WO2021243925A1 - ZIF-8纳米材料在降解广谱突变p53蛋白中的应用 - Google Patents

ZIF-8纳米材料在降解广谱突变p53蛋白中的应用 Download PDF

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WO2021243925A1
WO2021243925A1 PCT/CN2020/122708 CN2020122708W WO2021243925A1 WO 2021243925 A1 WO2021243925 A1 WO 2021243925A1 CN 2020122708 W CN2020122708 W CN 2020122708W WO 2021243925 A1 WO2021243925 A1 WO 2021243925A1
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zif
fitc
nanocrystals
degradation
add
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温龙平
张云娇
黄晓婉
王连生
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华南理工大学
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Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to the application of a ZIF-8 nano material in degrading a broad-spectrum mutant p53 protein.
  • the p53 gene is the gene with the highest correlation with human tumors so far. Different from traditional tumor factors in human cancers where the expression level is down-regulated or gene deleted, p53 is mutated in most tumor cells. The results of genome sequencing of different human tumor cells showed that more than 50% of all malignant tumors will have mutations in this gene, and the p53 mutation profiles observed in different types of tumors are often inconsistent. Unlike wild-type p53, which is maintained at a low level under normal physiological levels, mutated p53 (mutp53) will diffuse or accumulate in cells in a large amount and is difficult to degrade.
  • the mutated p53 not only loses its ability as a tumor suppressor gene, but also often produces many new functions (gain-of-function, GOF) to promote the occurrence and development of cancer. Such as causing the instability of the genome, promoting the development, deterioration, and metastasis of tumors, anti-apoptosis, and resistance to chemotherapy and radiotherapy.
  • GOF gain-of-function
  • mutp53 Due to the presence of high levels of mutp53 in tumor cells, it enhances the degradation of mutp53 in tumor cells, which is expected to provide an ideal solution for tumor treatment targeting mutp53.
  • small molecules that induce the degradation of mutp53 through the proteasome pathway such as the Hsp90 inhibitor 17-AAG and ganetespib, NSC59984, and statins; there are also substances that induce the degradation of mutp53 through the autophagy pathway, such as MCB-613, SAHA, Gambogic acid and Zn(II)-curc, etc.
  • current methods rely almost entirely on small molecules, and the substances identified so far that can degrade mutp53 usually lack sufficient specificity and low toxicity to normal cells.
  • Metal organic frameworks are a new class of hybrid porous materials, which are composed of metal ions and organic ligands self-assembly.
  • MOF materials have been widely used in many fields including medicine.
  • nano-scale zeolite imidazole ester-8 (ZIF-8) with zinc as the metal junction and 2-methylimidazole as the linking molecule is the best studied one.
  • ZIF-8 nanomaterials have good biocompatibility and are widely used for drug delivery, especially for the delivery of anti-cancer drugs.
  • the research on the degradation of mutp53 by MOF material for tumor treatment is still a blank at home and abroad.
  • the purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide an application of ZIF-8 nanomaterials in degrading broad-spectrum mutant p53 proteins.
  • ZIF-8 nanomaterials in the degradation of a broad-spectrum mutant p53 protein, wherein the ZIF-8 nanomaterials are ZIF-8 nanocrystals, ZIF-8 nanocrystals containing dyes, and pH-responsive At least one of peptide-modified ZIF-8 nanomaterials.
  • the dye in the dye-carrying ZIF-8 nanocrystal is at least one of sulfo-cy5 and FITC (fluorescein 5-isothiocyanate).
  • the pH-responsive peptide is at least one of Z1-RGD peptide and FITC-Z1-RGD peptide.
  • the sequence of the Z1-RGD peptide is: RGDGGAHPHSDKLVPPR.
  • the sequence of the FITC-Z1-RGD peptide is: FITC-RGDGGAHPHSDKLVPPR.
  • the dye-carrying ZIF-8 nanocrystals are at least one of ZIF-8@sulfo-cy5 and ZIF-8@FITC.
  • the pH-responsive peptide-modified ZIF-8 nanomaterials are P-ZIF-8, FITC-P-ZIF-8, FITC-ZIF-8@sulfo-cy5 and P-ZIF-8@FITC. At least one; 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:
  • mixed solution I Add Zn(NO 3 ) 2 ⁇ 6H 2 O to the solvent and mix uniformly by ultrasonic to obtain mixed solution I; add 2-methylimidazole to the solvent and mix uniformly by ultrasonic to obtain mixed solution II (2-methylimidazole Solution); The mixed solution II is added dropwise to the mixed solution I, stirred, centrifuged to collect the precipitate, washed, and vacuum dried to obtain 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 8.0 water is adjusted with NaOH.
  • the Zn (NO 3) 2 ⁇ 6H 2 O per ml of solvent is used in an amount ratio of 0.02 ⁇ 0.04g Zn (NO 3) 2 ⁇ 6H 2 O calculated.
  • the conditions of the ultrasound are: 600W ultrasound for 2 to 3 minutes; preferably 600W ultrasound for 2 minutes.
  • the dosage of the 2-methylimidazole is calculated based on the ratio of 0.045 to 0.2g 2-methylimidazole per milliliter of solvent.
  • the mass ratio of the 2-methylimidazole to Zn(NO 3 ) 2 ⁇ 6H 2 O is 1-2:0.1-1.
  • the conditions of the stirring are: stirring at 2000 rpm for 10 to 20 minutes; preferably stirring at 2000 rpm for 15 minutes.
  • the centrifugation conditions are preferably: 4°C, 7000-10000g centrifugation for 10 minutes.
  • Said washing is washing with more than one of methanol, ethanol and water; preferably washing with a 50% aqueous ethanol solution by volume; more preferably washing with a 50% ethanol aqueous solution by volume more than 3 times; Remove as much as possible the excess 2-methylimidazole adsorbed on the surface of the nanocrystals.
  • the P-ZIF-8 nanocrystals are preferably prepared by the following method:
  • the ZIF-8 nanocrystals and the Z1-RGD peptide are mixed and added to HEPES buffer, vortexed for 30 seconds, incubated at room temperature, centrifuged, and washed to obtain P-ZIF-8 nanocrystals.
  • the mass ratio of the ZIF-8 nanocrystals to the Z1-RGD peptide is 5:1.
  • the formula of the HEPES buffer is: 50mM HEPES-NaOH, 150mM NaCl, pH 7.0.
  • the incubation time is preferably about 2 hours.
  • the centrifugation conditions are preferably: 4°C, 7000-10000g centrifugation for 10 minutes.
  • the FITC-P-ZIF-8 nanocrystals are preferably prepared by the following method:
  • the mass ratio of the ZIF-8 nanocrystals and FITC-Z1-RGD peptide is 5:1.
  • the formula of the HEPES buffer is: 50mM HEPES-NaOH, 150mM NaCl, pH 7.0.
  • the incubation time is preferably about 2 hours.
  • the centrifugation conditions are preferably: 4°C, 7000-10000g centrifugation for 10 minutes.
  • the ZIF-8@sulfo-cy5 nanocrystals are preferably prepared by the following method:
  • the solvent is water; preferably water with a pH of 8.0.
  • the pH 8.0 water is adjusted with NaOH.
  • the Zn (NO 3) 2 ⁇ 6H 2 O per ml of solvent is used in an amount ratio of 0.02 ⁇ 0.04g Zn (NO 3) 2 ⁇ 6H 2 O calculated; per ml of solvent ratio is preferably 0.025gZn (NO 3 ) 2 ⁇ 6H 2 O calculation.
  • the conditions of the ultrasound are: 600W ultrasound for 2 to 3 minutes; preferably 600W ultrasound for 2 minutes.
  • the dosage of sulfo-cy5 is calculated based on 5mg sulfo-cy5 per milliliter of solvent.
  • the amount of 2-methylimidazole is calculated based on the ratio of 0.045 to 0.2g 2-methylimidazole per milliliter of solvent; preferably, it is calculated based on the ratio of 0.2g 2-methylimidazole per milliliter of solvent.
  • the mass ratio of Zn(NO 3 ) 2 ⁇ 6H 2 O, sulfo-cy5 and 2-methylimidazole is 10:1:100.
  • the conditions of the stirring are: stirring at 2000 rpm for 10 to 20 minutes; preferably stirring at 2000 rpm for 15 minutes.
  • the centrifugation conditions are preferably: 4°C, 7000-10000g centrifugation for 10 minutes.
  • Said washing is washing with more than one of methanol, ethanol and water; preferably washing with a 50% aqueous ethanol solution by volume; more preferably washing with a 50% ethanol aqueous solution by volume more than 3 times; Remove as much as possible the excess 2-methylimidazole adsorbed on the surface of the nanocrystals.
  • the FITC-ZIF-8@sulfo-cy5 nanocrystals are preferably prepared by the following method:
  • the mass ratio of the ZIF-8@sulfo-cy5 nanocrystal to the FITC-Z1-RGD peptide is 5:1.
  • the formula of the HEPES buffer is: 50mM HEPES-NaOH, 150mM NaCl, pH 7.0.
  • the incubation time is preferably about 2 hours.
  • the centrifugation conditions are preferably: 4°C, 7000-10000g centrifugation for 10 minutes.
  • the ZIF-8@FITC nanocrystals are preferably prepared by the following method:
  • the solvent is methanol.
  • the Zn (NO 3) 2 ⁇ 6H 2 O per ml of solvent is used in an amount ratio of 0.02 ⁇ 0.04g Zn (NO 3) 2 ⁇ 6H 2 O calculated; per ml of solvent ratio is preferably 0.02gZn (NO 3 ) 2 ⁇ 6H 2 O calculation.
  • the conditions of the ultrasound are: 600W ultrasound for 2 to 3 minutes; preferably 600W ultrasound for 2 minutes.
  • the dosage of the fluorescein 5-isothiocyanate solution is calculated based on the ratio of 2 mg fluorescein 5-isothiocyanate solution (FITC) per milliliter of solvent.
  • the amount of 2-methylimidazole is calculated based on the ratio of 0.045 to 0.2g 2-methylimidazole per milliliter of solvent; preferably, it is calculated based on the ratio of 0.2g 2-methylimidazole per milliliter of solvent.
  • the mass ratio of the Zn(NO 3 ) 2 ⁇ 6H 2 O, 5-isothiocyanate solution (FITC) and 2-methylimidazole is 1:0.002:2.2.
  • the conditions of the stirring are: stirring at 2000 rpm for 10 to 20 minutes; preferably stirring at 2000 rpm for 15 minutes.
  • the centrifugation conditions are preferably: 4°C, 7000-10000g centrifugation for 10 minutes.
  • the washing is preferably washing with methanol; more preferably, washing with methanol 3 times or more to remove as much as possible the excess 2-methylimidazole adsorbed on the surface of the nanocrystals.
  • the P-ZIF-8@FITC nanocrystals are preferably prepared by the following method:
  • the mass ratio of the ZIF-8@FITC nanocrystals and Z1-RGD peptide is 5:1.
  • the formula of the HEPES buffer is: 50mM HEPES-NaOH, 150mM NaCl, pH 7.0.
  • the incubation time is preferably about 2 hours.
  • the centrifugation conditions are preferably: 4°C, 10000g centrifugation for 10 minutes.
  • the mutant p53 protein is a p53 protein encoded by a partial base mutation of the p53 gene; preferably at least one of p53 protein mutants S241F, E285K, Y220C, R249S, R280K, R248W, R175H, R273H and G245C .
  • the application environment is an in vitro environment.
  • the effective concentration of the ZIF-8 nanomaterial is 25-150 ⁇ g/mL; preferably 25-100 ⁇ g/mL; more preferably 100 ⁇ g/mL.
  • the degradation time is 0.5-8 hours; preferably 4-8 hours.
  • the application of the ZIF-8 nanomaterials in degrading the broad-spectrum mutant p53 protein is to directly add ZIF-8 nanomaterials or add pretreated ZIF-8 nanomaterials; the pretreatment is: ZIF-8
  • the nanomaterials are incubated at a pH of 5.5-7.5 for 0-12 hours (not including 0).
  • the pH value is preferably 5.5 to 6.5; more preferably 6.5.
  • Said products include medicines, kits, degradation inhibitors and the like.
  • ZIF-8 nanomaterials as a broad-spectrum mutant p53 protein degradation agent in the preparation of anti-tumor drugs.
