CN113350522B - Platinum-copper alloy/chloride ion transporter composite tumor treatment preparation and preparation method and application thereof - Google Patents

Platinum-copper alloy/chloride ion transporter composite tumor treatment preparation and preparation method and application thereof Download PDF

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CN113350522B
CN113350522B CN202110557260.9A CN202110557260A CN113350522B CN 113350522 B CN113350522 B CN 113350522B CN 202110557260 A CN202110557260 A CN 202110557260A CN 113350522 B CN113350522 B CN 113350522B
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李翔
陈彤
傅译可
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ZJU Hangzhou Global Scientific and Technological Innovation Center
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Abstract

The invention relates to a platinum-copper alloy/chloride ion transporter composite tumor treatment preparation, and a preparation method and application thereof. The preparation has the advantages of good controllability, small side effect and the like, and is beneficial to realizing high-efficiency tumor treatment effect.

Description

Platinum-copper alloy/chloride ion transporter composite tumor treatment preparation and preparation method and application thereof
Technical Field
The invention relates to the technical field of tumor treatment preparations, in particular to a platinum-copper alloy/chloride ion transporter composite tumor treatment preparation as well as a preparation method and application thereof.
Background
Tumors are one of the most important killers threatening human health and life, and the current main methods for clinically treating the tumors comprise surgical methods, chemotherapy, radiotherapy and the like, but all have defects and disadvantages of different degrees.
Recently, our group has developed a new therapeutic approach, electrokinetic therapy, that is effective in treating cancer. The electrodynamic therapy utilizes electrocatalytic reaction of platinum nanoparticles under an electric field to induce active oxygen by triggering water molecule decomposition, and is used for treating cancer.
Compared with current active oxygen-based therapeutic methods, electrokinetic therapy has a series of advantages such as feasibility in remote control, dependence on hydrogen peroxide and oxygen, little side effects, and the like. According to previous studies, the mechanism of electrically triggering the generation of reactive oxygen species is as follows: in a neutral environment, both water molecules and chloride ions favor absorption at the platinum (111) surface. Under the action of an electric field, a Faraday cage effect appears on the platinum nanoparticles. The hydroxide bonds in the water molecules are broken only in the presence of chloride ions. This indicates that chloride ion is the main driving force for the electron dissociation of water on platinum nanoparticles. However, intracellular chloride concentrations are typically 5-40mM, much lower than extracellular chloride concentrations (-120 mM). In addition, autophagy is a process of cell self-renewal that contributes to the resistance of cells to various therapies. Autophagy is one of the obstacles in tumor therapy. In addition, the defense against intracellular antioxidants is also one of the obstacles in reactive oxygen-based therapeutic approaches. Glutathione is one of the antioxidants in the cell that can scavenge excess reactive oxygen species, thereby impairing all reactive oxygen-based therapies, including electrokinetic therapies. Thus, glutathione depletion is a widely adopted method for enhancing reactive oxygen-based therapy. Currently, there are several strategies for depleting glutathione in tumor cells. For example, inhibitors of γ -glutamylcysteine synthetase are used to inhibit upstream pathway synthesis of glutathione. Another strategy is that the oxidant converts the glutathione produced to glutathione disulfide by a redox reaction. For example, the oxidizing metal ions are consumed for glutathione by a redox reaction. However, the electrokinetic performance is greatly affected due to the surface characteristics of platinum nanoparticles. Therefore, adding a functional factor to the surface of the platinum nanoparticles, doping metal ions or compounding other metal oxides on the platinum nanoparticles may impair the effect of the electromotive force. In view of the above, there is still a lack of an electrodynamic therapeutic agent that can promote the production of active oxygen by increasing the chloride ion concentration and inhibit autophagy and glutathione consumption.
Disclosure of Invention
The embodiment of the application aims to provide a platinum-copper alloy/chloride ion transporter composite tumor treatment preparation, and a preparation method and application thereof, so as to solve the problems of low intracellular chloride ion concentration, less active oxygen generation, autophagy resistance and poor tumor treatment effect caused by antioxidant defense in the related technology.
According to the first aspect of the embodiment of the invention, the preparation method of the platinum-copper alloy/chloride ion transporter composite tumor treatment preparation is provided, and the chloride ion transporter is loaded on the surface of the platinum-copper alloy nanoparticle through electrostatic interaction.
