Cerium-based targeted nano-drug as well as application and preparation method thereof

Abstract

The invention discloses a cerium-based targeted nano-drug as well as application and a preparation method thereof. According to the preparation method, a water-soluble glucose oxidase solution, a cerium ion solution and a fat-soluble tirapazamine solution are mixed together through a carrier-free coordination driven self-assembly technology, and the carrier-free coordination self-driven cerium-based targeted nano-drug is obtained through a functional group coordination bond. The platelet membrane is further efficiently prepared in a repeated freezing and thawing and differential centrifugation mode, the platelet membrane and the cerium-based targeted nano-drug are prepared into the platelet membrane bionic cerium-based targeted nano-drug in a co-extrusion mode, and the targeting property and the biological safety of the cerium-based targeted nano-drug can be effectively enhanced. The prepared medicine is good in stability, excellent in biocompatibility, low in toxicity, high in targeting performance and high in tumor killing performance.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a cerium-based targeted nanomedicine and an application and preparation method thereof. Background Art

[0002] In recent years, nanomedicines based on reactive oxygen species (ROS) enhancement strategies have attracted much attention in tumor treatment, but their functional integration and precise delivery still face multiple technical barriers. 3+ ) has Fenton-like catalytic activity and can specifically catalyze overexpressed hydrogen peroxide (H2O2) in the tumor microenvironment (TME) to generate highly toxic hydroxyl radicals (·OH). However, traditional cerium-based nanosystems rely on carriers such as silica and liposomes to load drugs, resulting in low drug loading efficiency and poor size uniformity, and the introduction of carriers can trigger nonspecific drug release. In addition, a single cerium-based catalytic function is difficult to meet the needs of synergistic treatment, and there is an urgent need to develop carrier-free, multi-component synergistic self-assembly technology.

[0003] As a key component for ROS enhancement, glucose oxidase (GOx) can achieve dual regulation by catalyzing glucose oxidation: on the one hand, it consumes glucose in tumor cells to induce "starvation therapy", and on the other hand, it generates H2O2 and reduces the local pH value, which is an excellent material for enhancing the efficiency of cerium-based catalysis. At the same time, the efficacy of hypoxia-activated prodrug tirapazamine (TPZ) depends on the hypoxia condition of the tumor. Cerium-based materials can work closely with GOx, and GOx can consume oxygen to enhance the anti-tumor therapeutic effect of TPZ, thereby achieving synergistic anti-tumor treatment. Therefore, the development of GOx, Ce 3+ The preparation method of synthesizing nanomedicines using TPZ as the basic unit through carrier-free coordination self-assembly technology is feasible and promising.

[0004] Traditional nanomedicines still have problems such as insufficient targeting, rapid immune clearance, short circulation half-life, and poor biocompatibility. To improve tumor targeting, platelet membrane biomimetic modification technology has attracted widespread attention due to its surface CD47 protein (mediating immune escape) and P-selectin (targeting CD44 receptors on tumor blood vessels). The expression of CD47 protein on the surface of platelet membranes can significantly reduce the risk of nanomedicines being cleared by the immune system, thereby achieving immune escape and prolonging blood circulation time. P-selectin expressed on the surface of platelet membranes can actively target tumor sites, breaking through the limitations of traditional nanodelivery systems that passively target tumors, and achieving efficient enrichment of nanomedicines at tumor sites. In addition, platelets come from the organism itself, have low immunogenicity, and good biosafety, making them excellent natural delivery carriers. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a cerium-based targeted nanomedicine and its application and preparation method, which solves the problems of low drug loading efficiency and poor size uniformity of traditional cerium-based nanosystems in the above-mentioned background technology, and further solves the problems of insufficient targeting of nanomedicines, rapid immune clearance, short circulation half-life, and poor biocompatibility.

[0006] One of the technical solutions adopted by the present invention to solve its technical problems is: providing a method for preparing a cerium-based targeted nanomedicine, wherein a glucose oxidase solution, a cerium ion solution, and a tirapazamine solution are added in sequence while stirring, the stirring temperature is 20-40°C, and the reaction time is 2-4 hours, to prepare a carrier-free coordinated self-driven cerium-based targeted nanomedicine.

[0007] In a preferred embodiment of the present invention, the glucose oxidase solution is a deionized aqueous solution of glucose oxidase, and the concentration of glucose oxidase in the solution is 1.5-2.5 mg / mL.

[0008] In a preferred embodiment of the present invention, the cerium ion solution is prepared by dissolving cerium trichloride (CeCl3), cerium hydroxide (Ce(OH)3), and cerium bromide (CeBr3) in deionized water, and the cerium ion concentration in the solution is 1.5-2.5 mg / mL.