  • the enzyme inhibitor is at least one of a ubiquitinase inhibitor and a tyrosine protein kinase inhibitor; the ubiquitinase inhibitor can inhibit the degradation of mutp53 caused by ZIF-8 nanomaterials, ZIF-8 nano.
  • the material-induced degradation of mutp53 in ES-2 cells can be effectively inhibited by the endocytosis inhibitor Genistein, and TPEN can inhibit the degradation of mutp53 induced by ZIF-8 nanomaterials.
  • the ubiquitinase inhibitor is ubiquitinase inhibitor PYR-41.
  • the effective concentration of the ubiquitinase inhibitor PYR-41 is 5 ⁇ mol/L.
  • the tyrosine protein kinase inhibitor is Genistein, a tyrosine protein kinase inhibitor.
  • the effective concentration of the tyrosine protein kinase inhibitor Genistein is 50 ⁇ mol/L.
  • the effective concentration of the TPEN (N, N, N', N'-tetrakis (2-pyridylmethyl) ethylene diamine) is 50 ⁇ mol/L.
  • the present invention has the following advantages and effects:
  • the present invention provides an application of ZIF-8 nanomaterials in the degradation of a broad-spectrum mutant p53 protein.
  • ZIF-8 can selectively trigger the degradation of the mutant p53 protein in the mutant p53 cell line. The decrease is due to degradation rather than a decrease in protein synthesis.
  • ZIF-8 nanomaterials effectively degrade mutp53 mutants to varying degrees, including Y220C, R249S, R175H, R248W, R273H and G245C.
  • the ZIF-8 nano material of the present invention can significantly reduce the toxicity in the cell line after being modified with a pH-responsive peptide, and has a better therapeutic effect on tumors.
  • the ubiquitinase inhibitor PYR-41 can inhibit the degradation of mutp53 induced by ZIF-8, while the deubiquitinase inhibitor PR-619 does not affect the degradation of mutp53 induced by ZIF-8
  • the process relies on the ubiquitination modification of mutp53.
  • Figure 1 is the western blot detection result of ZIF-8 in SK-BR-3 and ES-2 cells that induced mup53 degradation (in the figure, "-" is the control PBS buffer, and “+” is the ZIF-8 nanoparticles) .
  • Figure 2 is a graph showing the fluorescence results of ZIF-8 triggering mup53 degradation in ES-2 cells.
  • Figure 3 is a western blot test result of the degradation of mup53 triggered by ZIF-8 without affecting the synthesis pathway of mutp53.
  • Figure 4 is a western blot test result of the necessity of the framework structure of ZIF-8 to degrade mutp53.
  • Figure 5 is a western blot test result of the dose-dependent effect of the degradation of mutant p53 triggered by ZIF-8.
  • Figure 6 is a graph showing the results of western blot detection of the time-dependent effect of the degradation of mutant p53 triggered by ZIF-8.
  • Figure 7 is a western blot test result of ZIF-8 not causing degradation of wild-type p53.
  • Figure 8 is a diagram showing the western blot detection results of the pathway by which ZIF-8 triggers the degradation of mutp53 in ES-2 cells.
  • Figure 9 is a western blot test result of the increase of K48 ubiquitination level in ES-2 cells triggered by ZIF-8.
  • Fig. 10 is a diagram showing the results of western blot detection in which the degradation of mutp53 in cells triggered by ZIF-8 requires the participation of ubiquitinase.
  • Figure 11 is a western blot verification result that ZIF-8 induced degradation of mutp53 in cells can be effectively inhibited by the endocytosis inhibitor Genistein.
  • Figure 12 is a graph showing the result of fluorescence of the effect of ZIF-8 on the level of Zn 2+ in cell acid lysosomes.
  • Figure 13 is a graph showing the results of FACS detection of the effects of ZIF-8 and C-ZIF-8 on intracellular Zn 2+ levels.
  • Figure 14 is a graph showing the results of FACS detection in which ZIF-8 triggers an increase in Zn 2+ levels in cells, which can be effectively inhibited by the metal chelator TPEN.
  • Figure 15 is a western blot verification result that ZIF-8 induced degradation of mutp53 in cells can be effectively inhibited by the metal chelator TPEN.
  • Figure 16 is a dose-dependent fluorescence result of ZIF-8 causing intracellular Zn 2+ increase.
  • Figure 17 is a dose-dependent FACS test result of ZIF-8 causing intracellular Zn 2+ increase.
  • Figure 18 is a western blot verification result of the correlation between the decomposition of ZIF-8 and the release of Zn 2+ and the induced degradation of mutp53.
  • Figure 19 is a graph of the fluorescence results of testing the inclusion body escape behavior of ZIF-8 in ES-2 cells.
  • Example 20 is a transmission electron micrograph of ZIF-8 and P-ZIF-8 obtained in Example 1.
  • Figure 21 is a graph showing the test results of ZIF-8 and P-ZIF-8 having pH-sensitive Zn 2+ release behavior.
  • Figure 22 is a western blot test result comparing the degradation behavior of mutp53 by ZIF-8 and P-ZIF-8 under pH 6.5 conditions.
  • Figure 23 is a graph showing the cell viability test results of HEK 293 and ARPE-19 cells after ZIF-8 and P-ZIF-8 were pre-incubated at pH 7.4 and pH 6.5; A is HEK 293 cell; B is ARPE- 19 cells.
  • Figure 24 is a graph showing the results of fluorescence detection comparing the ability of ZIF-8 and P-ZIF-8 to enter ES-2 cells.
  • Figure 25 is a graph of FACS detection results comparing the ability of ZIF-8 and P-ZIF-8 to release Zn 2+ in cells.
  • Figure 26 is a western blot test result comparing the ability of ZIF-8 and P-ZIF-8 to degrade mutant p53 in ES-2 cells.
  • FIG 27 is a ZIF-8 IC 50 in a variety of cells and the relationship analysis mutp53 degradation percentage FIG.
  • Figure 28 is a graph showing the results of ICP detection of ZIF-8 and P-ZIF-8 in animals.
  • Figure 29 is a graph showing changes in tumor volume after ZIF-8 and P-ZIF-8 treatment of ES-2 tumor-bearing mice.
  • Figure 30 is a graph showing changes in tumor weight after ZIF-8 and P-ZIF-8 treatment of ES-2 tumor-bearing mice.
  • Figure 31 is a diagram showing the western blot detection results of p53 protein levels in tumor tissues of ES-2 tumor-bearing mice treated with ZIF-8 and P-ZIF-8.
  • Figure 32 is a graph showing changes in tumor volume after P-ZIF-8 treatment of MCF-7 tumor-bearing mice.
  • Figure 33 is a graph of tumor volume changes after ZIF-8 and P-ZIF-8 treatment of the PDX tumor model.
  • Figure 34 is a graph showing changes in tumor weight after ZIF-8 and P-ZIF-8 treatment of the PDX tumor model.
  • Figure 35 is a photograph of the tumor after ZIF-8 and P-ZIF-8 treatment of the PDX tumor model.
  • Figure 36 is a graph showing the results of immunohistochemical staining of tumor tissues after ZIF-8 and P-ZIF-8 treatment of the PDX tumor model.
  • the present invention will be further described in detail below in conjunction with examples, but the implementation of the present invention is not limited thereto.
  • the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.
  • the test methods that do not indicate specific experimental conditions in the following examples are usually in accordance with conventional experimental conditions or in accordance with experimental conditions recommended by the manufacturer.
  • the reagents and raw materials used in the present invention are all commercially available.
  • P-ZIF-8 nanocrystals 5 mg of the ZIF-8 powder prepared above was mixed with 1 mg of Z1-RGD peptide (sequence RGDGGAHPHSDKLVPPR, purchased from Qiangyao Biotechnology). Add 1 mL of HEPES buffer (50 mM HEPES-NaOH, 150 mM NaCl, pH 7.0) and vortex for 30 seconds. After incubating for 2 hours at room temperature, unbound peptides were removed by centrifugation (10000 g, 4°C, 10 minutes). Before use, the prepared P-ZIF-8 nanoparticles were washed 3 times with HEPES buffer.
  • HEPES buffer 50 mM HEPES-NaOH, 150 mM NaCl, pH 7.0
  • FITC-P-ZIF-8 nanocrystals In order to prepare FITC-P-ZIF-8 nanocrystals, the same method as the preparation of P-ZIF-8 was used, and FITC-Z1-RGD peptide (sequence FITC-RGDGGAHPHSDKLVPPR, purchased from Qiangyao Biotechnology) was used instead of Z1-RGD, The rest of the steps are the same.
  • FITC-ZIF-8@sulfo-cy5 nanocrystals refer to the preparation method of P-ZIF-8, replace ZIF-8 nanocrystals with ZIF-8@sulfo-cy5, and replace Z1-RGD with FITC-Z1-RGD peptides , The rest of the steps are the same.
  • SKBR-3 cells purchased from ATCC
  • ES-2 cells purchased from ATCC
  • SKBR-3 cells purchased from ATCC
  • ES-2 cells purchased from ATCC
  • ES-2 cells Inoculate ES-2 cells in a 24-well cell culture plate pre-placed with circular slides, with a density of about 3 ⁇ 5 ⁇ 10 4 cells/well, culture overnight, and set aside. Before adding the sample, the cells were changed into fresh McCoy's 5A medium, and 50 ⁇ g/mL ZIF-8 nanoparticles (prepared in Example 1) were added. The blank control was an equal volume of PBS buffer (Cont). The cells were cultured for 6 hours, and then subjected to immunofluorescence detection. The results are shown in Figure 2, ZIF-8 nanoparticles can reduce the level of mutant p53 in ES-2 cells.
  • the degradation percentage of mutp53 of each mutant in 9 different mutant p53 cell lines is based on western blot data and calculated by the following formula: (1-ZIF-8 treatment of p53 and GAPDH gray value ratio / PBS buffer treatment The gray value ratio of p53 to GAPDH) ⁇ 100%.
  • the gray value of the band obtained by Western blot was obtained using Photoshop software.
  • ZIF-8 effectively degrades 9 mutp53 mutants to varying degrees, including six hotspot mutants, namely Y220C, R249S, R175H, R248W, R273H and G245C. in,
  • S241F Arg at position 241 of p53 gene (Gene ID: 7157) is mutated to Phe;
  • E285K The 285th Glu mutation of p53 gene is Lys
  • Y220C Tyr mutation at position 220 of p53 gene is Cys
  • R249S mutation of codon 249 of p53 gene AGG ⁇ AGT/Arg ⁇ Ser (R249S);
  • R280K Arg at position 280 of p53 gene is mutated to Lys
  • R248W Arg at position 248 of p53 gene is mutated to Trp;
  • R175H Arg at position 175 of p53 gene is mutated to His
  • R273H The Arg at position 273 of the p53 gene is mutated to His;
  • G245C The 245th Gly mutation of p53 gene is Cys
  • Wild Type Wild type.
  • Mutp53 1 9 mutants p53 in each mutant cell line degradation percentage calculated by the following formula: (p53 after treatment gradation value proportional to GAPDH gradation 1-ZIF-8 / PBS buffer solution after treatment of p53 and GAPDH Value ratio) ⁇ 100%.
  • Example 10 Test whether ZIF-8 reduces the level of wild-type p53
  • the wild-type p53 cell line A549 (purchased from ATCC) cells and MCF-7 (purchased from ATCC) cells were seeded in a 24-well cell culture plate with a density of about 3 ⁇ 5 ⁇ 10 4 cells/well, and cultured overnight. use. Before adding samples, change the cells to fresh McCoy's 5A medium, and then add 50 ⁇ g/mL ZIF-8 (prepared in Example 1) respectively. The blank control is an equal volume of PBS buffer (Cont). Cell culture After 6h, western blot detection was then performed. The results are shown in Figure 7. In A549 and MCF-7, the level of wild-type p53 protein increased rather than decreased after ZIF-8 treatment, indicating that ZIF-8 selectively degrades mutp53.
  • ZIF-8 prepared in Example 1
  • MG-132 10 ⁇ M, purchased from Selleck
  • 50 ⁇ g/mL ZIF-8 + proteasome inhibitor PS-341 10 ⁇ M, purchased from Selleck
  • the blank control was an equal volume of PBS buffer (Cont). After the cells were cultured for 6 hours, they were then detected by western blot. The results are shown in Figure 8. MG-132 and PS-341 completely inhibited the ability of ZIF-8 to degrade mutp53, indicating that ZIF-8 is the proteasome-induced degradation of mutp53.