Further, the platinum-copper alloy nanoparticles are prepared by a solvothermal method, and polyethylene glycol (PEG) is used as a stabilizer.
Further, the preparation method of the platinum-copper alloy nanoparticles comprises the following steps:
dissolving platinum acetylacetonate and copper acetylacetonate in oleylamine to obtain a solution 1;
dissolving Cetyl Trimethyl Ammonium Bromide (CTAB) in oleylamine to obtain a solution 2;
mixing the solution 2 and the solution 1, transferring the mixture to a reaction kettle, and putting the reaction kettle into an oven for reaction;
naturally cooling to room temperature after the reaction is finished, taking out, centrifugally separating a reaction product, washing with acetone, and then centrifuging to obtain a centrifugal product;
and adding polyethylene glycol (PEG) into the centrifugal product, stirring, and centrifuging to obtain the platinum-copper alloy nanoparticles.
Further, the concentration range of the acetylacetone platinum in the oleylamine is 1-6mg/mL, the concentration range of the acetylacetone copper in the oleylamine is 10-20mg/mL, and the concentration range of the CTAB in the oleylamine is 50-100 mg/mL.
Further, the volume ratio of the solution 2 to the solution 1 is (1-4):2, and the reaction conditions of the reaction kettle placed in the oven are 150-.
Further, the mass ratio of the centrifugation product to the polyethylene glycol PEG is (1-10): 1, stirring for 12-48 h.
Further, the preparation method of the chloride ion transporter comprises the following steps:
adding 3, 4-diethoxy-3-cyclobutene-1, 2-diketone and zinc trifluoromethanesulfonate into a toluene-tetrahydrofuran mixed solution for reaction;
after the reaction, adding 4-trifluoromethyl aniline, and magnetically stirring;
and (3) cooling to form a precipitate after magnetic stirring, filtering, washing and drying to obtain the chloride ion transporter.
Further, the concentration range of the 3, 4-diethoxy-3-cyclobutene-1, 2-dione in the mixed solution is 50-150mg/mL, the concentration range of the zinc trifluoromethanesulfonate in the mixed solution is 20-60mg/mL, the volume ratio of toluene to tetrahydrofuran in the toluene-tetrahydrofuran mixed solution is (15-25):1, and the volume ratio of the 4-trifluoromethylaniline to the toluene-tetrahydrofuran mixed solution is (0.05-0.1): 1.
further, the mass ratio of the chloride ion transporters to the platinum-copper alloy nanoparticles is (0.04-0.8): 1.
According to a second aspect of the embodiments of the present invention, there is provided a platinum-copper alloy/chloride ion transporter composite tumor therapeutic preparation prepared by the preparation method of the first aspect.
According to a third aspect of the embodiments of the present invention, there is provided an application of the platinum-copper alloy/chloride ion transporter composite tumor treatment preparation prepared by the preparation method of the first aspect in preparing a tumor treatment drug.
The technical scheme provided by the embodiment of the application can have the following beneficial effects:
the preparation provided by the embodiment of the invention is platinum-copper alloy nanoparticles (PtCu) 3 ) The preparation method adopts a solvothermal method, polyethylene glycol (PEG) is used as a stabilizer, and a Chloride Ion Transporter (CIT) is loaded. The preparation can catalyze the water in tissue fluid to decompose, generate active oxygen with cytotoxicity, respond to hydrogen peroxide in a tumor microenvironment to generate hydroxyl free radicals with cytotoxicity, and kill tumor cells. Furthermore, the preparation can consume Glutathione (GSH) to prevent active oxygen from being eliminated by tumor cells. In addition, the loaded chloride ion transporter can improve the content of intracellular chloride ions, so that the electrokinetic performance is improved, the lysosome function can be damaged, and the autophagy of cells is inhibited, so that the resistance of tumor cells to treatment is weakened. The preparation can realize more efficient tumor killing, and has important significance in tumor treatment.
In the invention, the enhanced electrokinetic therapeutic effect is realized by loading the chloride ion transporters on the surfaces of the platinum-copper alloy nanoparticles with uniform size and good dispersibility. To date, a platinum-copper alloy/chloride ion transporter complex tumor therapeutic formulation for synergistic electrokinetic therapy has not been developed in the art. The invention fills the blank, overcomes the existing defects of the electrodynamic force and enhances the treatment effect of the electrodynamic force.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and together with the description, serve to explain the principles of the application.
