Synthesis method and application of tumor microenvironment GSH / pH dual response type copper-based self-assembly nanoparticles

Abstract

The invention discloses a synthesis method and application of tumor microenvironment GSH / pH dual response type copper-based self-assembly nanoparticles, and belongs to the technical field of nano material preparation. The preparation method comprises the following steps: self-assembling copper ions, 3, 3 '-dithio-bis-propionyl hydrazide and a copper chaperonin inhibitor DCAC50, and carrying out surface modification by using F-127, so as to obtain the copper-based self-assembled nanoparticles. The material can respond to GSH / acidic pH of TME, released Cu < 2 + > is reduced into Cu under the action of high-concentration GSH, a large amount of active oxygen is generated through a Fenton-like reaction while copper death is triggered, and oxidative stress in cells is amplified; the released DCAC50 inhibitor can block Cu efflux mediated by copper chaperonin Atox1, and copper ion outflow is inhibited. Through double regulation and control of copper metabolism kinetics, tumor cell copper death is synergistically induced, oxidative stress is enhanced, and the efficient anti-tumor effect is achieved.

Description

Technical Field

[0001] This invention relates to the field of nanomaterial preparation technology, specifically to a method for synthesizing and applying GSH / pH dual-responsive copper-based self-assembled nanoparticles for tumor microenvironments. Background Technology

[0002] Cancer is one of the leading diseases threatening human health. According to a report by the International Agency for Research on Cancer, it is estimated that 20 million new cancer cases and more than 10 million cancer deaths will occur in the next 20 years. Chemotherapy is the main clinical treatment for cancer, but its effectiveness gradually decreases or even becomes ineffective due to drug resistance developed by cancer cells during treatment. Furthermore, the low targeting of drugs leads to poor treatment results and is accompanied by neurotoxic side effects. Surgery causes pain and carries the risk of metastasis. Radiotherapy can effectively control cancer to some extent, but patients are prone to developing drug resistance, which further affects its efficacy. Therefore, exploring new treatment modalities to compensate for the shortcomings of existing treatment methods has significant clinical research value and guiding significance.

[0003] Chemodynamic therapy (CDT) is an emerging cancer treatment strategy that utilizes Fenton or Fenton-like reactions to generate hydroxyl radicals (×OH) in the lesion area, which can kill cancer cells, thereby inducing apoptosis and necrosis. Compared with traditional chemotherapy, CDT's local activation properties reduce systemic toxicity (such as myelosuppression and hair loss); at the same time, its self-energizing mechanism requires no additional stimulation (such as light or ultrasound), simplifying the treatment process. Based on these advantages, CDT has become a research hotspot in precision oncology in recent years.

[0004] Copper death (Cuproptosis) is a newly discovered form of programmed cell death in recent years. It is caused by excessive accumulation of intracellular copper ions or dysregulation of copper metabolism, leading to cellular dysfunction and ultimately cell death. However, copper death can only be induced when the intracellular copper ion concentration reaches a certain level. Atox1, a copper chaperone protein, is a small metal chaperone protein ubiquitously found in the cytoplasm of eukaryotic cells. It can selectively bind reduced copper ions (Cu... + This process precisely delivers copper ions to the copper ion efflux pumps ATP7A / ATP7B, thereby preventing excessive accumulation of copper ions in the cytoplasm and maintaining intracellular copper homeostasis. However, this also leads to the failure of copper-mediated antitumor therapy. DC_AC50 is a small molecule inhibitor targeting the copper chaperone protein Atox1. It specifically binds to Atox1, blocking its interaction with Cu. + Combining, thereby inhibiting Cu + Excretion promotes the release of free Cu within tumor cells. +Large accumulations of DC_AC50 induce copper death in tumor cells and amplify oxidative stress. However, its poor water solubility, short half-life as a small molecule drug, and rapid metabolism in vivo limit its clinical application. Therefore, designing a nanodelivery system that can efficiently deliver drugs to cancer cells is of great significance.