  • the ubiquitinase inhibitor PYR-41 can inhibit the degradation of mutp53 induced by ZIF-8, while the deubiquitinase inhibitor PR-619 does not affect the degradation of mutp53 induced by ZIF-8.
  • the process relies on the ubiquitination modification of mutp53.
  • Example 14 To test whether endocytosis of cells is necessary for ZIF-8 to degrade mutp53 in cells
  • Example 16 Test the effect of ZIF-8 and C-ZIF-8 on the level of intracellular Zn 2+
  • ZIF-8 can cause an increase in intracellular Zn 2+ concentration, while C-ZIF-8 does not cause an increase in intracellular Zn 2+.
  • TPEN can completely inhibit the increase of intracellular Zn 2+ caused by ZIF-8.
  • Example 18 Test whether the degradation of mutp53 induced by ZIF-8 is related to the level of Zn 2+ in the cell
  • Example 19 Test that ZIF-8 causes a dose-dependent increase in intracellular Zn 2+
  • Example 20 testing the behavior of ZIF-8 causing intracellular Zn 2+ increase
  • ZIF-8 prepared in Example 1
  • the blank control is an equal volume of PBS Buffer.
  • trypsinization, washing with PBS buffer, and FACS (Flow Cytometry Fluorescence Sorting Technology) detection The results are shown in Figure 17, as time goes by, ZIF-8 causes a gradual increase in intracellular Zn 2+.
  • Example 21 Test whether the decomposition and release of Zn 2+ by ZIF-8 is related to the degradation of mutp53 caused by ZIF-8
  • the degree of collapse is the data of zinc release in ZIF-8 (pH 5.5). ZIF-8 releases zinc after collapse.
  • the specific measurement method is the same as that in Example 24). With the extension of ZIF-8, the ability of ZIF-8 to induce degradation of mutp53 gradually decreases.
  • Example 24 test the pH sensitivity of ZIF-8 and P-ZIF-8 to release Zn 2+ behavior
  • zinc release (%) 100 ⁇ mr/143.2, where mr is the total content of zinc in the supernatant ( ⁇ g), and 143.2 is the content of zinc in 0.5mg ZIF-8 ( ⁇ g) .
  • mr is the total content of zinc in the supernatant
  • 143.2 is the content of zinc in 0.5mg ZIF-8 ( ⁇ g) .
  • Figure 21 At pH 5.5 and pH 6.5, the modification of Z1-RGD peptide reduced the rate of ZIF-8 releasing Zn 2+ in vitro, and the inhibitory effect was strongest at pH 6.5.
  • Example 25 test comparison of the behavior of ZIF-8 and P-ZIF-8 in degradation of mutp53 under pH 6.5 conditions
  • PBS buffer NA
  • Example 26 Comparison of the killing ability of ZIF-8 and P-ZIF-8 on normal cells under the conditions of pH 7.4 and pH 6.5
  • HEK 293 cells from ATCC
  • ARPE-19 cells from ATCC
  • HEK 293 cells from ATCC
  • HEK 293 cells from ATCC
  • ARPE-19 cells from ATCC
  • a concentration of 50 ⁇ g/mL both are pretreated at pH 7.4 and pH 6.5 for 12h
  • the blank control is an equal volume of PBS buffer. Treat for 24h, and then perform MTT detection.
  • Incubation at pH 6.5 for 12 hours significantly increased the toxicity of ZIF-8 to HEK 293 and ARPE-19 cells, but the modification of Z1-RGD peptide significantly reduced the toxicity in these two cell lines. toxicity.
  • ZIF-8@FITC prepared in Example 2
  • P-ZIF-8@FITC prepared in Example 2
  • the blank control was an equal volume of PBS buffer. After 4 hours of treatment, the cells were washed twice with PBS buffer.
  • ZIF-8 prepared in Example 1
  • P-ZIF-8 prepared in Example 1
  • Z1-RGD sequence RGDGGAHPHSDKLVPPR, purchased from Qiangyao Biotechnology
  • the blank control is an equal volume of PBS buffer (Cont). After 2h of treatment, trypsin digestion, washing with PBS buffer, and FACS (Flow Cytometry Fluorescence Sorting Technology) detection. The results are shown in Figure 25. Compared with the ZIF-8 group, the fluorescence signal of the P-ZIF-8 group is stronger, indicating that the modified P-ZIF-8 of the Z1-RGD peptide has a stronger ability to release Zn 2+.
  • Example 30 The relationship between the IC 50 of ZIF-8 in cells and the degradation percentage of mutp53
  • Inoculate 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)
  • the density is about 1 ⁇ 2 ⁇ 10 4 /well, cultured overnight, set aside.
  • Example 31 Exploring the pharmacokinetics of ZIF-8 and P-ZIF-8 in animals
  • mice Female Balb/c mice (purchased from Beijing Weitong Lihua, weighing about 20g) were divided into two groups, and the two groups were injected with ZIF-8 (20mg/kg) and P-ZIF-8 (20mg/kg) (implemented) Prepared in Example 1), blood was taken from the orbital venous plexus at different times (0, 2, 4, 8, 16, 24h), and the zinc content in the blood was detected by ICP. The results are shown in Figure 28. The pharmacokinetics of ZIF-8 and P-ZIF-8 in mice are similar, and there is no significant difference.
  • Example 32 Detecting the effect of ZIF-8 and P-ZIF-8 on inhibiting tumor growth in ES-2 animal tumor models
  • ES-2 cells 1 ⁇ 10 7 were injected subcutaneously into the right ribs of female Balb/c Nude nude mice (purchased from Beijing Weitong Lihua, weighing about 20 g) to construct an ES-2 tumor model.
  • ES-2 tumor model 100 ⁇ L ES-2 cells (1 ⁇ 10 7 ) were injected subcutaneously into the right ribs of female Balb/c Nude nude mice (purchased from Beijing Weitong Lihua, weighing about 20 g) to construct an ES-2 tumor model.
  • follow-up experiments were performed when the tumor volume reached 100mm 3.
  • the obtained ES-2 tumor-bearing mice were divided into four groups with six mice in each group. Each group was treated with the following treatments, PBS buffer treatment, Z1-RGD peptide (4mg/kg) (sequence RGDGGAHPHSDKLVPPR, purchased from Qiangyao Biotechnology) treatment, ZIF-8 (20mg/kg) (prepared in Example 1) Treatment, P-ZIF-8 (20mg/kg) (prepared in Example 1) treatment. It was administered intravenously twice a week, and the tumor volume changes in mice were recorded during the treatment (Figure 29). After 17 days of treatment (two doses per week for the first two weeks and one dose on the 17th day in the third week), the mice were sacrificed and the tumors were stripped and weighed ( Figure 30). It can be seen that both the ZIF-8 treatment group and the P-ZIF-8 treatment group have the effect of inhibiting tumor growth in mice, and the P-ZIF-8 treatment group has the best therapeutic effect.
  • Example 33 Detection of the effect of ZIF-8 and P-ZIF-8 on the degradation of mutp53 in the ES-2 tumor model
  • mice For the ES-2 animal tumor model in Example 32, after 17 days of treatment, the mice were sacrificed to remove the tumor and weighed, chopped, mixed thoroughly with a tissue homogenizer, centrifuged, and the supernatant was taken to prepare the tumor tissue. The samples are then subjected to western blot testing. The results are shown in Figure 31 (numbers 1-6 in the figure indicate six mice in each group). Both the ZIF-8 treatment group and the P-ZIF-8 treatment group can reduce the level of mutp53 in tumor tissues, and P-ZIF The degradation effect of -8 treatment group is better.
  • Example 34 Detect whether P-ZIF-8 can inhibit tumor growth in the MCF-7 animal tumor model
  • MCF-7 cells 1 ⁇ 10 7
  • mice purchased from Beijing Weitong Lihua, weighing about 20 g
  • the obtained MCF-7 tumor-bearing mice were divided into two groups with six mice in each group. Each group was subjected to the following treatments, PBS buffer treatment, and P-ZIF-8 (20 mg/kg) (prepared in Example 1) treatment. It was administered intravenously twice a week, and the tumor volume changes in mice were recorded during the treatment. As can be seen in Figure 32, the P-ZIF-8 treatment group has no obvious effect of inhibiting tumor growth in mice compared with the PBS control group.
  • Example 35 Detection of the effect of ZIF-8 and P-ZIF-8 on inhibiting tumor growth in a PDX tumor model
  • PDX tumor model A specimen of human breast invasive ductal carcinoma (ER+PR+Her3+, grade III, 55 years old, female) was obtained from N searching Hospital (Guangzhou, Guangdongzhou, China). The full-length p53 coding sequence was sequenced after RT-PCR performed at the Beijing Genomics Institute to verify the mutp53 status (Y220C) of the tumor. Experiments conducted using patient-sourced materials comply with all relevant ethical regulations, and with the patient’s informed consent, the operations were performed in accordance with the approved guidelines established by the Human Research Object Protection Committee of the Ethics Committee of South China University of Technology.
  • the tumor tissue obtained after the operation was cut into 2 ⁇ 3mm 3 pieces and implanted in situ into the breast fat of female NOD/SCID mice (purchased from Beijing Weitong Lihua, 6-8 weeks old, weighing about 20g) Mat in.
  • the tumor volume reaches about 800mm 3
  • the mice with the third-generation xenotransplantation were randomly divided into four groups, each with 7 mice, and the follow-up experiment was performed when the tumor grew to about 100 mm 3.
  • Each group was treated with the following treatments: PBS buffer treatment, Z1-RGD peptide (4mg/kg) (sequence RGDGGAHPHSDKLVPPR, purchased from Qiangyao Biotechnology) treatment, ZIF-8 (20mg/kg) treatment, P-ZIF-8 (20mg/kg) treatment.
  • the administration was intravenously administered twice a week, and the tumor volume changes in mice were recorded during the treatment (Figure 33). After 27 days of treatment (two doses per week for the first three weeks and one dose on the 24th day in the fourth week), the sacrificed mice were taken to take pictures of the tumors ( Figure 34) and weighed ( Figure 35). It can be seen that both the ZIF-8 treatment group and the P-ZIF-8 treatment group have the effect of inhibiting tumor growth in mice, and the P-ZIF-8 treatment group has the best therapeutic effect.
  • Example 36 Detection of the effect of ZIF-8 and P-ZIF-8 on the degradation of mutp53 in the PDX tumor model
  • Example 35 After the PDX tumor-bearing mice in Example 35 were treated for 27 days, the tumors of each group were embedded in paraffin and sliced, and then sliced in xylene and graded alcohol (concentrations of 100%, 95%, 80%, 70%, respectively). Volume percentage) Dewaxing and rehydration in the solution. The sections were stained with anti-p53 antibody (Santa Cruz), and the nuclei were stained with hematoxylin. The results are shown in Figure 36. Both ZIF-8 and P-ZIF-8 can significantly reduce the level of mutp53 in the PDX tumor model.

