Preparation method and anti-tumor application of spherical nanomaterial with TME response based on copper death

Abstract

The application discloses a preparation method of a nanometer material based on a "sandwich" structure of copper death and anti-tumor application of the nanometer material. The nanometer material is prepared by taking hydroxypropyl methyl cellulose, chitosan and sodium alginate as raw materials, and adopting combination of elisamod and copper acetate to form elisamod-copper (ESCu) which is wrapped in the nanometer material, so as to obtain spherical nanometer material CHA@ESCu. Experiments prove that EScu can specifically combine with FDX1 in mitochondria, so as to transport copper ions to mitochondria of tumor cells, and induce copper (II) accumulation. FDX1 reduces Cu (II) to Cu (I) which is more toxic, DLAT further combines with Cu (I) to induce DLAT oligomerization, so that the insoluble DLAT increases, and protein stress induces tumor cell death. The CHA@ESCu raw material is low in price, simple in preparation process and excellent in anti-tumor effect, and provides a new treatment idea for tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the preparation of a copper death-based nanomaterial with TME response and its anti-tumor application. Background Technology

[0002] Breast cancer is the most common malignant tumor among women worldwide, and its prevention and treatment face a severe challenge. In China, the incidence of breast cancer is increasing at a rate of 3%-4% annually, with approximately 420,000 new cases and over 120,000 deaths in 2022, and a trend towards affecting younger women. Current clinical treatments for breast cancer mainly include surgical resection, radiotherapy, chemotherapy, and targeted / immunotherapy. Although early-stage breast cancer can achieve high cure rates with these methods, patients with advanced or metastatic breast cancer still face core challenges such as treatment resistance, high risk of recurrence, and low survival rates.

[0003] With the development of biomedicine, nanomaterials have become an important tool for cancer treatment. Based on the high permeability and retention effect (EPR effect) of tumor blood vessels, nanomaterials can passively target tumor sites. On the other hand, the modifiability of nanomaterials allows for the modification of their surfaces with antibodies, ligands, or peptides, enabling them to specifically recognize tumor cell surface markers and actively target tumor sites. Based on these two targeting effects, nanomaterials preferentially concentrate at tumor sites rather than normal tissues, thus significantly reducing side effects compared to traditional treatments. Clinical data shows that the concentration of nanomedicines at tumor sites can be 10-100 times higher than that of traditional drugs, thereby reducing the exposure of normal cells.

[0004] In 2022, Tsvetkov et al. discovered that elesclomol transports copper ions into tumor cells. Cu(II) is reduced to Cu(I) by FDX1 in tumor cells, leading to decreased Fe-S cluster protein synthesis. Cu(I) further binds to DLAT, causing DLAT oligomerization and an increase in insoluble DLAT. Ultimately, protein stress induces tumor cell death. This cell death mechanism was formally defined as copper death (Cuproptosis), and it has officially entered the public eye as a cell death mechanism. Copper death, due to its independent apoptosis pathway, can effectively circumvent drug resistance. However, current copper death-based nanomaterials suffer from complex preparation processes and high raw material costs. There is a need for a method to synthesize copper death-based nanomaterials with low-cost raw materials and a simple preparation process. Summary of the Invention

[0005] To overcome existing problems, this invention provides a simple method for synthesizing nanomaterials based on copper death and its application in anti-tumor therapy.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides an antitumor nanomaterial based on copper death. The antitumor nanomaterial is constructed by encapsulating ESCu, a substance formed by hydroxypropyl methylcellulose (HPMC), chitosan, and sodium alginate, using these biomolecules as raw materials. ESCu is obtained by ultrasonic-assisted dissolution of irismo and copper acetate in ethanol.

[0007] The present invention also provides a method for preparing the aforementioned antitumor nanomaterial based on promoting copper death, comprising the following steps: (1) Dissolve hydroxypropyl methylcellulose and chitosan in deionized water and heat and stir until the solution is clear and transparent; (2) Iliximab and copper acetate were dissolved in ethanol and dissolved with ultrasonic assistance until the solution was clear and transparent to obtain ESCu solution; (3) Add ESCu solution to the solution obtained in step one and heat and stir.

