CN113750246B - Application of ZIF-8 nanomaterial in degradation of broad-spectrum mutant p53 protein - Google Patents

Application of ZIF-8 nanomaterial in degradation of broad-spectrum mutant p53 protein Download PDF

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CN113750246B
CN113750246B CN202010498057.4A CN202010498057A CN113750246B CN 113750246 B CN113750246 B CN 113750246B CN 202010498057 A CN202010498057 A CN 202010498057A CN 113750246 B CN113750246 B CN 113750246B
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zif
cells
degradation
nanocrystals
mutant
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CN113750246A (en
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温龙平
张云娇
黄晓婉
王连生
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South China University of Technology SCUT
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Abstract

The invention discloses an application of ZIF-8 nano material in degradation of broad-spectrum mutant p53 protein. According to the invention, ZIF-8 nano materials can selectively trigger degradation of mutant p53 protein in a mutant p53 cell line, the triggered degradation of the mutp53 level is caused by degradation rather than protein synthesis, and the toxicity in the cell line can be obviously reduced after being modified by a peptide with pH responsiveness (Z1-RGD peptide), so that the treatment effect on tumors is better. The invention also discovers that the ubiquitin enzyme inhibitor, the tyrosine protein kinase inhibitor and the N, N, N ', N' -tetra (2-picolyl) ethylenediamine can inhibit the degradation of the mutant p53 protein induced by ZIF-8, and provides an effective way for the research of the degradation of the mutant p53 protein by ZIF-8 nano materials.

Description

Application of ZIF-8 nanomaterial in degradation of broad-spectrum mutant p53 protein
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to an application of a ZIF-8 nanomaterial in degradation of broad-spectrum mutant p53 protein.
Background
The p53 gene is the gene found to date to have the highest relevance to human tumors. Unlike traditional tumor factors, which often have conditions of down-regulation of expression or deletion of genes in human cancers, p53 is mutated in most tumor cells. Results from genomic sequencing of different human tumor cells showed that mutations in this gene occurred in more than 50% of all malignant tumors, and that the p53 mutation profile observed in different types of tumors tended to be inconsistent. Unlike wild-type p53, which is maintained at a lower level at normal physiological levels, mutated p53 (mutp 53) is heavily dispersed or aggregated in cells and is difficult to degrade. More importantly, mutated p53 not only loses its ability to act as an oncogene, but also tends to produce many new functions (GOFs) that promote the development and progression of cancer. If the genome is unstable, the development, the deterioration, the metastasis, the apoptosis resistance, the chemotherapy and radiotherapy tolerance and the like of the tumor are promoted.
Due to the presence of high levels of mutp53 in tumor cells, enhanced degradation of mutp53 in tumor cells is expected to provide a desirable solution for tumor therapy targeting mutp 53. Currently, there are small molecules that induce the degradation of mutp53 via the proteasome pathway, such as the Hsp90 inhibitor 17-AAG and ganetespib, NSC59984, and statins, among others; there are also substances inducing the degradation of mutp53 by the autophagy pathway, such as MCB-613, SAHA, gambogic acid and Zn (II) -curc, etc. However, current methods rely almost entirely on small molecules, and substances identified so far that are capable of degrading mutp53 often lack sufficient specificity and low toxicity to normal cells.
Metal Organic Frameworks (MOFs) are an emerging class of hybrid porous materials that are built up by self-assembly of metal ions and organic ligands. In recent years, MOF materials have found widespread use in many fields including medicine. Of the many types of MOF materials, nanoscale zeolite imidazole ester-8 (ZIF-8), which uses zinc as the metal node and 2-methylimidazole as the linker molecule, is the best studied one. The ZIF-8 nano material has good biocompatibility and is widely used for drug delivery, in particular for delivering anticancer drugs. However, the research on tumor treatment by degrading mutp53 with MOF material is still blank at home and abroad.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art and provides an application of a ZIF-8 nanomaterial in degradation of broad-spectrum mutant p53 protein.
The aim of the invention is achieved by the following technical scheme:
the application of the ZIF-8 nanomaterial in degradation of broad-spectrum mutant p53 protein, wherein the ZIF-8 nanomaterial is at least one of a ZIF-8 nanocrystal, a dye-entrapped ZIF-8 nanocrystal and a peptide-modified ZIF-8 nanomaterial with pH responsiveness.
The dye in the dye-entrapped ZIF-8 nanocrystals is at least one of sulfo-cy5 and FITC (fluorescein 5-isothiocyanate).
The peptide with pH responsiveness is at least one of Z1-RGD peptide and FITC-Z1-RGD peptide.
The sequence of the Z1-RGD peptide is as follows: RGDGGAHPHSDKLVPPR.
The sequence of the FITC-Z1-RGD peptide is as follows: FITC-RGDGGAHPHSDKLVPPR.
The ZIF-8 nanocrystals for entrapping the dye are at least one of ZIF-8@sulfo-cy5 and ZIF-8@FITC.
The ZIF-8 nanomaterial with pH responsiveness is at least one of P-ZIF-8, FITC-ZIF-8@sulfo-cy5 and P-ZIF-8@FITC; more preferably at least one of P-ZIF-8 and P-ZIF-8@FITC.
The ZIF-8 nanocrystals are preferably prepared by the following method:
zn (NO) 3 ) 2 ·6H 2 Adding O into a solvent, and uniformly mixing by ultrasonic to obtain a mixed solution I; adding 2-methylimidazole into a solvent, and uniformly mixing by ultrasonic to obtain a mixed solution II (2-methylimidazole solution); and (3) dropwise adding the mixed solution II into the mixed solution I, stirring, centrifugally collecting precipitate, washing, and drying in vacuum to obtain the ZIF-8 nanocrystals.
The solvent is at least one of water and methanol.
The water is preferably water with a pH of 8.0.
The pH value of the water with the pH value of 8.0 is regulated by NaOH.
Said Zn (NO) 3 ) 2 ·6H 2 The dosage of O is 0.02-0.04 g Zn (NO) per milliliter solvent 3 ) 2 ·6H 2 O is calculated.
The ultrasonic conditions are as follows: 600W ultrasonic for 2-3 min; preferably 600W for 2 minutes.
The dosage of the 2-methylimidazole is calculated according to the proportion of 0.045-0.2 g of 2-methylimidazole in each milliliter of solvent.
The 2-methylimidazole and Zn (NO 3 ) 2 ·6H 2 The mass ratio of O is 1-2:0.1-1.
The stirring conditions are as follows: stirring at 2000rpm for 10-20 minutes; preferably 2000rpm for 15 minutes.
The centrifugation conditions are preferably as follows: centrifuge at 4℃and 7000-10000 g for 10 minutes.
The washing is carried out by adopting more than one of methanol, ethanol and water; preferably, the washing is carried out by adopting an ethanol water solution with the volume percentage of 50 percent; more preferably, the washing is performed for more than 3 times by adopting an ethanol water solution with the volume percentage of 50%; to remove as much as possible the excess 2-methylimidazole adsorbed on the nanocrystal surface.
The P-ZIF-8 nanocrystals are preferably prepared by the following method:
mixing the ZIF-8 nanocrystals with the Z1-RGD peptide, adding HEPES buffer solution, vortex shaking for 30 seconds, incubating at room temperature, centrifuging, and washing to obtain the P-ZIF-8 nanocrystals.
The mass ratio of the ZIF-8 nanocrystals to the Z1-RGD peptide is 5:1.
The HEPES buffer solution comprises the following components: 50mM HEPES-NaOH,150mM NaCl,pH 7.0.
The incubation time is preferably about 2 hours.
The centrifugation conditions are preferably as follows: centrifuge at 4℃and 7000-10000 g for 10 minutes.
The FITC-P-ZIF-8 nanocrystals are preferably prepared by the following method:
mixing the ZIF-8 nanocrystals with FITC-Z1-RGD peptide, adding HEPES buffer, vortex shaking for 30 seconds, incubating at room temperature, centrifuging, and washing to obtain FITC-P-ZIF-8 nanocrystals.
The mass ratio of the ZIF-8 nanocrystals to the FITC-Z1-RGD peptide is 5:1.
The HEPES buffer solution comprises the following components: 50mM HEPES-NaOH,150mM NaCl,pH 7.0.
