparation of a drug or a treatment system for treating cancer.

Further, the application is to prepare a multifunctional nanomedicine UiO-66@GOx@Ag with near-infrared absorption and photothermal conversion properties, to realize the integrated construction of cancer starvation therapy, photothermal synergistic therapy and drug delivery.

Further, the preparation method of the multifunctional nanomedicine UiO-66@GOx@Ag includes:

The nano-silver solution was stirred and reacted with UiO-66@GOx, and the resulting solution was freeze-dried to obtain UiO-66@GOx@Ag.

Further, the preparation method of the nano-silver solution comprises the following steps: ultrasonically dissolving polyvinylpyrrolidone (PVP) in deionized water, adding silver nitrate, mixing uniformly, and then adding sodium borohydride solution dropwise under stirring at room temperature. After the reaction is completed, washing is required. The product was centrifuged at 12000 rpm for 20 minutes. Then wash with ethanol for 3 times, wash with deionized water for 3 times, and redisperse the product in deionized water to obtain nano-silver solution.

Further, the stirring reaction for preparing UiO-66@GOx@Ag is carried out at room temperature; the stirring rate is 15rad to 30rad, preferably 20rad; the stirring time is 30min to 60min, preferably 40min; the stirring rate and stirring In this time interval, silver nanoparticles can be fully anchored on the surface of MOFs@GOx.

Further, the cancer includes lung cancer, cervical cancer, breast cancer, liver cancer, chronic myelogenous leukemia and acute monocytic leukemia, ovarian cancer, kidney cancer, brain cancer, colon cancer or gastric cancer.

Compared with the prior art, the present invention has the following significant advantages and beneficial effects:

(1) Innovatively use GOx, an endogenous substance of human cells, to modify MOFs materials. This method has a good modification effect on various MOFs materials. The modified MOFs materials have a uniform dispersion size and can be dispersed in water for a long time. It will not decompose, has excellent stability, and can be used as a drug carrier to load one or more other drugs or biomolecules. The technical method is easy to operate, requires less equipment, has good general adaptability and scalability, and is conducive to large-scale production.

(2) Simultaneously realize the effective unification and combination of carrier modification and anticancer active substance loading. GOx is an endogenous biological enzyme without any toxicity itself. In addition to improving the stability and dispersion of MOFs, it can also be used as a drug for treating cancer. It consumes a large amount of glucose in cancer cells, realizes starvation therapy, and avoids the need for chemical drugs. The resulting damage to body functions can achieve targeted damage to cancer cells, greatly reducing the side effects of drugs.

(3) The controllable loading of silver nanoparticles was efficiently realized, and a multifunctional nanomedicine with near-infrared absorption and photothermal conversion properties was prepared. The nanomedicine has low toxicity and good biocompatibility, and is easily taken up by cells. And it can maintain good GOx release and catalytic glucose degradation performance in cells. Under the irradiation of near-infrared light, it can exert excellent starvation and photothermal synergistic therapeutic performance. Both cell and animal experiments have confirmed that the nano-medicine can significantly reduce cancer cells Survival rate, improve the effect of cancer treatment.

Description of drawings

Figure 1. Transmission electron micrographs of UiO-66.

Fig. 2. TEM images of UiO-66@GOx.

Fig. 3. TEM images of UiO-66@GOx@Ag.

Fig. 4. X-ray photoelectron spectroscopy of UiO-66@GOx@Ag.

Figure 5. Anticancer activity data of different concentrations of GOx and UiO-66@GOx@Ag under near-infrared light irradiation.

Figure 6. The 14-day tumor volume change data of mice injected with PBS, GOx, and UiO-66@GOx@Ag, respectively.

Figure 7. Dispersion and particle size data of MOFs materials before and after GOx modification.

detailed description

The present application will be described in further detail below in the form of embodiments, so that those skilled in the art can practice the present application. It is to be understood that other embodiments may be utilized, and that appropriate changes may be made without departing from the spirit or scope of the application. To avoid detail not necessary to enable those skilled in the art to practice the application, the description may omit certain information known to those skilled in the art. Accordingly, the following detailed description should not be taken in a limiting sense, and the scope of the present invention is defined only by the appended claims. The following examples facilitate a better understanding of the present application, but are not intended to limit the scope of the present application.