FIG. 1 is an electron microscope image of Pt-Cu alloy nanoparticles in an example of the present invention, in which (A) is a transmission electron microscope image, and (B) is a high-resolution transmission electron microscope image.
FIG. 2 is an energy spectrum of Pt-Cu alloy nanoparticles in an example of the present invention.
Fig. 3 is an XRD spectrum of pt-cu alloy nanoparticles in the example of the present invention.
FIG. 4 is a dynamic light scattering particle size analysis of platinum-copper alloy nanoparticles in an aqueous solution according to an embodiment of the present invention.
Fig. 5 is an uv-vis absorption spectrum of pt-cu alloy nanoparticles with 100mM hydrogen peroxide catalyzed oxidation of TMB in reaction buffer (pH 6) in an example of the invention.
FIG. 6 is a graph showing that DTNB is used as a capturing agent of a thiol group (-SH) in GSH to verify the glutathione consumption capacity of platinum-copper alloy nanoparticles in the example of the present invention.
FIG. 7 is a graph showing the effect of the platinum-copper alloy nanoparticles on the degradation of MB under the action of an electric field in the example of the present invention, wherein (A) is the MB degradation rate under different conditions (platinum-copper particles: 100. mu.g mL) -1 And outputting current: 10mA, [ MB ]]：2.5×10- 5 M) and (B) are degradation rate curves of the platinum-copper alloy nanoparticles with different concentrations under the action of an electric field, and (C) is a degradation rate curve of the platinum-copper alloy nanoparticles under different chloride ion concentrations.
FIG. 8 shows PtCu in an embodiment of the present invention 3 ，PtCu 3 -PEG and PtCu 3 Zeta potential of PEG @ CIT NP.
FIG. 9 shows PtCu in an embodiment of the invention 3 PtCu at different mass ratios of PEG to CIT 3 UV absorption Spectroscopy of PEG @ CIT
FIG. 10 shows PtCu in an embodiment of the present invention 3 Different mass ratios of PEG to CIT, CIT in PtCu 3 Loading and loading on PEG NPsEfficiency.
FIG. 11 shows the results of in vitro cell therapy in accordance with an embodiment of the present invention, wherein (A) is PtCu concentrations 3 Cytotoxicity of PEG nanoparticles on 4T1 cells and 7702 cells, (B) PtCu at different concentrations 3 Killing of 4T1 cells by PEG nanoparticles under the action of hydrogen peroxide and an electric field applied by hydrogen peroxide, (C) at different concentrations of PtCu 3 -PEG nanoparticles and PtCu 3 Killing condition of PEG @ CIT composite particles on 4T1 cells under the action of electric field, wherein (D) is PtCu with different concentrations 3 -killing of 4T1 cells by PEG @ CIT composite particles under the action of hydrogen peroxide and an electric field.
FIG. 12 is a graph depicting intracellular ROS production in cells following various treatments using DCFH-DA as a ROS probe in an example of the present invention.
FIG. 13 shows PtCu concentrations in different embodiments of the present invention 3 Clearance of PEG NP from GSH in 4T1 cells,
FIG. 14 is a cytofluorescence image of an embodiment of the present invention, in which (A) MQAE is used as a probe pair with PtCu 3 Monitoring intracellular chloride ion concentration after 0, 2, 4, 6 hours incubation of PEG @ CIT NP, (B) use of SBFI-AM as probe pair with PtCu in the present examples 3 Monitoring intracellular sodium ion concentration after 0, 2, 4, 6 hours incubation of PEG @ CIT NPs,
FIG. 15 is a Western blot of an example of the invention, in which (A) is control, energization only, PtCu 3 -PEG nanoparticles and PtCu 3 Western blot of four groups of samples energized with PEG nanoparticles, (B) quantitative ratios of LC3 II and p62 expression levels to β -actin, (C) control, addition of CIT, PtCu only 3 -PEG nanoparticles and PtCu 3 -western blot of PEG @ CIT composite particles, (D) quantitative ratio of LC3 II expression level and p62 expression level to β -actin.
FIG. 16 is a fluorescent image of cells pretreated with fluorescein-tetramethylrhodamine-labeled dextran and then incubated with different samples for 12 hours in an example of the invention.
Fig. 17 shows the results of Balb/C mouse 4T1 tumor model treatment according to the present invention, (A) the schematic flow chart of the actual operation of the method of the present invention in tumor treatment, (B) the average tumor volume growth curve of different treatment groups, (C) the tumor weight of different treatment groups after 14 days of treatment cycle, and (D) the tumor photographs of different treatment groups after 14 days of treatment cycle.