The incubation time is preferably about 2 hours.
The centrifugation conditions are preferably as follows: centrifuge at 4℃and 7000-10000 g for 10 minutes.
The ZIF-8@sulfo5 nanocrystals are preferably prepared by the following method:
Zn (NO) 3 ) 2 ·6H 2 O is added into the solutionIn the agent, uniformly mixing by ultrasonic to obtain a mixed solution A; adding sulfo-cy5 into a solvent, and uniformly mixing by ultrasonic to obtain a mixed solution B; adding 2-methylimidazole into a solvent, and uniformly mixing by ultrasonic to obtain a mixed solution C (2-methylimidazole solution); and (3) sequentially dripping the mixed solution B and the mixed solution C into the mixed solution A, stirring, centrifugally collecting the precipitate, washing, and drying in vacuum to obtain the ZIF-8@sulfo-cy5 nanocrystals.
The solvent is water; preferably water at a pH of 8.0.
The pH value of the water with the pH value of 8.0 is regulated by NaOH.
Said Zn (NO) 3 ) 2 ·6H 2 The dosage of O is 0.02-0.04 g Zn (NO) per milliliter solvent 3 ) 2 ·6H 2 O is calculated; preferably 0.025g Zn (NO) per ml solvent 3 ) 2 ·6H 2 O is calculated.
The ultrasonic conditions are as follows: 600W ultrasonic for 2-3 min; preferably 600W for 2 minutes.
The dosage of the sulfo-cy5 is calculated according to the ratio of 5mg sulfo-cy5 in each milliliter of solvent.
The dosage of the 2-methylimidazole is calculated according to the proportion of 0.045-0.2 g of 2-methylimidazole in each milliliter of solvent; preferably 0.2g of 2-methylimidazole per ml of solvent.
Said Zn (NO) 3 ) 2 ·6H 2 The mass ratio of O, sulfo-cy5 to 2-methylimidazole is 10:1:100.
The stirring conditions are as follows: stirring at 2000rpm for 10-20 minutes; preferably 2000rpm for 15 minutes.
The centrifugation conditions are preferably as follows: centrifuge at 4℃and 7000-10000 g for 10 minutes.
The washing is carried out by adopting more than one of methanol, ethanol and water; preferably, the washing is carried out by adopting an ethanol water solution with the volume percentage of 50 percent; more preferably, the washing is performed for more than 3 times by adopting an ethanol water solution with the volume percentage of 50%; to remove as much as possible the excess 2-methylimidazole adsorbed on the nanocrystal surface.
The FITC-ZIF-8@sulfo-cy5 nanocrystals are preferably prepared by the following method:
and mixing the ZIF-8@sulfo-cy5 nanocrystals with FITC-Z1-RGD peptide, adding HEPES buffer solution, vortex shaking for 30 seconds, incubating at room temperature, centrifuging, and washing to obtain the ZIF-8@sulfo-cy5 nanocrystals.
The mass ratio of the ZIF-8@sulfo-cy5 nanocrystals to the FITC-Z1-RGD peptide is 5:1.
The HEPES buffer solution comprises the following components: 50mM HEPES-NaOH,150mM NaCl,pH 7.0.
The incubation time is preferably about 2 hours.
The centrifugation conditions are preferably as follows: centrifuge at 4℃and 7000-10000 g for 10 minutes.
The ZIF-8@FITC nanocrystals are preferably prepared by the following method:
Zn (NO) 3 ) 2 ·6H 2 Adding O into a solvent, and uniformly mixing by ultrasonic waves to obtain a mixed solution D; adding a fluorescein 5-isothiocyanate solution (FITC) into a solvent, and uniformly mixing by ultrasonic to obtain a mixed solution E; adding 2-methylimidazole into a solvent, and uniformly mixing by ultrasonic to obtain a mixed solution F (2-methylimidazole solution); and (3) sequentially dripping the mixed solution F and the mixed solution E into the mixed solution D, stirring, centrifugally collecting the precipitate, washing, and drying in vacuum to obtain the ZIF-8@FITC nanocrystals.
The solvent is methanol.
Said Zn (NO) 3 ) 2 ·6H 2 The dosage of O is 0.02-0.04 g Zn (NO) per milliliter solvent 3 ) 2 ·6H 2 O is calculated; preferably 0.02g Zn (NO) per ml solvent 3 ) 2 ·6H 2 O is calculated.
The ultrasonic conditions are as follows: 600W ultrasonic for 2-3 min; preferably 600W for 2 minutes.
The dosage of the fluorescein 5-isothiocyanate solution (FITC) is calculated according to the proportion of 2mg of the fluorescein 5-isothiocyanate solution (FITC) per milliliter of solvent.
The dosage of the 2-methylimidazole is calculated according to the proportion of 0.045-0.2 g of 2-methylimidazole in each milliliter of solvent; preferably 0.2g of 2-methylimidazole per ml of solvent.
Said Zn (NO) 3 ) 2 ·6H 2 The mass ratio of O, 5-isothiocyanate solution (FITC) to 2-methylimidazole was 1:0.002:2.2.
The stirring conditions are as follows: stirring at 2000rpm for 10-20 minutes; preferably 2000rpm for 15 minutes.
The centrifugation conditions are preferably as follows: centrifuge at 4℃and 7000-10000 g for 10 minutes.
The washing is preferably performed by methanol; more preferably, the washing with methanol is performed 3 times or more to remove as much as possible the excess 2-methylimidazole adsorbed on the nanocrystal surface.
The P-ZIF-8@FITC nanocrystals are preferably prepared by the following method:
and mixing the ZIF-8@FITC nanocrystals with the Z1-RGD peptide, adding HEPES buffer, vortex shaking for 30 seconds, incubating at room temperature, centrifuging, and washing to obtain the P-ZIF-8@FITC nanocrystals.
The mass ratio of the ZIF-8@FITC nanocrystals to the Z1-RGD peptide is 5:1.
The HEPES buffer solution comprises the following components: 50mM HEPES-NaOH,150mM NaCl,pH 7.0.
The incubation time is preferably about 2 hours.
The centrifugation conditions are preferably as follows: centrifuge at 4℃for 10 min with 10000 g.
The mutant p53 protein is p53 protein coded after partial basic groups on the p53 gene are mutated; preferably at least one of the p53 protein mutants S241F, E285K, Y220C, R249S, R280K, R248W, R175H, R273H and G245C.
The environment of the application is in vitro.
The effective concentration of the ZIF-8 nanomaterial is 25-150 mug/mL; preferably 25 to 100. Mu.g/mL; more preferably 100. Mu.g/mL.
The degradation time is 0.5-8 hours; preferably 4 to 8 hours.
The application of the ZIF-8 nanomaterial in degrading broad-spectrum mutant p53 protein is that the ZIF-8 nanomaterial is directly added or the pretreated ZIF-8 nanomaterial is added; the pretreatment is as follows: the ZIF-8 nanomaterial is incubated at a pH of 5.5-7.5 for 0-12 hours (excluding 0).
The pH value is preferably 5.5-6.5; more preferably 6.5.
Application of ZIF-8 nanomaterial in preparing products for degrading broad-spectrum mutant p53 protein.
The products include medicines, kits, degradation inhibitors and the like.
The ZIF-8 nano material is used as a broad-spectrum mutation p53 protein degradation agent in the preparation of antitumor drugs.
Use of an enzyme inhibitor or TPEN (N, N' -tetrakis (2-picolyl) ethylenediamine) in the preparation of a product for inhibiting ZIF-8 nanomaterial degradation of a broad-spectrum mutant p53 protein; wherein the enzyme inhibitor is at least one of a ubiquitin enzyme inhibitor and a tyrosine protein kinase inhibitor; the ubiquitin enzyme inhibitor can inhibit the degradation of mutp53 induced by the ZIF-8 nanomaterial, the degradation of mutp53 in ES-2 cells induced by the ZIF-8 nanomaterial can be effectively inhibited by the endocytic inhibitor Genistein, and TPEN can inhibit the degradation of mutp53 induced by the ZIF-8 nanomaterial.
The ubiquitin enzyme inhibitor is a ubiquitinase inhibitor PYR-41.
The effective concentration of the ubiquitinase inhibitor PYR-41 is 5 mu mol/L.
The tyrosine protein kinase inhibitor is tyrosine protein kinase inhibitor Genistein.
