Antitumor nano material based on copper death promotion and preparation method and application thereof

Abstract

The invention discloses an anti-tumor nano material based on copper death promotion as well as a preparation method and application of the anti-tumor nano material. The anti-tumor nano material is constructed by taking PCN-Cu as a metal organic framework compound and merging Pt nano particles with stable polyvinylpyrrolidone into the PCN-Cu, and the anti-tumor nano material is named as Pt-coated PCN-Cu. Experiments prove that the Pt-coated PCN-Cu can improve the concentration of copper ions in tumor cells, promote the lipoic acylation aggregation process of mitochondrial related proteins, promote degradation and proteotoxicity reaction of Fe-S cluster proteins and induce copper death of the tumor cells. The Pt-coated PCN-Cu has the advantages of being simple in preparation process, green, economical, low in cost, capable of being produced on a large scale and the like, has a good anti-tumor effect, expands a novel treatment mode for promoting tumor cell copper death, and provides a new technical means for tumor treatment.

Description

Technical Field

[0001] The invention relates to an anti-tumor nano material based on promoting copper death and a preparation method and application thereof, belonging to the technical field of nano medicines for tumor treatment. Background Art

[0002] The essential micronutrient copper (Cu) plays a key catalytic role in a variety of biological processes such as mitochondrial respiration, anti-oxidation, and biosynthesis. Under physiological conditions, the intracellular copper ion concentration is maintained at a relatively low range; moderate increases in copper ion concentrations can cause cytotoxicity and even lead to cell death. Copper ion deficiency or accumulation can have adverse effects on human health, and the inability to maintain copper ion balance is associated with a variety of pathological conditions, including Menkes syndrome, Wilson's disease, neurodegenerative diseases, cancer, and cardiovascular disease.

[0003] The role of copper in cancer progression has long been a subject of research, and copper may be involved in the activation of cell proliferation-related signaling pathways. Compared with normal tissue cells, tumor cells generally have a higher demand for copper. In addition, copper ion concentrations are increased in tumor tissue and serum of patients with various types of cancer. Among them, increased serum copper ion levels are associated with tumor staging and disease progression in patients with colorectal cancer, lung cancer, and breast cancer. Changes in copper ion homeostasis in tumor tissues and the important role that copper ions may play in promoting tumor progression will bring new ideas and methods to tumor treatment.

[0004] At present, research on tumor therapy based on copper death can be divided into two main methods. The first method is to use copper ion carriers, such as Disulfiram and Elesclomol (ES), to transport copper ions to cells and induce copper death. However, due to changes in copper ion metabolism and the effectiveness of copper ion carriers, it is still challenging to achieve copper death in tumor cells. The second method is to use sonosensitizers / photosensitizers in combination with copper ion carriers to form nanoparticles. Further, the nanoparticles release copper ions in the tumor area through external stimulation, inducing cellular copper death. However, the stability and biosafety of nanoparticles have greatly limited practical applications. Therefore, the development of a copper death inducer that is independent of copper ion carriers and has high stability is of vital importance in practical applications and is also an important avenue for further research.

[0005] Surgical resection, radiotherapy, chemotherapy and immunotherapy are the most common tumor treatments, and the combination of multiple treatments is still the most effective tumor treatment. Based on this, it is extremely important to design and develop a nanomaterial tumor treatment method based on the plasticity and multifunctionality of nanomaterials that promotes copper death. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide an anti-tumor nano material based on promoting copper death with good anti-tumor effect, and a preparation method and application thereof.

[0007] In order to solve the above technical problems, the present invention adopts the following technical means:

[0008] The invention provides an anti-tumor nano material based on promoting copper death. The anti-tumor nano material is constructed by taking PCN-Cu as a metal organic framework compound and incorporating Pt nanoparticles stabilized by polyvinyl pyrrolidone into PCN-Cu; wherein the PCN-Cu is obtained by dissolving tetracarboxylphenylporphyrin copper, zirconium oxychloride octahydrate and benzoic acid in an N,N-dimethylformamide solution, and heating, stirring, cooling and centrifuging.