[0005] Furthermore, compared to normal tissues, tumors develop a unique tumor microenvironment (TME) during their development. This TME is characterized by overexpression of glutathione (GSH), weak acidity (pH 6.5–6.8), oxygen deficiency (hypoxia), and high expression of hydrogen peroxide (H2O2). These characteristics not only promote tumor angiogenesis and metastasis but may also induce drug resistance, leading to treatment failure. Therefore, developing a nanomaterial that can specifically respond to the TME, efficiently induce copper death in tumor cells, and simultaneously amplify the intracellular oxidative stress cascade has significant research value and application potential. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to provide a copper-based self-assembled nanoparticle with dual GSH / pH response, which synergistically enhances the intracellular copper ion concentration in tumor cells through a "source-source and resource-saving" strategy. The copper-based nanoparticles respond to the GSH / acidic pH of the tumor microenvironment (TME), releasing Cu... 2+ Under high concentrations of GSH, the copper ion is reduced to Cu⁺, triggering copper death and generating a large amount of reactive oxygen species through a Fenton-like reaction, amplifying intracellular oxidative stress. Furthermore, the released DC_AC50 inhibitor blocks Atox1-mediated Cu⁺ efflux, inhibiting copper ion outflow. This strategy achieves highly effective anti-tumor effects by dually regulating copper metabolism kinetics, synergistically inducing copper death in tumor cells and enhancing oxidative stress. This system not only effectively overcomes the resistance problem of single-agent chemotherapy but also breaks through the key bottleneck of insufficient metal ion utilization in traditional CDT by improving copper ion delivery efficiency.

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: The first aspect of the present invention is a GSH / pH dual-responsive copper-based self-assembled nanoparticle for tumor microenvironment, wherein the copper-based self-assembled nanoparticle is composed of copper ions Cu 2+ It was obtained by self-assembly of 3,3'-dithiobispropionylhydrazide (TPH) and copper chaperone protein inhibitor DC_AC50, and surface modified with F-127.

[0008] In one alternative embodiment, the diameter of the copper-based self-assembled nanoparticles is 100-120 nm.

[0009] The second aspect of the present invention is a method for synthesizing GSH / pH dual-responsive copper-based self-assembled nanoparticles for tumor microenvironments as described in the first aspect, comprising the following steps: Step 1: Dissolve TPH in deionized water to obtain solution A; dissolve DC_AC50 in DMSO to obtain solution B; Step 2: Under vigorous stirring, add copper chloride aqueous solution and solution B to solution A in sequence, shake on a shaker, and the solution changes from light blue to dark green. After centrifugation and washing with deionized water, the solution is resuspended in anhydrous ethanol. Step 3: Dissolve F127 in anhydrous ethanol and mix it with the solution obtained in Step 2. Sonicate in an ice bath and stir, then rotary evaporate to obtain the copper-based self-assembled nanoparticles.

[0010] In one optional embodiment, the stirring speed in step 2 is 800~1200 rpm.

[0011] In one optional implementation, the shaking temperature in step 2 is 37°C, the speed is 100 rpm, and the time is 12 h.

[0012] In one optional implementation, the centrifugation speed in step 2 is 12,000 rpm and the time is 10 min.

[0013] In one alternative implementation, the mass ratio of the dark green product to F127 in step 3 is 1:3.

[0014] In one optional embodiment, the stirring temperature in step 3 is room temperature, the stirring speed is 400~600 rpm, and the stirring time is 2 h.

