CN115252644A

CN115252644A - Preparation method and application of nano-drug for enhancing anti-tumor effect by synergistic starvation therapy/chemodynamic therapy

Info

Publication number: CN115252644A
Application number: CN202210840566.XA
Authority: CN
Inventors: 于倩倩; 徐蒙蒙; 王林格
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2022-07-18
Filing date: 2022-07-18
Publication date: 2022-11-01
Anticipated expiration: 2042-07-18
Also published as: CN115252644B

Abstract

The invention belongs to the technical field of biological medicine, and provides self-sufficient O₂And H₂O₂And the nano enzyme medicine can enhance the anti-tumor capability by cooperating with the hunger therapy/the chemical power therapy. Construction of nano-enzyme catalytic medicine ZnO by bottom-up method₂@ Au @ ZIF-67, decomposed in a weakly acidic environment in tumors to release a catalyst Co²⁺Exposed ZnO₂And H₂Reaction of O to O₂And H₂O₂Can relieve the hypoxia in the tumor and cooperate with the subminiature Au nano-particlesCatalyzing glucose to produce H₂O₂And the glucose consumption of the tumor cells is accelerated, so that the tumor cells are in a severe hunger state. At the same time, H is generated₂O₂Takes part in Fenton-like reaction again and is Co²⁺Catalyzes the production of highly toxic OH, thereby improving the chemokinetic treatment. The implementation of the nano-enzyme provides a rich reference experience for the synthesis of a subsequent series of nano-enzymes and the application of multi-mode synergistic treatment on cancer.

Description

Preparation method and application of nano-medicament for enhancing anti-tumor effect by using cooperative hunger therapy/chemodynamic therapy

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a preparation method of an anti-tumor nano enzyme medicine for synergetic chemodynamics and hunger therapy.

Technical Field

In recent years, with the rapid development of nanotechnology and the recent progress of various technologies, many emerging methods have been used for cancer treatment, and the most potential application of these methods is nanomedicine technology. The characteristics of the nano-drugs, including the nano-scale size effect, have higher surface area/volume ratio and excellent drug slow-release capacity, so that the nano-drugs can better reach tumor tissues and complete the treatment effect. Some monotherapy nanomedicines have been used clinically or in preliminary clinical studies, including chemotherapy, radiotherapy, gene therapy, photothermal therapy, photodynamic therapy, magnetocaloric therapy, immunotherapy and some other non-mainstream therapies. However, in many experimental studies, a single treatment regimen does not completely eliminate the tumor and has no effect on preventing metastasis and recurrence of the cancer. Therefore, designing and preparing multifunctional anti-tumor nano-drugs with a combination of multiple modes to realize higher anti-tumor effect becomes a promising strategy.

In addition, tumor Microenvironment (TME), as the "soil" for Tumor growth, has complexity, diversity, heterogeneity and dynamics, and has characteristics of acidity, hypoxia and higher osmotic pressure different from normal tissues, which affects the efficacy of the nano-drug. In recent years, two very important means for realizing microenvironment response comprehensive treatment and personalized treatment by utilizing the regulation of TME are provided.

Of the numerous therapeutic approaches, chemokinetic therapy (CDT) in combination with starvation therapy (PTT) has become an effective approach for treating cancer. According to previous studies and reports, the metal ion Fe²⁺、Co²⁺、Cu²⁺And Mn²⁺Has excellent catalytic activity for Fenton reaction. Can effectively decompose H in the acidic microenvironment of the tumor₂O₂Is a highly toxic hydroxyl radical (·)OH), promoting apoptosis or necrosis of tumor cells. However, H in tumors₂O₂The level of (A) is too low to generate sufficient OH, and thus satisfactory catalytic effect cannot be obtained. Therefore, it is necessary to introduce H into the CDT catalyst₂O₂To improve the anticancer efficiency. Meanwhile, when the material with catalytic property such as ultra-small Au particles is accumulated in a tumor area, sensitive glucose in tumor tissues is degraded into H₂O₂Cutting off the nutrient source of the tumor, and inhibiting the growth of the tumor by using the hunger therapy. The invention is expected to synergize the treatment of the cancer through the cooperative linkage, and provides a new treatment strategy for the treatment of the cancer.