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Abstract

一种ZIF-8纳米材料在降解广谱突变p53蛋白中的应用。发现ZIF-8纳米材料可以选择性的引发突变p53细胞系中突变p53蛋白的降解，该引发的mutp53水平的降低是由于降解而不是蛋白质合成的降低，且发现经过具有pH响应性的肽(Z1-RGD肽)修饰后，能够显著降低在细胞系中的毒性，对肿瘤的治疗效果更好。还发现泛素化酶抑制剂，酪氨酸蛋白激酶抑制剂，以及N,N,N',N'-四(2-吡啶甲基)乙二胺能够抑制ZIF-8引发的突变p53蛋白的降解，为ZIF-8纳米材料降解突变p53蛋白的研究提供了有效途径。

Description

ZIF-8纳米材料在降解广谱突变p53蛋白中的应用 技术领域
本发明属于生物医学领域，特别涉及一种ZIF-8纳米材料在降解广谱突变p53蛋白中的应用。
背景技术
p53基因是迄今发现与人类肿瘤相关性最高的基因。不同于传统的肿瘤因子在人类癌症中通常存在表达量下调或基因删除的状况，p53在绝大多数肿瘤细胞中会发生突变。对不同的人类肿瘤细胞进行基因组测序的结果表明，在所有恶性肿瘤中50％以上会出现该基因的突变，并且在不同类型肿瘤内观察到的p53突变谱往往是不一致的。与野生型p53在正常生理水平下会维持在较低水平不同，突变后的p53(mutp53)会大量弥散或聚集在细胞中，难以降解。更重要的是，突变后的p53不仅丧失了作为抑癌基因的能力，还往往产生许多新的功能(gain-of-function，GOF)，促进癌症发生发展。如导致基因组的不稳定，促进肿瘤的发展、恶化、转移，抗凋亡，化疗放疗耐受等。
由于肿瘤细胞中高水平mutp53的存在，因此增强对肿瘤细胞中mutp53的降解作用，有望为以mutp53为靶点的肿瘤治疗提供较理想的解决方案。目前，有通过蛋白酶体途径诱导mutp53降解的小分子，例如Hsp90抑制剂17-AAG和ganetespib，NSC59984，和他汀类药物等；也有通过自噬途径诱导mutp53降解的物质，例如MCB-613，SAHA，藤黄酸和Zn(II)-curc等。但是，当前的方法几乎完全依赖小分子，而且到目前为止鉴定出的能够降解mutp53的物质通常缺乏足够的特异性和对正常细胞的低毒性。
金属有机框架(MOFs)是一类新兴的混合多孔材料，由金属离子和有机配体自组装构成。近年来，MOF材料在包括医学在内的许多领域得到广泛应用。在许多类型的MOF材料中，以锌为金属结点，以2-甲基咪唑为连接分子的纳米级沸石咪唑酯-8(ZIF-8)是研究得最好的一种。ZIF-8纳米材料具有良好的生物相容性，被广泛用于药物输送，特别是用于输送抗癌药物。但是对于MOF材料降解mutp53进行肿瘤治疗的研究国内外还是一片空白。
发明内容
本发明的目的在于克服现有技术的缺点与不足，提供一种ZIF-8纳米材料在降解广谱突变p53蛋白中的应用。
本发明的目的通过下述技术方案实现：
一种ZIF-8纳米材料在降解广谱突变p53蛋白中的应用，其中，所述的ZIF-8纳米材料为ZIF-8纳米晶体、包载染料的ZIF-8纳米晶体和具有pH响应性的肽修饰的ZIF-8纳米材料中的至少一种。
所述的包载染料的ZIF-8纳米晶体中的染料为sulfo-cy5和FITC(荧光素5-异硫氰酸酯)中的至少一种。
所述的具有pH响应性的肽为Z1-RGD肽和FITC-Z1-RGD肽中的至少一种。
所述的Z1-RGD肽的序列为：RGDGGAHPHSDKLVPPR。
所述的FITC-Z1-RGD肽的序列为：FITC-RGDGGAHPHSDKLVPPR。
所述的包载染料的ZIF-8纳米晶体为ZIF-8@sulfo-cy5和ZIF-8@FITC中的至少一种。
所述的具有pH响应性的肽修饰的ZIF-8纳米材料为P-ZIF-8，FITC-P-ZIF-8，FITC-ZIF-8@sulfo-cy5和P-ZIF-8@FITC中的至少一种；更优选为P-ZIF-8和P-ZIF-8@FITC 中的至少一种。
所述的ZIF-8纳米晶体优选为通过如下方法制备得到：
将Zn(NO 3) 2·6H 2O加入到溶剂中，超声混合均匀，得到混合溶液I；将2-甲基咪唑加入到溶剂中，超声混合均匀，得到混合溶液II(2-甲基咪唑溶液)；将混合溶液II滴加到混合溶液I中，搅拌，离心收集沉淀物，洗涤，真空干燥，得到ZIF-8纳米晶体。
所述的溶剂为水和甲醇中的至少一种。
所述的水优选为pH 8.0的水。
所述的pH 8.0的水为采用NaOH调节。
所述的Zn(NO 3) 2·6H 2O的用量为按每毫升溶剂配比0.02～0.04g Zn(NO 3) 2·6H 2O计算。
所述的超声的条件为：600W超声2～3分钟；优选为600W超声2分钟。
所述的2-甲基咪唑的用量为按每毫升溶剂配比0.045～0.2g 2-甲基咪唑计算。
所述的2-甲基咪唑与Zn(NO 3) 2·6H 2O的质量比为1～2:0.1～1。
所述的搅拌的的条件为：2000rpm搅拌10～20分钟；优选为2000rpm搅拌15分钟。
所述的离心的条件优选为：4℃，7000～10000g离心10分钟。
所述的洗涤为采用甲醇、乙醇和水中的一种以上进行洗涤；优选为采用体积百分比为50％的乙醇水溶液进行洗涤；更优选为采用体积百分比为50％的乙醇水溶液洗涤3次以上；以尽可能除去纳米晶体表面吸附着的过量的2-甲基咪唑。
所述的P-ZIF-8纳米晶体优选为通过如下方法制备得到：
将上述ZIF-8纳米晶体与Z1-RGD肽混合后加入HEPES缓冲液，涡旋震荡30秒，在室温下进行孵育，再离心，洗涤得到P-ZIF-8纳米晶体。
所述的ZIF-8纳米晶体与Z1-RGD肽的质量比为5:1。
所述的HEPES缓冲液的配方为：50mM HEPES-NaOH，150mM NaCl，pH 7.0。
所述的孵育的时间优选为2小时左右。
所述的离心的条件优选为：4℃，7000～10000g离心10分钟。
所述的FITC-P-ZIF-8纳米晶体优选为通过如下方法制备得到：
将上述ZIF-8纳米晶体与FITC-Z1-RGD肽混合后加入HEPES缓冲液，涡旋震荡30秒，在室温下进行孵育，再离心，洗涤得到FITC-P-ZIF-8纳米晶体。
所述的ZIF-8纳米晶体与FITC-Z1-RGD肽的质量比为5:1。
所述的HEPES缓冲液的配方为：50mM HEPES-NaOH，150mM NaCl，pH 7.0。
所述的孵育的时间优选为2小时左右。
所述的离心的条件优选为：4℃，7000～10000g离心10分钟。
所述的ZIF-8@sulfo-cy5纳米晶体优选为通过如下方法制备得到：
将Zn(NO 3) 2·6H 2O加入到溶剂中，超声混合均匀，得到混合溶液A；将sulfo-cy5加入到溶剂中，超声混合均匀，得到混合溶液B；将2-甲基咪唑加入到溶剂中，超声混合均匀，得到混合溶液C(2-甲基咪唑溶液)；将混合溶液B和混合溶液C依次滴加到混合溶液A中，搅拌，离心收集沉淀物，洗涤，真空干燥，得到ZIF-8@sulfo-cy5纳米晶体。
所述的溶剂为水；优选为pH 8.0的水。
所述的pH 8.0的水为采用NaOH调节。
所述的Zn(NO 3) 2·6H 2O的用量为按每毫升溶剂配比0.02～0.04g Zn(NO 3) 2·6H 2O计算；优选为按每毫升溶剂配比0.025gZn(NO 3) 2·6H 2O计算。
所述的超声的条件为：600W超声2～3分钟；优选为600W超声2分钟。
所述的sulfo-cy5的用量为按每毫升溶剂配比5mg sulfo-cy5计算。
所述的2-甲基咪唑的用量为按每毫升溶剂配比0.045～0.2g 2-甲基咪唑计算；优选为按每毫升溶剂配比0.2g 2-甲基咪唑计算。
所述的Zn(NO 3) 2·6H 2O、sulfo-cy5和2-甲基咪唑的质量比为10:1:100。
所述的搅拌的的条件为：2000rpm搅拌10～20分钟；优选为2000rpm搅拌15分钟。
所述的离心的条件优选为：4℃，7000～10000g离心10分钟。
所述的洗涤为采用甲醇、乙醇和水中的一种以上进行洗涤；优选为采用体积百分比为50％的乙醇水溶液进行洗涤；更优选为采用体积百分比为50％的乙醇水溶液洗涤3次以上；以尽可能除去纳米晶体表面吸附着的过量的2-甲基咪唑。
所述的FITC-ZIF-8@sulfo-cy5纳米晶体优选为通过如下方法制备得到：
将上述ZIF-8@sulfo-cy5纳米晶体与FITC-Z1-RGD肽混合后加入HEPES缓冲液，涡旋震荡30秒，在室温下进行孵育，再离心，洗涤得到ZIF-8@sulfo-cy5纳米晶体。
所述的ZIF-8@sulfo-cy5纳米晶体与FITC-Z1-RGD肽的质量比为5:1。
所述的HEPES缓冲液的配方为：50mM HEPES-NaOH，150mM NaCl，pH 7.0。
所述的孵育的时间优选为2小时左右。
所述的离心的条件优选为：4℃，7000～10000g离心10分钟。
所述的ZIF-8@FITC纳米晶体优选为通过如下方法制备得到：
将Zn(NO 3) 2·6H 2O加入到溶剂中，超声混合均匀，得到混合溶液D；将荧光素5-异硫氰酸酯溶液(FITC)加入到溶剂中，超声混合均匀，得到混合溶液E；将2-甲基咪唑加入到溶剂中，超声混合均匀，得到混合溶液F(2-甲基咪唑溶液)；将混合溶液F和混合溶液E依次滴加到混合溶液D中，搅拌，离心收集沉淀物，洗涤，真空干燥，得到ZIF-8@FITC纳米晶体。
所述的溶剂为甲醇。
所述的Zn(NO 3) 2·6H 2O的用量为按每毫升溶剂配比0.02～0.04g Zn(NO 3) 2·6H 2O计算；优选为按每毫升溶剂配比0.02gZn(NO 3) 2·6H 2O计算。
所述的超声的条件为：600W超声2～3分钟；优选为600W超声2分钟。
所述的荧光素5-异硫氰酸酯溶液(FITC)的用量为按每毫升溶剂配比2mg荧光素5-异硫氰酸酯溶液(FITC)计算。
所述的2-甲基咪唑的用量为按每毫升溶剂配比0.045～0.2g 2-甲基咪唑计算；优选为按每毫升溶剂配比0.2g 2-甲基咪唑计算。
所述的Zn(NO 3) 2·6H 2O、5-异硫氰酸酯溶液(FITC)和2-甲基咪唑的质量比为1:0.002:2.2。
所述的搅拌的的条件为：2000rpm搅拌10～20分钟；优选为2000rpm搅拌15分钟。
所述的离心的条件优选为：4℃，7000～10000g离心10分钟。
所述的洗涤优选为采用甲醇进行洗涤；更优选为采用甲醇洗涤3次以上，以尽可能除去纳米晶体表面吸附着的过量的2-甲基咪唑。
所述的P-ZIF-8@FITC纳米晶体优选为通过如下方法制备得到：
将ZIF-8@FITC纳米晶体与Z1-RGD肽混合后加入HEPES缓冲液，涡旋震荡30秒，在室温下进行孵育，再离心，洗涤得到P-ZIF-8@FITC纳米晶体。
所述的ZIF-8@FITC纳米晶体与Z1-RGD肽的质量比为5:1。
所述的HEPES缓冲液的配方为：50mM HEPES-NaOH，150mM NaCl，pH 7.0。
所述的孵育的时间优选为2小时左右。
所述的离心的条件优选为：4℃，10000g离心10分钟。
所述的突变p53蛋白为p53基因上的部分碱基发生突变后编码的p53蛋白；优选为p53蛋白突变体S241F，E285K，Y220C，R249S，R280K，R248W，R175H，R273H和G245C中的至少一种。
所述的应用的环境为体外环境。
所述的ZIF-8纳米材料的有效浓度为25～150μg/mL；优选为25～100μg/mL；更优选为100μg/mL。
所述的降解的时间为0.5～8小时；优选为4～8小时。