[0015] The third aspect of the present invention is the use of the tumor microenvironment GSH / pH dual-responsive copper-based self-assembled nanoparticles described in the first aspect in any one or more of the following: (1) Preparation of copper-based nanomedicine delivery system; (2) Preparation of drugs that induce copper death in tumor cells; (3) Preparation of anti-tumor drugs.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a method for synthesizing GSH / pH dual-responsive copper-based self-assembled nanoparticles for tumor microenvironments. This method utilizes copper ions (Cu... 2+ Cu-MOF nanoparticles were formed by self-assembly of 3,3'-dithiobispropionylhydrazine (TPH) and DC_AC50, and then surface-modified with F127. The resulting nanomaterials release Cu in response to the glutathione / acidic tumor microenvironment. 2+And the copper chaperone protein inhibitor DC_AC50, in which released Cu 2+ To "open source" increase the amount of Cu in tumor cells 2+ The content, under the action of GSH with high expression in TME, is reduced to Cu. + While triggering copper death, it generates a large amount of reactive oxygen species through a Fenton-like reaction, amplifying intracellular oxidative stress and further exerting its tumor-killing effect; DC_AC50, on the other hand, blocks Cu by inhibiting the copper chaperone protein Atox1. + By "reducing the outflow pathway," the concentration of intracellular copper ions can be effectively increased through a "reducing the outflow and increasing the inflow" strategy. This induces copper death in tumor cells while amplifying oxidative stress, thereby achieving a combined treatment strategy of chemotherapy and chemokinetics. Attached Figure Description

[0017] Figure 1 This is a transmission electron microscope image of the GSH / pH dual-responsive copper-based self-assembled nanoparticles for the tumor microenvironment synthesized in Example 1 of this invention.

[0018] Figure 2 This is a transmission electron microscopy (TEM) elemental distribution diagram of the GSH / pH dual-responsive copper-based self-assembled nanoparticles for the tumor microenvironment synthesized in Example 1 of this invention.

[0019] Figure 3 The Fourier transform infrared spectrum is shown for the GSH / pH dual-responsive copper-based self-assembled nanoparticles for tumor microenvironment synthesized in Example 1 of this invention.

[0020] Figure 4 The XPS spectra of the GSH / pH dual-responsive copper-based self-assembled nanoparticles for the tumor microenvironment synthesized in Example 1 of this invention are shown. Figure (a) shows the full spectrum of elements in the nanoparticles, and Figure (b) shows the X-ray photoelectron spectrum of Cu.

[0021] Figure 5 The Cu-based self-assembled nanoparticles of the tumor microenvironment, synthesized in Example 1 of this invention, exhibit Cu under different conditions. 2+ And the cumulative release curve of DC_AC50.

[0022] Figure 6 These are TEM images of the tumor microenvironment GSH / pH dual-responsive copper-based self-assembled nanoparticles synthesized in Example 1 of this invention, incubated under different conditions for 24 h.

[0023] Figure 7 This study investigates the generation of reactive oxygen species after co-incubation of the GSH / pH dual-responsive copper-based self-assembled nanoparticles for tumor microenvironment synthesized in Example 1 of this invention with hydrogen peroxide.

[0024] Figure 8This study investigates the GSH consumption of the GSH / pH dual-responsive copper-based self-assembled nanoparticles synthesized in Example 1 of this invention.

[0025] Figure 9 This study investigates the cytotoxicity of the GSH / pH dual-responsive copper-based self-assembled nanoparticles synthesized in Example 1 of this invention against CCK-8.

[0026] Figure 10 This study investigates the cell viability and mortality of the GSH / pH dual-responsive copper-based self-assembled nanoparticles synthesized in Example 1 of this invention.

[0027] Figure 11 The image shows the immunofluorescence of copper death-related proteins in the GSH / pH dual-responsive copper-based self-assembled nanoparticles synthesized in Example 1 of this invention. Figure (a) shows the immunofluorescence of the Fe-S cluster protein ferroredoxin (FDX1), and Figure (b) shows the immunofluorescence of dihydrolipoamide S-acetyltransferase (DLAT). Detailed Implementation