[0008] (4) Add sodium alginate to the solution in step three, heat and stir, then centrifuge and discard the supernatant. Collect the yellow solid, which is CHA@ESCu.

[0009] Preferably, in step (1), the mass ratio of hydroxypropyl methylcellulose to chitosan is 1:1, the heating temperature is 80°C, the stirring speed is 300 rpm, and the stirring time is 6 h.

[0010] Preferably, the mass ratio of irismo and copper acetate in step (2) is 1:1, and the ultrasonic time is 30 min.

[0011] Preferably, the heating temperature in step (3) is 80°C and the heating time is 12 h.

[0012] Preferably, the heating temperature in step (4) is 80°C, the stirring speed is 500 rpm, the stirring time is 12 h, the sodium alginate dosage is 20 mg, the centrifugation speed is 10000 rpm, and the centrifugation time is 10 min.

[0013] The present invention also provides the aforementioned copper death-based tumor treatment application.

[0014] Preferably, the concentration of the antitumor nanomaterial based on promoting copper death is 10–50 nM, more preferably 10–30 nM.

[0015] Compared with the prior art, the present invention has the following significant advantages.

[0016] This invention uses biomacromolecules as raw materials, which are inexpensive and widely available, overcoming the shortcomings of existing technologies where raw materials are difficult to obtain and expensive. A one-pot method is employed to prepare spherical nanomaterials, optimizing the complexity of the preparation process for copper death-based nanomaterials. In the microenvironment of tumor cells, the electrostatic interaction between the outer sodium alginate layer and chitosan weakens, releasing the internally encapsulated ESCu, inducing tumor cell death. This results in a copper death-based nanomaterial with a TME response. Attached Figure Description

[0017] Figure 1 Here is a high-resolution transmission electron microscope image of CHA@ESCu; Figure 2 HAADF-STEM spectra and corresponding elemental spectra of CHA@ESCu; Figure 3 X-ray photoelectron spectroscopy of CHA@ESCu; Figure 4 The IC50 value of CHA@ESCu was obtained by MTT assay. Figure 5 Western blot results of MCF-7 cells treated with elixomo (ES), CHA@ES, and CHA@ESCu for copper death-related markers; Figure 6 This is a grayscale analysis of proteins that are associated with copper death. Detailed Implementation

[0018] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] The present invention will be further described below through specific embodiments: Example 1: Preparation steps and characterization of CHA@ESCu, a spherical nanomaterial with TME response based on copper death.

[0020] This embodiment provides a spherical nanomaterial with TME response based on copper death, and its preparation method includes the following steps: Step 1: Accurately weigh 100 mg of hydroxypropyl methylcellulose and 100 mg of chitosan and dissolve them in deionized water. Stir at 300 rpm at 80°C until the solution is clear and transparent.

[0021] Step 2: Weigh 10 mL of ethanol for later use. Accurately weigh 20 mg of ilisimo and 20 mg of copper acetate, mix them, add them to the ethanol, and sonicate for 30 min to assist dissolution, thus preparing ESCu.

[0022] Step 3: Use a 10 mL syringe to draw up the ESCu solution and add it dropwise to the solution from Step 1. Stir at 80℃ and 300 rpm for 12 h, and set aside.

[0023] Step 4: Accurately weigh 20 mg of sodium alginate and add it to the solution from Step 3. Stir at 80°C for 12 h. Centrifuge at 10000 rpm for 10 min, discard the supernatant, and the resulting solid is CHA@ESCu.

[0024] The obtained solid was dispersed in deionized water, and its morphology was analyzed by transmission electron microscopy. The results are as follows: Figure 1 As shown, CHA@ESCu exhibits as uniformly sized and well-dispersed spherical nanomaterials with a diameter of approximately 250 nm. The loading of ESCu was confirmed by X-ray photoelectron spectroscopy (XPS), with results as follows... Figure 2 As shown, the XPS spectrum reveals peaks for Cu and S, indicating successful loading of ESCu.

[0025] Example 2: Analysis of CHA@ESCu ICs by MTT assay 50 value.