所述的ZIF-8纳米材料在降解广谱突变p53蛋白中的应用，为直接加入ZIF-8纳米材料或加入预处理后的ZIF-8纳米材料；所述的预处理为：将ZIF-8纳米材料在pH值为5.5～7.5下孵育0～12小时(不包括0)。
所述的pH值优选为5.5～6.5；更优选为6.5。
ZIF-8纳米材料在制备降解广谱突变p53蛋白的产品中的应用。
所述的产品包括药物，试剂盒、降解抑制剂等。
ZIF-8纳米材料作为广谱突变p53蛋白降解剂在制备抗肿瘤药物中的应用。
酶抑制剂或TPEN(N,N,N',N'-四(2-吡啶甲基)乙二胺)在制备抑制ZIF-8纳米材料降解广谱突变p53蛋白的产品中的应用；其中，所述的酶抑制剂为泛素化酶抑制剂和酪氨酸蛋白激酶抑制剂中的至少一种；泛素化酶抑制剂能够抑制ZIF-8纳米材料引发的mutp53的降解，ZIF-8纳米材料引发的ES-2细胞中的mutp53的降解可以被内吞抑制剂Genistein有效抑制，TPEN能够抑制ZIF-8纳米材料引发的mutp53的降解。
所述的泛素化酶抑制剂为泛素化酶抑制剂PYR-41。
所述的泛素化酶抑制剂PYR-41的有效浓度为5μmol/L。
所述的酪氨酸蛋白激酶抑制剂为酪氨酸蛋白激酶抑制剂Genistein。
所述的酪氨酸蛋白激酶抑制剂Genistein的有效浓度为50μmol/L。
所述的TPEN(N,N,N',N'-四(2-吡啶甲基)乙二胺)的有效浓度为50μmol/L。
本发明相对于现有技术具有如下的优点及效果：
(1)本发明中提供一种ZIF-8纳米材料在降解广谱突变p53蛋白中的应用，ZIF-8可以选择性的引发突变p53细胞系中突变p53蛋白的降解，该引发的mutp53水平的降低是由于降解而不是蛋白质合成的降低。ZIF-8纳米材料在不同程度上有效地降解mutp53突变体，包括Y220C，R249S，R175H，R248W，R273H和G245C等。本发明中ZIF-8纳米材料经过具有pH响应性的肽修饰后，能够显著降低在细胞系中的毒性，对肿瘤的治疗效果更好。
(2)本发明中发现泛素化酶抑制剂PYR-41能够抑制ZIF-8引发的mutp53的降解，而去泛素化酶抑制剂PR-619不影响，说明ZIF-8引发的mutp53的降解过程依赖于mutp53的泛素化修饰。
(3)本发明中发现ZIF-8引发的细胞中的mutp53的降解可以被内吞抑制剂Genistein有效抑制，说明细胞的内吞对于ZIF-8引发的降解是必须的。
(4)本发明中发现ZIF-8处理后，在细胞中观察到酸性溶酶体中Zn 2+升高，而TPEN能够完全抑制ZIF-8引起的细胞内Zn 2+升高，并且进一步证明TPEN能够抑制ZIF-8引发的mutp53的降解，说明ZIF-8引发的mutp53的降解依赖于细胞内的Zn 2+升高。
附图说明
图1是ZIF-8在SK-BR-3和ES-2细胞中引发mup53降解的western blot检测结果图(图中，“-”为对照PBS缓冲液，“+”为ZIF-8纳米颗粒)。
图2是ZIF-8在ES-2细胞中引发mup53降解的荧光结果图。
图3是ZIF-8引发mup53降解不影响mutp53的合成途径的western blot检测结果图。
图4是ZIF-8的框架结构对降解mutp53的必要性的western blot检测结果图。
图5是ZIF-8引发突变p53降解的剂量依赖效应的western blot检测结果图。
图6是ZIF-8引发突变p53降解的时间依赖效应的western blot检测结果图。
图7是ZIF-8不引发野生型p53降解的western blot检测结果图。
图8是ZIF-8引发ES-2细胞中mutp53降解的途径的western blot检测结果图。
图9是ZIF-8引发ES-2细胞中K48泛素化水平升高的western blot检测结果图。
图10是ZIF-8引发细胞中的mutp53降解需要泛素化酶的参与的western blot检测结果图。
图11是ZIF-8引发细胞中的mutp53降解可被内吞抑制剂Genistein有效抑制的western blot验证结果图。
图12是ZIF-8对细胞酸性溶酶体中Zn 2+水平影响的荧光结果图。
图13是ZIF-8和C-ZIF-8对细胞内Zn 2+水平影响的FACS检测结果图。
图14是ZIF-8引发细胞中的Zn 2+水平升高可被金属螯合剂TPEN有效抑制的FACS检测结果图。
图15是ZIF-8引发细胞中的mutp53降解可被金属螯合剂TPEN有效抑制的western blot验证结果图。
图16是ZIF-8引起细胞内Zn 2+升高具有剂量依赖性的荧光结果图。
图17是ZIF-8引起细胞内Zn 2+升高具有剂量依赖性的FACS检测结果图。
图18是ZIF-8分解释放Zn 2+与引发的mutp53降解相关的western blot验证结果图。
图19是测试ZIF-8在ES-2细胞中存在内含体逃逸行为的荧光结果图。
图20是实施例1得到的ZIF-8和P-ZIF-8透射电子显微镜照片图。
图21是ZIF-8和P-ZIF-8具有pH敏感性释放Zn 2+行为的测试结果图。
图22是ZIF-8和P-ZIF-8在pH 6.5条件下降解mutp53的行为比较的western blot检测结果图。
图23是ZIF-8和P-ZIF-8在pH 7.4和pH 6.5条件预孵育后，处理HEK 293和ARPE-19细胞的细胞活力检测结果图；其中，A为HEK 293细胞；B为ARPE-19细胞。
图24是ZIF-8和P-ZIF-8进入ES-2细胞的能力比较的荧光检测结果图。
图25是ZIF-8和P-ZIF-8在细胞中释放Zn 2+能力比较的FACS检测结果图。
图26是ZIF-8和P-ZIF-8在ES-2细胞中降解突变p53能力比较的western blot检测结果图。
图27是ZIF-8在多种细胞中的IC 50与mutp53降解百分率的关系分析图。
图28是ZIF-8和P-ZIF-8在动物体内的ICP检测结果图。
图29是ZIF-8和P-ZIF-8治疗ES-2荷瘤鼠后的肿瘤体积变化图。
图30是ZIF-8和P-ZIF-8治疗ES-2荷瘤鼠后的肿瘤重量变化图。
图31是ZIF-8和P-ZIF-8治疗ES-2荷瘤鼠后的肿瘤组织中p53蛋白水平的western blot检测结果图。
图32是P-ZIF-8治疗MCF-7荷瘤鼠后的肿瘤体积变化图。
图33是ZIF-8和P-ZIF-8治疗PDX肿瘤模型后的肿瘤体积变化图。
图34是ZIF-8和P-ZIF-8治疗PDX肿瘤模型后的肿瘤重量变化图。
图35是ZIF-8和P-ZIF-8治疗PDX肿瘤模型后的肿瘤拍照图。
图36是ZIF-8和P-ZIF-8治疗PDX肿瘤模型后的肿瘤组织的免疫组化染色结果图。
具体实施方式
下面结合实施例对本发明作进一步详细的描述，但本发明的实施方式不限于此。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。下列实施例中未注明具体实验条件的试验方法，通常按照常规实验条件或按照制造厂所建议的实验条件。除非特别说明，本发明所用试剂和原材料均可通过市售获得。
实施例1、ZIF-8、P-ZIF-8、FITC-P-ZIF-8、ZIF-8@sulfo-cy5、FITC-ZIF-8@sulfo-cy5纳米晶体的制备
(1)制备ZIF-8纳米晶体
在常温下，在50mL的玻璃反应瓶中加入0.2gZn(NO 3) 2·6H 2O、4.8mL H 2O(pH 8.0， 用NaOH调节)和一粒磁子，然后在一个15mL的EP管中加入2g 2-甲基咪唑和10mL H 2O。瓶盖盖紧后，分别将两种混合液放在超声仪中超声(600W，两分钟)使原料混合均匀，然后将混匀的2-甲基咪唑溶液缓慢滴加到玻璃反应瓶中。磁力搅拌(2000rpm)反应15分钟后，离心收集沉淀物，并用含50％(v/v)乙醇的去离子水溶液洗涤3次，以尽可能除去纳米晶体表面吸附着的过量的2-甲基咪唑。最终制备的ZIF-8纳米晶体(ZIF-8纳米颗粒)在室温下真空干燥，并以粉末形式在4℃下储存。
(2)制备P-ZIF-8纳米晶体
为了制备P-ZIF-8纳米晶体，将上述制备的5mg ZIF-8粉末与1mg Z1-RGD肽(序列为RGDGGAHPHSDKLVPPR，购自于强耀生物)混合。加入1mL HEPES缓冲液(50mM HEPES-NaOH，150mM NaCl，pH 7.0)并涡旋震荡30秒。在室温下孵育2小时后，通过离心(10000g，4℃，10分钟)去除未结合的肽。使用前，将制备好的P-ZIF-8纳米颗粒再用HEPES缓冲液洗涤3次。
(3)制备FITC-P-ZIF-8纳米晶体
为了制备FITC-P-ZIF-8纳米晶体，采用与制备P-ZIF-8相同的方法，用FITC-Z1-RGD肽(序列为FITC-RGDGGAHPHSDKLVPPR，购自于强耀生物)替代Z1-RGD，其余步骤相同。
(4)制备ZIF-8@sulfo-cy5纳米晶体
为了制备ZIF-8@sulfo-cy5纳米晶体，在常温下，在50mL的玻璃反应瓶中加入0.2g Zn(NO 3) 2·6H 2O、0.8mL H 2O(pH 8.0，用NaOH调节)和一粒磁子，然后在一个EP管中加入20mg的sulfo-cy5(购自于成都栢尔康生物)和4mL的H 2O，另一个EP管中加入2g2-甲基咪唑和10mL H 2O。瓶盖盖紧后，分别将三种混合液放在超声仪中超声(600W，两分钟)2分钟使原料混合均匀，然后先将sulfo-cy5溶液缓慢滴加到玻璃反应瓶中，反应瓶中溶液逐渐变为蓝色，混匀后将2-甲基咪唑溶液也缓慢滴加到玻璃反应瓶中。磁力搅拌(2000rpm)反应15分钟后，离心收集沉淀物，并用含50％(v/v)乙醇的去离子水溶液洗涤3次，以尽可能除去纳米晶体表面吸附着的过量的2-甲基咪唑。最终制备的ZIF-8@sulfo-cy5纳米晶体在室温下真空干燥，并以粉末形式在4℃下储存。
(5)制备FITC-ZIF-8@sulfo-cy5纳米晶体
为了制备FITC-ZIF-8@sulfo-cy5纳米晶体，参考P-ZIF-8的制备方法，将ZIF-8@sulfo-cy5替代ZIF-8纳米晶体，将FITC-Z1-RGD肽替代Z1-RGD，其余步骤相同。
实施例2、ZIF-8、ZIF-8@FITC、P-ZIF-8@FITC纳米晶体的制备
(1)制备ZIF-8纳米晶体
在常温下，在50mL的玻璃反应瓶中加入0.15g Zn(NO 3) 2·6H 2O、7.3mL甲醇和一粒磁子，然后在一个15mL的EP管中加入0.33g 2-甲基咪唑和7.3mL甲醇溶液。瓶盖盖紧后，分别将两种混合液放在超声仪中超声(600W，两分钟)2分钟使原料混合均匀，然后将2-甲基咪唑溶液缓慢滴加到玻璃反应瓶中。磁力搅拌(2000rpm)反应5分钟后，离心(7000转/分钟，离心10分钟)收集沉淀物，并用甲醇溶液洗涤3次，以尽可能除去纳米晶体表面吸附着的过量的2-甲基咪唑。最终制备的ZIF-8纳米晶体在室温下真空干燥，并以粉末形式在4℃下储存。
(2)制备ZIF-8@FITC纳米晶体
为了制备ZIF-8@FITC纳米晶体，在常温下，在50mL的玻璃反应瓶中加入0.15g Zn(NO 3) 2·6H 2O、7.15mL甲醇和一粒磁子，然后在一个EP管中加入0.3mg的荧光素5-异硫氰酸酯溶液(FITC，购自Sigma)和0.15mL的甲醇，另一个EP管中加入0.33g 2-甲基咪唑和7.3mL甲醇。瓶盖盖紧后，分别将三种混合液放在超声仪中超声2分钟使原料混合均匀，然后将2-甲基咪唑溶液缓慢滴加到玻璃反应瓶中，后续步骤与上述在甲醇中合 成ZIF-8纳米晶体的步骤相同。
(3)制备P-ZIF-8@FITC纳米晶体
为了制备P-ZIF-8@FITC纳米晶体，使用与实施例1中制备P-ZIF-8纳米晶体相同的方法，用ZIF-8@FITC纳米晶体替代ZIF-8纳米晶体。
