Metal nano-material, preparation method thereof and application of metal nano-material in tumor resistance

Abstract

The invention discloses a metal nano-material and a preparation method and application thereof in tumor resistance, and relates to the field of metal nano-materials, linoleic acid modified polydopamine coated nano-iron metal nano-materials are constructed by taking nano-iron as a core, coating a polydopamine layer and modifying linoleic acid, the preparation method is simple and convenient to operate, the condition is mild, the cost is low, and the metal nano-material is suitable for industrial production. The repeatability is good. And in the application of tumor treatment, excellent performance is shown. Due to the photo-thermal performance, local high temperature can be generated under irradiation of near-infrared light, tumor cells are directly killed, and drug release is promoted; the nano iron powder participates in an iron death process, and cooperates with linoleic acid to promote lipid peroxide accumulation and activate an iron or copper death pathway; linoleic acid can also improve the anti-tumor function of CD8 + T cells through metabolic reprogramming, and efficient cooperation of photothermal therapy, ferroptosis and immunotherapy is achieved. The material effectively overcomes the limitation of traditional single treatment, remarkably improves the treatment effect, reduces the side effects of the whole body, and provides an innovative solution for multi-mode combined treatment of tumors.

Description

Technical Field

[0001] The present invention relates to the field of metal nanomaterials, and particularly to a metal nanomaterial, a preparation method thereof, and an application thereof in anti-tumor treatment, and more particularly to a linoleic acid-modified polydopamine-coated nano-iron powder metal nanomaterial, a preparation method thereof, and an application thereof in anti-tumor treatment. Background Art

[0002] In recent years, the improvement of cancer prevention and treatment levels has increased the survival rate of patients, but treatment resistance and drug shortage are still the core problems. Although the combination of traditional therapies and immunotherapy has made progress, it has not changed the poor prognosis of solid tumors. The emerging PDT, PTT, PIT, CDT, and SDT therapies bring new hope, and the multi-modal combination therapy has significant effects. For example, the combination of chemotherapy, photothermal, and photodynamic therapies with metal-organic frameworks can achieve efficient tumor clearance with good biocompatibility, which promotes the design of simple and controllable multi-modal drug delivery systems to become a research hotspot.

[0003] Photothermal therapy uses near-infrared laser irradiation to generate local high temperature, kill tumor cells, and promote drug release. As a new generation of PTT reagent, PDA has the advantages of good biodegradability, no long-term toxicity, and high photothermal conversion efficiency. However, single PTT is limited by heat diffusion, with a narrow treatment range and high temperature prone to damage normal tissues, making it difficult to completely remove tumors.

[0004] Ferroptosis is a programmed cell death triggered by iron-dependent lipid peroxidation, which is of great significance for tumor suppression. The intracellular iron ion concentration, ROS level, and lipid peroxidation accumulation affect ferroptosis of tumor cells. However, due to tumor immune escape and cell drug resistance, the single ferroptosis treatment has poor effects. Iron-based nanomaterials such as Fe3O4 can enhance oxidative stress through the Fenton reaction, but they rely on an acidic microenvironment (pH < 5), and the burst release of free Fe 2+ will cause iron overload in normal tissues.

[0005] Linoleic acid (LA) has attracted much attention because it can induce ferroptosis. It can destroy tumor cell membranes by promoting the accumulation of lipid peroxides and activate the ferroptosis pathway. However, its clinical application faces two major problems: poor stability, the unsaturated double bonds are easily auto-oxidized under oxygen, light, and high temperature, producing toxic hydroperoxides, resulting in the inactivation of active ingredients and systemic inflammation; low delivery efficiency, strong hydrophobicity, easy to form aggregates in aqueous environments, hindering enrichment in tumor tissues, and may also cause vascular embolism. Currently, linoleic acid is mostly ingested through diet, which cannot fundamentally solve the application limitations. Researchers have explored the following improvement methods:

[0006] 1) Liposome encapsulation technology: According to the Chinese invention patent publication number CN115252779B, "An Oxidized Liposome for Efficiently Inducing Ferroptosis in Tumor Cells, Its Preparation Method, and Application," linoleic acid is first peroxidized and then encapsulated in liposomes. The liposome structure prevents oxidation during transport, and the surface cRGD peptide can target tumor cells. Through membrane fusion, lipid peroxides accumulate excessively on the tumor cell membrane, inducing ferroptosis.