[0026] CHA@ESCu was added to cell culture medium at different concentrations (0.01, 0.1, 1, 10, 100, 1000 nM), 100 μL per well, with three replicates per group. A blank control group (cell-free, culture medium only) and a negative control group (drug concentration 0) were also included. Cells and the compound were incubated at 37°C, 5% CO2 for 24 h. After incubation, 20 μL of MTT solution was added to each well for an additional 4 h of incubation. After incubation, 150 μL of DMSO was added to each well, and the 96-well plate was shaken for 10 min. The absorbance (OD value) of each well was measured at 570 nm using a microplate reader, and cell viability was calculated using the following formula: Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of negative control group - OD value of blank group) * 100%. A CHA@ESCu dose-cell viability curve was plotted, and the IC50 value was calculated. The results are shown below. Figure 3 As shown, the IC50 value of CHA@ESCu is 35.60 nM.

[0027] Example 3 uses Western blot experiments to verify that CHA@ESCu can induce copper death.

[0028] MCF-7 cells were seeded in 6-well plates (1×10⁶ cells per well) and divided into 4 groups for Western blot analysis: (1) PBS; (2) ES; (3) CHA@ES; (4) CHA@ESCu (25 μL of drug was administered to each sample at a concentration of 50 nM). After incubation for 24 h, the cells were collected by centrifugation and lysed with 80 μL of pre-chilled RIPA cell lysis buffer (containing 1% 100 nM PMSF). Subsequently, the cells were collected by centrifugation at 12,000 rpm for 15 min at 4°C.

[0029] Protein concentration was determined using the BCA Protein Assay Kit (P0011, Beyotime). 20 μg protein samples were loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) plate and separated, then transferred to a PVDF membrane using a gel electrophoresis apparatus (Bio-Rad mini, USA). After blocking with 5% skim milk for 1 hour, the membrane was incubated overnight at 4°C with primary antibody. Subsequently, the PVDF membrane was washed three times, incubated with HRP (A0208, Beyotime)-labeled secondary antibody at room temperature for 2 hours, and finally washed three times with TBST. Results are shown below. Figure 4 , Figure 5 As shown, the expression of DLAT and FDX1 proteins in the copper death-apoptosis pathway was significantly decreased, indicating that CHA@ESCu can induce tumor cell apoptosis through copper death.

Claims

1. A copper death-based antitumor nanomaterial, characterized in that... The backbone material is a biological macromolecule with high biosafety. The "sandwich" structured spherical nanomaterials are synthesized by "one-pot" method using hydroxypropyl methylcellulose, chitosan and sodium alginate, and ESCu is encapsulated in them to obtain CHA@ESCu. The preparation process is simple.

2. The method for preparing nanomaterials as described in claim 1, characterized in that, Includes the following steps: (1) Dissolve hydroxypropyl methylcellulose and chitosan in deionized water and heat and stir until the solution is clear and transparent; (2) Iliximab and copper acetate were dissolved in ethanol and dissolved with ultrasonic assistance until the solution was clear and transparent to obtain ESCu solution; (3) Add ESCu solution to the solution obtained in step one and heat and stir.

3. (4) Add sodium alginate to the solution in step 3, heat and stir, then centrifuge and discard the supernatant. Collect the yellow solid, which is CHA@ESCu.

4. The preparation method according to claim 2, characterized in that, The mass ratio of hydroxypropyl methylcellulose, chitosan and sodium alginate in step (1) is 1:1, the heating temperature is 80℃, the stirring speed is 300 rpm, and the stirring time is 6 h.

5. The preparation method according to claim 2, characterized in that, In step (2), the mass ratio of ilismole to copper acetate is 1:1, and the ultrasonic time is 30 min.

6. The preparation method according to claim 2, characterized in that, In step (3), the heating temperature is 80℃ and the stirring time is 12 h.

7. The preparation method according to claim 2, characterized in that, In step (4), the heating temperature is 80℃, the stirring speed is 500 rpm, the stirring time is 12 h, the sodium alginate dosage is 20 mg, the centrifugation speed is 10000 rpm, and the centrifugation time is 10 min.

8. The application of the copper death-based TME-responsive nanomaterials as described in claim 1 in tumor therapy.

9. The application according to claim 6, characterized in that, The concentration at which the copper death-based TME-responsive nanomaterial exerts its antitumor effect is 10–50 nM.

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