[0009] In a preferred embodiment of the present invention, the tirapazamine solution is prepared by dissolving tirapazamine in an organic solvent C, wherein the organic solvent C comprises one of methanol, ethanol, dichloromethane, ether, and dimethyl sulfoxide (the organic solvent C is subsequently removed by vacuum rotary evaporation), and the concentration of tirapazamine in the solution is 0.2-0.8 mg / mL.

[0010] In a preferred embodiment of the present invention, the concentration ratio of glucose oxidase, cerium ion and tirapazamine is 4:4:1.

[0011] The second technical solution adopted by the present invention to solve its technical problem is: providing a cerium-based targeted nanomedicine prepared by the above method; the cerium-based targeted nanomedicine can be used to prepare active targeting and synergistic anti-tumor drugs, including platelet membrane biomimetic drugs.

[0012] The third technical solution adopted by the present invention to solve the technical problem is: a method for preparing a platelet membrane biomimetic cerium-based targeted nanomedicine, comprising the following steps:

[0013] S1. Glucose oxidase solution, cerium ion solution, and tirapazamine solution are added in sequence while stirring at a temperature of 20-40° C. and a reaction time of 2-4 h to prepare a carrier-free coordinated self-driven cerium-based targeted nanomedicine;

[0014] S2, platelet membrane preparation by repeated freeze-thaw and differential centrifugation;

[0015] S3. The cerium-based targeted nanomedicine and the platelet membrane are mixed and co-extruded to obtain the platelet membrane biomimetic cerium-based targeted nanomedicine.

[0016] In a preferred embodiment of the present invention, the method for preparing the platelet membrane includes the following steps: taking blood from an experimental animal, suspending it in solution A, repeatedly centrifuging it at 150×g at room temperature to remove excess blood cells, collecting the platelet precipitate by centrifugation at 800×g, and resuspending it in solution B, freezing it at -80°C for 3 hours, repeatedly freezing and thawing it at room temperature for 3 times, and collecting it by centrifugation at 4000×g at 4°C to obtain the platelet membrane.

[0017] In a preferred embodiment of the present invention, the solution A comprises a phosphate buffer containing EDTA·2Na, and the solution B comprises a phosphate buffer containing a protease inhibitor.

[0018] In a preferred embodiment of the present invention, the platelet membrane is mixed with the cerium-based targeted nanomedicine at a concentration of 0.1-10 mg / mL and then ultrasonicated with an ultrasonic power of 100-300 W. The platelet membrane biomimetic cerium-based targeted nanomedicine is obtained by co-extrusion under the condition that the pore size of the microporous filter membrane is 0.22-0.8 μm.

[0019] The fourth technical solution adopted by the present invention to solve its technical problem is: providing a platelet membrane bionic cerium-based targeted nanomedicine prepared by the above method, the concentration of the prepared platelet membrane bionic cerium-based targeted nanomedicine is 0.5-1.5 mg / mL, and the drug shape is square and the particle size is distributed between 100-300 nm.

[0020] Compared with the background technology, this technical solution has the following advantages:

[0021] 1. The method of the present invention uses glucose oxidase, cerium ions, and tirapazamine as building blocks. A water-soluble glucose oxidase solution, a cerium ion solution, and a fat-soluble tirapazamine solution are mixed together through a carrier-free coordination-driven self-assembly technique. A carrier-free coordination-driven cerium-based targeted nanomedicine is obtained through functional group coordination bonds. This overcomes the problems of poor anticancer efficacy and low tumor targeting efficiency in existing carrier-free nanomedicine synthesis technologies.

[0022] 2. This invention further efficiently prepared platelet membranes through repeated freeze-thaw cycles and differential centrifugation, and successfully obtained platelet membrane-mimicking cerium-based targeted nanomedicines by co-extrusion with cerium-based targeted nanomedicines, solving the problems of insufficient targeting and biosafety of existing nanomedicines.

[0023] 3. The platelet membrane biomimetic cerium-based targeted nanomedicine prepared by the present invention has a square shape and a particle size distribution between 100-300nm. It has good stability and excellent biocompatibility, can efficiently target tumors, and has more outstanding value in terms of synergistic tumor killing.