[0009] The present invention also provides a method for preparing the anti-tumor nanomaterial based on promoting copper death, comprising the following steps:

[0010] (1) dissolving tetracarboxyphenylporphyrin copper, zirconium oxychloride octahydrate and benzoic acid in an N,N-dimethylformamide solution, and obtaining PCN-Cu nanoparticles by heating, stirring, cooling and centrifugation;

[0011] (2) dissolving polyvinyl pyrrolidone in methanol, adding chloroplatinic acid thereto, refluxing the mixture with air, removing methanol, precipitating with acetone, and centrifuging to obtain Pt nanoparticles. The obtained Pt nanoparticles are further purified with chloroform and hexane, and redispersed in N,N-dimethylformamide solution;

[0012] (3) The PCN-Cu nanoparticles prepared in step (1) are mixed with the Pt nanoparticles prepared in step (2), heated, stirred, cooled, and centrifuged. After washing multiple times with N,N-dimethylformamide solution, an anti-tumor nanomaterial based on promoting copper death is obtained, which is named Pt@PCN-Cu.

[0013] Among them, preferably, the mass ratio of tetracarboxyphenylporphyrin copper, zirconium oxychloride octahydrate and benzoic acid described in step (1) is 1.1:3:29, the heating temperature is 90°C, the stirring speed is 300rpm, the time is 5h, the centrifugal speed is 12000rpm, and the centrifugal time is 20min.

[0014] Preferably, the amount of polyvinyl pyrrolidone used in step (2) is 266 mg, the amount of methanol used is 90 mL, the amount of 50 mM chloroplatinic acid used is 10 mL, the air reflux time is 3 h, the precipitation time is 3 h, the centrifugal speed is 6000 rpm, and the centrifugal time is 5 min.

[0015] Preferably, the heating temperature in step (3) is 90° C., the stirring speed is 300 rpm, the time is 5 h, the centrifugal speed is 12000 rpm, and the centrifugal time is 20 min.

[0016] The present invention also provides the use of the anti-tumor nanomaterial based on promoting copper death in the preparation of drugs for improving and / or treating tumors.

[0017] Among them, preferably, the concentration of the anti-tumor nanomaterial based on promoting copper death is 10 to 100 nM, more preferably 10 to 50 nM, and further preferably 10 to 25 nM.

[0018] Compared with the prior art, the present invention has the following significant advantages:

[0019] The present invention obtains an anti-tumor nanomaterial Pt@PCN-Cu based on promoting copper death through a simple synthesis method, and applies the anti-tumor nanomaterial to a mouse pancreatic cancer tumor-bearing model, showing a good anti-tumor effect. Therefore, the present invention provides a new treatment method for promoting copper death of tumor cells, which improves the existing tumor treatment model. The anti-tumor nanomaterial based on promoting copper death of the present invention has the advantages of simple preparation process, green economy, environmental friendliness, low cost and mass production, and based on the development of new nanomedicines, it promotes the research on the molecular mechanism of promoting copper death tumor treatment, integrates the concept of multi-modal treatment, and brings more hope of survival to tumor patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The synthetic route of the anti-tumor nanomaterial Pt@PCN-Cu based on promoting copper death of the present invention;

[0021] Figure 2 Transmission electron microscopy image of Pt@PCN-Cu;

[0022] Figure 3 is the X-ray diffraction pattern of Pt@PCN-Cu;

[0023] Figure 4 is the X-ray photoelectron spectrum of Pt@PCN-Cu;

[0024] Figure 5 This is the graph of copper ion content in PANC-1 cells after drug treatment;

[0025] Figure 6 The effect of Pt@PCN-Cu on the survival rate of PANC-1 cells;

[0026] Figure 7 This is the WB result of Pt@PCN-Cu on copper death-related markers of PANC-1 cells;

[0027] Among them, ①PBS, ②ES, ③CuCl2+ES, ④Pt@PCN-Cu;