Disclosure of Invention

The invention provides a self-supporting O₂And H₂O₂And the nano enzyme medicine for enhancing the anti-tumor capacity by cooperating with the hunger therapy/the chemodynamic therapy, firstly, the nano enzyme medicine ZnO is constructed by the method from bottom to top₂@ Au @ ZIF-67. In a weakly acidic environment within the tumor, znO₂@ Au @ ZIF-67 is decomposed to rapidly release Fenton-like catalyst Co²⁺Exposed ZnO₂And H₂O(H⁺) Reaction to form O₂And H₂O₂Generation of O₂Can relieve the oxygen deficiency in the tumor and cooperate with the subminiature Au nano-particles to catalyze the glucose to generate H₂O₂Accelerating the glucose consumption of tumor cells and the exhaustion of energy metabolism, so that the tumor cells are in a severe hunger state. At the same time, H is generated₂O₂Takes part in Fenton-like reaction again and is Co²⁺Catalyzing the formation of highly toxic OH, thereby improving the chemokinetic treatment. The implementation of the nano-enzyme provides a rich reference experience for the synthesis of a subsequent series of nano-enzymes and the application of multi-mode synergistic treatment on cancer.

Another object of the present invention is to provide a method for preparing the above nanoenzyme system.

The final purpose of the invention is to provide an application of the above mentioned nanoenzyme system in the anti-tumor aspect.

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

a method for preparing nanometer enzyme medicine with synergistic effect of hunger therapy/chemodynamic therapy for enhancing antitumor ability comprises preparing ZnO by chemical method₂Nano particles, then obtaining ZnO by in-situ reduction through a seed crystal growth method₂@ Au nanoparticles, finally in ZnO₂Growth of ZIF-67 on the surface of @ Au to obtain ZnO₂@ au @ zif-67, achieves more efficient tumor killing by combining chemo-dynamic therapy/starvation therapy.

A method for preparing a nano enzyme medicament for enhancing anti-tumor capability by synergic starvation therapy/chemodynamic therapy comprises the following steps:

s1, preparing ZnO₂Nanoparticle: green preparation of ZnO by using underwater Leidenfrost nano-chemical method₂Zn (CH)₃COO)₂And H₂O₂Mixing, heating to obtain ZnO₂Nanoparticles;

s2, preparing ZnO₂@ Au nanoparticles: znO obtained from S1₂Mixing the nano particles with DMF solution, adding sodium borohydride solution and HAuCl₄Solution to obtain ZnO₂@ Au nanoparticles;

s3, preparing ZnO₂@ Au @ ZIF-67 nanoparticles: the ZnO obtained above is subjected to₂Dispersing the @ Au nanoparticles in an organic solvent, and then adding Co (NO)₃)₂·6H₂O, and marking the mixed solution as A; dissolving 2-methylimidazole and triethylamine in an organic solvent to obtain a clear solution, which is marked as B; finally mixing and stirring the A solution and the B solution to obtain ZnO₂@ Au @ ZIF-67 nanoparticles.

Zn (CH) in the step S1₃COO)₂In a concentration of 10-100mM, said Zn (CH)₃COO)₂And H₂O₂The volume ratio of (A) is 10-50.

ZnO obtained in step S1₂The particle size of the nano particles is 5-250nm. Preferably 50 to 200 nm.

The heating reaction temperature of the step S1 is 250-350 ℃, and the reaction time is 15-30S.

In step S2, the ZnO₂Mass volume of nanoparticles to DMFThe ratio is 1-200 mg: 1-50 mL; preferably 50 to 150:5 to 30mL.

In the step S2, the sodium borohydride solution is 0.2-1 mg/mL and HAuCl₄The concentration of the solution is 1-3 mmol/L;

the volume ratio of the sodium borohydride solution to the DMF in the step S2 is 1-50: 2 to 10, preferably 2 to 20:2 to 10; the HAuCl₄The volume ratio of the solution to DMF is 1-50: 2 to 4, preferably 1 to 10:2 to 10.

In step S3, the ZnO₂The mass volume ratio of @ Au to the organic solvent is 10-100 mg: 10-50 mL; the Co (NO)₃)₂·6H₂O and ZnO₂The mass ratio of @ Au is 5-20:10 to 100; preferably 8 to 20:20 to 70.

In the step S2, the mass-to-volume ratio of the 2-methylimidazole, the triethylamine and the organic solvent is 5-20 mg: 1-10 μ l:10 to 50mL.

And the organic solvent in the step S3 is at least one of methanol, ethanol, acetone and DMF.

The stirring time of the solution A and the solution B in the step S3 is 12 to 36 hours, and preferably 24 hours.

