阅读说明：本技术 一种用于肿瘤靶向的沸石复合纳米酶及其制备方法与应用 (Zeolite composite nanoenzyme for tumor targeting and preparation method and application thereof ) 是由 潘友长 塔吉亚娜 于 2021-08-16 设计创作，主要内容包括：本发明公开了一种用于肿瘤靶向的沸石复合纳米酶及其制备方法与应用,所述用于肿瘤靶向的沸石复合纳米酶按照质量份数计,由乳酸氧化酶4～6份、核糖核酸内切酶4～7份、脱氧核糖核酸内切酶2～4份、人血清白蛋白1～2份、谷胱甘肽1～2份、硫酸亚铁铵2～3份、连氮二铵盐造影剂1～2份组成,本纳米药物的制备方法简单,通过聚合可以控制重复单元数,两种聚合物的用量比和浓度都可调,可方便调节硼替佐米药物的负载量,本纳米药物除了可通过硼替佐米进行化疗外,还可进行光热治疗和光声成像,各部分之间协同作用,为肿瘤治疗过程的实时诊断提供指导。(The invention discloses a zeolite composite nano enzyme for tumor targeting and a preparation method and application thereof, the zeolite composite nanoenzyme for tumor targeting is composed of, by mass, 4-6 parts of lactate oxidase, 4-7 parts of endoribonuclease, 2-4 parts of endoribonuclease, 1-2 parts of human serum albumin, 1-2 parts of glutathione, 2-3 parts of ferrous ammonium sulfate and 1-2 parts of azinium diammonium contrast agent, and the preparation method of the nano-drug is simple, the number of the repeating units can be controlled through polymerization, the dosage ratio and the concentration of the two polymers can be adjusted, the nano-drug can conveniently adjust the loading capacity of the bortezomib drug, can be used for performing photothermal therapy and photoacoustic imaging besides chemotherapy through the bortezomib, and provides guidance for real-time diagnosis in the tumor treatment process due to the synergistic effect of all parts.)

1. A preparation method of zeolite composite nanoenzyme for tumor targeting is characterized by comprising the following steps:

1, preparing a mixed enzyme solution by using lactate oxidase, endoribonuclease and endodeoxyribonuclease: weighing lactate oxidase, endoribonuclease and endodeoxyribonuclease in sequence according to the mass parts; mixing and uniformly stirring the weighed lactate oxidase, endoribonuclease and endodeoxyribonuclease to obtain a mixed solution; placing the mixed solution in an ultrasonic device for ultrasonic dispersion to obtain a mixed enzyme solution for later use;

2, preparation of human serum albumin: collecting venous blood of healthy people; extracting total RNA from the collected blood to synthesize total cDNA; amplifying a human serum albumin gene by using cDNA as a template and adopting a PCR technology, and expressing the human serum albumin gene by using a recombinant engineering host cell to obtain human serum albumin for later use; wherein, the recombinant engineering host cell is yeast, CHO cell, plant, insect, bacteria or animal cell, and the human serum albumin is transferred into the body of the plant and the animal to be expressed by plasmid DNA vector expression, virus vector expression or transgenic technology;

3, preparing reduced human serum albumin by using human serum albumin and glutathione: carrying out cation exchange chromatography on the human serum albumin prepared in the step two to obtain a primary product, carrying out anion exchange chromatography on the primary product, and carrying out membrane sterilization to obtain reduced human serum albumin for later use; wherein the conditions of the cation exchange chromatography are as follows: balancing the chromatographic column by using a balance buffer solution at a flow rate of 60-220 cm/h, wherein the balance buffer solution comprises 15-25 mM NaAc, 25-30 mM NaCl, 0.4-1 mM EDTA-2Na and reduced glutathione, and the pH value is 5.0; the anion exchange chromatography conditions are as follows: balancing a chromatographic column by using a balance buffer solution at a flow rate of 60-220 cm/h, wherein the components of the balance buffer solution are 30-60 mM phosphate, 1-6 mM EDTA-2Na and reduced glutathione, the conductance is 2.5-9 mS/cm, and the pH is 6.1-7.9;