The effective concentration of the tyrosine protein kinase inhibitor Genistein is 50 mu mol/L.
The effective concentration of TPEN (N, N, N ', N' -tetra (2-picolyl) ethylenediamine) is 50 mu mol/L.
Compared with the prior art, the invention has the following advantages and effects:
(1) The invention provides an application of ZIF-8 nano material in degrading broad-spectrum mutant p53 protein, ZIF-8 can selectively trigger degradation of mutant p53 protein in mutant p53 cell line, and the triggered reduction of mutp53 level is caused by degradation rather than reduction of protein synthesis. ZIF-8 nanomaterials effectively degrade mutp53 mutants to varying degrees, including Y220C, R249S, R175H, R248W, R273H, G245C, and the like. The ZIF-8 nanomaterial disclosed by the invention can be subjected to peptide modification with pH responsiveness, so that the toxicity in a cell line can be obviously reduced, and the treatment effect on tumors is better.
(2) According to the invention, the ubiquitinase inhibitor PYR-41 can inhibit the degradation of mutp53 caused by ZIF-8, while the deubiquitinase inhibitor PR-619 is not affected, which shows that the degradation process of mutp53 caused by ZIF-8 depends on the ubiquitination modification of mutp 53.
(3) In the invention, the degradation of mutp53 in ZIF-8-induced cells can be effectively inhibited by an endocytosis inhibitor Genistein, which indicates that endocytosis of the cells is necessary for the degradation induced by ZIF-8.
(4) In the present invention, it was found that Zn in acid lysosomes was observed in cells after ZIF-8 treatment 2+ Elevated, while TPEN is capable of completely inhibiting intracellular Zn caused by ZIF-8 2+ Elevated, and further demonstrated that TPEN was able to inhibit ZIF-8-induced degradation of mutp53, suggesting that ZIF-8-induced degradation of mutp53 is dependent on intracellular Zn 2+ Raised.
Drawings
FIG. 1 is a graph of the result of western blot detection of the degradation of mup53 induced by ZIF-8 in SK-BR-3 and ES-2 cells (in the figure, "-" is control PBS buffer, "+" is ZIF-8 nanoparticle).
FIG. 2 is a graph of the fluorescence results of ZIF-8 in ES-2 cells initiating the degradation of mup 53.
FIG. 3 is a graph of the result of western blot detection of the synthetic pathway of mutp53 not affected by the degradation of mutp53 induced by ZIF-8.
FIG. 4 is a graph of the result of western blot detection of the necessity of the framework structure of ZIF-8 to degrade mutp 53.
FIG. 5 is a graph of the result of western blot analysis of the dose-dependent effect of ZIF-8-induced degradation of mutant p 53.
FIG. 6 is a graph of the result of western blot detection of the time-dependent effect of ZIF-8 induced degradation of mutant p 53.
FIG. 7 is a graph showing the result of western blot detection of ZIF-8 without causing degradation of wild type p 53.
FIG. 8 is a graph of the result of western blot analysis of pathways by which ZIF-8 triggers the degradation of mutp53 in ES-2 cells.
FIG. 9 is a graph of the result of western blot detection of elevated levels of K48 ubiquitination in ZIF-8-primed ES-2 cells.
FIG. 10 is a graph showing the result of western blot detection of the involvement of ubiquitinase in the initiation of mutp53 degradation in ZIF-8 cells.
FIG. 11 is a graph showing the result of western blot verification that degradation of mutp53 in ZIF-8-induced cells can be effectively inhibited by the endocytic inhibitor Genistein.
FIG. 12 shows the Zn content of ZIF-8 pair cell acid lysosomes 2+ Fluorescence results for horizontal effects.
FIG. 13 shows the pair of intracellular Zn in ZIF-8 and C-ZIF-8 2+ FACS test results graph of horizontal effects.
FIG. 14 is Zn in ZIF-8 primed cells 2+ FACS test results graphs in which elevated levels can be effectively inhibited by the metal chelator TPEN.
FIG. 15 is a graph showing the result of western blot verification that mutp53 degradation in ZIF-8-induced cells can be effectively inhibited by metal chelator TPEN.
FIG. 16 shows that ZIF-8 causes intracellular Zn 2+ The dose-dependent fluorescence results were plotted.
FIG. 17 shows that ZIF-8 causes intracellular Zn 2+ Dose-dependent FACS test results plots were elevated.
FIG. 18 shows the decomposition of ZIF-8 to release Zn 2+ Western blot validation results plots associated with induced mutp53 degradation.
FIG. 19 is a graph of fluorescence results of testing ZIF-8 for inclusion body escape behavior in ES-2 cells.
FIG. 20 is a transmission electron micrograph of ZIF-8 and P-ZIF-8 obtained in example 1.
FIG. 21 shows that ZIF-8 and P-ZIF-8 release Zn with pH sensitivity 2+ And (5) testing result graphs of behaviors.
FIG. 22 is a graph of the result of western blot analysis comparing the behavior of ZIF-8 and P-ZIF-8 in degrading mutp53 at pH 6.5.
FIG. 23 is a graph showing the results of cell viability assays of HEK 293 and ARPE-19 cells treated after pre-incubation of ZIF-8 and P-ZIF-8 at pH 7.4 and pH 6.5; wherein A is HEK 293 cells; b is ARPE-19 cells.
FIG. 24 is a graph of fluorescence detection results of comparison of the ability of ZIF-8 and P-ZIF-8 to enter ES-2 cells.
FIG. 25 shows the release of Zn in cells by ZIF-8 and P-ZIF-8 2+ FACS test result graph of capacity comparison.
FIG. 26 is a graph showing the result of western blot analysis comparing the ability of ZIF-8 and P-ZIF-8 to degrade mutant P53 in ES-2 cells.
FIG. 27 is an IC of ZIF-8 in various cells 50 Graph of the relationship with the percent degradation of mutp 53.
FIG. 28 is a graph showing the results of ICP measurement of ZIF-8 and P-ZIF-8 in animals.
FIG. 29 is a graph showing tumor volume change in ES-2 tumor-bearing mice treated with ZIF-8 and P-ZIF-8.
FIG. 30 is a graph showing the change in tumor weight of ZIF-8 and P-ZIF-8 treated ES-2 tumor-bearing mice.
FIG. 31 is a graph showing the results of western blot detection of P53 protein levels in tumor tissue of ES-2 tumor-bearing mice treated with ZIF-8 and P-ZIF-8.
FIG. 32 is a graph showing tumor volume change in MCF-7 tumor-bearing mice treated with P-ZIF-8.
FIG. 33 is a graph of tumor volume change following treatment of PDX tumor models with ZIF-8 and P-ZIF-8.
FIG. 34 is a graph of tumor weight change after treatment of PDX tumor models with ZIF-8 and P-ZIF-8.
FIG. 35 is a photograph of tumors after ZIF-8 and P-ZIF-8 treatment with a PDX tumor model.
FIG. 36 is a graph of immunohistochemical staining of tumor tissue following treatment of the PDX tumor model with ZIF-8 and P-ZIF-8.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The reagents and starting materials used in the present invention are commercially available unless otherwise specified.
Example 1 preparation of FITC-ZIF-8, P-ZIF-8, FITC-P-ZIF-8, ZIF-8@sulfo5, FITC-ZIF-8@sulfo5 nanocrystals
(1) Preparation of ZIF-8 nanocrystals
Into a 50mL glass reaction flask, 0.2g of Zn (NO) was charged at room temperature 3 ) 2 ·6H 2 O、4.8mL H 2 O (pH 8.0, adjusted with NaOH) and a particle of magnetic material, 2g of 2-methylimidazole and 10mL of H were then added to a 15mL EP tube 2 O. After the bottle cap is tightly covered, respectively placing the two mixed solutions in an ultrasonic instrument for ultrasonic treatment (600W for two minutes) to uniformly mix the raw materials, and then slowly dripping the uniformly mixed 2-methylimidazole solution into a glass reaction bottle. After 15 minutes of reaction with magnetic stirring (2000 rpm), the precipitate was collected by centrifugation and washed 3 times with a deionized water solution containing 50% (v/v) ethanol to remove as much as possible the excess 2-methylimidazole adsorbed on the nanocrystal surface. The finally prepared ZIF-8 nanocrystals (ZIF-8 nanoparticles) were dried in vacuo at room temperature and stored as a powder at 4 ℃.