Detailed Description
Reference will now be made in detail to the exemplary embodiments, examples of which are illustrated in the accompanying drawings. The following description refers to the accompanying drawings in which the same numbers in different drawings represent the same or similar elements unless otherwise indicated. The implementations described in the following exemplary examples do not represent all implementations consistent with the present application. Rather, they are merely examples of apparatus and methods consistent with certain aspects of the present application, as detailed in the appended claims.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.
The invention is further described with reference to the following figures and specific examples.
Example 1:
the embodiment provides a preparation method of a platinum-copper alloy/chloride ion transporter composite tumor treatment preparation, and the method loads a chloride ion transporter on the surface of platinum-copper alloy nanoparticles through electrostatic interaction.
The platinum-copper alloy nanoparticles are prepared by a solvothermal method, and the polyethylene glycol (PEG) is used as a stabilizer, and specifically the preparation method of the platinum-copper alloy nanoparticles comprises the following steps:
dissolving 10mg of platinum acetylacetonate and 100mg of copper acetylacetonate in 10mL of oleylamine, and magnetically stirring for 2 hours to obtain a solution 1;
dissolving 500mg of cetyltrimethylammonium bromide (CTAB) in 10mL of oleylamine to obtain a solution 2;
mixing the solution 2 and the solution 1 according to the volume ratio of 1:2, transferring the mixture into a 50mL stainless steel reaction kettle, sealing, putting the reaction kettle into a 150 ℃ oven for reaction for 24 hours;
naturally cooling to room temperature after the reaction is finished, taking out, centrifugally separating a reaction product at 12000rpm, washing the reaction product for multiple times by using acetone, and then centrifuging to obtain a centrifugal product;
dissolving the centrifugal product and polyethylene glycol (PEG) in water according to the mass ratio of 1:1, stirring for 12h, and centrifuging to obtain the platinum-copper alloy nanoparticles.
In this example, the preparation method of the chloride ion transporter is as follows:
adding 3, 4-diethoxy-3-cyclobutene-1, 2-diketone and zinc trifluoromethanesulfonate into a toluene-tetrahydrofuran mixed solution for reaction;
after the reaction, adding 4-trifluoromethyl aniline, and magnetically stirring;
and (3) cooling to form a precipitate after magnetic stirring, filtering, washing and drying to obtain the chloride ion transporter.
Further, the concentration of the 3, 4-diethoxy-3-cyclobutene-1, 2-dione in the mixed solution is 50mg/mL, the concentration range of the zinc trifluoromethanesulfonate in the mixed solution is 20mg/mL, the volume ratio of toluene to tetrahydrofuran in the toluene-tetrahydrofuran mixed solution is 15:1, and the volume ratio of the 4-trifluoromethylaniline to the toluene-tetrahydrofuran mixed solution is 0.05: 1.
the mass ratio of the chloride ion transporter to the platinum-copper alloy nanoparticles is 0.04: 1.
Example 2:
the preparation method of the platinum-copper alloy nanoparticles comprises the following steps:
dissolving 60mg of platinum acetylacetonate and 200mg of copper acetylacetonate in 10mL of oleylamine, and magnetically stirring for 2 hours to obtain a solution 1;
dissolving 1000mg of cetyltrimethylammonium bromide (CTAB) in 10mL of oleylamine to obtain a solution 2;
mixing the solution 2 and the solution 1 according to a volume ratio of 2: 1, mixing, transferring the mixture to a 50mL stainless steel reaction kettle, sealing, putting the reaction kettle into a 200 ℃ oven for reaction for 72 hours;
naturally cooling to room temperature after the reaction is finished, taking out, centrifugally separating a reaction product at 12000rpm, washing the reaction product for multiple times by using acetone, and then centrifuging to obtain a centrifugal product;
dissolving the centrifugal product and polyethylene glycol PEG in water according to the mass ratio of 1:10, stirring for 48h, and centrifuging to obtain the platinum-copper alloy nanoparticles.
In this example, the preparation method of the chloride ion transporter is as follows:
adding 3, 4-diethoxy-3-cyclobutene-1, 2-dione and zinc trifluoromethanesulfonate into a toluene-tetrahydrofuran mixed solution for reaction;
after the reaction, adding 4-trifluoromethyl aniline, and magnetically stirring;
and (3) cooling to form a precipitate after magnetic stirring, filtering, washing and drying to obtain the chloride ion transporter.