4, mixing the mixed enzyme solution with ammonium ferrous sulfate to prepare the composite nano enzyme: mixing the prepared mixed enzyme solution with ammonium ferrous sulfate, and uniformly stirring to obtain a mixture; dropwise adding deionized water into the mixture, uniformly stirring, and performing ultrasonic dispersion for 30-50 min to obtain a dispersion liquid; magnetically stirring the dispersion under nitrogen atmosphere; carrying out ultrafiltration centrifugation on the stirred dispersion liquid, and collecting a centrifugal product to obtain the composite nano enzyme for later use;

5, preparation of composite nanoenzyme for tumor targeting using composite nanoenzyme: putting the composite nano enzyme prepared in the third step into a water bath heating device for water bath heating; magnetically stirring the composite nano enzyme in the heating process and adding the azine diammonium salt contrast agent during stirring; continuing heating for 3-5 min after the magnetic stirring is finished, and finishing stirring to obtain a composite nano enzyme solution; and (3) placing the composite nano enzyme solution in a drying oven for low-temperature drying to obtain the composite nano enzyme for tumor targeting.

2. The method for preparing zeolite-composited nanoenzyme for tumor targeting as claimed in claim 1, wherein in step 2, the extracting total RNA from the collected blood to synthesize total cDNA includes: the collected blood is centrifuged to obtain nucleated cells in the blood, total RNA is extracted, and total cDNA is synthesized from the extracted RNA.

3. The method for preparing the zeolite composite nanoenzyme for tumor targeting according to claim 1, wherein the rotation speed for centrifuging the collected blood is 1300-1400 r/min, the centrifugation temperature is 5-10 ℃, and the centrifugation time is 15-20 min.

4. The method for preparing zeolite-complexed nanoenzyme for tumor targeting according to claim 1, wherein the extracting total RNA further comprises: RNA detection and integrity verification were performed.

5. The method for preparing zeolite-complexed nanoenzyme for tumor targeting according to claim 1, wherein the total cDNA is synthesized by extracted RNA comprising: cDNA acquisition by reverse transcription

6. The preparation method of the zeolite composite nanoenzyme for tumor targeting according to claim 1, wherein the temperature of the water bath heating in step 5 is 35-40 ℃.

7. The preparation method of the zeolite composite nanoenzyme for tumor targeting according to claim 1, wherein in the step 5, the low-temperature drying temperature is 20-30 ℃ and the drying time is 8-10 h.

8. The zeolite composite nanoenzyme for tumor targeting prepared by the preparation method of the zeolite composite nanoenzyme for tumor targeting according to any one of claims 1 to 7, wherein the zeolite composite nanoenzyme for tumor targeting comprises, by mass, 4 to 6 parts of lactate oxidase, 4 to 7 parts of endoribonuclease, 2 to 4 parts of endoribonuclease, 1 to 2 parts of human serum albumin, 1 to 2 parts of glutathione, 2 to 3 parts of ferrous ammonium sulfate, and 1 to 2 parts of azinium salt contrast agent.

9. Use of the tumor-targeted complex nanoenzyme of any one of claims 1 to 8 in the manufacture of a medicament.

Technical Field

The invention relates to the technical field of nano-pharmaceutical preparations, in particular to a zeolite composite nano-enzyme for tumor targeting and a preparation method and application thereof.