[0007] 2) Nanoparticle co-loading system: For example, the Chinese invention patent application with publication number CN114288265A, "Nanoparticles containing cisplatin-linoleic acid and SN38-linoleic acid, preparation method and application," combines linoleic acid with cisplatin and SN38 and encapsulates them in nanoparticles, isolating the linoleic acid from the outside world and reducing the risk of oxidation;

[0008] 3) Chemical modification strategies: For example, Chinese invention patent application publication number CN111484501A, "Hydroxycamptothecin Linoleate Small Molecule Prodrug and Its Self-Assembled Nanoparticle Construction," proposes esterifying linoleic acid with 2,2'-dithiodiethanol under the catalysis of p-toluenesulfonic acid to change its chemical properties and reduce oxidation. For example, Chinese invention patent application publication number CN118388569A, "An Amphiphilic Dopamine Derivative, Targeted Nanocarrier, and Preparation Method Thereof," proposes using EDC·HCl and HOBt as catalysts to covalently link linoleic acid to dopamine (DA) molecules through an esterification reaction to form linoleic acid dopamine (DAO), significantly improving drug delivery stability. Furthermore, studies have shown that the binding of hydrophobic molecules (such as linoleic acid) to polydopamine (PDA) relies on Michael addition / Schiff base reactions and hydrogen bonding / electrostatic interactions mediated by bridging molecules (such as lysine) to achieve stable binding of PDA to linoleic acid.

[0009] The combined strategy of PTT therapy and ferroptosis induction shows unique advantages, but the strategy of combining linoleic acid and PTT still has several key issues that need to be overcome:

[0010] 1) Multimodal synergistic mechanism: How to integrate the hyperthermic effect of PTT, lipid peroxidation caused by ferroptosis, and the immune response to linoleic acid to form a "thermal-chemical-immune" synergistic network;

[0011] 2) Spatiotemporal controlled release: How to respond to the tumor microenvironment and synchronously regulate the release of photothermal agents, iron ions, and linoleic acid, especially to avoid the explosive release of free Fe2+;

[0012] 3) Targeted delivery system: Traditional methods rely on bridging molecules or catalysts, which increases the complexity of preparation. In addition, due to covalent or non-covalent bonding, the chemical structure or spatial conformation of linoleic acid is changed, affecting the reactivity of its double bond.

[0013] Based on this, the present invention aims to construct a multifunctional nanocarrier with both photothermal performance, iron ion sustained-release ability, and linoleic acid protection effect, breaking through the limitations of traditional methods, constructing a stable and biocompatible nanocarrier, and providing an innovative solution for multimodal combined tumor therapy. Summary of the Invention

[0014] In view of this, the purpose of the present invention is to provide a linoleic acid-modified polydopamine-coated nanoiron powder metal nanomaterial, its preparation method, and its application in anti-tumor.

[0015] The purpose of the present invention is achieved through the following technical solutions:

[0016] <First aspect>

[0017] The present invention provides a preparation method of a linoleic acid-modified polydopamine-coated nanoiron powder metal nanomaterial, including the following steps:

[0018] Prepare a pre-coated material with a nanometal coated with polydopamine (PDA), and the average particle size of the pre-coated material is not greater than 200 nm;

[0019] Disperse the pre-coated material in a linoleic acid solution, and perform ultrasonic treatment to disperse it evenly to obtain a mixed solution containing linoleic acid-modified nanoparticles. The dosage ratio of the pre-coated material to the linoleic acid solution is (0.05 - 1) mg : (1 - 2) mL, and the concentration of the linoleic acid solution is 100 - 1000 μM.

[0020] As an embodiment, the nanometal is nano-Fe or nano-Cu.

[0021] As an embodiment, the pre-coated material is Fe@PDA or Cu@PDA.

[0022] As an embodiment, the nanoparticles containing linoleic acid modification are nanoparticles with linoleic acid modified outside the pre-coated material.

[0023] In some embodiments, the nanoparticles are LA-Fe@PDA nanoparticles with linoleic acid modified outside Fe@PDA.

[0024] As an embodiment, the average particle size of the pre-coated material is not greater than 200 nm.

[0025] In some embodiments, the average particle size of the Fe@PDA material is not greater than 100 nm.

[0026] As an embodiment, the dosage ratio of the pre-coated material to the linoleic acid solution is (0.2 - 1) mg : 1 mL.

[0027] In some embodiments, the dosage ratio of the Fe@PDA material to the linoleic acid solution is (0.2 - 1) mg: 1 mL.