[0024] 4. The platelet membrane biomimetic cerium-based targeted nanomedicine of the present invention is easily soluble in deionized water solution, suitable for intravenous injection, and has the advantages of long circulation, low toxicity and good therapeutic effect. After the platelet membrane-modified nanomedicine enters the body, it can effectively reduce the phagocytosis of macrophages in the human reticuloendothelial system (RES), prolong the drug's circulation time in the body, and give it tumor targeting. After the biomimetic nanomedicine is delivered to the tumor tissue through the active targeting effect mediated by the platelet membrane and is internalized, its core-shell structure dissociates under the dual stimulation of the lysosomal acidic microenvironment (pH 5.0) and the high concentration of reduced GSH, achieving the effect of GOx, Ce 3+ The biomimetic nanomedicine prepared by this method can significantly enhance the anti-tumor effect through a three-level synergistic anti-tumor effect. Furthermore, the biomimetic system can avoid the loss of drug activity, facilitating drug storage and transportation. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a transmission electron microscopy image of the platelet membrane biomimetic cerium-based targeted nanomedicine of Example 1;

[0026] Figure 2 This is a diagram of the hydrated particle size of the platelet membrane biomimetic cerium-based targeted nanomedicine of Examples 1-3;

[0027] Figure 3 This is a transmission electron microscopy image of the platelet membrane biomimetic cerium-based targeted nanomedicine of Example 2;

[0028] Figure 4 This is a transmission electron microscopy image of the platelet membrane biomimetic cerium-based targeted nanomedicine of Example 3;

[0029] Figure 5 This is a transmission electron microscopy image of the platelet membrane biomimetic cerium-based targeted nanomedicine of Example 4;

[0030] Figure 6 This is a diagram of the hydrated particle size of the platelet membrane biomimetic cerium-based targeted nanomedicine of Examples 4-6;

[0031] Figure 7 This is a transmission electron microscopy image of the platelet membrane biomimetic cerium-based targeted nanomedicine of Example 5;

[0032] Figure 8 This is a transmission electron microscope image of the platelet membrane biomimetic cerium-based targeted nanomedicine of Example 6. DETAILED DESCRIPTION

[0033] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is described in more detail below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to these embodiments.

[0034] The Chinese and English names of the present invention correspond to the following:

[0035] Glucose oxidase: Glucose oxidase;

[0036] Tirapazamine: Tirapazamine, molecular formula (Hill notation) is C7H6N4O2

[0037] In the following examples, the solution A is a phosphate buffer containing EDTA·2Na, and the solution B is a phosphate buffer containing a protease inhibitor.

[0038] Example 1

[0039] The present embodiment provides a method for preparing a cerium-based targeted nanomedicine, comprising the following steps:

[0040] S1. Glucose oxidase and cerium ions were dissolved in deionized water (the concentration of both aqueous solutions was 2 mg / mL), and tirapazamine was dissolved in methanol (the concentration was 1 mg / mL). The mixture was stirred magnetically at 300 rpm for 4 h at room temperature. After the mixing reaction, the methanol was removed, and deionized water was added for dilution to obtain an aqueous solution of cerium-based targeted nanomedicine.

[0041] The cerium-based targeted nanomedicine of this embodiment is used to further prepare platelet membrane biomimetic cerium-based targeted nanomedicine, and the preparation method is as follows:

[0042] S2. Take the blood of the experimental animals, suspend it in solution A, centrifuge it repeatedly at 150×g at room temperature to remove excess blood cells, collect the platelet precipitate by centrifugation at 800×g, resuspend it in solution B, freeze it at -80℃ for 3h, thaw it at room temperature, freeze and thaw it repeatedly 3 times, and collect the platelet membrane by centrifugation at 4000×g at 4℃.

[0043] S3. After mixing the platelet membrane and the cerium-based targeted nanomedicine, ultrasonication was performed (ultrasound power of 300 W, ultrasonication time of 10 s), and the platelet membrane biomimetic cerium-based targeted nanomedicine was obtained by co-extrusion using a filter (0.4 μm).

[0044] Example 2

[0045] The difference between this embodiment and embodiment 1 is that:

[0046] S1. Glucose oxidase and cerium ions were dissolved in deionized water (the concentration of both aqueous solutions was 2 mg / mL), and tirapazamine was dissolved in ethanol (the concentration was 0.5 mg / mL). The mixture was stirred magnetically at 300 rpm for 4 h at room temperature. After the mixing reaction, the ethanol was removed, and deionized water was added for dilution to obtain a cerium-based targeted nanodrug aqueous solution.

[0047] S2. Take the blood of the experimental animals, suspend it in solution A, centrifuge it repeatedly at 150×g at room temperature to remove excess blood cells, collect the platelet precipitate by centrifugation at 800×g, resuspend it in solution B, freeze it at -80℃ for 3h, thaw it at room temperature, freeze and thaw it repeatedly 3 times, and collect the platelet membrane by centrifugation at 4000×g at 4℃.