实施例3、测试ZIF-8纳米颗粒对SKBR-3细胞和ES-2细胞中突变p53降解的能力
将SKBR-3细胞(购自于ATCC)和ES-2细胞(购自于ATCC)接种在24孔细胞培养板中，密度约3～5×10 4cell/孔，过夜培养，待用。在加样前，先将细胞换入新鲜的McCoy's 5A培养基(购自于Gibco)，加入50μg/mL的ZIF-8纳米颗粒(先用McCoy's 5A培养基溶解实施例1制得的ZIF-8纳米颗粒，使其浓度为50μg/mL，再加入细胞中；下同)，空白对照为等体积的PBS缓冲液。细胞培养6h后，然后进行western blot检测。结果如图1所示，ZIF-8纳米颗粒可以引发SKBR-3细胞和ES-2细胞中突变p53蛋白的降解。
实施例4、测试ZIF-8纳米颗粒对ES-2细胞中突变p53降解的能力
将ES-2细胞接种在预前放有圆形爬片的24孔细胞培养板中，密度约3～5×10 4cell/孔，过夜培养，待用。在加样前，先将细胞换入新鲜的McCoy's 5A培养基，加入50μg/mL的ZIF-8纳米颗粒(实施例1制得)，空白对照为等体积的PBS缓冲液(Cont)。细胞培养6h后，然后进行免疫荧光检测。结果如图2所示，ZIF-8纳米颗粒可以降低ES-2细胞中的突变p53水平。
实施例5、测试ZIF-8纳米颗粒是否影响突变p53的合成
将ES-2细胞接种在24孔细胞培养板中，密度约3～5×10 4cell/孔，过夜培养，待用。在加样前，先将细胞换入新鲜的McCoy's 5A培养基，然后在指定的小时(0、2、4、8h)内，在不存在或存在ZIF-8(50μg/mL)(实施例1制得)的情况下，用环己酰亚胺(CHX，20μM)处理，然后进行western blot检测。结果如图3所示，ZIF-8与CHX联合处理后mutp53水平的加速下降，说明ZIF-8引发的mutp53水平的降低是由于降解而不是蛋白质合成的降低。
实施例6、测试ZIF-8的框架结构对降解突变p53是否是重要的
将ES-2细胞接种在24孔细胞培养板中，密度约3～5×10 4cell/孔，过夜培养，待用。在加样前，先将细胞换入新鲜的McCoy's 5A培养基，然后分别加入50μg/mL的ZIF-8(实施例1制得)和经800℃高温煅烧2h后的ZIF-8(C-ZIF-8)，阴性对照为加入等体积的PBS缓冲液(Cont)。细胞培养6h后，然后进行western blot检测。结果如图4所示，C-ZIF-8失去了降解mutp53的能力，说明ZIF-8的框架结构对于ZIF-8引发的mutp53的降解是至关重要的。
实施例7、测试ZIF-8降解突变p53的剂量依赖效应
将ES-2细胞接种在24孔细胞培养板中，密度约3～5×10 4cell/孔，过夜培养，待用。在加样前，先将细胞换入新鲜的McCoy's 5A培养基，然后分别加入25、50、100、150μg/mL的ZIF-8(实施例1制得)，阴性对照为加入等体积的PBS缓冲液。细胞培养6h后，然后进行western blot检测。结果如图5所示，ZIF-8以剂量依赖性方式引起mutp53降解，在25μg/mL处观察到显著降解，在100μg/mL处观察到最大降解。
实施例8、测试ZIF-8降解突变p53的时间依赖效应
将ES-2细胞接种在24孔细胞培养板中，密度约3～5×10 4cell/孔，过夜培养，待用。 在加样前，先将细胞换入新鲜的McCoy's 5A培养基，然后加入浓度为50μg/mL的ZIF-8(实施例1制得)，分别处理2h、4h、6h、8h，空白对照为等体积的PBS缓冲液。细胞培养2h、4h、6h、8h后，进行western blot检测。结果如图6所示，ZIF-8以时间依赖性方式引起mutp53降解，在处理4h时观察到显著降解，在处理8h时观察到最大降解。
实施例9、测试ZIF-8降解不同mutp53的能力
将9个不同突变p53细胞系ES-2、BT-474、BxPC-3、BT-549、MDA-MB-231、SK-BR-3、MIA PaCa-2、MDA-MB-468和NCI-H596细胞(均来自ATCC)，以及4个野生型p53细胞系MCF-7，A549，HCT116和NCI-H446(均来自ATCC)分别接种在24孔细胞培养板中，密度约3～5×10 4cell/孔，过夜培养，待用。分别加入50μg/mL的ZIF-8(实施例1制得)，空白对照为等体积的PBS缓冲液，细胞培养6h后，然后进行western blot检测。9个不同突变p53细胞系中每种突变体的mutp53降解百分比均基于western blot数据，并通过以下公式计算：(1-ZIF-8处理后p53与GAPDH的灰度值比例/PBS缓冲液处理后p53与GAPDH的灰度值比例)×100％。Western blot得到的条带的灰度值使用Photoshop软件获得。
结果如表1所示，ZIF-8在不同程度上有效地降解了9个mutp53突变体，包括六个热点突变体，即Y220C，R249S，R175H，R248W，R273H和G245C。其中，
S241F：p53基因(Gene ID:7157)第241位Arg突变为Phe；
E285K：p53基因的第285位Glu突变为Lys；
Y220C：p53基因的第220位Tyr突变为Cys；
R249S：p53基因的第249位密码子突变AGG→AGT/Arg→Ser(R249S)；
R280K：p53基因的第280位Arg突变为Lys；
R248W：p53基因的第248位Arg突变为Trp；
R175H：p53基因的第175位Arg突变为His；
R273H：p53基因的第273位Arg突变为His；
G245C：p53基因的第245位Gly突变为Cys；
Wild Type：野生型。
表1.ZIF-8和P-ZIF-8的Mutp53降解百分比和IC 50
Figure PCTCN2020122708-appb-000001
19种突变体p53细胞系中每种突变体的Mutp53降解百分比按下面公式计算：(1-ZIF-8处理后 p53与GAPDH的灰度值比例/PBS缓冲液处理后p53与GAPDH的灰度值比例)×100％。
2IC50来自于24小时细胞活力的数据分析。
实施例10、测试ZIF-8是否降低野生型p53的水平
将野生型p53细胞系A549(购自于ATCC)细胞和MCF-7(购自于ATCC)细胞接种在24孔细胞培养板中，密度约3～5×10 4cell/孔，过夜培养，待用。在加样前，先将细胞换入新鲜的McCoy's 5A培养基，然后分别加入50μg/mL的ZIF-8(实施例1制得)，空白对照为等体积的PBS缓冲液(Cont)，细胞培养6h后，然后进行western blot检测。结果如图7所示，A549和MCF-7中，野生型p53蛋白的水平在ZIF-8处理后增加而不是减少，表明ZIF-8是选择性地降解mutp53。
实施例11、测试ZIF-8降解细胞中mutp53的途径
将ES-2细胞接种在24孔细胞培养板中，密度约3～5×10 4cell/孔，过夜培养，待用。在加样前，先将细胞换入新鲜的McCoy's 5A培养基，分别加入浓度为50μg/mL的ZIF-8(实施例1制得)、50μg/mL的ZIF-8+蛋白酶体抑制剂MG-132(10μM，购自于Selleck)、50μg/mL的ZIF-8+蛋白酶体抑制剂PS-341(10μM，购自于Selleck)，空白对照为等体积的PBS缓冲液(Cont)。细胞培养6h后，然后进行western blot检测。结果如图8所示，MG-132和PS-341完全抑制了ZIF-8降解mutp53的能力，说明ZIF-8是通过蛋白酶体途径引发的mutp53的降解。
实施例12、测试ZIF-8对细胞中mutp53泛素化水平的影响
将ES-2细胞接种在6孔细胞培养板中，密度约1～3×10 5cell/孔，过夜培养，待用。在加样前，先将细胞换入新鲜的McCoy's 5A培养基，分别加入浓度为50μg/mL的ZIF-8(实施例1制得)、10μM的MG-132(购自于Selleck)，50μg/mL的ZIF-8+MG-132(10μM，购自于Selleck)，空白对照为等体积的PBS缓冲液(Cont)。细胞培养6h后，用PBS缓冲液清洗细胞两次，然后使用胰酶消化，随后使用p53抗体(购自Santa cruz biotechnology)进行免疫共沉淀，最后进行western blot检测。结果如图9所示，ZIF-8增强了mutp53的K48位点的泛素化水平。
实施例13、测试ZIF-8降解细胞中的mutp53是否需要泛素化酶的参与
将ES-2细胞接种在24孔细胞培养板中，密度约3～5×10 4cell/孔，过夜培养，待用。在加样前，先将细胞换入新鲜的McCoy's 5A培养基，加入浓度为50μg/mL的ZIF-8(实施例1制得)、50μg/mL的ZIF-8+泛素化酶抑制剂PYR-41(5μM，购自于Selleck)、50μg/mL的ZIF-8+去泛素化酶抑制剂PR-619(5μM，购自于Selleck)，空白对照为等体积的PBS缓冲液(Cont)。细胞培养6h后，然后进行western blot检测。结果如图10所示，泛素化酶抑制剂PYR-41能够抑制ZIF-8引发的mutp53的降解，而去泛素化酶抑制剂PR-619不影响，说明ZIF-8引发的mutp53的降解过程依赖于mutp53的泛素化修饰。
实施例14、测试细胞的内吞对于ZIF-8降解细胞中的mutp53是否是必须的
将ES-2细胞接种在24孔细胞培养板中，密度约3～5×10 4cell/孔，过夜培养，待用。在加样前，先将细胞换入新鲜的McCoy's 5A培养基，加入浓度为50μg/mL的ZIF-8(实施例1制得)、50μg/mL的ZIF-8+50μM Genistein(酪氨酸蛋白激酶抑制剂，购自于Sigma)、50μM Genistein。空白对照为等体积的PBS缓冲液(Cont)。细胞培养6h后，然后进行western blot检测。结果如图11所示，ZIF-8引发的ES-2细胞中的mutp53的降解可以被内吞抑制剂Genistein有效抑制，说明细胞的内吞对于ZIF-8引发的降解是必须的。
实施例15、测试ZIF-8对细胞酸性溶酶体中Zn 2+水平的影响
将ES-2细胞接种在96孔细胞培养板中，密度约1～2×10 4cell/孔，过夜培养，待用。在加样前，先将细胞换入新鲜的McCoy's 5A培养基，加入浓度为50μg/mL的ZIF-8(实施例1制得)，空白对照为等体积的PBS缓冲液(Cont)。处理2h后，用PBS缓冲液清洗两次细胞。加入含有Zn 2+绿色离子荧光探针FluoZin-3 AM(1μM，Invitrogen)和溶酶体红色荧光探针LysoTracker(1μM，Invitrogen)的新鲜培养基，37℃培养条件下处理30min，PBS缓冲液清洗后使用共聚焦显微镜(Nikon，Ti-E Al)成像。结果如图12所示，ZIF-8处理2小时后，在ES-2细胞中观察到酸性溶酶体中Zn 2+升高。
实施例16、测试ZIF-8和C-ZIF-8对细胞内Zn 2+水平的影响
将ES-2细胞接种在24孔细胞培养板中，密度约3～5×10 4cell/孔，过夜培养，待用。在加样前，先在无血清培养基中洗涤两次，并与1μM FluoZin-3 AM(Invitrogen)在37℃孵育40分钟。用无酚红的DMEM培养基洗涤细胞两次后，分别加入浓度为50μg/mL的ZIF-8(实施例1制得)和C-ZIF-8(制备方法同实施例6)，空白对照为等体积的PBS缓冲液(Cont)。处理2h后，用PBS缓冲液清洗两次细胞。胰酶消化，PBS缓冲液清洗后进行FACS(流式细胞荧光分选技术)检测。结果如图13所示，ZIF-8能引发细胞内Zn 2+浓度升高，而C-ZIF-8不会引起细胞内Zn 2+升高。