(2) Preparation of P-ZIF-8 nanocrystals
To prepare the P-ZIF-8 nanocrystals, 5mg of ZIF-8 powder prepared above was mixed with 1mg of the Z1-RGD peptide (SEQ ID NO: RGDGGAHPHSDKLVPPR, available from Yu Jiangyao organisms). 1mL HEPES buffer (50 mM HEPES-NaOH,150mM NaCl,pH 7.0) was added and vortexed for 30 seconds. After incubation for 2 hours at room temperature, unbound peptide was removed by centrifugation (10 g,4 ℃,10 min). The prepared P-ZIF-8 nanoparticles were washed 3 times with HEPES buffer before use.
(3) Preparation of FITC-P-ZIF-8 nanocrystals
To prepare FITC-P-ZIF-8 nanocrystals, the same procedure as for the preparation of P-ZIF-8 was used, substituting the Z1-RGD with the FITC-Z1-RGD peptide (sequence FITC-RGDGGAHPHSDKLVPPR, available from Yu Jiangyao organisms) and the remaining steps were the same.
(4) Preparation of ZIF-8@sulfo-cy5 nanocrystals
To prepare ZIF-8@sulfo5 nanocrystals, 0.2g Zn (NO) was added to a 50mL glass reactor at ambient temperature 3 ) 2 ·6H 2 O、0.8mL H 2 O (pH 8.0, adjusted with NaOH) and a particle of magnetic material, then 20mg of sulfocy 5 (from Chengdu Corp. Kang Shengwu) and 4mL of H were added to an EP tube 2 O, another EP tube was charged with 2g of 2-methylimidazole and 10mL of H 2 O. After the bottle caps are tightly covered, respectively placing the three mixed solutions in an ultrasonic instrument for ultrasonic treatment (600W for two minutes) for 2 minutes to uniformly mix the raw materials, then slowly dripping the sulfo-cy5 solution into a glass reaction bottle, gradually changing the solution in the reaction bottle into blue, and slowly dripping the 2-methylimidazole solution into the glass reaction bottle after uniformly mixing. After 15 minutes of reaction with magnetic stirring (2000 rpm), the precipitate was collected by centrifugation and washed 3 times with a deionized water solution containing 50% (v/v) ethanol to remove as much as possible the excess 2-methylimidazole adsorbed on the nanocrystal surface. The finally prepared ZIF-8@sulfo5 nanocrystals were dried in vacuo at room temperature and stored as a powder at 4 ℃.
(5) Preparation of FITC-ZIF-8@sulfo-cy5 nanocrystals
For the preparation of FITC-ZIF-8@sulfo-cy5 nanocrystals, reference was made to the preparation method of P-ZIF-8, with ZIF-8@sulfo-cy5 replacing ZIF-8 nanocrystals, FITC-Z1-RGD peptide replacing Z1-RGD, the remaining steps being identical.
Example 2 preparation of ZIF-8, ZIF-8@FITC, P-ZIF-8@FITC nanocrystals
(1) Preparation of ZIF-8 nanocrystals
Into a 50mL glass reaction flask, 0.15g of Zn (NO) was charged at room temperature 3 ) 2 ·6H 2 O, 7.3mL of methanol and one magnetic particle, then 0.33g of 2-methylimidazole and 7.3mL of methanol solution were added to a 15mL EP tube. After the bottle cap is tightly covered, the two mixed solutions are respectively placed in an ultrasonic instrument for ultrasonic treatment (600W for two minutes) for 2 minutes to uniformly mix the raw materials, and then the 2-methylimidazole solution is slowly dripped into a glass reaction bottle. After reacting for 5 minutes with magnetic stirring (2000 rpm), the precipitate was collected by centrifugation (7000 rpm, 10 minutes) and washed 3 times with methanol solution to remove nano-particles as much as possibleExcess 2-methylimidazole adsorbed on the crystal surface. The finally prepared ZIF-8 nanocrystals were dried in vacuo at room temperature and stored as a powder at 4 ℃.
(2) Preparation of ZIF-8@FITC nanocrystals
To prepare ZIF-8@FITC nanocrystals, 0.15g Zn (NO) was added at ambient temperature to a 50mL glass reactor flask 3 ) 2 ·6H 2 O, 7.15mL of methanol and one magnetic particle, then 0.3mg of fluorescein 5-isothiocyanate solution (FITC, available from Sigma) and 0.15mL of methanol were added to one EP tube, and 0.33g of 2-methylimidazole and 7.3mL of methanol were added to the other EP tube. After the bottle cap is tightly covered, respectively placing the three mixed solutions in an ultrasonic instrument for ultrasonic treatment for 2 minutes to uniformly mix the raw materials, then slowly dripping the 2-methylimidazole solution into a glass reaction bottle, and the subsequent steps are the same as the step of synthesizing ZIF-8 nanocrystals in methanol.
(3) Preparation of P-ZIF-8@FITC nanocrystals
To prepare the P-ZIF-8@FITC nanocrystals, the same procedure as used for the preparation of the P-ZIF-8 nanocrystals in example 1 was used, substituting ZIF-8@FITC nanocrystals for ZIF-8 nanocrystals.
Example 3 testing ZIF-8 nanoparticles for their ability to degrade mutant p53 in SKBR-3 cells and ES-2 cells
SKBR-3 cells (from ATCC) and ES-2 cells (from ATCC) were seeded in 24-well cell culture plates at a density of about 3 to 5X 10 4 cell/well, overnight culture, ready for use. Prior to loading, cells were replaced with fresh McCoy's 5A medium (available from Gibco) and 50. Mu.g/mL of ZIF-8 nanoparticles were added (ZIF-8 nanoparticles prepared in example 1 were first solubilized with McCoy's 5A medium to a concentration of 50. Mu.g/mL and then added to the cells; the same applies below), and the blank was an equal volume of PBS buffer. After 6h of cell culture, western blot detection was then performed. As shown in FIG. 1, ZIF-8 nanoparticles can trigger the degradation of mutant p53 proteins in SKBR-3 cells and ES-2 cells.
Example 4 testing ZIF-8 nanoparticles for their ability to degrade mutant p53 in ES-2 cells
Seeding ES-2 cells on a circular slideIn 24-well cell culture plates, the density is about 3 to 5X 10 4 cell/well, overnight culture, ready for use. Prior to loading, cells were replaced with fresh McCoy's 5A medium, 50. Mu.g/mL ZIF-8 nanoparticles (prepared in example 1) were added, and the blank was an equal volume of PBS buffer (Cont). After 6h of cell culture, immunofluorescence detection was then performed. As shown in FIG. 2, ZIF-8 nanoparticles can reduce mutant p53 levels in ES-2 cells.
Example 5 test whether ZIF-8 nanoparticles affect the Synthesis of mutant p53
ES-2 cells were seeded in 24-well cell culture plates at a density of about 3 to 5X 10 4 cell/well, overnight culture, ready for use. Prior to loading, the cells were replaced with fresh McCoy's 5A medium, then treated with cycloheximide (CHX, 20. Mu.M) in the absence or presence of ZIF-8 (50. Mu.g/mL) (prepared in example 1) for the indicated hours (0, 2, 4, 8 h), and then subjected to western blot detection. As shown in FIG. 3, the accelerated decrease in mutp53 levels after ZIF-8 and CHX combined treatment indicated that the decrease in mutp53 levels induced by ZIF-8 was due to degradation rather than protein synthesis.
Example 6 testing whether the framework structure of ZIF-8 is important for degrading mutant p53
ES-2 cells were seeded in 24-well cell culture plates at a density of about 3 to 5X 10 4 cell/well, overnight culture, ready for use. Prior to loading, the cells were replaced with fresh McCoy's 5A medium, and then 50. Mu.g/mL ZIF-8 (prepared in example 1) and ZIF-8 (C-ZIF-8) calcined at 800℃for 2h, respectively, were added, with negative control being the addition of an equal volume of PBS buffer (Cont). After 6h of cell culture, western blot detection was then performed. As a result, as shown in FIG. 4, the C-ZIF-8 lost the ability to degrade mutp53, indicating that the framework structure of ZIF-8 is critical for the degradation of mutp53 by ZIF-8.