[0048] S3. After mixing the platelet membrane and the cerium-based targeted nanomedicine, ultrasonication was performed (ultrasound power of 300 W, ultrasonication time of 10 s), and the platelet membrane biomimetic cerium-based targeted nanomedicine was obtained by co-extrusion using a filter (0.4 μm).

[0049] Example 3

[0050] The difference between this embodiment and embodiment 1 is that:

[0051] S1. Glucose oxidase and cerium ions were dissolved in deionized water (the concentration of both aqueous solutions was 1.5 mg / mL), and tirapazamine was dissolved in dimethyl sulfoxide (concentration was 0.5 mg / mL). The mixture was stirred magnetically at 300 rpm for 4 h at room temperature. After the mixing reaction, dimethyl sulfoxide was removed, and deionized water was added for dilution to obtain a cerium-based targeted nanodrug aqueous solution.

[0052] S2. Take the blood of the experimental animals, suspend it in solution A, centrifuge it repeatedly at 150×g at room temperature to remove excess blood cells, collect the platelet precipitate by centrifugation at 800×g, resuspend it in solution B, freeze it at -80℃ for 3h, thaw it at room temperature, freeze and thaw it repeatedly 3 times, and collect the platelet membrane by centrifugation at 4000×g at 4℃.

[0053] S3. After mixing the platelet membrane and the cerium-based targeted nanomedicine, the mixture was ultrasonicated (ultrasonic power of 300 W, ultrasonic time of 10 s), and the biomimetic nanomedicine was prepared by co-extrusion using a filter (0.4 μm).

[0054] Example 4

[0055] The difference between this embodiment and embodiment 1 is that:

[0056] S1. Glucose oxidase and cerium ions were dissolved in deionized water (the concentration of both aqueous solutions was 2 mg / mL), and tirapazamine was dissolved in methanol (the concentration was 0.5 mg / mL). The mixture was stirred magnetically at 300 rpm for 3 h at room temperature. After the mixing reaction, the methanol was removed, and deionized water was added for dilution to obtain a cerium-based targeted nanodrug aqueous solution.

[0057] S2. Take the blood of the experimental animals, suspend it in solution A, centrifuge it repeatedly at 150×g at room temperature to remove excess blood cells, collect the platelet precipitate by centrifugation at 800×g, resuspend it in solution B, freeze it at -80℃ for 3h, thaw it at room temperature, freeze and thaw it repeatedly 3 times, and collect the platelet membrane by centrifugation at 4000×g at 4℃.

[0058] S3. After mixing the platelet membrane and the cerium-based targeted nanomedicine, the mixture was ultrasonicated (ultrasonic power of 300 W, ultrasonic time of 10 s), and the biomimetic nanomedicine was prepared by co-extrusion using a filter (0.22 μm).

[0059] Example 5

[0060] The difference between this embodiment and embodiment 1 is that:

[0061] S1. Glucose oxidase and cerium ions were dissolved in deionized water (the concentration of both aqueous solutions was 3 mg / mL), and tirapazamine was dissolved in ethanol (the concentration was 1 mg / mL). The mixture was stirred magnetically at 300 rpm for 2 h at room temperature. After the mixing reaction, the ethanol was removed, and deionized water was added for dilution to obtain a cerium-based targeted nanodrug aqueous solution.

[0062] S2. Take the blood of the experimental animals, suspend it in solution A, centrifuge it repeatedly at 150×g at room temperature to remove excess blood cells, collect the platelet precipitate by centrifugation at 800×g, resuspend it in solution B, freeze it at -80℃ for 3h, thaw it at room temperature, freeze and thaw it repeatedly 3 times, and collect the platelet membrane by centrifugation at 4000×g at 4℃.

[0063] S3. After mixing the platelet membrane and the cerium-based targeted nanomedicine, the mixture was ultrasonicated (ultrasonic power of 300 W, ultrasonic time of 10 s), and the biomimetic nanomedicine was prepared by co-extrusion using a filter (0.22 μm).

[0064] Example 6

[0065] The difference between this embodiment and embodiment 1 is that:

[0066] S1. Glucose oxidase and cerium ions were dissolved in deionized water (the concentration of both aqueous solutions was 2 mg / mL), and tirapazamine was dissolved in dimethyl sulfoxide (concentration was 0.5 mg / mL). The mixture was stirred magnetically at 300 rpm for 2 h at room temperature. After the mixing reaction, dimethyl sulfoxide was removed, and deionized water was added for dilution to obtain a cerium-based targeted nanodrug aqueous solution.