Further, the concentration of the 3, 4-diethoxy-3-cyclobutene-1, 2-dione in the mixed solution is 150mg/mL, the concentration range of the zinc trifluoromethanesulfonate in the mixed solution is 60mg/mL, the volume ratio of toluene to tetrahydrofuran in the toluene-tetrahydrofuran mixed solution is 25:1, and the volume ratio of the 4-trifluoromethylaniline to the toluene-tetrahydrofuran mixed solution is 0.1: 1.
the mass ratio of the chloride ion transporter to the platinum-copper alloy nanoparticles is 0.8: 1.
Example 3:
the platinum-copper alloy nano particles are prepared by a solvothermal method, and PEG is used as a stabilizer
41.4mg of platinum acetylacetonate and 138.4mg of copper acetylacetonate were dissolved in 10mL of oleylamine (solution 1) and magnetically stirred for 2 hours. At the same time, 700mg CTAB was weighed out and dissolved in 10mL oleylamine (solution 2). Then, solution 2 was transferred to solution 1, and the mixed solution was transferred to a 50mL stainless steel reaction vessel and sealed. The reaction kettle is put into an oven at 170 ℃ for reaction for 48 hours. Naturally cooling to room temperature, taking out, centrifugally separating reaction products at 12000rpm, washing for 3 times by using acetone, and centrifuging to obtain the platinum-copper alloy nanoparticles.
FIG. 1 is an electron microscope image of Pt-Cu alloy nanoparticles in an example of the present invention, wherein (A) is a transmission electron microscope image, from which it can be seen that the nanoparticles have a relatively uniform size distribution. (B) The obtained spacing between crystal planes of the crystal lattice stripes is 0.21nm, 0.19nm and 0.13nm respectively.
FIG. 2 is a spectrum diagram of Pt-Cu alloy nanoparticles in an example of the present invention, which shows the existence of Pt and Cu elements.
FIG. 3 is an XRD pattern of Pt-Cu alloy nanoparticles in an example of the present invention, corresponding to the cubic Pt-Cu alloy of JCPDS card (JCPDS No. 035-1358).
FIG. 4 is a dynamic light scattering particle size analysis of platinum-copper alloy nanoparticles in an aqueous solution according to an embodiment of the present invention. The figure is a hydrodynamic radius plot before and after platinum copper alloy modification of PEG and after chloride transporter loading. The platinum particles synthesized by the method have good dispersibility in aqueous solution.
The platinum-copper alloy nano particles synthesized by the method have the advantages of uniform size, small particle size and high specific surface area, and are convenient for subsequent surface catalytic reaction. Has good stability in aqueous solution, and lays a foundation for treatment in tumor models.
Example 4:
the platinum-copper alloy nanoparticles are proved to have the capability of generating hydroxyl radicals in response to hydrogen peroxide by utilizing TMB (tetramethylbenzidine) color reaction. And (4) verifying the glutathione consumption capacity of the platinum-copper alloy nanoparticles by using DTNB. The degradation of methylene blue is utilized to prove the generation of active oxygen, and the influence of the concentration of the platinum-copper alloy and the concentration of chloride ions on the generation of the active oxygen is explored. The alternating electric field can effectively reduce the pH change of tissues around the tumor caused by electrode reaction and reduce the side effect of the electrode. Therefore, square wave alternating current is adopted when active oxygen is generated under the action of an exploration electric field, the output current is 10mA, and the frequency is 10 mHz.
Fig. 5 is an ultraviolet-visible absorption spectrum of pt-cu alloy nanoparticles catalyzed by TMB oxidation with 100mM hydrogen peroxide in reaction buffer (pH 6) according to an embodiment of the present invention, from which it can be seen that pt-cu alloy can generate hydroxyl radicals under acidic conditions and hydrogen peroxide.
FIG. 6 is a graph showing that DTNB is used as a capture agent for thiol groups (-SH) in GSH to verify the glutathione-consuming capacity of platinum-copper alloy nanoparticles in the example of the present invention.