Background

In recent years, the targeted nano-drug is widely developed and applied to tumor treatment due to the advantages of strong targeting capability, low toxic and side effects and the like. The hydroxyapatite is the main component of human teeth and has the characteristics of excellent biocompatibility, biological safety and acid response. Therefore, hydroxyapatite is used as a nano carrier, and the drug-loaded hydroxyapatite nano material is designed and prepared for targeted tumor therapy. The drug-loaded hydroxyapatite DOX @ HAP is prepared by regulating and controlling the conditions and parameters of the synthesis process of the hydroxyapatite nano material. Further, GGT enzyme targeted fluorescent dye, polypeptide cRGD and hyaluronic acid molecules of three targeted tumor markers are respectively adopted to perform surface modification on the hydroxyapatite nano material, so that 3 tumor targeted nano medicines are constructed for tumor treatment. (1) GGT enzyme targeted nano-drug DDHAP: the tumor marker gamma-glutamyltranspeptidase (GGT) is a glycoprotein that is overexpressed on the surface of most tumor cell membranes. The paper designs and synthesizes a GGT enzyme targeted fluorescent dye DFA1, and modifies the dye on the surface of a nano-drug through hydrogen bond action to construct a GGT enzyme targeted nano-drug DDHAP. The nano-drug enters tumor cells after targeting recognition of GGT enzyme through dye DFA1, and releases the drug in the tumor slightly acidic environment. Ratiometric fluorescence imaging enables dynamic real-time monitoring of the drug release process. The nano-drug has obvious treatment effect on tumor cells and tissues over-expressed by GGT enzyme in cell, tissue and mouse tumor model experiments. (2) The cRGD targeting-nucleic acid binding nano-drug cRDHAP: the cyclic peptide cRGD has high affinity with the tumor marker integrin α v β 3. The action mechanism of the chemotherapeutic drug adriamycin is that the adriamycin is embedded into cell nucleus to inhibit the synthesis of DNA and RNA. The paper designs and synthesizes an example of RNA-conjugated fluorescent dye Na-RNA, and sequentially modifies the dye, PEG-cRGD and PEG to the surface of the nano-drug to construct cRGD targeting-nucleic acid conjugated nano-drug cRDHAP. The nano-drug enters tumor cells after the integrin alpha v beta 3 is identified by cRGD, and PEG is removed in the tumor micro-acid environment and the loaded drug is released. The dye Na-RNA on the surface of the nano-drug reacts with RNA in cell nucleus, so that the accumulation amount of the nano-drug in the cell nucleus is increased, and the chemotherapy effect is enhanced. The nano-drug has obvious tumor inhibition effect in a mouse tumor inhibition model of integrin alpha v beta 3 over-expression tumor cells. (3) The oligohyaluronic acid modified composite nano-drug oHA-DOX @ MSNs/HAP: the hyaluronic acid has a binding effect with a tumor marker CD44 protein, and the strength of the binding effect is closely related to the molecular weight of the hyaluronic acid.

Disclosure of Invention

The invention aims to provide a zeolite composite nano enzyme for tumor targeting and a preparation method and application thereof, so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme:

the preparation method of the zeolite composite nanoenzyme for targeting the tumor comprises the following steps:

1, preparing a mixed enzyme solution by using lactate oxidase, endoribonuclease and endodeoxyribonuclease: weighing lactate oxidase, endoribonuclease and endodeoxyribonuclease in sequence according to the mass parts; mixing and uniformly stirring the weighed lactate oxidase, endoribonuclease and endodeoxyribonuclease to obtain a mixed solution; placing the mixed solution in an ultrasonic device for ultrasonic dispersion to obtain a mixed enzyme solution for later use;

2, preparation of human serum albumin: collecting venous blood of healthy people; extracting total RNA from the collected blood to synthesize total cDNA; amplifying a human serum albumin gene by using cDNA as a template and adopting a PCR technology, and expressing the human serum albumin gene by using a recombinant engineering host cell to obtain human serum albumin for later use; wherein, the recombinant engineering host cell is yeast, CHO cell, plant, insect, bacteria or animal cell, and the human serum albumin is transferred into the body of the plant and the animal to be expressed by plasmid DNA vector expression, virus vector expression or transgenic technology;