A nano enzyme medicine for improving the antineoplastic power by cooperative hunger therapy/chemical dynamic therapy is prepared by the above method.

An application of a nanometer enzyme medicine for enhancing anti-tumor ability by coordinating with starvation therapy/chemodynamic therapy in preparing anti-tumor materials.

Due to the adoption of the technical scheme, the invention has the technical progress that:

the implementation and application of the invention break through the limitation of monotherapy, overcome the defects of the catalysis substrate of the nano-enzyme, low catalytic activity, poor specificity and selectivity and the like through a series of cascade reactions, realize multi-mode synergistic treatment, and provide a high-efficiency and safe nano-medicament for realizing the comprehensive treatment strategy.

2, the implementation of the invention improves the effective tumor enrichment of the nano-drug: at present, the nano-materials can be enriched in tumor regions through an EPR effect. However, most of materials are still gathered in organs such as liver, spleen and the like in the material transportation process, and the nano materials need to be further modified or disguised, so that the in vivo circulation of nano enzyme is increased by introducing the nano materials which are degraded in response to pH or other internal and external stimuli, the premature degradation in the delivery process is prevented, and the tumor targeting efficiency is improved.

3, the implementation of the cascade reaction nanoenzyme of the invention: the cascade reaction nanoenzyme is synthesized from top to bottom through simple design, the defect of insufficient endogenous substrates is effectively overcome, the catalytic reaction is more thorough, and the treatment effect is greatly improved.

4, the implementation of the invention provides a new method and thought for the synthesis of the multimode nano enzyme: through reasonable design and construction, a single nano platform has multiple application functions, and the multiple treatment modes are mutually cooperated, so that the limitation of the single treatment mode is effectively overcome, and the treatment effect of the nano enzyme is remarkably improved.

5, znO of the invention₂The synthesis reagent is non-toxic and harmless, is green and environment-friendly, and the method is convenient and efficient.

6, the anti-tumor nano-drug has good biocompatibility and shows obvious biological safety; meanwhile, the hunger therapy and the chemo-kinetic therapy are combined to show remarkable anti-tumor activity.

Drawings

FIG. 1 is the ZnO prepared in example 1₂，ZnO₂@ Au and ZnO₂Transmission electron microscope picture of @ Au @ ZIF-67 nanoenzyme, wherein a) is nano-particle ZnO obtained after completion of S1₂B) and c) are nano-particles ZnO obtained after S2 is finished₂@ Au, d) is the nanoparticle ZnO obtained after S3 is completed₂@Au@ZIF-67。

FIG. 2 is a schematic diagram of the reaction activity of the nanoenzyme in solution, wherein a) is ZnO₂A schematic representation of the catalytic process of @ Au @ ZIF-67, b) is ZnO in the presence of hydrogen peroxide₂Detection of the formation of OH by catalysis of @ Au @ ZIF-67 c) is ZnO in the presence of glucose₂@ Au @ ZIF-67 catalysis of glucose consumption detection, d) is O under acidic conditions₂The resulting detection.

FIG. 3 is the results of the nano-enzyme in cell and animal experiments, wherein a) is the biocompatibility test of the nano-enzyme, b) is the chemokinetics and starvation therapy combined toxicity study of the nano-enzyme on tumor cells, and c) is the combined anti-tumor effect study of the nano-carrier on transplanted tumors in mice.

Detailed Description

The invention constructs the nano enzyme catalysis medicine ZnO by a bottom-up method₂@ Au @ ZIF-67. Fenton-like catalyst Co is quickly released by weak acid decomposition of tumor microenvironment²⁺Exposed ZnO₂And H₂O(H⁺) Reaction to form O₂And H₂O₂Generation of O₂Can relieve the oxygen deficiency in the tumor and cooperate with the Au nano-particles to catalyze the glucose to generate H₂O₂And the glucose consumption and energy depletion of tumor cells are accelerated, so that the tumor cells are in a severe hunger state. At the same time, H is generated₂O₂Participate in Fenton-like reaction and Co again²⁺The reaction produces highly toxic OH, thereby improving the chemokinetic treatment. Finally, the purposes of inhibiting the growth of the tumor and eliminating the tumor are achieved, and the implementation of the nano enzyme provides abundant reference experience for the synthesis of a series of subsequent nano enzymes and the application of multi-mode synergistic treatment on the cancer. In the embodiment of the invention, the normal temperature/room temperature and the unspecified temperature are both 20-35 ℃.