MOF-808, MOF-5, HKUST-1, MIL-125, NH ₂ -MIL-125 described in the present invention were purchased from commercially available products.

Example 1 Preparation of Metal Organic Framework UiO-66

Dissolve 750mg of 1,4-phthalic acid in 15mL of DMF ultrasonically until the solution is clear; 315mg of zirconium oxychloride octahydrate is mechanically stirred and dissolved in 45mL of DMF until the solution is clear; after mixing the above two solutions evenly, add 9.6mL of ice Acetic acid, sonicate for 1-2min, then transfer the solution to a 100mL reactor, and react at 90°C for 18h. After the reaction was completed, the product was centrifuged at a rotation speed of 11000 rpm for 10 min. Then solvent exchange was performed with DMF and methanol for 3 times, and finally the centrifuged product was dried in an oven at a drying temperature of 60°C for 12 hours to obtain a metal organic framework UiO-66.

Embodiment 2 Modification of glucose oxidase GOx

4 mg of glucose oxidase was ultrasonically dissolved in 5 mL of deionized water, and 2 mg of metal-organic framework UiO-66 was ultrasonically dispersed in the glucose oxidase solution. Subsequently, the reaction was stirred with a magnet, the stirring rate was 20 rad, the stirring temperature was room temperature, and the stirring time was 1 h. After the reaction was completed, the product was centrifuged at a rotation speed of 11000 rpm for 10 min. Then it was washed with deionized water three times, and finally the centrifuged product was freeze-dried to obtain UiO-66@GOx. The transmission electron microscope photo is shown in Figure 2. The results show that the MOFs modified with GOx are uniformly dispersed and in a monodisperse state.

Apply the same modification method to complete the modification of other MOFs materials applicable to the present invention, and obtain MOF-808@GOx, MOF-5@GOx, HKUST-1@GOx, MIL-125@GOx, NH ₂ -MIL-125@GOx . The dispersion photos of the obtained GOx-modified MOFs materials and their particle size data before and after GOx-modification are shown in Figure 7. The results show that the unmodified MOFs material has poor dispersibility in water and is very easy to agglomerate, and the particle size after agglomeration is large; the dispersibility of the MOFs material modified by GOx is greatly improved, and it will not agglomerate in water for a long time. The particle size is close to that of monodisperse pure MOFs nanoparticles, which confirms that it is a monodisperse nanomaterial in water after modification, especially the particle size of the modified UiO-66@GOx is below 100nm. It can be endocytosed by tumor cells more rapidly and has a significant EPR effect.

The loading of embodiment 3 nano-silver particles

44.9mg of polyvinylpyrrolidone was ultrasonically dissolved in 30mL of deionized water, 5.1mg of silver nitrate was dissolved in 5mL of deionized water, and then the two solutions were mixed evenly; 3.0mg of sodium borohydride was dissolved in 10mL of deionized water, and then immediately added dropwise to the above mixture solution; after the reaction was completed, the product was centrifuged at 12000rpm for 20min; then washed 3 times with ethanol and 3 times with deionized water; the product was redispersed in 5mL deionized water to obtain a nano-silver solution.

2mg UiO-66@GOx was ultrasonically dispersed in the nano-silver solution prepared in (1), and then the reaction was stirred with a magneton at a stirring rate of 20rad, stirring temperature was room temperature, and stirring time was 40min; after the reaction was completed, the product was centrifuged , the rotation speed was 11000rpm, the centrifugation time was 10min, and then washed three times with deionized water, and finally the centrifuged product was freeze-dried to obtain UiO-66@GOx@Ag.

The transmission electron microscope photos shown in Fig. 3 show that silver nanoparticles are evenly dispersed on the surface of UiO-66, and UiO-66 and silver nanoparticles have no obvious agglomeration.

The X-ray photoelectron spectroscopy data shown in Figure 4 show that, in addition to the carbon (286.0eV), oxygen (532.1eV) and zirconium (181.1eV, 331.4eV, 349.0eV) elements in UiO-66, the nitrogen in GOx (400.0eV) elements, and silver (368.1eV, 373.9eV) elements in silver nanoparticles were also detected, confirming that both GOx and Ag were modified and loaded on the surface of UiO-66.