FIG. 7 is a graph showing the effect of the Pt-Cu alloy nanoparticles on the degradation of MB under the action of an electric field in the example of the present invention, wherein (A) is the MB degradation rate under different conditions (Pt-Cu particles: 100. mu.g mL- 1 And outputting current: 10mA, [ MB ]]：2.5×10- 5 M), comparing three conditions of not performing any treatment, only applying an electric field without adding platinum nanoparticles and only adding platinum nanoparticles without an external electric field, the platinum nanoparticles can be proved to have the effect of degrading methylene blue under the external direct-current electric field. (B) The degradation rate curve of MB under the action of an electric field is shown in the specification. As the concentration of material increases, the degradation rate of MB increases. It was demonstrated that in the present invention, platinum-copper alloy nanoparticles are one of the key factors affecting the generation of active oxygen. (C) Is a curve of the rate of degradation of MB by platinum-copper alloy nanoparticles at different chloride ion concentrations. The degradation rate of MB increases with increasing chloride ion concentration, indicating that chloride ion concentration is also one of the key factors affecting active oxygen production.
Example 5:
the platinum-copper alloy nanoparticles are loaded with chloride ion transporters through electrostatic action, and the ratio of the platinum-copper alloy to the chloride ion transporters (CIT/PtCu) is changed 3 (w/w) ═ 0.8,0.4,0.2,0.08,0.04) to explore the optimum loading conditions.
Different amounts of CIT were dissolved in DMSO, diluted 1000-fold and added to 1mg/mL of PtCu 3 -PEG solution in water, stirring for 12h, then centrifuging, washing twice with water, removing excess CIT.
FIG. 8 shows PtCu in an embodiment of the present invention 3 ，PtCu 3 -PEG and PtCu 3 Zeta potential of PEG @ CIT NP, demonstrating that CIT can be electrostatically loaded to PtCu 3 The above.
FIG. 9 shows PtCu in an embodiment of the invention 3 PtCu at different mass ratios of PEG to CIT 3 UV absorption spectrum of PEG @ CIT, it can be seen that the UV curve absorbs at 360nm as the ratio of CIT to Pt-Cu alloy increasesThe peak intensity gradually increases and then does not increase again, indicating that the load of the CIT gradually increases and then reaches the maximum load.
FIG. 10 shows PtCu in an embodiment of the present invention 3 Different mass ratios of PEG and CIT, CIT at PtCu 3 Loading and loading efficiency on PEG NPs. As can be seen from the above, when CIT/PtCu 3 When the (w/w) is 0.2, the loading efficiency of the CIT is the maximum, and the loading condition is the optimal loading condition adopted by us.
Example 6:
the embodiment provides an application of the platinum-copper alloy/chloride ion transporter composite tumor treatment preparation prepared by the preparation method in preparation of tumor treatment medicines. The prepared platinum-copper alloy/chloride ion transporter composite tumor treatment preparation is dispersed in water to obtain a tumor treatment medicament or is directly used as the tumor treatment medicament.
Example 7:
FIG. 11 shows the results of in vitro cell therapy in accordance with one embodiment of the present invention, wherein (A) is PtCu at various concentrations 3 The cytotoxicity of PEG nanoparticles on 4T1 cells and 7702 cells shows that the platinum nanoparticles in the concentration range of 0-50 mu g/mL have no obvious cytotoxicity. (B) For different concentrations of PtCu 3 The killing condition of the PEG nanoparticles on the 4T1 cells under the action of the hydrogen peroxide and the hydrogen peroxide plus the electric field shows that the cells are killed more strongly under the double action of the electric field and the hydrogen peroxide. (C) At different concentrations of PtCu 3 -PEG nanoparticles and PtCu 3 The killing condition of the PEG @ CIT composite particles on 4T1 cells under the action of an electric field indicates that CIT has an enhancement effect on cell killing. (D) For different concentrations of PtCu 3 The killing condition of the PEG @ CIT composite particles on 4T1 cells under the action of hydrogen peroxide and an electric field can be found by comparison, and PtCu can be obtained under the same concentration of the platinum-copper alloy 3 The PEG @ CIT composite particles have the strongest killing effect on cells under the dual actions of hydrogen peroxide and an electric field, and the enhanced electrodynamic force treatment effect is realized.
FIG. 12 is a graph depicting intracellular ROS production in cells following various treatments using DCFH-DA as a ROS probe in an example of the present invention. From this it can be seen that PtCu 3 -PEGThe @ CIT composite particle has the most obvious green fluorescence in cells under the dual actions of hydrogen peroxide and an electric field.