Example 7 testing of dose-dependent Effect of ZIF-8 degrading mutant p53
ES-2 cells were seeded in 24-well cell culture plates at a density of about 3 to 5X 10 4 cell/well, overnight culture, ready for use. Before loading, the cells were replaced with fresh McCoy's 5A cultureBased on, then 25, 50, 100, 150. Mu.g/mL ZIF-8 (prepared in example 1) was added, respectively, with negative control being the addition of an equal volume of PBS buffer. After 6h of cell culture, western blot detection was then performed. The results are shown in FIG. 5, ZIF-8 causes degradation of mutp53 in a dose-dependent manner, with significant degradation observed at 25 μg/mL and maximum degradation observed at 100 μg/mL.
Example 8 testing of the time dependent Effect of ZIF-8 degrading mutant p53
ES-2 cells were seeded in 24-well cell culture plates at a density of about 3 to 5X 10 4 cell/well, overnight culture, ready for use. Prior to loading, the cells were replaced with fresh McCoy's 5A medium, and ZIF-8 (prepared in example 1) was added at a concentration of 50. Mu.g/mL, and treated for 2h, 4h, 6h, 8h, respectively, with an equal volume of PBS buffer as a blank. After culturing the cells for 2 hours, 4 hours, 6 hours and 8 hours, western blot detection is carried out. The results are shown in FIG. 6, ZIF-8 caused mutp53 degradation in a time-dependent manner, with significant degradation observed at 4h of treatment and maximum degradation observed at 8h of treatment.
Example 9 testing ZIF-8 ability to degrade different mutps 53
9 different mutant p53 cell lines ES-2, BT-474, bxPC-3, BT-549, MDA-MB-231, SK-BR-3, MIA PaCa-2, MDA-MB-468 and NCI-H596 cells (all from ATCC), and 4 wild-type p53 cell lines MCF-7, A549, HCT116 and NCI-H446 (all from ATCC) were inoculated into 24-well cell culture plates, respectively, at a density of about 3 to 5X 10 4 cell/well, overnight culture, ready for use. ZIF-8 (prepared in example 1) was added at a concentration of 50. Mu.g/mL, and the blank was incubated with an equal volume of PBS buffer for 6 hours, followed by western blot detection. The percent mutp53 degradation for each mutant in the 9 different mutant p53 cell lines was based on western blot data and calculated by the following formula: (ratio of gray scale values of p53 and GAPDH after 1-ZIF-8 treatment/ratio of gray scale values of p53 and GAPDH after PBS buffer treatment). Times.100%. The grey values of the bands obtained by Western blot were obtained using Photoshop software.
The results are shown in Table 1, ZIF-8 degraded 9 mutp53 mutants, including six hot spot mutants, Y220C, R249S, R175H, R248W, R273H and G245C, to varying degrees. Wherein,
S241F: arg at position 241 of the p53 Gene (Gene ID: 7157) was mutated to Phe;
E285K: glu 285 of the p53 gene is mutated to Lys;
Y220C: the Tyr at position 220 of the p53 gene is mutated to Cys;
R249S: AGG- & gtAGT/Arg- & gtSer (R249S) of the 249 th codon of the p53 gene;
R280K: arg 280 of the p53 gene is mutated to Lys;
R248W: arg 248 of the p53 gene is mutated into Trp;
R175H: arg 175 of the p53 gene is mutated to His;
R273H: arg at position 273 of p53 gene is mutated to His;
G245C: gly 245 of p53 gene is mutated into Cys;
wild Type: wild type.
TABLE 1 Mutp53 degradation percentage and IC for ZIF-8 and P-ZIF-8 50
1 The percent Mutp53 degradation for each mutant in the 9 mutant p53 cell lines was calculated as follows: (ratio of gray scale values of p53 and GAPDH after 1-ZIF-8 treatment/ratio of gray scale values of p53 and GAPDH after PBS buffer treatment). Times.100%.
2 IC50 was derived from data analysis of 24 hour cell viability.
Example 10 test whether ZIF-8 reduces wild type p53 levels
Wild-type p53 cell line A549 (from ATCC) cells and MCF-7 (from ATCC) cells were seeded in 24-well cell culture plates at a density of about 3 to 5X 10 4 cell/well, overnight culture, ready for use. Before sample addition, the cells were replaced with fresh McCoy's 5A medium, then 50. Mu.g/mL ZIF-8 (prepared in example 1) was added, the blank was equal volume of PBS buffer (Cont), the cells were cultured for 6h, and then western blot was performedAnd (5) measuring. As shown in FIG. 7, the levels of wild-type p53 protein increased rather than decreased in A549 and MCF-7 following ZIF-8 treatment, indicating that ZIF-8 selectively degraded mutp53.
Example 11 testing of the pathway of ZIF-8 degradation of mutp53 in cells
ES-2 cells were seeded in 24-well cell culture plates at a density of about 3 to 5X 10 4 cell/well, overnight culture, ready for use. Prior to loading, cells were replaced with fresh McCoy's 5A medium, and ZIF-8 (prepared in example 1), 50. Mu.g/mL of the ZIF-8+ proteasome inhibitor MG-132 (10. Mu.M, available from Selleck), 50. Mu.g/mL of the ZIF-8+ proteasome inhibitor PS-341 (10. Mu.M, available from Selleck) and an equal volume of PBS buffer (Cont) were added to the cells, respectively. After 6h of cell culture, western blot detection was then performed. As shown in FIG. 8, MG-132 and PS-341 completely inhibited the ability of ZIF-8 to degrade mutp53, indicating that ZIF-8 is a proteasome-initiated degradation of mutp53.
Example 12 testing of the effect of ZIF-8 on the level of ubiquitination of mutp53 in cells
ES-2 cells were seeded in 6-well cell culture plates at a density of about 1 to 3X 10 5 cell/well, overnight culture, ready for use. Prior to loading, cells were replaced with fresh McCoy's 5A medium, and ZIF-8 (prepared in example 1) at a concentration of 50. Mu.g/mL, 10. Mu.M MG-132 (available from Selleck), 50. Mu.g/mL ZIF-8+MG-132 (10. Mu.M available from Selleck) and an equal volume of PBS buffer (Cont) were added to the cells. After 6h of cell culture, the cells were washed twice with PBS buffer, then digested with pancreatin, then co-immunoprecipitated with p53 antibody (purchased from Santa cruz biotechnology), and finally subjected to western blot detection. As shown in FIG. 9, ZIF-8 enhances the level of ubiquitination at the K48 site of mutp 53.
Example 13 testing whether mutp53 in ZIF-8 degrading cells requires the involvement of ubiquitin enzymes
ES-2 cells were seeded in 24-well cell culture plates at a density of about 3 to 5X 10 4 cell/well, overnight culture, ready for use. Before the sample addition, the cells were replaced with fresh McCoy's 5A medium and ZIF-8 was added at a concentration of 50. Mu.g/mL(prepared in example 1), 50. Mu.g/mL of the ZIF-8+ ubiquitin enzyme inhibitor PYR-41 (5. Mu.M, available from Selleck), 50. Mu.g/mL of the ZIF-8+ deubiquitinase inhibitor PR-619 (5. Mu.M, available from Selleck) and the blank was an equal volume of PBS buffer (Cont). After 6h of cell culture, western blot detection was then performed. As shown in FIG. 10, the ubiquitinase inhibitor PYR-41 was able to inhibit the ZIF-8-induced degradation of mutp53, while the deubiquitinase inhibitor PR-619 was unaffected, indicating that the ZIF-8-induced degradation of mutp53 was dependent on ubiquitination modification of mutp 53.
Example 14 test whether endocytosis of cells was necessary for ZIF-8 degradation of mutp53 in cells
ES-2 cells were seeded in 24-well cell culture plates at a density of about 3 to 5X 10 4 cell/well, overnight culture, ready for use. Prior to loading, cells were replaced with fresh McCoy's 5A medium and ZIF-8 (prepared in example 1) was added at a concentration of 50. Mu.g/mL, 50. Mu.g/mL ZIF-8+50. Mu.M Genistein (tyrosine protein kinase inhibitor from Sigma) and 50. Mu.M Genistein. The blank was an equal volume of PBS buffer (Cont). After 6h of cell culture, western blot detection was then performed. As shown in FIG. 11, the degradation of mutp53 in ZIF-8-induced ES-2 cells was effectively inhibited by the endocytosis inhibitor Genistein, indicating that endocytosis of the cells was essential for ZIF-8-induced degradation.