[0028] Figure 8 The effect of Pt@PCN-Cu on LDH of PANC-1 cells;

[0029] Fig. 9 The effect of Pt@PCN-Cu on ATP in PANC-1 cells;

[0030] Fig.10 This is a flow chart of the pancreatic cancer tumor-bearing model;

[0031] Fig.11 This is a diagram of tumor size under different treatment conditions in a pancreatic cancer tumor-bearing model;

[0032] Fig.12 This is a graph showing the changes in tumor volume over time in a pancreatic cancer tumor-bearing model;

[0033] Fig.13 This is a HE staining image of pancreatic cancer. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with specific embodiments, but the embodiments are only used to illustrate the present invention and do not constitute any limitation to the protection scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

[0035] Tetracarboxyphenylporphyrin copper (TCPP-Cu), zirconium oxychloride octahydrate (ZrOCl2·8(H2O)), benzoic acid, N,N-dimethylformamide solution, polyvinyl pyrrolidone, methanol, chloroplatinic acid, acetone, chloroform, and hexane were purchased from China National Pharmaceutical Group Co., Ltd.

[0036] C57 / BL6 mice were obtained from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0037] Example 1 Preparation and characterization of anti-tumor nanomaterial Pt@PCN-Cu based on promoting copper death

[0038] according to Figure 1 The method shown in the figure is used to prepare Pt@PCN-Cu. The specific steps are as follows:

[0039] (1) In N,N-dimethylformamide solution (20 mL), tetracarboxyphenylporphyrin copper (22 mg), zirconium oxychloride octahydrate (60 mg) and benzoic acid (580 mg) were dissolved and magnetically stirred at 90 °C for 5 h at a speed of 300 rpm. After the solution was cooled to room temperature, PCN-Cu nanoparticles were collected by centrifugation (12000 rpm, 20 min) and washed with N,N-dimethylformamide solution several times. The obtained PCN-Cu was then redispersed in N,N-dimethylformamide solution for storage;

[0040] (2) Polyvinyl pyrrolidone (266 mg) was dissolved in methanol (90 mL), and then chloroplatinic acid (10 mL, 50 mM) was added to the solution. The mixture was refluxed in air for 3 h. The methanol was then removed using a rotary evaporator, and the Pt nanoparticles were precipitated by acetone for 3 h and collected by centrifugation (6000 rpm, 5 min). The obtained Pt nanoparticles were further purified with chloroform and hexane and then redispersed in N,N-dimethylformamide solution;

[0041] (3) The PCN-Cu nanoparticles prepared in step (1) were mixed with the Pt nanoparticles prepared in step (2), and the mixture was stirred (300 rpm) at 90°C for 5 h. Pt@PCN-Cu was then obtained by centrifugation and washing with N,N-dimethylformamide solution for multiple times. The obtained Pt@PCN-Cu was redispersed in N,N-dimethylformamide solution and stored for later use.

[0042] The formation of Pt@PCN-Cu was confirmed by transmission electron microscopy. Figure 2 As shown, Pt@PCN-Cu exhibits a uniform morphology with an average size of 200 nm. Figure 3 is the X-ray diffraction pattern of Pt@PCN-Cu. In addition, X-ray photoelectron spectroscopy analysis was performed to explore the valence state and composition of Pt@PCN-Cu. Figure 4 As shown, X-ray photoelectron spectroscopy shows that Pt@PCN-Cu is composed of Cu, C, N, Zr and Pt elements.

[0043] Example 2 Evaluation of the in vitro anti-tumor effect of Pt@PCN-Cu

[0044] PANC-1 cells (human pancreatic cancer cells) were cultured at 1×10 6The cells were seeded in 6-well plates at a density of 100 cells / well and cultured overnight. PANC-1 cells were then treated with PBS, Elesclomol (ES, 50 nM), CuCl2+ES (5 μM+50 nM) and Pt@PCN-Cu (20 nM), respectively. After incubation at 37 °C for 4 h, the cells were washed three times with PBS and acidified with nitric acid. The copper ion content of the cell lysate was determined by atomic absorption spectroscopy. The results are shown in Figure 2. Figure 5 As shown, compared with the PBS treatment group, the copper ion content in PANC-1 cells in the Pt@PCN-Cu treatment group was significantly increased.