Example 8:
FIG. 13 shows PtCu concentrations in different embodiments of the present invention 3 Clearance of PEG NP from GSH in 4T1 cells, with PtCu 3 Increase in PEG nanoparticle concentration, lower GSH content in the cells, indicating PtCu 3 PEG nanoparticles are key to the depletion of GSH.
FIG. 14 is a fluorescence diagram of cells in accordance with an embodiment of the present invention, in which (A) MQAE is used as a probe pair with PtCu 3 Intracellular chloride concentration was monitored after 0, 2, 4, 6 hours incubation with PEG @ CIT NP, and fluorescence was quenched by MQAE in the presence of chloride. As can be seen from the graph, the green fluorescence intensity decreased with increasing time, indicating an increase in intracellular chloride ion concentration, demonstrating the ability of CIT to increase intracellular chloride ion concentration. (B) To use SBFI-AM as a probe pair with PtCu 3 After the PEG @ CIT NPs are incubated for 0, 2, 4 and 6 hours, the intracellular sodium ion concentration is monitored, the SBFI-AM shows green fluorescence when meeting the sodium ions, and as can be seen from the figure, the green fluorescence intensity is increased along with the increase of time, which indicates that the intracellular sodium ion concentration is increased, and the CIT is proved to transfer chloride ions and simultaneously increase the intracellular sodium ion concentration.
FIG. 15 is a Western blot of a control, current-only, PtCu sample in an example of the invention 3 -PEG nanoparticles and PtCu 3 Western blot of four groups of samples energized with PEG nanoparticles, (B) quantitative ratios of LC3 II expression levels and p62 expression levels to β -actin, from which it can be seen that PtCu 3 The high expression of LC3 II protein and the low expression of p62 protein of PEG nanoparticle electrified group indicate that the active oxygen generated by electrokinetic force can induce the autophagy of tumor cells and help the tumor cells escape from treatment. (C) For comparison, only CIT and PtCu were added 3 -PEG nanoparticles and PtCu 3 Western blot of the PEG @ CIT composite particles, (D) is the quantitative ratio of LC3 II expression level and p62 expression level to beta-actin, from which it can be seen that the group LC3 II protein with CIT is highly expressed, but the p62 protein is also highly expressed, indicating that CIT can be inhibited from self-expressionPhagocytosis, and inhibition is in the late stage of autophagy.
FIG. 16 is a fluorescent image of cells pretreated with fluorescein-tetramethylrhodamine-labeled dextran and then incubated with different samples for 12 hours in an example of the invention. Wherein fluorescein is pH responsive and exhibits green fluorescence at high pH, while tetramethylrhodamine is pH independent. As can be seen, CIT and PtCu 3 Treatment with PEG @ CIT resulted in significant green fluorescence, indicating an increase in lysosomal pH. Therefore, it is suggested that CIT can increase the lysosomal pH, destroy the activity of lysosomal cathepsins in cells, thereby inhibiting the autophagy process, and effectively weaken the resistance of tumor cells to therapy.
Example 9:
taking a mouse breast cancer 4T1 subcutaneous solid tumor model as an example, 100 μ l of an aqueous solution of platinum nanoparticles (4mg/mL) was injected into a mouse body by intravenous injection, after 24 hours, square wave ac with a frequency of 10mHz was applied to the tumor region and inserted into the center of the tumor through an electrode, an electrode was applied in a manner of being applied in a skin-surrounding manner around the tumor, and the current was continuously applied for 10 minutes at a dose of 5mA of the system current, fig. 17 (a) is a schematic diagram of an actual operation flow when the method of the present invention is used for tumor treatment.
For the initial 4T1 subcutaneous model tumor size of 500mm 3 Left and right Balb/c mice were treated, and 35 mice were randomly divided into 7 groups of 5 mice each. Tumor size was measured every two days after treatment and according to the formula: tumor volume (length x width) 2 ) The/2 was calculated, and (B) in fig. 17 is the average tumor volume growth curve of the different treatment groups. It can be seen that the tumor treated by the platinum-copper alloy composite particles loaded with the chloride ion transporters through the square wave alternating current is basically unchanged, and the tumor can still continuously grow only after the group of the platinum-copper alloy nanoparticles is subjected to the action of the electric field, although the growth speed is not as fast as that of other groups. While the remaining groups of tumors grew rapidly. The platinum-copper alloy composite particles loaded with the chloride ion transporters have the best tumor treatment effect under the action of an electric field. FIG. 17 (C) shows the tumor weights of the different treatment groups after the 14-day treatment period, and FIG. 17 (D) shows the tumor weights of the different treatment groups after the 14-day treatment periodFrom the photographs of the tumors of the same treatment group, (C) and (D) in fig. 17, it can be seen that the tumor weight and volume of the composite particle electric field group are minimal, which also indicates that the composite particles under the square wave ac electric field indeed have excellent tumor treatment effect.