实施例17、测试ZIF-8处理后对细胞内Zn 2+水平的影响
将ES-2细胞接种在24孔细胞培养板中，密度约3～5×10 4cell/孔，过夜培养，待用。在加样前，先在无血清培养基中洗涤两次，并与1μM FluoZin-3 AM(Invitrogen)在37℃孵育40分钟。用无酚红的DMEM培养基洗涤细胞两次后，分别加入浓度为50μg/mL的ZIF-8(实施例1制得)、50μg/mL的ZIF-8+TPEN(N,N,N',N'-四(2-吡啶甲基)乙二胺)(50μM，Selleck)、TPEN(50μM，Selleck)，空白对照为等体积的PBS缓冲液(Cont)。处理2h后，用PBS缓冲液清洗两次细胞。胰酶消化，PBS缓冲液清洗后进行FACS(流式细胞荧光分选技术)检测。结果如图14所示，TPEN能够完全抑制ZIF-8引起的细胞内Zn 2+升高。
实施例18、测试ZIF-8引发的mutp53的降解是否与细胞内的Zn 2+水平有关
将ES-2细胞接种在24孔细胞培养板中，密度约3～5×10 4cell/孔，过夜培养，待用。在加样前，先将细胞换入新鲜的McCoy's 5A培养基，分别加入浓度为50μg/mL的ZIF-8(实施例1制得)、50μg/mL的ZIF-8+TPEN(50μM，Selleck)、TPEN(50μM，Selleck)，空白对照为等体积的PBS缓冲液(Cont)。细胞培养6h后，然后进行western blot检测。结果如图15所示，TPEN能够抑制ZIF-8引发的mutp53的降解，说明ZIF-8引发的mutp53的降解依赖于细胞内的Zn 2+升高。
实施例19、测试ZIF-8引起细胞内Zn 2+升高具有剂量依赖性
将ES-2细胞接种在96孔细胞培养板中，密度约1～2×10 4cell/孔，过夜培养，待用。在加样前，先在无血清培养基中洗涤两次，并与1μM FluoZin-3 AM(Invitrogen)在37℃孵育40分钟。用无酚红的DMEM培养基洗涤细胞两次后，分别加入浓度分别为25μg/mL、50μg/mL、75μg/mL和100μg/mL的ZIF-8(实施例1制得)，空白对照为等体积的PBS缓冲液。处理2h后，PBS缓冲液清洗后使用荧光显微镜(Nikon,DS-Fi3)成像。结果如图16所示，随着浓度的升高，细胞中绿色荧光信号逐渐增强，说明ZIF-8引起细胞内Zn 2+升高具有剂量依赖性。
实施例20、测试ZIF-8引起细胞内Zn 2+升高的行为
将ES-2细胞接种在24孔细胞培养板中，密度约3～5×10 4cell/孔，过夜培养，待用。在加样前，先在无血清培养基中洗涤两次，并与1μM FluoZin-3 AM(Invitrogen)在37℃孵育40分钟。用无酚红的DMEM培养基洗涤细胞两次后，分别在0.5h、1h、3h和5h时加入浓度为50μg/mL的ZIF-8(实施例1制得)，空白对照为等体积的PBS缓冲液。最后胰酶消化，PBS缓冲液清洗后进行FACS(流式细胞荧光分选技术)检测。结果如图17所示，随着时间的延长，ZIF-8引起细胞内Zn 2+逐渐升高。
实施例21、测试ZIF-8分解释放Zn 2+是否与ZIF-8引起的mutp53降解相关
将ES-2细胞接种在24孔细胞培养板中，密度约3～5×10 4cell/孔，过夜培养，待用。在加样前，先将细胞换入新鲜的McCoy's 5A培养基，加入浓度为50μg/mL的ZIF-8(ZIF-8在pH 5.5下预处理0、1、3、6和12h)(实施例1制得)，空白对照为等体积的PBS缓冲液(NA)。细胞培养6h后，然后进行western blot检测。结果如图18所示(图中，崩塌程度为ZIF-8(pH 5.5)中的锌释放的数据，ZIF-8崩塌后释放出锌，具体测定方法同实施例24)，随着预处理时间的延长，ZIF-8诱导mutp53降解的能力逐渐降低。
实施例22、测试ZIF-8纳米颗粒存在内含体逃逸行为
将ES-2细胞接种在共聚焦小皿中，密度约5～10×10 4cell/孔，过夜培养，待用。在加样前，先将细胞换入新鲜的McCoy's 5A培养基。在指定时间内，加入浓度为50μg/mL的FITC-ZIF-8@sulfo-cy5(实施例1制得)，空白对照为等体积的PBS缓冲液。处理2h后，用PBS缓冲液清洗两次细胞。加入含有溶酶体红色荧光探针LysoTracker(1μM，Invitrogen)的新鲜培养基，37℃培养条件下处理30min，然后PBS缓冲液清洗细胞两次，最后使用共聚焦显微镜(Nikon，Ti-E Al)成像。
结果如图19所示(图中：“ZIF-8(cy5)”使用包裹在纳米颗粒内的sulfo-cy5染料发出的红色荧光代表ZIF-8纳米颗粒；“ZIF-8(FITC)”使用包载在纳米颗粒表面的绿色荧光FITC-Z1肽代表ZIF-8纳米颗粒；“Lysosome”表示细胞中的溶酶体结构)，FITC-ZIF-8@sulfo-cy5处理1h后，在ES-2细胞中观察到大多数绿色和红色荧光共定位，表明纳米粒子完整无损，它们进一步与溶酶体的蓝色荧光共定位(合并图像中为白色)，表明它们进入到酸性内含体中。但是到了2h时，合并后的图像中出现了粉红色和红色荧光，分别表明与酸性内含体和不与酸性内含体共定位的ZIF-8分解释放出来的sulfo-cy5。另外，还可以在蓝色染色的结构外发现黄色荧光，这表明完整或部分分解的ZIF-8纳米颗粒存在内含体逃逸的行为。
实施例23、ZIF-8和P-ZIF-8纳米晶体的表征
实施例1中ZIF-8(实施例1制得)和P-ZIF-8(实施例1制得)纳米晶体的透射电子显微镜照片如图20所示，从图中可以看出所得ZIF-8和P-ZIF-8纳米晶体尺寸均匀、分散性好，每个颗粒的平均粒径为90nm。
实施例24、测试ZIF-8和P-ZIF-8的pH敏感性释放Zn 2+行为
分别将0.5mg ZIF-8(实施例1制得)和P-ZIF-8(实施例1制得)粉末加入到1640培养基中，剧烈涡旋，使ZIF-8或P-ZIF-8粉末悬浮在1640培养基中，然后用HCl溶液将pH值调节至7.4、6.5和5.5。在37℃下振荡孵育一定时间(0，1h，3h，6h，12h，24h)后，将样品离心(10000×g，4℃，10分钟)。通过ICP-MS测定上清液中的锌含量，表示从纳米颗粒分解释放的锌。锌释放百分比的计算公式为：锌释放(％)＝100×mr/143.2，其中mr是上清液中锌的总含量(μg)，143.2是0.5mg ZIF-8中的锌的含量(μg)。结果如 图21所示，在pH 5.5和pH 6.5时，Z1-RGD肽的修饰降低了ZIF-8在体外释放Zn 2+的速率，在pH 6.5时抑制效应最强。
实施例25、测试ZIF-8和P-ZIF-8在pH 6.5条件下降解mutp53的行为比较
将ES-2细胞接种在24孔细胞培养板中，密度约3～5×10 4cell/孔，过夜培养，待用。在加样前，先将细胞换入新鲜的McCoy's 5A培养基，分别加入浓度为50μg/mL的ZIF-8和P-ZIF-8(均在pH 6.5条件下预处理0、3、6和12h)(实施例1制得)，空白对照为等体积的PBS缓冲液(NA)。细胞培养6h后，然后进行western blot检测。结果如图22所示(图中，崩塌程度为ZIF-8(pH 6.5)和P-ZIF-8(pH 6.5)释放锌的数据，具体测定方法同实施例24)，未修饰的ZIF-8在pH 6.5下孵育12小时，mutp53降解能力降低了210％，但是Z1-RGD修饰的P-ZIF-8几乎完全消除了这种不良的降解降低作用。
实施例26、ZIF-8和P-ZIF-8在pH 7.4和pH 6.5条件下对正常细胞的杀伤能力比较
将HEK 293细胞(来自于ATCC)、ARPE-19细胞(来自于ATCC)分别接种在96孔细胞培养板中，密度约1～2×10 4cell/孔，过夜培养，待用。在加样前，先将细胞换入新鲜培养基DMEM，分别加入浓度为50μg/mL的ZIF-8和P-ZIF-8(均分别在pH 7.4和pH 6.5条件下预处理12h)(实施例1制得)，空白对照为等体积的PBS缓冲液。处理24h，然后进行MTT检测。结果如图23所示，在pH 6.5条件下孵育12小时显著增加了ZIF-8对HEK 293和ARPE-19细胞的毒性，但Z1-RGD肽的修饰显著降低了在这两种细胞系中的毒性。
实施例27、ZIF-8和P-ZIF-8入胞的能力比较
将ES-2细胞接种在共聚焦小皿中，密度约5～10×10 4cell/孔，过夜培养，待用。在加样前，先将细胞换入新鲜的McCoy's 5A培养基。分别加入浓度为50μg/mL的ZIF-8@FITC(实施例2制得)和P-ZIF-8@FITC(实施例2制得)，空白对照为等体积的PBS缓冲液。处理4h后，用PBS缓冲液清洗两次细胞。加入含有细胞核蓝色染料Hoechst(2μg/mL，Invitrogen)的新鲜McCoy's 5A培养基，常温条件下孵育10min，PBS缓冲液清洗细胞两次，然后使用共聚焦显微镜(Nikon，Ti-EAl)成像。结果如图24所示，与ZIF-8组相比，P-ZIF-8组的绿色荧光更强，说明Z1-RGD肽的修饰能使ZIF-8更有效地进入到ES-2细胞中。
实施例28、ZIF-8和P-ZIF-8释放Zn 2+能力比较
将ES-2细胞接种在24孔细胞培养板中，密度约3～5×10 4cell/孔，过夜培养，待用。在加样前，先在无血清培养基中洗涤两次，并与1μM FluoZin-3 AM(Invitrogen)在37℃孵育40分钟。用无酚红的DMEM培养基洗涤细胞两次后，分别加入浓度为50μg/mL的ZIF-8(实施例1制得)，浓度为50μg/mL的P-ZIF-8(实施例1制得)以及浓度为10μg/mL的Z1-RGD(序列为RGDGGAHPHSDKLVPPR，购于强耀生物)，空白对照为等体积的PBS缓冲液(Cont)。处理2h后，胰酶消化，PBS缓冲液清洗后进行FACS(流式细胞荧光分选技术)检测。结果如图25所示，与ZIF-8组相比，P-ZIF-8组的荧光信号更强，说明Z1-RGD肽的修饰后的P-ZIF-8释放Zn 2+能力更强。
实施例29、ZIF-8和P-ZIF-8降解突变p53能力比较
将ES-2细胞接种在24孔细胞培养板中，密度约3～5×10 4cell/孔，过夜培养，待用。在加样前，先将细胞换入新鲜的McCoy's 5A培养基，分别加入浓度为40μg/mL的ZIF-8(实施例1制得)，40μg/mL的P-ZIF-8(实施例1制得)以及浓度为8μg/mL的Z1-RGD(序 列为RGDGGAHPHSDKLVPPR，购于强耀生物)，空白对照为等体积的PBS缓冲液(Cont)。细胞培养6h后，然后进行western blot检测。结果如图26所示，与ZIF-8组相比，P-ZIF-8组能更有效的引起ES-2细胞内突变p53的降解。
实施例30、ZIF-8在细胞中的IC 50与mutp53降解百分率的关系
将ES-2、BT-474、BxPC-3、BT-549、MDA-MB-231、SK-BR-3、MIA PaCa-2、MDA-MB-468和NCI-H596细胞(均来自ATCC)接种在96孔细胞培养板中，密度约1～2×10 4/孔，过夜培养，待用。分别50μg/mL的ZIF-8(实施例1制得)，空白对照为等体积的PBS缓冲液，细胞培养24h后，然后进行MTT检测。使用GraphPadPrism软件计算ZIF-8在上述细胞系中的IC 50，并将得到的数据与对应细胞系中mutp53降解百分率(实施例9得到)用GraphPadPrism软件进行回归分析。结果如图27所示，ZIF-8在9种表达不同mutp53的细胞中的IC 50与mutp53降解百分率呈负相关。