Verification Example

1. MTT experiment: UiO-66@GOx@Ag anticancer activity test

HeLa cells were seeded in 96-well plates, GOx and UiO-66@GOx@Ag were added 24 hours later to form a corresponding concentration gradient, cultured after adding medium, and irradiated with near-infrared light after 6 hours. Cell viability was detected after 24 hours.

The results shown in Figure 5 show that at the same concentration, GOx has almost no toxicity to HeLa cells, and when the concentration of UiO-66@GOx@Ag+NIR reaches 0.04 μg/mL, the survival rate of HeLa cells is close to 0, which significantly inhibits cancer cells. growth, showing excellent therapeutic effects.

2. Animal experiments:

Healthy female nude mice (body weight 25 g) were subcutaneously injected with a cell suspension containing 1×10 ⁶ cancer cells. When the tumor size reached about 100 mm ³ , the tumor mice were divided into three groups (3 mice in each group). The first group was subcutaneously injected with 200 μL of pure GOx, the second group was subcutaneously injected with UiO-66@GOx@Ag, and then irradiated with near-infrared light 12 hours later, the concentrations of GOx and UiO-66@GOx@Ag were both 1 mg/ml, The control group was injected with 200 μL PBS (0.01 M). The drug was injected once a day for 13 days, and the tumor volume of the mice was regularly recorded once a day. Tumor volume (V) was determined by the following formula: V= ^0.5xy2 , where x and y are the length and width of the tumor, respectively.

The results shown in Figure 6 show that UiO-66@GOx@Ag can significantly inhibit tumor growth under near-infrared light irradiation, while simple injection of GOx is similar to injection of PBS and cannot inhibit tumor growth.

Claims (10)

1. A nano-drug carrier with anticancer activity, characterized in that the drug carrier is a metal-organic framework MOFs nano-drug carrier MOFs@GOx modified by glucose oxidase GOx. 2. The nano drug carrier according to claim 1, wherein the MOFs are selected from UiO-66, MOF-808, MOF-5, HKUST-1, MIL-125, NH ₂ -MIL-125. 3. The nano-drug carrier according to claim 1, wherein the MOFs@GOx is selected from UiO-66@GOx, MOF-808@GOx, MOF-5@GOx, HKUST-1@GOx, MIL -125@GOx, NH ₂ -MIL-125@GOx. 4. A preparation method of the nano-medicine carrier as described in any one of claims 1-3, characterized in that, the method comprises the steps of: Glucose oxidase is mixed with MOFs materials, stirred and reacted to obtain glucose oxidase-modified MOFs, namely MOFs@GOx. 5. The preparation method according to claim 4, characterized in that, in the mixing process, the specific operation is to place MOFs in an aqueous solution of glucose oxidase, and then perform ultrasonic treatment, and the ultrasonic power is 200 W to 400 W. 6. The preparation method according to claim 4, characterized in that, for the stirring reaction, the stirring rate is 15 rad to 30 rad; the stirring time is 40 min to 90 min. 7. The preparation method according to claim 4, wherein the nano drug carrier is specifically UiO-66@GOx, and its specific preparation method comprises: Step 1: Dissolve 1,4-phthalic acid and zirconium oxychloride octahydrate separately and then mix them. Add glacial acetic acid to the mixture, and then carry out hydrothermal synthesis reaction in the reaction kettle to obtain UiO-66; Step 2: Put UiO-66 in the glucose oxidase aqueous solution, then sonicate, and then stir the reaction to obtain glucose oxidase-modified UiO-66, which is UiO-66@GOx. 8. An application of the nano-drug carrier according to any one of claims 1-3, characterized in that, the application of the nano-drug carrier in the preparation of a drug or treatment system for treating cancer. 9. The application method according to claim 8, characterized in that the application is specifically the preparation of a multifunctional nanomedicine UiO-66@GOx@Ag with near-infrared absorption and photothermal conversion properties. 10. The application method according to claim 9, wherein the multifunctional nanomedicine UiO-66@GOx@Ag is prepared by the following steps: The nano-silver solution and UiO-66@GOx were stirred together for reaction, and the resulting solution was freeze-dried to obtain UiO-66@GOx@Ag; the stirring reaction was carried out at room temperature; the stirring rate was 15 rad~30 rad; the stirring time was 30 min~60 min.

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