The present invention will be described in further detail with reference to the following examples:

example 1

5mL of hydrogen peroxide were mixed with 50mL of aqueous zinc acetate (70 mM) in a glass petri dish and then rapidly transferred to a high temperature plate at a temperature of 300 ℃. After 30s, the solution gradually changed from colorless to milky white, and then the solution was centrifuged at 10000rpm to obtain ZnO with a particle size of about 100nm₂Nanoparticles. 100mg of ZnO₂The nanoparticles were dispersed in 10mL DMF, stirred well, and 4mL of 0.5mg/mL sodium borohydride solution and 2mL of 1mmol/L HAuCl were added using a micro syringe pump₄Simultaneously injecting the solution into the above solution, stirring the mixed solution at room temperature for about 8h to obtain ZnO₂@ Au nanoparticles. In the ultrasonic wave50mg of ZnO under treatment₂@ Au was dispersed in 20mL of methanol, and then 12mg of Co (NO) was dissolved in the above solution₃)₂·6H₂O, to give a mixture solution (denoted as a). Next, 13mg of 2-methylimidazole and 6. Mu.L of triethylamine were dissolved in 20mL of methanol under the same ultrasonic treatment to obtain a clear solution (described as B). Finally, solution B was poured into solution a and stirred for 24h, the product was isolated by centrifugation and washed 3 times with methanol. The product is dried in vacuum for 4 hours at 50 ℃ to obtain ZnO₂@ Au @ ZIF-67 nanoparticles.

Example 2

5ml of hydrogen peroxide was mixed with 50ml of aqueous zinc acetate (50 mM) in a glass petri dish and then rapidly transferred to a high temperature plate at a temperature of 300 ℃. After 30s, the solution was gradually changed from colorless to milky white by the naked eye. Then centrifuging at 10000rpm to obtain ZnO with particle size of about 200nm₂Nanoparticles. 150mg of ZnO₂The nanoparticles were dispersed in 20mL DMF, stirred well, and then 8mL of 0.5mg/mL sodium borohydride solution, and 4mL of 1mmol/L HAuCl were added using a micro syringe pump₄Injecting the solution into the above solution simultaneously, and stirring the mixed solution at room temperature for about 8 hr to obtain ZnO₂@ Au nanoparticles. 50mg of ZnO was treated with ultrasonic waves₂@ Au was dispersed in 20mL of methanol, and then 12mg of Co (NO) was dissolved in the above solution₃)₂·6H₂O, to give a mixture solution (denoted as a). Then, 13mg of 2-methylimidazole and 6. Mu.L of triethylamine were dissolved in 20mL of methanol under the same sonication, to obtain a clear solution (described as B). Finally, solution B was poured into solution a and stirred for 24h. The product was isolated by centrifugation and washed 3 times with methanol. Vacuum drying the product at 50 ℃ for 4h to obtain ZnO₂@ Au @ ZIF-67 nanoparticles.

Application examples

FIG. 1 results implementation procedure:

the ZnO prepared in example 1 was weighed separately₂，ZnO₂@ Au and ZnO₂@ Au @ ZIF-67.0 mg in 1ml ethanol solution, ultrasonically dispersing for 10min, respectively taking 5 μ L of the above dispersion with a pipette, dropping on a 100 mesh carbon support film copper net, and filteringAnd (5) placing the mixture in a vacuum drying oven and drying. Then, the morphology of the nanoparticles was obtained by using a transmission electron microscope (TEM, JEOL JEM-2011), and the experimental result is shown in fig. 1.

FIG. 2 results implementation procedure:

(1) ZnO prepared in example 1₂@ Au (0.05 mg/mL) and ZnO₂@ Au @ ZIF-67 (0.05 mg/mL) nanoparticles, 3', 5' - -tetramethylbenzidine (TMB, 2 mM) and H at a concentration of 1.0mM₂O₂Adding into buffer solution with pH of 6.0, incubating the mixture in water bath (37 deg.C) for 4min, and detecting ZnO with ultraviolet-visible spectrophotometer₂@ Au and ZnO₂@ Au @ ZIF-67 nanoenzyme catalysis H₂O₂The experimental results are shown in FIG. 2 a) and FIG. 2 b).

(2) ZnO prepared in example 1₂@ Au (0.05 mg/mL) nanoparticles, glucose oxidase (GOx) were mixed with a glucose solution (50 mM), respectively, and then 3, 5-dinitrosalicylic acid solution (1 mL) was added, and ZnO was detected by UV-visible spectrophotometer₂The reaction condition of the glucose solution is catalyzed by the @ Au and glucose oxidase nano-particles, and the experimental result is shown in FIG. 2 c).