[0067] S2. Take the blood of the experimental animals, suspend it in solution A, centrifuge it repeatedly at 150×g at room temperature to remove excess blood cells, collect the platelet precipitate by centrifugation at 800×g, resuspend it in solution B, freeze it at -80℃ for 3h, thaw it at room temperature, freeze and thaw it repeatedly 3 times, and collect the platelet membrane by centrifugation at 4000×g at 4℃.

[0068] S3. After mixing the platelet membrane and the cerium-based targeted nanomedicine, the mixture was ultrasonicated (ultrasonic power of 300 W, ultrasonic time of 10 s), and the biomimetic nanomedicine was prepared by co-extrusion using a filter (0.22 μm).

[0069] Electron microscopic images and hydrated particle size diagrams of the platelet membrane-mimetic cerium-based targeted nanomedicine prepared in the above examples show that the drug particle size is distributed between 100-300 nm and has good stability. The platelet membrane-mimetic cerium-based targeted nanomedicine prepared in the present invention has advantages that bare drugs lack, such as low toxicity, targeting, and high efficacy. The platelet membrane-mimetic cerium-based targeted nanomedicine preparation prepared in the present invention is suitable for intravenous injection and has the advantages of targeting, long circulation, low toxicity, and good therapeutic effect.

[0070] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a cerium-based targeted nanomedicine, characterized in that: Glucose oxidase solution, cerium ion solution and tirapazamine solution were added in sequence while stirring at a stirring temperature of 20-40° C. and a reaction time of 2-4 h to prepare a carrier-free coordinated self-driven cerium-based targeted nanomedicine.

2. The method for preparing a cerium-based targeted nanomedicine according to claim 1, wherein: The glucose oxidase solution is a deionized aqueous solution of glucose oxidase, and the concentration of glucose oxidase in the solution is 1.5-2.5 mg / mL; the cerium ion solution is prepared by dissolving cerium trichloride, cerium hydroxide or cerium bromide in deionized water, and the concentration of cerium ions in the solution is 1.5-2.5 mg / mL; the tirapazamine solution is prepared by dissolving tirapazamine in an organic solvent C, and the organic solvent C includes one of methanol, ethanol, dichloromethane, ether and dimethyl sulfoxide, and the concentration of tirapazamine in the solution is 0.2-0.8 mg / mL.

3. The method for preparing a cerium-based targeted nanomedicine according to claim 1, wherein: The concentration ratio of glucose oxidase, cerium ion and tirapazamine is 4:4:

1.

4. A cerium-based targeted nanomedicine, characterized in that: The invention is prepared by the method according to any one of claims 1 to 3.

5. The use of a cerium-based targeted nanomedicine according to claim 4, characterized in that: Used to prepare active-targeted, synergistic anti-tumor drugs, including platelet membrane biomimetic drugs.

6. A method for preparing a platelet membrane biomimetic cerium-based targeted nanomedicine, characterized by: The steps include: S1. Glucose oxidase solution, cerium ion solution, and tirapazamine solution are added in sequence while stirring at a temperature of 20-40° C. and a reaction time of 2-4 h to prepare a carrier-free coordinated self-driven cerium-based targeted nanomedicine; S2, platelet membrane preparation by repeated freeze-thaw and differential centrifugation; S3. The cerium-based targeted nanomedicine and the platelet membrane are mixed and co-extruded to obtain the platelet membrane biomimetic cerium-based targeted nanomedicine.

7. The method for preparing a platelet membrane biomimetic cerium-based targeted nanomedicine according to claim 6, characterized in that: The platelet membrane preparation method comprises the following steps: blood from an experimental animal is collected, suspended in solution A, and repeatedly centrifuged at 150×g at room temperature to remove excess blood cells until the red precipitate is reduced. The platelet precipitate is collected by centrifugation at 800×g for 15 minutes, resuspended in solution B, frozen at -80°C for 3 hours, repeatedly frozen and thawed at room temperature three times, and then centrifuged at 4000×g for 10 minutes at 4°C to obtain the platelet membrane.

8. The method for preparing a platelet membrane biomimetic cerium-based targeted nanomedicine according to claim 7, characterized in that: The solution A includes a phosphate buffer containing EDTA·2Na, and the solution B includes a phosphate buffer containing a protease inhibitor.

9. The method for preparing a platelet membrane biomimetic cerium-based targeted nanomedicine according to claim 6, characterized in that: After the platelet membrane is mixed with the cerium-based targeted nanomedicine with a concentration of 0.1-10 mg / mL, the platelet membrane biomimetic cerium-based targeted nanomedicine is prepared by co-extrusion under the condition that the pore size of the microporous filter membrane is 0.22-0.8 μm.

10. A platelet membrane biomimetic cerium-based targeted nanomedicine, characterized by: The method is prepared according to any one of claims 7 to 9.

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