3, preparing reduced human serum albumin by using human serum albumin and glutathione: carrying out cation exchange chromatography on the human serum albumin prepared in the step two to obtain a primary product, carrying out anion exchange chromatography on the primary product, and carrying out membrane sterilization to obtain reduced human serum albumin for later use; wherein the conditions of the cation exchange chromatography are as follows: balancing the chromatographic column by using a balance buffer solution at a flow rate of 60-220 cm/h, wherein the balance buffer solution comprises 15-25 mM NaAc, 25-30 mM NaCl, 0.4-1 mM EDTA-2Na and reduced glutathione, and the pH value is 5.0; the anion exchange chromatography conditions are as follows: balancing a chromatographic column by using a balance buffer solution at a flow rate of 60-220 cm/h, wherein the components of the balance buffer solution are 30-60 mM phosphate, 1-6 mM EDTA-2Na and reduced glutathione, the conductance is 2.5-9 mS/cm, and the pH is 6.1-7.9;

4, mixing the mixed enzyme solution with ammonium ferrous sulfate to prepare the composite nano enzyme: mixing the prepared mixed enzyme solution with ammonium ferrous sulfate, and uniformly stirring to obtain a mixture; dropwise adding deionized water into the mixture, uniformly stirring, and performing ultrasonic dispersion for 30-50 min to obtain a dispersion liquid; magnetically stirring the dispersion under nitrogen atmosphere; carrying out ultrafiltration centrifugation on the stirred dispersion liquid, and collecting a centrifugal product to obtain the composite nano enzyme for later use;

5, preparation of composite nanoenzyme for tumor targeting using composite nanoenzyme: putting the composite nano enzyme prepared in the third step into a water bath heating device for water bath heating; magnetically stirring the composite nano enzyme in the heating process and adding the azine diammonium salt contrast agent during stirring; continuing heating for 3-5 min after the magnetic stirring is finished, and finishing stirring to obtain a composite nano enzyme solution; and (3) placing the composite nano enzyme solution in a drying oven for low-temperature drying to obtain the composite nano enzyme for tumor targeting.

Further, in the step 1, the frequency of ultrasonic dispersion is 45-50 kHz, and the time of ultrasonic dispersion is 15-20 min.

Further, in step 2, the extracting total RNA from the collected blood to synthesize total cDNA comprises: the collected blood is centrifuged to obtain nucleated cells in the blood, total RNA is extracted, and total cDNA is synthesized from the extracted RNA.

Further, the rotation speed of centrifuging the collected blood is 1300-1400 r/min, the centrifugation temperature is 5-10 ℃, and the centrifugation time is 15-20 min.

Further, the extracting total RNA further comprises: RNA detection and integrity verification were performed.

Further, the synthesis of total cDNA from the extracted RNA comprises: cDNA acquisition was performed by reverse transcription.

Further, in the step 5, the water bath heating temperature is 35-40 ℃.

Further, in the step 5, the low-temperature drying temperature is 20-30 ℃, and the drying time is 8-10 hours.

Compared with the prior art, the invention has the beneficial effects that:

the preparation method of the nano-drug is simple, the number of the repeating units can be controlled through polymerization, the dosage ratio and the concentration of the two polymers can be adjusted, and the loading capacity of the bortezomib drug can be conveniently adjusted.

The nano-drug can be used for chemotherapy through bortezomib, photo-thermal therapy and photoacoustic imaging, and all parts have synergistic effect, so that guidance is provided for real-time diagnosis in the tumor treatment process.

Detailed Description

The technical solution of the present invention will be described in further detail with reference to specific embodiments.

The zeolite composite nanoenzyme for tumor targeting provided by the embodiment of the invention comprises, by mass, 4-6 parts of lactate oxidase, 4-7 parts of endoribonuclease, 2-4 parts of endoribonuclease, 1-2 parts of human serum albumin, 1-2 parts of glutathione, 2-3 parts of ferrous ammonium sulfate and 1-2 parts of azinium diammonium salt contrast agent.

1. Preparing a mixed enzyme solution by using lactate oxidase, endoribonuclease and endodeoxyribonuclease;

2. preparing human serum albumin;

3. preparing reduced human serum albumin by using human serum albumin and glutathione;

4. mixing the mixed enzyme solution with ammonium ferrous sulfate to prepare composite nano enzyme;

5. the preparation of the composite nanoenzyme for tumor targeting is performed using the composite nanoenzyme.