Example 15 testing of ZIF-8 vs. Zn in cell acid lysosomes 2+ Influence of level
ES-2 cells were seeded in 96-well cell culture plates at a density of about 1 to 2X 10 4 cell/well, overnight culture, ready for use. Prior to loading, cells were replaced with fresh McCoy's 5A medium and ZIF-8 (prepared in example 1) was added at a concentration of 50. Mu.g/mL, with the blank being an equal volume of PBS buffer (Cont). After 2h of treatment, the cells were washed twice with PBS buffer. Adding Zn 2+ Fresh medium of the green ion fluorescent probe Fluozin-3 AM (1. Mu.M, invitrogen) and the lysosome red fluorescent probe LysoTracker (1. Mu.M, invitrogen) was treated at 37℃for 30min, washed with PBS buffer and imaged using a confocal microscope (Nikon, ti-E Al). As a result, as shown in FIG. 12, ZIF-8 was treated for 2 hoursZn in acid lysosomes was observed in ES-2 cells 2+ Raised.
Example 16 testing of intracellular Zn by ZIF-8 and C-ZIF-8 pairs 2+ Influence of level
ES-2 cells were seeded in 24-well cell culture plates at a density of about 3 to 5X 10 4 cell/well, overnight culture, ready for use. Prior to loading, the samples were washed twice in serum-free medium and incubated with 1. Mu.M Fluozin-3 AM (Invitrogen) for 40 minutes at 37 ℃. After washing the cells twice with a phenol red-free DMEM medium, ZIF-8 (prepared in example 1) and C-ZIF-8 (prepared in the same manner as in example 6) were added at a concentration of 50. Mu.g/mL, respectively, and the blank was an equal volume of PBS buffer (Cont). After 2h of treatment, the cells were washed twice with PBS buffer. Pancreatin digestion, washing with PBS buffer, and FACS (flow cytometry fluorescence sorting technique) detection. As shown in FIG. 13, ZIF-8 was able to trigger intracellular Zn 2+ Elevated concentration without causing intracellular Zn by C-ZIF-8 2+ Raised.
Example 17 testing of intracellular Zn after ZIF-8 treatment 2+ Influence of level
ES-2 cells were seeded in 24-well cell culture plates at a density of about 3 to 5X 10 4 cell/well, overnight culture, ready for use. Prior to loading, the samples were washed twice in serum-free medium and incubated with 1. Mu.M Fluozin-3 AM (Invitrogen) for 40 minutes at 37 ℃. After washing the cells twice with a phenol red-free DMEM medium, ZIF-8 (prepared in example 1) and ZIF-8+TPEN (N, N, N ', N' -tetrakis (2-picolyl) ethylenediamine) (50. Mu.M, selleck) were added at a concentration of 50. Mu.g/mL, respectively, and the blank was an equal volume of PBS buffer (Cont). After 2h of treatment, the cells were washed twice with PBS buffer. Pancreatin digestion, washing with PBS buffer, and FACS (flow cytometry fluorescence sorting technique) detection. As shown in FIG. 14, TPEN was able to completely inhibit intracellular Zn induced by ZIF-8 2+ Raised.
Example 18 test whether ZIF-8-induced degradation of mutp53 was associated with intracellular Zn 2+ Level of
ES-2 cells were seeded in 24-well cell culture plates at a density of about 3 to 5X 10 4 cell/well overnight cultureNourishing for later use. Prior to loading, cells were replaced with fresh McCoy's 5A medium, and ZIF-8 (prepared in example 1), 50. Mu.g/mL ZIF-8+TPEN (50. Mu.M, selleck), TPEN (50. Mu.M, selleck) and the blank control was an equal volume of PBS buffer (Cont) were added at 50. Mu.g/mL, respectively. After 6h of cell culture, western blot detection was then performed. As shown in FIG. 15, TPEN was able to inhibit the degradation of mutp53 by ZIF-8, suggesting that the degradation of mutp53 by ZIF-8 is dependent on intracellular Zn 2+ Raised.
Example 19 testing ZIF-8 causes intracellular Zn 2+ Elevation has dose dependency
ES-2 cells were seeded in 96-well cell culture plates at a density of about 1 to 2X 10 4 cell/well, overnight culture, ready for use. Prior to loading, the samples were washed twice in serum-free medium and incubated with 1. Mu.M Fluozin-3 AM (Invitrogen) for 40 minutes at 37 ℃. After washing the cells twice with phenol red-free DMEM medium, ZIF-8 (prepared in example 1) was added at concentrations of 25. Mu.g/mL, 50. Mu.g/mL, 75. Mu.g/mL and 100. Mu.g/mL, respectively, with an equal volume of PBS buffer as a blank. After 2h of treatment, PBS buffer was washed and imaged using a fluorescence microscope (Nikon, DS-Fi 3). As shown in FIG. 16, the green fluorescence signal in the cells was gradually increased with increasing concentration, indicating that ZIF-8 caused intracellular Zn 2+ Elevation was dose dependent.
Example 20 testing ZIF-8 causes intracellular Zn 2+ Behavior of elevation
ES-2 cells were seeded in 24-well cell culture plates at a density of about 3 to 5X 10 4 cell/well, overnight culture, ready for use. Prior to loading, the samples were washed twice in serum-free medium and incubated with 1. Mu.M Fluozin-3 AM (Invitrogen) for 40 minutes at 37 ℃. After washing the cells twice with phenol red-free DMEM medium, ZIF-8 (prepared in example 1) was added at a concentration of 50. Mu.g/mL at 0.5h, 1h, 3h and 5h, respectively, with an equal volume of PBS buffer as a blank. Finally, pancreatin digestion, PBS buffer solution cleaning, and FACS (flow cytometry fluorescence sorting technology) detection are carried out. As shown in FIG. 17, ZIF-8 causes intracellular Zn with the lapse of time 2+ Gradually rise.
Implementation of the embodimentsExample 21 testing of decomposition of ZIF-8 to release Zn 2+ Whether or not to correlate with Mutp53 degradation by ZIF-8
ES-2 cells were seeded in 24-well cell culture plates at a density of about 3 to 5X 10 4 cell/well, overnight culture, ready for use. Prior to loading, cells were replaced with fresh McCoy's 5A medium and ZIF-8 (ZIF-8 pretreated at pH5.5 for 0, 1, 3, 6 and 12 h) at a concentration of 50. Mu.g/mL was added (prepared in example 1) and the blank was an equal volume of PBS buffer (NA). After 6h of cell culture, western blot detection was then performed. The results are shown in FIG. 18 (in the graph, the degree of collapse is the data of zinc release in ZIF-8 (pH 5.5), and zinc is released after ZIF-8 has collapsed, and the ability of ZIF-8 to induce mutp53 degradation gradually decreases with the duration of pretreatment, as in example 24).
Example 22 testing ZIF-8 nanoparticles for inclusion escape behavior
The ES-2 cells are inoculated into a confocal cuvette with a density of about 5 to 10X 10 4 cell/well, overnight culture, ready for use. Cells were replaced with fresh McCoy's 5A medium prior to loading. FITC-ZIF-8@sulfo5 (prepared in example 1) was added at a concentration of 50 μg/mL over a specified period of time, with a blank control of equal volume of PBS buffer. After 2h of treatment, the cells were washed twice with PBS buffer. Fresh medium containing lysosome red fluorescent probe LysoTracker (1 μm, invitrogen) was added, treated at 37 ℃ for 30min, then PBS buffer was used to wash the cells twice, and finally imaged using confocal microscopy (Nikon, ti-E Al).