[0045] In order to evaluate the in vitro anti-tumor effects of various drugs, MTT assay was performed on PANC-1 cells. The specific operation was as follows: PANC-1 cells were seeded in a 96-well plate at a cell density of 5000 cells / well and cultured for 24 hours. PANC-1 cells were incubated with ES, CuCl2 (5μM) + ES and Pt@PCN-Cu in the concentration range of 0.1, 0.25, 0.5, 1, 2.5, 10, 20, 50 to 100nM for 24 hours. Then, MTT (10μL) was added to the wells. After incubation for 4 hours, 10% SDS (100μL) was added to each well and incubated for another 12 hours. The absorbance was measured at 570nm. The results are as shown in the figure. Figure 6 As shown, the IC50 of Pt@PCN-Cu against PANC-1 cells was 10.18 nM.

[0046] PANC-1 cells were divided into four groups for Western Blot experiments: ①PBS, ②ES, ③CuCl2+ES, ④Pt@PCN-Cu. The specific operation was as follows: PANC-1 cells were 1×10 6 Cells were seeded in 6-well plates at a density of 100 cells / well and cultured overnight. PANC-1 cells were then treated with PBS, Elesclomol (ES, 50nM), CuCl2+ES (5μM+50nM) and Pt@PCN-Cu (20nM), respectively. After incubation at 37°C for 24h, the cell lysate was added to the culture dish to extract cell protein, and the protein concentration was determined using a BCA protein assay kit. 20μg of protein sample was added to each lane and separated by electrophoresis on a 12.5% ​​polyacrylamide gel and transferred to a PVDF membrane using a gel electrophoresis apparatus. After blocking with 5% skim milk for 1h, the PVDF membrane was incubated with DLAT, LIAS, FDX1 and β-actin antibodies at 4°C overnight. Subsequently, the PVDF membrane was washed three times and incubated with HRP-conjugated antibodies for 1h at room temperature. 200μL of ECL chemiluminescent reagent was added to the PVDF membrane, and Western Blot images were obtained using a gel imaging system. The results are shown in Figure 2. Figure 7As shown, the expression of DLAT, FDX1, and LIAS proteins decreased after cells were treated with Pt@PCN-Cu, suggesting that Pt@PCN-Cu may induce copper death in PANC-1 cells.

[0047] PANC-1 cells were seeded into 96-well plates (1×10 4 The PANC-1 cells were then incubated with PBS, Elesclomol (ES, 50 nM), CuCl2+ES (5 μM+50 nM), and Pt@PCN-Cu (20 nM) at 37 °C for 24 h. The concentrations of extracellular LDH and intracellular ATP in PANC-1 cells were determined by LDH and ATP detection kits, respectively, according to the instructions provided by the manufacturer. The results are shown in Figure 8 and Fig. 9 As shown, Pt@PCN-Cu can effectively induce PANC-1 cell membrane damage and significantly reduce the ATP content in PANC-1 cells.