[0028] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] DC_AC50 was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China); Copper chloride dihydrate was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). F127 was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Example 1: Synthesis of CuTF-DC_AC50NPs Prepare a 0.4 M copper chloride aqueous solution for later use; Add 200 mg DC_AC50 to 0.6 mL DMSO and sonicate until completely dissolved for later use. Take 38.08 mg of TPH, add 4 mL of deionized water, and ultrasonically disperse in an ultrasonic disperser until completely dissolved to form a clear and transparent solution. Add 0.2 mL of pre-prepared copper chloride aqueous solution and DC_AC50 solution under stirring speed of 800~1200 rpm, and the solution turns light blue. Shake the above mixed solution in a shaker at 37℃ and 100 rpm for 12 h, and the solution changes from light blue to dark green. Wash three times by high-speed centrifugation in deionized water (speed of 12000 rpm) and resuspend in anhydrous ethanol. Dissolve F127 in anhydrous ethanol at a mass ratio of the above product:F127=1:3 and mix with the above solution. Ultrasonic bath on ice for 10 min and stir at room temperature at 400~600 rpm for 2 h. Rotary evaporation for 10 min yields copper-based self-assembled nanoparticles, labeled CuTF-DC_AC50 NPs. Resuspend the nanoparticles in deionized water and store at 4 ℃.

[0031] Example 2: Characterization of CuTF-DC_AC50NPs Figure 1 This is a transmission electron microscope (TEM) image of the GSH / pH dual-responsive copper-based self-assembled nanoparticles for the tumor microenvironment synthesized in Example 1 of this invention. The sample was dropped onto a 300-mesh copper grid covered with a carbon film, allowed to dry naturally, and then observed under a TEM. The image shows the morphology and size of the synthesized nanoparticles; they are uniformly distributed, with a diameter of 100–120 nm, and are spherical.

[0032] Figure 2 This is a transmission electron microscopy (TEM) elemental distribution image of the GSH / pH dual-responsive copper-based self-assembled nanoparticles for the tumor microenvironment synthesized in Example 1 of this invention. The sample was dropped onto a 300-mesh nickel grid covered with a carbon film, allowed to dry naturally, and then observed under a TEM. The image shows that the synthesized nanoparticles contain copper, sulfur, nitrogen, oxygen, and fluorine, indicating the successful synthesis of the copper-based self-assembled nanoparticles and the successful loading of DC_AC50.

[0033] Figure 3 This is the Fourier transform infrared (FTIR) spectrum of the GSH / pH dual-responsive copper-based self-assembled nanoparticles for the tumor microenvironment synthesized in Example 1 of this invention. CuT powder, DC_AC50 powder, CuT-DC_AC50 powder, and CuTF-DC_AC50 powder were mixed with potassium bromide and pressed into tablets. The FTIR spectra of the samples were acquired using a Vertex PerkinElmer 580 BIR spectrophotometer (Bruker). As shown in the figure, CuT-DC_AC50 exhibits the following response at 3308 cm⁻¹. -1The characteristic vibration at this location is attributed to the NH stretching vibration related to the amide bond; while its vibration at 1600 cm⁻¹ is... -1 The characteristic vibrations at this point are attributed to the stretching vibrations of C=C, and these phenomena confirm that the DC_AC50 has been successfully loaded. The sample at 2888 cm⁻¹... -1 The characteristic peak at the location is attributed to -CH stretching, proving the successful modification of F127.

[0034] Figure 4 This is the XPS spectrum of the GSH / pH dual-responsive copper-based self-assembled nanoparticles for the tumor microenvironment synthesized in Example 1 of this invention. Figure (a) shows the full spectrum of elements in the nanoparticles, and Figure (b) shows the X-ray photoelectron spectrum of Cu. As shown in Figure (a), the synthesized nanoparticles contain the elements Cu, C, S, N, and O; according to Figure (b), the main ionic state of copper in the nanoparticles is Cu. 2+ .

[0035] Example 3: Performance Study of CuTF-DC_AC50NPs Figure 5 The Cu-based self-assembled nanoparticles of the tumor microenvironment, synthesized in Example 1 of this invention, exhibit Cu under different conditions. 2+ The cumulative release curves of DC_AC50 were obtained. 10 mg of nanoparticles were placed in 10 mL of phosphate buffer under different conditions. At different time points, 2 mL of the release medium was collected by centrifugation, and the Cu content was determined by ICP-MS. 2+ The content of DC_AC50 in the nanoparticles was determined by steady-state transient fluorescence spectroscopy, and the same volume of phosphate buffer was added under the same conditions to ensure that the total volume of the release medium remained constant. As shown in Figure (a), after incubation at pH 6.5 and pH 7.4 + GSH for 24 h, the Cu content in the nanoparticles... 2+ Cu released 12% and 42% respectively under pH 6.5 + GSH conditions. 2+ The release rate reached 69%, while only 3% was released at pH 7.4. As shown in Figure (b), after incubation for 24 h at pH 6.5 and pH 7.4 + GSH, the nanoparticles released 23% and 54% of DC_AC50, respectively. At pH 6.5 + GSH, the DC_AC50 release reached 85%, while only 5% was released at pH 7.4. These results indicate that the nanoparticles exhibit pH and GSH-dependent Cu release. 2+ And the release behavior of DC_AC50.