The results are shown in FIG. 19 (in the figure, "ZIF-8 (cy 5)" representing ZIF-8 nanoparticles using red fluorescence from the sulfo-cy5 dye entrapped within the nanoparticles, "ZIF-8 (FITC)" representing ZIF-8 nanoparticles using green fluorescence FITC-Z1 peptide entrapped on the nanoparticle surface, "Lysome" representing Lysosome structure in the cells), and after 1h treatment, most of the green and red fluorescence co-localized in ES-2 cells was observed, indicating that the nanoparticles were intact, which were further co-localized with the blue fluorescence of lysosomes (white in the combined image), indicating that they entered into acidic inclusion bodies. However, by 2 hours, pink and red fluorescence appeared in the combined images, indicating that ZIF-8, which was co-localized with and not with the acidic inclusion bodies, respectively, interpreted as released sulfocy 5. In addition, yellow fluorescence could also be found outside the blue-stained structure, indicating the behavior of the fully or partially decomposed ZIF-8 nanoparticles with inclusion body escape.
Characterization of examples 23, ZIF-8 and P-ZIF-8 nanocrystals
The transmission electron micrographs of ZIF-8 (prepared in example 1) and P-ZIF-8 (prepared in example 1) nanocrystals in example 1 are shown in FIG. 20, from which it can be seen that the resulting ZIF-8 and P-ZIF-8 nanocrystals were uniform in size and good in dispersibility, with each particle having an average particle size of 90nm.
Example 24 testing of pH sensitivity of ZIF-8 and P-ZIF-8 to release Zn 2+ Behavior
0.5mg of ZIF-8 (prepared in example 1) and P-ZIF-8 (prepared in example 1) powders, respectively, were added to 1640 medium, vortexed vigorously to suspend the ZIF-8 or P-ZIF-8 powders in 1640 medium, and then the pH was adjusted to 7.4, 6.5 and 5.5 with HCl solution. After incubation with shaking for a certain period of time (0, 1h,3h,6h,12h,24 h) at 37℃the samples were centrifuged (10000 Xg, 4℃for 10 min). The zinc content in the supernatant was determined by ICP-MS and indicated the release of zinc from nanoparticle decomposition. The calculation formula of the zinc release percentage is as follows: zinc release (%) =100×mr/143.2, where mr is the total content of zinc (μg) in the supernatant, and 143.2 is the content of zinc (μg) in 0.5mg ZIF-8. As shown in FIG. 21, modification of the Z1-RGD peptide reduced in vitro Zn release by ZIF-8 at pH 5.5 and pH 6.5 2+ At a pH of 6.5.
Example 25 comparison of the behavior of test ZIF-8 and P-ZIF-8 to degrade mutp53 at pH 6.5
ES-2 cells were seeded in 24-well cell culture plates at a density of about 3 to 5X 10 4 cell/well, overnight culture, ready for use. Prior to loading, cells were replaced with fresh McCoy's 5A medium and ZIF-8 and P-ZIF-8 (both pretreated at pH 6.5 for 0, 3, 6 and 12 h) at 50. Mu.g/mL, respectively, were added (prepared in example 1) and the blank was an equal volume of PBS buffer (NA). Thin and fine After 6h of cell culture, western blot detection was then performed. The results are shown in FIG. 22 (in the graph, the degree of collapse is the data for zinc release from ZIF-8 (pH 6.5) and P-ZIF-8 (pH 6.5), and the specific assay is the same as in example 24), unmodified ZIF-8 was incubated at pH 6.5 for 12 hours with a 210% decrease in mutp53 degradation, but Z1-RGD modified P-ZIF-8 almost completely eliminated this undesirable degradation reduction.
Example 26, ZIF-8 and P-ZIF-8 have a higher killing potential against normal cells at pH 7.4 and pH 6.5
HEK 293 cells (from ATCC) and ARPE-19 cells (from ATCC) were inoculated into 96-well cell culture plates, respectively, at a density of about 1.about.2X10 4 cell/well, overnight culture, ready for use. Before loading, cells were replaced with fresh medium DMEM, ZIF-8 and P-ZIF-8 (prepared in example 1) were added at 50. Mu.g/mL, respectively, and pretreated for 12h at pH 7.4 and pH 6.5, respectively, with an equal volume of PBS buffer as a blank. Treatment is carried out for 24 hours, and then MTT detection is carried out. The results are shown in FIG. 23, where incubation for 12 hours at pH 6.5 significantly increased the toxicity of ZIF-8 to HEK 293 and ARPE-19 cells, but modification of the Z1-RGD peptide significantly reduced toxicity in both cell lines.
Example 27 comparison of the ability of ZIF-8 and P-ZIF-8 to enter cells
The ES-2 cells are inoculated into a confocal cuvette with a density of about 5 to 10X 10 4 cell/well, overnight culture, ready for use. Cells were replaced with fresh McCoy's 5A medium prior to loading. ZIF-8@FITC (prepared in example 2) and P-ZIF-8@FITC (prepared in example 2) were added at a concentration of 50. Mu.g/mL, respectively, and the blank was an equal volume of PBS buffer. After 4h of treatment, the cells were washed twice with PBS buffer. Fresh McCoy's 5A medium containing the nuclear blue dye Hoechst (2. Mu.g/mL, invitrogen) was added, incubated for 10min at ambient temperature, and the cells were washed twice with PBS buffer and then imaged using a confocal microscope (Nikon, ti-E Al). As shown in FIG. 24, the green fluorescence of the P-ZIF-8 group was stronger than that of the ZIF-8 group, indicating that modification of the Z1-RGD peptide resulted in more efficient entry of ZIF-8 into ES-2 cells.
Example 28, ZIF-8 and P-ZIF-8 release Zn 2+ Comparison of competence
ES-2 cells were seeded in 24-well cell culture plates at a density of about 3 to 5X 10 4 cell/well, overnight culture, ready for use. Prior to loading, the samples were washed twice in serum-free medium and incubated with 1. Mu.M Fluozin-3 AM (Invitrogen) for 40 minutes at 37 ℃. After washing the cells twice with phenol red-free DMEM medium, ZIF-8 (prepared in example 1) at a concentration of 50. Mu.g/mL, P-ZIF-8 (prepared in example 1) at a concentration of 50. Mu.g/mL and Z1-RGD (sequence RGDGGAHPHSDKLVPPR, available from Jiang Yao organisms) at a concentration of 10. Mu.g/mL were added, respectively, with a blank of equal volume of PBS buffer (Cont). After 2h of treatment, pancreatin digestion, washing with PBS buffer and FACS (flow cytometry fluorescence sorting technique) detection were carried out. As shown in FIG. 25, the fluorescence signal of the P-ZIF-8 group was stronger than that of the ZIF-8 group, indicating that the modified P-ZIF-8 of the Z1-RGD peptide released Zn 2+ The capability is stronger.
Example 29 comparison of the ability of ZIF-8 and P-ZIF-8 to degrade mutant P53
ES-2 cells were seeded in 24-well cell culture plates at a density of about 3 to 5X 10 4 cell/well, overnight culture, ready for use. Prior to loading, cells were replaced with fresh McCoy's 5A medium, ZIF-8 (prepared in example 1) at a concentration of 40. Mu.g/mL, P-ZIF-8 (prepared in example 1) at a concentration of 40. Mu.g/mL, and Z1-RGD (sequence RGDGGAHPHSDKLVPPR, available from Jiang Yao organisms) at a concentration of 8. Mu.g/mL were added, respectively, and the blank was an equal volume of PBS buffer (Cont). After 6h of cell culture, western blot detection was then performed. As a result, as shown in FIG. 26, the P-ZIF-8 group was able to more effectively cause degradation of the mutant P53 in ES-2 cells than the ZIF-8 group.
Example 30 IC of ZIF-8 in cells 50 Relationship to the percent degradation of mutp53
ES-2, BT-474, bxPC-3, BT-549, MDA-MB-231, SK-BR-3, MIA PaCa-2, MDA-MB-468 and NCI-H596 cells (all from ATCC) were seeded in 96-well cell culture plates at a density of about 1 to 2X 10 4 And (3) culturing overnight for later use. ZIF-8 (prepared in example 1) at 50. Mu.g/mL each, a blank was an equal volume of PBS buffer, and after cell culture for 24h, MTT assay was performed. Calculation of ZIF-8 Using GraphPad Prism software IC in cell line 50 And the resulting data were analyzed by regression with the percent degradation of mutp53 in the corresponding cell line (obtained in example 9) using GraphPad Prism software. As shown in FIG. 27, ZIF-8 IC in 9 cells expressing different mutp53 50 Is inversely related to the percent degradation of mutp 53.