[0048] Example 3 Evaluation of the anti-tumor effect of Pt@PCN-Cu in vivo

[0049] according to Fig.10 The KPC pancreatic cancer model was used as a model tumor model. KPC tumor cells (seeding density of 5×10 6 The KPC tumor cells were derived from ATCC, number CRL-2539, and were routinely raised until tumors (approximately 100 mm) were formed. 3 ) mice. The mice with tumors were divided into 4 groups and 200 μL of drugs (PBS, ES, CuCl2+ES or Pt@PCN-Cu) were administered through the tail vein. The drug concentration for each group of mice was 5 mg / kg: the first group (PBS); the second group (ES); the third group (CuCl2+ES); the fourth group (Pt@PCN-Cu); the changes in the subcutaneous tumor volume of the mice were observed and counted on days 3, 6, 9, 12 and 15 after administration. On the 15th day after administration, the mice were sampled and the tumors were dissected for analysis. Fig.11 As shown in Figure 2, the tumors in the Pt@PCN-Cu group were significantly smaller than those in the other groups. During the entire treatment period, the tumor volumes of each group changed over time. Fig.12 As shown, the tumor volumes of mice in the PBS group, ES group, and CuCl2+ES group continued to increase, while the tumor size of mice in the Pt@PCN-Cu group did not change significantly, indicating that Pt@PCN-Cu can effectively inhibit the growth of mouse tumors. Fig.13 This is the HE staining image of pancreatic cancer-bearing mice. The tumor cell apoptosis in the Pt@PCN-Cu group mice was significant, indicating that Pt@PCN-Cu has significant anti-tumor ability.

Claims

1. An anti-tumor nanomaterial based on promoting copper death, It is characterized in that The anti-tumor nanomaterial is constructed by taking PCN-Cu as a metal organic framework compound and incorporating Pt nanoparticles stabilized by polyvinyl pyrrolidone into PCN-Cu; wherein the PCN-Cu is obtained by dissolving tetracarboxyphenylporphyrin copper, zirconium oxychloride octahydrate and benzoic acid in an N,N-dimethylformamide solution, and then heating, stirring, cooling and centrifuging.

2. A method for preparing an anti-tumor nanomaterial based on promoting copper death according to claim 1, It is characterized in that The following steps are involved: (1) dissolving tetracarboxyphenylporphyrin copper, zirconium oxychloride octahydrate and benzoic acid in an N,N-dimethylformamide solution, and obtaining PCN-Cu nanoparticles by heating, stirring, cooling and centrifugation; (2) dissolving polyvinyl pyrrolidone in methanol, adding chloroplatinic acid thereto, refluxing the mixture with air, removing methanol, precipitating with acetone, and centrifuging to obtain Pt nanoparticles. The obtained Pt nanoparticles are further purified with chloroform and hexane, and redispersed in N,N-dimethylformamide solution; (3) The PCN-Cu nanoparticles prepared in step (1) are mixed with the Pt nanoparticles prepared in step (2), heated, stirred, cooled, and centrifuged. After washing multiple times with N,N-dimethylformamide solution, an anti-tumor nanomaterial based on promoting copper death is obtained, which is named Pt@PCN-Cu.

3. The preparation method according to claim 2, It is characterized in that The mass ratio of tetracarboxyphenylporphyrin copper, zirconium oxychloride octahydrate and benzoic acid described in step (1) is 1.1:3:29, the heating temperature is 90°C, the stirring speed is 300rpm, the time is 5h, the centrifugal speed is 12000rpm, and the centrifugal time is 20min.

4. The preparation method according to claim 2, It is characterized in that The amount of polyvinyl pyrrolidone used in step (2) is 266 mg, the amount of methanol used is 90 mL, the amount of 50 mM chloroplatinic acid used is 10 mL, the air reflux time is 3 h, the precipitation time is 3 h, the centrifugal speed is 6000 rpm, and the centrifugal time is 5 min.

5. The preparation method according to claim 2, It is characterized in that The heating temperature in step (3) is 90° C., the stirring speed is 300 rpm, the time is 5 h, the centrifugal speed is 12000 rpm, and the centrifugal time is 20 min.

6. Use of the anti-tumor nanomaterial based on promoting copper death as claimed in claim 1 in the preparation of drugs for improving and / or treating tumors.

7. The use according to claim 6, It is characterized in that The concentration of the anti-tumor nano material based on promoting copper death is 10-100 nM.

8. The use according to claim 7, It is characterized in that The concentration of the anti-tumor nano material based on promoting copper death is 10-50nM.

9. The use according to claim 8, It is characterized in that When the concentration of the anti-tumor nanomaterial based on promoting copper death is 10-25nM.

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