[0036] Figure 6These are TEM images of the tumor microenvironment GSH / pH dual-responsive copper-based self-assembled nanoparticles synthesized in Example 1 of this invention, incubated under different conditions for 24 h. The nanoparticles were incubated with phosphate buffers under different conditions for 24 h, and the morphological changes of the samples were observed under an electron microscope. As shown in the figure, the morphology of the nanoparticles did not change significantly in PBS buffer at pH 7.4, but they were destroyed and decomposed in PBS buffer at pH 7.4 + GSH and pH 6.5. The structural destruction was more pronounced under pH 6.5 + GSH conditions, indicating that the nanoparticles can responsively degrade under glutathione and acidic conditions.

[0037] Figure 7 This study investigates the generation of reactive oxygen species (ROS) from the GSH / pH dual-responsive copper-based self-assembled nanoparticles synthesized in Example 1 of this invention after co-incubation with hydrogen peroxide. The ability of the nanoparticles to generate ·OH under glutathione and acidic conditions was evaluated using methylene blue (MB) indicator. As shown in the figure, after co-incubation with H₂O₂, the absorbance of MB at 664 nm decreased with increasing nanoparticle concentration, indicating that the nanoparticles can react with hydrogen peroxide to generate ·OH, and this reaction is enhanced with increasing concentration.

[0038] Figure 8 This study investigates the GSH consumption of GSH- and pH-responsive copper-based self-assembled nanoparticles synthesized in Example 1 of this invention. The ability of the nanoparticles to consume glutathione was detected using 5,5'-dithiomethyl (2-nitrobenzoic acid) (DTNB). As shown in the figure, the absorbance of DTNB at 412 nm decreased with increasing nanoparticle concentration, indicating a gradual decrease in GSH concentration and demonstrating the ability of the nanoparticles to consume GSH.

[0039] Example 4: In vitro antitumor activity of CuTF-DC_AC50NPs Figure 9 This study investigated the cytotoxicity of the GSH / pH dual-responsive copper-based self-assembled nanoparticles synthesized in Example 1 of this invention using CCK-8 assay. Different concentrations of nanoparticles were co-incubated with mouse colon cancer cells (CT26) under different conditions for 24 h, and the effect of the nanoparticles on cell viability was analyzed using the Cell Counting Kit-8 reagent. As shown in the figure, CT26 tumor cells exhibited the lowest cell viability and concentration-dependent cytotoxicity at pH 6.5 + GSH. The cytotoxicity was highest at a concentration of 200 μg / mL. -1 At that time, the nanoparticles caused the CT26 cell viability to decrease to 9.1%, indicating that the nanoparticles have a good cell-killing effect.

[0040] Figure 10This study investigated the cell viability and mortality staining of the GSH / pH dual-responsive copper-based self-assembled nanoparticles for the tumor microenvironment synthesized in Example 1 of this invention. Cells were treated under different grouping conditions, and viable and dead cells were observed using co-staining with calcein (AM, green fluorescence) and propidium iodide (PI, red fluorescence) to analyze the tumor-inhibiting effect of the nanoparticles. As shown in the figure, compared to the control group, slightly more red fluorescence was observed in the DC_AC50 and CuTF groups after cell treatment. However, a large number of dead cells (red fluorescence) were observed in the CuTF-DC_AC50 group after cell treatment, further demonstrating the excellent in vitro antitumor activity of CuTF-DC_AC50 NPs.