Example 31 investigation of pharmacokinetics of ZIF-8 and P-ZIF-8 in animals
Female Balb/c mice (purchased from Beijing velocin ritodrine, weighing about 20 g) were divided into two groups, and ZIF-8 (20 mg/kg) and P-ZIF-8 (20 mg/kg) (prepared in example 1) were injected into the two groups, and blood was collected from the orbital venous plexus at different times (0, 2, 4, 8, 16, 24 h), and the zinc content of the blood was measured by ICP. As shown in FIG. 28, the pharmacokinetics of ZIF-8 and P-ZIF-8 were similar in mice with no significant difference.
Example 32 detection of the Effect of ZIF-8 and P-ZIF-8 on inhibition of tumor growth in an ES-2 animal tumor model
A female Balb/c Nude mouse (purchased from Beijing velocide, weighing about 20 g) was subcutaneously injected with 100. Mu.L of ES-2 cells (1X 10) 7 ) And constructing an ES-2 tumor model. Tumor volume up to 100mm 3 Subsequent experiments were performed.
The obtained ES-2 tumor-bearing mice were divided into four groups of six. Each group was treated with PBS buffer, Z1-RGD peptide (4 mg/kg) (SEQ ID NO: RGDGGAHPHSDKLVPPR, from Yu Jiangyao organisms), ZIF-8 (20 mg/kg) (example 1), and P-ZIF-8 (20 mg/kg) (example 1). Mice tumor volume changes were recorded during treatment by intravenous injection 2 times per week (fig. 29). After the end of treatment for 17 days (twice weekly dosing for the first two weeks and 1 time dosing for the third week on day 17), the sacrificial mice were weighed for tumor removal (fig. 30). It can be seen that both ZIF-8 and P-ZIF-8 treated groups have an effect of inhibiting tumor growth in mice, and that the treatment effect of the P-ZIF-8 treated group is the best.
Example 33 detection of the Effect of ZIF-8 and P-ZIF-8 on degrading mutp53 in ES-2 tumor model
The ES-2 animal tumor model of example 32 was weighed after 17 days of treatment, sacrificed mice were minced after the tumor was peeled off, thoroughly mixed with a tissue homogenizer, centrifuged, and the supernatant was collected to prepare a tumor tissue sample, which was then subjected to western blot detection. As shown in FIG. 31 (in which the numbers 1-6 represent six mice in each group), both the ZIF-8 treated group and the P-ZIF-8 treated group were able to reduce the mutp53 level in tumor tissue, and the P-ZIF-8 treated group was more effective in degradation.
Example 34 detection of whether P-ZIF-8 inhibits tumor growth in an MCF-7 animal tumor model
100 μl MCF-7 cells (1×10) were subcutaneously injected in the right rib of female Balb/c Nude mice (purchased from Beijing velocin Lihua, weight about 20 g) 7 ) And constructing an MCF-7 tumor model. Tumor volume up to 100mm 3 Subsequent experiments were performed.
The obtained MCF-7 tumor-bearing mice were divided into two groups of six. Each of the following treatments was performed, namely, PBS buffer treatment and P-ZIF-8 (20 mg/kg) (prepared in example 1). The mice were given 2 times a week by intravenous injection and tumor volume changes were recorded during the course of treatment. As can be seen from the results in FIG. 32, the P-ZIF-8 treated group had no significant effect on inhibiting tumor growth in mice compared to the PBS control group.
Example 35 detection of the Effect of ZIF-8 and P-ZIF-8 on inhibition of tumor growth in PDX tumor model
Constructing a PDX tumor model: human breast invasive ductal carcinoma specimens (er+pr+her3+, grade III, 55 years old, female) were obtained from a southern hospital (guangzhou, guangdong, china). The mutp53 status of the tumor was verified by sequencing the full-length p53 coding sequence after sequencing the complete exome and RT-PCR performed at the beijing genome institute (Y220C). Experiments performed using patient-derived materials met all relevant ethical specifications and were conducted in accordance with approval guidelines set by the national institutes of the university of south China's ethics committee for human research object protection committee with informed consent of the patient. Cutting the tumor tissue obtained after the operation into 2-3 mm pieces 3 And implanted in situ into a mammary fat pad of female NOD/SCID mice (purchased from beijing velaihua, 6-8 weeks old, weighing about 20 g). When the tumor volume reaches about 800mm 3 Cutting the tumor xenograft into 2-3 mm pieces 3 And amplified according to the same implantation procedure (i.e. tumor xenografts were implanted into new NOD/SCID mice and repeated 3 times). Mice with third generation xenografts were randomly divided into four groups of 7 and grown to about 100mm in tumors 3 Subsequent experiments were performed. Each group was treated with PBS buffer, Z1-RGD peptide (4 mg/kg) (SEQ ID NO: RGDGGAHPHSDKLVPPR, from Yu Jiangyao organisms), ZIF-8 (20 mg/kg), and P-ZIF-8 (20 mg/kg). Mice tumor volume changes were recorded during treatment by intravenous injection 2 times per week (fig. 33). After the end of treatment for 27 days (twice weekly dosing for the first three weeks and once daily dosing for the fourth week on day 24), the sacrificed mice were tumor-stripped for photography (fig. 34) and weighed (fig. 35). It can be seen that both ZIF-8 and P-ZIF-8 treated groups have an effect of inhibiting tumor growth in mice, and that the treatment effect of the P-ZIF-8 treated group is the best.
Example 36 detection of the Effect of ZIF-8 and P-ZIF-8 on degradation of mutp53 in PDX tumor model
After 27 days of treatment in the PDX tumor-bearing mice of example 35, each group of tumors was sectioned after paraffin embedding, dewaxed and rehydrated in a solution of xylene and fractionated alcohol (concentrations 100%,95%,80%,70%, volume percent in this order). The sections were stained with anti-p 53 antibody (Santa Cruz) and the nuclei stained with hematoxylin. As shown in FIG. 36, both ZIF-8 and P-ZIF-8 significantly reduced the levels of mutp53 in PDX tumor models.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Sequence listing
<110> university of North China
Application of <120> ZIF-8 nanomaterial in degradation of broad-spectrum mutant p53 protein
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Z1-RGD peptide
<400> 1
Arg Gly Asp Gly Gly Ala His Pro His Ser Asp Lys Leu Val Pro Pro
1 5 10 15
Arg

Claims (4)

1. The application of ZIF-8 nanomaterial in preparing a product for degrading broad-spectrum mutant p53 protein is characterized in that: the ZIF-8 nanomaterial is at least one of a ZIF-8 nanocrystal and a peptide-modified ZIF-8 nanomaterial with pH responsiveness;
the application environment is in vitro;
the ZIF-8 nanocrystals are prepared by the following method:
zn (NO) 3 ) 2 ·6H 2 Adding O into a solvent, and uniformly mixing by ultrasonic to obtain a mixed solution I; adding 2-methylimidazole into a solvent, and uniformly mixing by ultrasonic to obtain a mixed solution II; dripping the mixed solution II into the mixed solution I, stirring, centrifugally collecting precipitate, washing, and vacuum drying to obtain ZIF-8 nanocrystals;
The peptide-modified ZIF-8 nanomaterial with pH responsiveness is P-ZIF-8 and is prepared by the following method:
mixing the ZIF-8 nanocrystals with the Z1-RGD peptide, adding HEPES buffer solution, vortex oscillating for 30 seconds, incubating at room temperature, centrifuging, and washing to obtain the P-ZIF-8 nanocrystals; wherein the sequence of the Z1-RGD peptide is as follows: RGDGGAHPHSDKLVPPR;
the mutant p53 protein is at least one of p53 protein mutants S241F, E285K, Y220C, R249S, R280K, R248W, R175H, R273H and G245C.
2. The use according to claim 1, characterized in that:
the 2-methylimidazole and Zn (NO 3 ) 2 ·6H 2 The mass ratio of O is 1-2:0.1-1;
the solvent is at least one of water and methanol;
the mass ratio of the ZIF-8 nanocrystals to the Z1-RGD peptide is 5:1.
3. The use according to claim 1, characterized in that:
the effective concentration of the ZIF-8 nanomaterial is 25-150 mug/mL;
the degradation time is 0.5-8 hours.
4. A use according to claim 3, characterized in that:
the effective concentration of the ZIF-8 nanomaterial is 25-100 mug/mL;
the degradation time is 4-8 hours.
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