In step 1, the preparation of the mixed enzyme solution by using lactate oxidase, endoribonuclease and endodeoxyribonuclease comprises:

1. weighing lactate oxidase, endoribonuclease and endodeoxyribonuclease according to the mass parts;

2. mixing and uniformly stirring the weighed lactate oxidase, endoribonuclease and endodeoxyribonuclease to obtain a mixed solution;

3. and placing the mixed solution in an ultrasonic device for ultrasonic dispersion to obtain a mixed enzyme solution.

In the step 3 provided by the embodiment of the invention, the frequency of ultrasonic dispersion is 45-50 kHz, and the time of ultrasonic dispersion is 15-20 min.

In step 2 provided in the embodiment of the present invention, the preparing human serum albumin includes:

1. collecting venous blood of healthy people;

2. extracting total RNA from the collected blood to synthesize total cDNA;

3. the cDNA is taken as a template, the human serum albumin gene is amplified by adopting a PCR technology, and the human serum albumin is obtained by recombinant engineering host cell expression.

In step 3 provided in the embodiments of the present invention, the extracting total RNA from the collected blood to synthesize total cDNA includes: the collected blood is centrifuged to obtain nucleated cells in the blood, total RNA is extracted, and total cDNA is synthesized from the extracted RNA.

The rotation speed for centrifuging the collected blood provided by the embodiment of the invention is 1300-1400 r/min, the centrifugation temperature is 5-10 ℃, and the centrifugation time is 15-20 min.

The method for extracting total RNA provided by the embodiment of the invention also comprises the following steps: RNA detection and integrity verification were performed.

The total cDNA synthesized by the extracted RNA provided by the embodiment of the invention comprises the following components: cDNA acquisition was performed by reverse transcription.

In step 3 provided by the embodiment of the present invention, the recombinant engineering host cell is yeast, CHO cell, plant, insect, bacteria or animal cell, and the human serum albumin is transferred to the plant and animal body for expression by plasmid DNA vector expression, viral vector expression or transgenic technology.

In step 1 provided in the embodiments of the present invention, the preparation of reduced human serum albumin using human serum albumin and glutathione includes:

1. mixing the prepared mixed enzyme solution with ammonium ferrous sulfate, and uniformly stirring to obtain a mixture;

2. dropwise adding deionized water into the mixture, uniformly stirring, and performing ultrasonic dispersion for 15-25 min to obtain a dispersion liquid;

3. magnetically stirring the dispersion under nitrogen atmosphere; and (4) carrying out ultrafiltration centrifugation on the stirred dispersion liquid, and collecting a centrifugal product to obtain the composite nano enzyme.

In step 4 provided by the embodiment of the present invention, the step of mixing the mixed enzyme solution with ammonium ferrous sulfate to prepare the composite nanoenzyme comprises:

1. mixing the prepared mixed enzyme solution with ammonium ferrous sulfate, and uniformly stirring to obtain a mixture;

2. dropwise adding deionized water into the mixture, uniformly stirring, and performing ultrasonic dispersion for 25-35 min to obtain a dispersion liquid;

3. magnetically stirring the dispersion under nitrogen atmosphere; and (4) carrying out ultrafiltration centrifugation on the stirred dispersion liquid, and collecting a centrifugal product to obtain the composite nano enzyme.

In step 5, the preparation of the zeolite-complexed nanoenzyme for tumor targeting using the preparation method includes:

1. placing the prepared composite nano enzyme in a water bath heating device for water bath heating; performing magnetic stirring on the composite nano-needle in the heating process, and adding a azine diammonium salt contrast agent during stirring;

2. continuing heating for 3-4 min after the magnetic stirring is finished, and finishing stirring to obtain a composite nano enzyme solution;

3. and (3) placing the composite nano enzyme liquid in a drying box for low-temperature drying to obtain the composite nano enzyme for tumor targeting.

In the step 5 provided by the embodiment of the invention, the water bath heating temperature is 35-40 ℃.

In the step 5 provided by the embodiment of the invention, the low-temperature drying temperature is 20-30 ℃, and the drying time is 8-10 h.

While the preferred embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.