(3) 20mg of ZnO prepared in example 1 were added₂@ Au @ ZIF-67 was dispersed in 20mL of a deoxygenated acetic acid buffer solution (0.1M, pH 6.0), and then the oxygen concentration was monitored every 10min for 120min with a portable oxygen dissolution apparatus. As a control, the oxygen concentration of 20mL of deoxygenated water was monitored under the same conditions. The results of the experiment are shown in FIG. 2 d).

FIG. 3 results implementation procedure

(1) HUVEC and 4T1 cells were seeded into 96-well plates and CO was determined under standard conditions (37 ℃ and 5%₂) And culturing for 24h. The culture medium was treated with different concentrations of ZnO prepared in example 1 at different pH₂@ Au @ ZIF-67 medium solution. Cells were cultured in an incubator for 12/24H, and after the incubation time was over, the relative cell viability was measured by performing a 3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2-H-tetrazolium (MTT) assay. The results of the experiments are shown in fig. 3 a) and 3 b).

(2) Kunming mice in the 4T1 tumor model were first randomized into 4 groups. (i) QuietIntravenous saline (200 mL); (ii) Intravenous ZnO₂(200mL，1.5mg kg^-1) B, carrying out the following steps of; (iii) Intravenous ZnO₂@Au(200mL，1.5mg kg^-1) (ii) a (iv) Intravenous ZnO₂@ZIF-67(200mL，1.5mg kg^-1) (v) intravenous injection of ZnO₂@Au@ZIF-67 (200mL，1.5mg kg^-1). The body weight change of the mice was recorded every 2 days for 15 days.

The above samples were all prepared from example 1, in which ZnO was present₂The preparation of @ ZIF-67 was carried out using the procedure described in example 1, but excluding the preparation from ZnO₂Preparation of ZnO from nanoparticles₂@ Au nanoparticles.

Claims

1. A preparation method of a nano enzyme medicament for enhancing anti-tumor capability by synergic hunger therapy/chemodynamic therapy is characterized by comprising the following steps:

s1, preparing ZnO₂Nanoparticle: green preparation of ZnO by using underwater Leidenfrost nano-chemical method₂From Zn (CH)₃COO)₂And H₂O₂Mixing, heating and reacting to obtain ZnO₂Nanoparticles;

s3, preparing ZnO₂@ Au @ ZIF-67 nanoparticles: znO obtained from S2₂@ Au nanoparticles dispersed in organic solvent followed by Co (NO) addition₃)₂·6H₂O, and marking the mixed solution as A; dissolving 2-methylimidazole and triethylamine in an organic solvent to obtain a clear solution, which is marked as B; finally mixing and stirring the A solution and the B solution to obtain ZnO₂@ Au @ ZIF-67 nanoparticles.

2. The method of claim 1, wherein:

zn (CH) in the step S1₃COO)₂In a concentration of 10-100mM, said Zn: (CH₃COO)₂And H₂O₂The volume ratio of (A) is 10-50.

3. The method of claim, wherein:

4. The method of claim, wherein: in step S2, the ZnO₂The mass-volume ratio of the nano particles to the DMF is 1-200 mg: 1-50 mL.

5. The method of claim, wherein: in the step S2, the sodium borohydride solution is 0.2-1 mg/mL and HAuCl₄The concentration of the solution is 1-3 mmol/L.

6. The method of claim, wherein: in the step S2, the volume ratio of the sodium borohydride solution to the DMF is 1-50₄The volume ratio of the solution to DMF is 1-50: 2 to 4.

7. The method of claim, wherein: in step S3, the ZnO₂The mass volume ratio of @ Au to methanol is 10-100 mg: 10-50 mL; the Co (NO)₃)₂·6H₂O and ZnO₂The mass ratio of @ Au is 5-20:10 to 100.

8. The method of claim, wherein: in the step S2, the mass volume ratio of the 2-methylimidazole to the triethylamine to the organic solvent is 5-20 mg: 1-10. Mu.l: 10-50 mL;

9. A nano enzyme medicament for enhancing anti-tumor capacity by cooperative hunger therapy/chemo-dynamic therapy, which is prepared by the method of any one of claims 1 to 8.

10. The use of the nanoenzyme drug for enhancing anti-tumor ability of the cooperative starvation therapy/chemokinetic therapy according to claim 9 in the preparation of an anti-tumor material.