Other embodiments of the present application will be apparent to those skilled in the art from consideration of the specification and practice of the present disclosure. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the application being indicated by the following claims.
It will be understood that the present application is not limited to the precise arrangements that have been described above and shown in the drawings, and that various modifications and changes may be made without departing from the scope thereof. The scope of the application is limited only by the appended claims.

Claims (8)

1. A preparation method of a platinum-copper alloy/chloride ion transporter composite tumor treatment preparation is characterized in that a chloride ion transporter is loaded on the surface of platinum-copper alloy nanoparticles through electrostatic interaction, the platinum-copper alloy nanoparticles are prepared through a solvothermal method, polyethylene glycol (PEG) is used as a stabilizer, and the preparation method of the platinum-copper alloy nanoparticles comprises the following steps:
dissolving platinum acetylacetonate and copper acetylacetonate in oleylamine to obtain a solution 1;
dissolving Cetyl Trimethyl Ammonium Bromide (CTAB) in oleylamine to obtain a solution 2;
mixing the solution 2 and the solution 1, transferring the mixture to a reaction kettle, and putting the reaction kettle into an oven for reaction;
naturally cooling to room temperature after the reaction is finished, taking out, centrifugally separating a reaction product, washing with acetone, and then centrifuging to obtain a centrifugal product;
and adding polyethylene glycol (PEG) into the centrifugal product, stirring, and centrifuging to obtain the platinum-copper alloy nanoparticles.
2. The method for preparing the platinum-copper alloy/chloride ion transporter composite tumor therapy preparation according to claim 1, wherein the concentration range of the platinum acetylacetonate in the oleylamine is 1-6mg/mL, the concentration range of the copper acetylacetonate in the oleylamine is 10-20mg/mL, and the concentration range of the CTAB in the oleylamine is 50-100 mg/mL.
3. The method for preparing the platinum-copper alloy/chloride ion transporter composite tumor therapy preparation as claimed in claim 1, wherein the volume ratio of the solution 2 to the solution 1 is (1-4):2, the reaction conditions of the reaction kettle placed in an oven are 150 ℃ and 200 ℃ and 24-72 h; the mass ratio of the centrifugal product to the polyethylene glycol PEG is (1-10): 1, stirring for 12-48 h.
4. The preparation method of the platinum-copper alloy/chloride ion transporter composite tumor therapeutic preparation according to claim 1, wherein the preparation method of the chloride ion transporter is as follows:
adding 3, 4-diethoxy-3-cyclobutene-1, 2-diketone and zinc trifluoromethanesulfonate into a toluene-tetrahydrofuran mixed solution for reaction;
after the reaction, adding 4-trifluoromethyl aniline, and magnetically stirring;
and (3) cooling to form a precipitate after magnetic stirring, filtering, washing and drying to obtain the chloride ion transporter.
5. The method for preparing a platinumrecopper alloy/chloride ion transporter composite tumor therapeutic preparation according to claim 4, wherein the concentration range of the 3, 4-diethoxy-3-cyclobutene-1, 2-dione in the mixed solution is 50-150mg/mL, the concentration range of the zinc trifluoromethanesulfonate in the mixed solution is 20-60mg/mL, the volume ratio of toluene to tetrahydrofuran in the toluene-tetrahydrofuran mixed solution is (15-25):1, and the volume ratio of the 4-trifluoromethylaniline to the toluene-tetrahydrofuran mixed solution is (0.05-0.1): 1.
6. the method for preparing the platinum-copper alloy/chloride ion transporter composite tumor therapeutic preparation according to claim 1, wherein the mass ratio of the chloride ion transporter to the platinum-copper alloy nanoparticles is (0.04-0.8): 1.
7. The platinum-copper alloy/chloride ion transporter complex tumor therapeutic preparation prepared by the preparation method of any one of claims 1 to 6.
8. The use of the platinum-copper alloy/chloride ion transporter composite tumor treatment preparation prepared by the preparation method of claim 1 in the preparation of a tumor treatment medicament.
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