[0041] Figure 11 This is an immunofluorescence image of copper death-related proteins in the GSH / pH dual-responsive copper-based self-assembled nanoparticles synthesized in Example 1 of this invention. To verify that the nanoparticles induce copper death in tumor cells, cells were treated with different grouping conditions, and the expression of copper death-related proteins was observed by immunofluorescence. As shown in Figure (a), compared with the control group, DC_AC50 group, and CuTF group, the fluorescence intensity of Fe-S cluster protein ferroredoxin (FDX1) in CuTF-DC_AC50 group was significantly reduced; as shown in Figure (b), compared with the control group, DC_AC50 group, and CuTF group, the fluorescence intensity of dihydrolipoamide S-acetyltransferase (DLAT) in CuTF-DC_AC50 group was significantly enhanced, indicating that CuTF-DC_AC50 NPs can disrupt the stability of FDX1 and effectively induce DLAT oligomerization, ultimately leading to copper death in tumor cells.

[0042] The above description is merely a detailed explanation of preferred embodiments of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, several improvements and additions can be made without departing from the spirit of the present invention, and all such improvements and additions should be included within the protection scope of the present invention.

Claims

1. A tumor microenvironment GSH / pH dual-responsive copper-based self-assembled nanoparticle, characterized in that, The copper-based self-assembled nanoparticles are composed of copper ions Cu 2+ It was obtained by self-assembly of 3,3'-dithiobispropionylhydrazide (TPH) and copper chaperone protein inhibitor DC_AC50, and surface modified with F-127.

2. The tumor microenvironment GSH / pH dual-responsive copper-based self-assembled nanoparticles according to claim 1, characterized in that, The diameter of the copper-based self-assembled nanoparticles is 100~120 nm.

3. The method for synthesizing GSH / pH dual-responsive copper-based self-assembled nanoparticles for tumor microenvironment as described in claim 1, characterized in that, Includes the following steps: Step 1: Dissolve TPH in deionized water to obtain solution A; dissolve DC_AC50 in DMSO to obtain solution B; Step 2: Under vigorous stirring, add copper chloride aqueous solution and solution B to solution A in sequence, shake on a shaker, and the solution changes from light blue to dark green. After centrifugation and washing with deionized water, the solution is resuspended in anhydrous ethanol. Step 3: Dissolve F127 in anhydrous ethanol and mix it with the solution obtained in Step 2. Sonicate in an ice bath and stir, then rotary evaporate to obtain the copper-based self-assembled nanoparticles.

4. The method for synthesizing GSH / pH dual-responsive copper-based self-assembled nanoparticles for tumor microenvironment according to claim 1, characterized in that, The stirring speed in step 2 is 800~1200 rpm.

5. The method for synthesizing GSH / pH dual-responsive copper-based self-assembled nanoparticles for tumor microenvironment according to claim 1, characterized in that, In step 2, the shaking temperature is 37°C, the speed is 100 rpm, and the time is 12 h.

6. The method for synthesizing GSH / pH dual-responsive copper-based self-assembled nanoparticles for tumor microenvironment according to claim 1, characterized in that, In step 2, the centrifugation speed is 12000 rpm and the time is 10 min.

7. The method for synthesizing GSH / pH dual-responsive copper-based self-assembled nanoparticles for tumor microenvironment according to claim 1, characterized in that, In step 3, the mass ratio of the dark green product to F127 is 1:

3.

8. The method for synthesizing GSH / pH dual-responsive copper-based self-assembled nanoparticles for tumor microenvironment according to claim 1, characterized in that, In step 3, the stirring temperature is room temperature, the stirring speed is 400~600 rpm, and the stirring time is 2 hours.

9. The application of the tumor microenvironment GSH / pH dual-responsive copper-based self-assembled nanoparticles according to claim 1 in any one or more of the following: (1) Preparation of copper-based nanomedicine delivery system; (2) Preparation of drugs that induce copper death in tumor cells; (3) Preparation of anti-tumor drugs.

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