实施例31、探究ZIF-8和P-ZIF-8在动物体内的药代动力学
雌性Balb/c小鼠(购自于北京维通利华，体重约20g)分为两组，两组分别注射ZIF-8(20mg/kg)和P-ZIF-8(20mg/kg)(实施例1制得)，不同时间(0、2、4、8、16、24h)点眼眶静脉丛取血，通过ICP检测血液中锌的含量。结果如图28所示，ZIF-8和P-ZIF-8在小鼠体内的药代动力学相似，没有明显差异。
实施例32、检测ZIF-8和P-ZIF-8在ES-2动物肿瘤模型中抑制肿瘤生长的效应
在雌性Balb/c Nude裸鼠(购自于北京维通利华，体重约20g)的右肋皮下注射100μL ES-2细胞(1×10 7)，构建ES-2肿瘤模型。肿瘤体积达到100mm 3时进行后续实验。
将获得的ES-2荷瘤鼠分为四组，每组六只。每组分别进行以下处理，PBS缓冲液处理、Z1-RGD肽(4mg/kg)(序列为RGDGGAHPHSDKLVPPR，购自于强耀生物)处理、ZIF-8(20mg/kg)(实施例1制得)处理、P-ZIF-8(20mg/kg)(实施例1制得)处理。每周静脉注射给药2次，治疗过程中记录小鼠肿瘤体积变化(图29)。治疗17天(前两周每周给药两次，第三周在第17天给药1次)结束后，牺牲小鼠剥取肿瘤称重(图30)。可以看到，ZIF-8处理组和P-ZIF-8处理组均有抑制小鼠肿瘤生长的效果，P-ZIF-8处理组的治疗效果最好。
实施例33、检测ZIF-8和P-ZIF-8在ES-2肿瘤模型中降解mutp53的效果
实施例32中的ES-2动物肿瘤模型，治疗17天结束后，牺牲小鼠剥取肿瘤称重后，切碎，使用组织匀浆仪充分混匀，离心，取上清，制备肿瘤组织的样品，随后进行western blot检测。结果如图31(图中序号1-6表示每组里面的六只小鼠)所示，ZIF-8处理组和P-ZIF-8处理组均能降低肿瘤组织中的mutp53水平，P-ZIF-8处理组的降解效果更好。
实施例34、检测P-ZIF-8在MCF-7动物肿瘤模型中是否可以抑制肿瘤生长
在雌性Balb/c Nude裸鼠(购自于北京维通利华，体重约20g)的右肋皮下注射100μL MCF-7细胞(1×10 7)，构建MCF-7肿瘤模型。肿瘤体积达到100mm 3时进行后续实验。
将获得的MCF-7荷瘤鼠分为两组，每组六只。每组分别进行以下处理，PBS缓冲液处理、P-ZIF-8(20mg/kg)(实施例1制得)处理。每周静脉注射给药2次，治疗过程中记录小鼠肿瘤体积变化。结果如图32可以看到，P-ZIF-8处理组与PBS对照组相比，没有明显的抑制小鼠肿瘤生长的效果。
实施例35、检测ZIF-8和P-ZIF-8在PDX肿瘤模型中抑制肿瘤生长的效应
构建PDX肿瘤模型：从南方医院(中国广东省广州市)获得了人乳腺浸润性导管癌标本(ER+PR+Her3+，III级，55岁，女性)。通过在北京基因组研究所进行的全外显子组测序以及RT-PCR后对全长p53编码序列进行测序，验证了该肿瘤的mutp53状态(Y220C)。使用患者来源的材料进行的实验符合所有相关的道德规范，并在得到患者知情同意的情况下，根据华南理工大学伦理委员会机构人类研究对象保护委员会制定的批准指南进行操作。将手术后获得的肿瘤组织切成2～3mm 3的碎片，并原位植入雌性NOD/SCID小鼠(购自于北京维通利华，6-8周龄，体重约20g)的乳腺脂肪垫中。当肿瘤体积达到约800mm 3，将肿瘤异种移植物切成2～3mm 3的切片，并按照相同的植入步骤进行扩增(即将肿瘤异种移植物植入新的NOD/SCID小鼠中，以此重复3次)。将具有第三代异种移植的小鼠随机分为四组，每组7只，并在肿瘤长至约100mm 3时进行后续实验。每组分别进行以下处理，PBS缓冲液处理、Z1-RGD肽(4mg/kg)(序列为RGDGGAHPHSDKLVPPR，购自于强耀生物)处理、ZIF-8(20mg/kg)处理、P-ZIF-8(20mg/kg)处理。每周静脉注射给药2次，治疗过程中记录小鼠肿瘤体积变化(图33)。治疗27天(前三周每周给药两次，第四周在第24天给药一次)结束后，牺牲小鼠剥取肿瘤拍照(图34)并进行称重(图35)。可以看到，ZIF-8处理组和P-ZIF-8处理组均有抑制小鼠肿瘤生长的效果，P-ZIF-8处理组的治疗效果最好。
实施例36、检测ZIF-8和P-ZIF-8在PDX肿瘤模型中降解mutp53的效果
在实施例35中PDX荷瘤鼠27天治疗后，将每组的肿瘤通过石蜡包埋后进行切片，然后在二甲苯和分级酒精(浓度依次为100％，95％，80％，70％，体积百分数)溶液中脱蜡并重新水化。使用抗p53抗体(Santa Cruz)对切片染色，并用苏木精将细胞核染色。结果如图36所示，ZIF-8和P-ZIF-8均能明显的降低PDX肿瘤模型中的mutp53水平。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Figure PCTCN2020122708-appb-000002

Claims (10)

  1. 一种ZIF-8纳米材料在降解广谱突变p53蛋白中的应用，其特征在于：所述的ZIF-8纳米材料为ZIF-8纳米晶体、包载染料的ZIF-8纳米晶体和具有pH响应性的肽修饰的ZIF-8纳米材料中的至少一种；
    所述的应用的环境为体外环境。
  2. 根据权利要求1所述的ZIF-8纳米材料在降解广谱突变p53蛋白中的应用，其特征在于：
    所述的包载染料的ZIF-8纳米晶体中的染料为sulfo-cy5和FITC中的至少一种；
    所述的具有pH响应性的肽为Z1-RGD肽和FITC-Z1-RGD肽中的至少一种；
    所述的Z1-RGD肽的序列为：RGDGGAHPHSDKLVPPR；
    所述的FITC-Z1-RGD肽的序列为：FITC-RGDGGAHPHSDKLVPPR。
  3. 根据权利要求1所述的ZIF-8纳米材料在降解广谱突变p53蛋白中的应用，其特征在于：
    所述的突变p53蛋白为p53蛋白突变体S241F，E285K，Y220C，R249S，R280K，R248W，R175H，R273H和G245C中的至少一种。
  4. 根据权利要求1～3任一项所述的ZIF-8纳米材料在降解广谱突变p53蛋白中的应用，其特征在于：
    所述的ZIF-8纳米晶体通过如下方法制备得到：
    将Zn(NO 3) 2·6H 2O加入到溶剂中，超声混合均匀，得到混合溶液I；将2-甲基咪唑加入到溶剂中，超声混合均匀，得到混合溶液II；将混合溶液II滴加到混合溶液I中，搅拌，离心收集沉淀物，洗涤，真空干燥，得到ZIF-8纳米晶体；
    所述的2-甲基咪唑与Zn(NO 3) 2·6H 2O的质量比为1～2:0.1～1；
    所述的溶剂为水和甲醇中的至少一种；
    所述的包载染料的ZIF-8纳米晶体为ZIF-8@sulfo-cy5和ZIF-8@FITC中的至少一种；
    所述的具有pH响应性的肽修饰的ZIF-8纳米材料为P-ZIF-8，FITC-P-ZIF-8，FITC-ZIF-8@sulfo-cy5和P-ZIF-8@FITC中的至少一种；
    所述的P-ZIF-8纳米晶体通过如下方法制备得到：
    将ZIF-8纳米晶体与Z1-RGD肽混合后加入HEPES缓冲液，涡旋震荡30秒，在室温下进行孵育，再离心，洗涤得到P-ZIF-8纳米晶体；
    所述的ZIF-8纳米晶体与Z1-RGD肽的质量比为5:1；
    所述的FITC-P-ZIF-8纳米晶体通过如下方法制备得到：
    将ZIF-8纳米晶体与FITC-Z1-RGD肽混合后加入HEPES缓冲液，涡旋震荡30秒，在室温下进行孵育，再离心，洗涤得到FITC-P-ZIF-8纳米晶体；
    所述的ZIF-8纳米晶体与FITC-Z1-RGD肽的质量比为5:1；
    所述的ZIF-8@sulfo-cy5纳米晶体通过如下方法制备得到：
    将Zn(NO 3) 2·6H 2O加入到溶剂中，超声混合均匀，得到混合溶液A；将sulfo-cy5加入到溶剂中，超声混合均匀，得到混合溶液B；将2-甲基咪唑加入到溶剂中，超声混合均匀，得到混合溶液C；将混合溶液B和混合溶液C依次滴加到混合溶液A中，搅拌，离心收集沉淀物，洗涤，真空干燥，得到ZIF-8@sulfo-cy5纳米晶体；
    所述的Zn(NO 3) 2·6H 2O、sulfo-cy5和2-甲基咪唑的质量比为10:1:100；
    所述的溶剂pH 8.0的水；
    所述的FITC-ZIF-8@sulfo-cy5纳米晶体通过如下方法制备得到：
    将上述ZIF-8@sulfo-cy5纳米晶体与FITC-Z1-RGD肽混合后加入HEPES缓冲液，涡旋震荡30秒，在室温下进行孵育，再离心，洗涤得到ZIF-8@sulfo-cy5纳米晶体；
    所述的ZIF-8@sulfo-cy5纳米晶体与FITC-Z1-RGD肽的质量比为5:1；
    所述的ZIF-8@FITC纳米晶体通过如下方法制备得到：
    将Zn(NO 3) 2·6H 2O加入到溶剂中，超声混合均匀，得到混合溶液D；将荧光素5-异硫氰酸酯 溶液加入到溶剂中，超声混合均匀，得到混合溶液E；将2-甲基咪唑加入到溶剂中，超声混合均匀，得到混合溶液F；将混合溶液F和混合溶液E依次滴加到混合溶液D中，搅拌，离心收集沉淀物，洗涤，真空干燥，得到ZIF-8@FITC纳米晶体；
    所述的溶剂为甲醇；
    所述的Zn(NO 3) 2·6H 2O、5-异硫氰酸酯溶液和2-甲基咪唑的质量比为1:0.002:2.2；
    所述的P-ZIF-8@FITC纳米晶体通过如下方法制备得到：
    将ZIF-8@FITC纳米晶体与Z1-RGD肽混合后加入HEPES缓冲液，涡旋震荡30秒，在室温下进行孵育，再离心，洗涤得到P-ZIF-8@FITC纳米晶体；
    所述的ZIF-8@FITC纳米晶体与Z1-RGD肽的质量比为5:1；
    所述的超声的条件均为：600W超声2～3分钟；
    所述的洗涤均为采用水或甲醇进行洗涤；
    所述的搅拌的的条件均为：2000rpm搅拌10～20分钟；
    所述的离心的条件均为：4℃，7000～10000g离心10分钟。
  5. 根据权利要求1～3任一项所述的ZIF-8纳米材料在降解广谱突变p53蛋白中的应用，其特征在于：
    所述的ZIF-8纳米材料的有效浓度为25～150μg/mL；
    所述的降解的时间为0.5～8小时。
  6. 根据权利要求5所述的ZIF-8纳米材料在降解广谱突变p53蛋白中的应用，其特征在于：
    所述的ZIF-8纳米材料的有效浓度为25～100μg/mL；
    所述的降解的时间为4～8小时。
  7. 权利要求1～6任一项中所述的ZIF-8纳米材料在制备降解广谱突变p53蛋白的产品中的应用。
  8. 权利要求1～6任一项中ZIF-8所述的纳米材料作为广谱突变p53蛋白降解剂在制备抗肿瘤药物中的应用。
  9. 酶抑制剂或N,N,N',N'-四(2-吡啶甲基)乙二胺在制备抑制权利要求1～6任一项中所述的ZIF-8纳米材料降解广谱突变p53蛋白的产品中的应用，其特征在于：所述的酶抑制剂为泛素化酶抑制剂和酪氨酸蛋白激酶抑制剂中的至少一种。
  10. 根据权利要求8所述的应用，其特征在于：
    所述的泛素化酶抑制剂为泛素化酶抑制剂PYR-41；
    所述的酪氨酸蛋白激酶抑制剂为酪氨酸蛋白激酶抑制剂Genistein。
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