[0028] As an embodiment, the solvent of the linoleic acid solution is RPMI1640 medium or PBS buffer.

[0029] As an embodiment, the preparation method of the pre-coated material is as follows:

[0030] Add the nano-metal into the Tris-HCl buffer solution, and under the protection of nitrogen, ultrasonically treat the mixture for mixing to obtain a suspension containing the nano-metal;

[0031] Add dopamine hydrochloride into the suspension containing the nano-metal, and electrically stir to promote the self-assembly reaction between dopamine hydrochloride and the surface of the nano-metal to form a suspension containing the pre-coated material;

[0032] Perform solid-liquid separation on the suspension containing the pre-coated material, collect the precipitate and wash it, and freeze-dry the precipitate to obtain the dried pre-coated material;

[0033] When the nano-metal is nano-Fe, the obtained pre-coated material is Fe@PDA;

[0034] When the nano-metal is nano-Cu, the obtained pre-coated material is Cu@PDA.

[0035] As an embodiment, the mass ratio of the nano-metal to dopamine hydrochloride is 1: (1.8 - 2.2).

[0036] In some embodiments, the mass ratio of the nano-Fe to dopamine hydrochloride is 1:2.

[0037] As an embodiment, the particle size of the nano-metal is 30 - 70 nm.

[0038] As an embodiment, the average particle size of the nano-Fe is 40 - 60 nm.

[0039] In some embodiments, the average particle size of the nano-Fe is 50 nm.

[0040] As an embodiment, the preparation method of the pre-coated material is as follows:

[0041] Dissolve the metal salt and dopamine hydrochloride in the Tris-HCl buffer solution, electrically stir and mix for reaction, and after the reaction is completed, centrifuge and freeze-dry to obtain the dried pre-coated material;

[0042] When the metal salt is a soluble iron salt, the obtained pre-coated material is Fe@PDA;

[0043] When the metal salt is a soluble copper salt, the obtained pre-coated material is Cu@PDA.

[0044] In some embodiments, the soluble copper salt is copper sulfate pentahydrate.

[0045] As an embodiment, the mass ratio of the metal salt to dopamine hydrochloride is 1:(0.8–1.2).

[0046] In some embodiments, the mass ratio of the copper salt to dopamine hydrochloride is 1:1.

[0047] As an embodiment, the pre-coated material is spherical.

[0048] As an embodiment, the valence states of iron in the Fe@PDA material are divalent and zero valence.

[0049] As an embodiment, the valence states of Cu in the Cu@PDA material are monovalent and zero valence.

[0050] As an embodiment, the time of ultrasonic treatment is 15–30 min.

[0051] In some embodiments, the time of ultrasonic treatment is 20–25 min.

[0052] As an embodiment, the rotation speed of the electric stirring is 600–800 rpm.

[0053] In some embodiments, the rotation speed of the electric stirring is 800 rpm.

[0054] As an embodiment, the solid-liquid separation is carried out by centrifugation.

[0055] As an embodiment, the centrifugation speed is 10000–12000 rpm, and the centrifugation time is 10–15 min.

[0056] In some embodiments, the centrifugation speed is 12000 rpm, and the centrifugation time is 10 min.

[0057] As an embodiment, the precipitate is washed with ultrapure water.

[0058] As an embodiment, the parameters of freeze-drying are -40–-50 °C, 1–5 Pa, 36 h–72 h.

[0059] In some embodiments, the parameters of freeze-drying are -50 °C, 2 Pa, 48 h.

[0060] As an embodiment, the pre-coated material is stored in a brown bottle filled with nitrogen.

[0061] <Second aspect>

[0062] The present invention provides a metal nanomaterial, which is prepared by the above method.

[0063] <Third aspect>

[0064] The present invention provides an application of a metal nanomaterial in the preparation of an anti-tumor preparation.

[0065] Compared with the prior art, the present invention has the following beneficial effects:

[0066] 1) The present invention provides a metal nanomaterial, its preparation method and its application in anti-tumor, and prepares a multifunctional nanomaterial integrating photothermal therapy, ferroptosis effect and immunotherapy - linoleic acid modified polydopamine-coated iron nanoparticles (LA-Fe@PDA), and constructs an anti-tumor treatment system with the synergistic effect of photothermal therapy, ferroptosis and immunotherapy. Photothermal therapy triggers local high temperature through near-infrared light irradiation, which not only directly kills tumor cells, but also improves the tumor microenvironment, thereby promoting the infiltration and activity of immune cells, providing favorable conditions for ferroptosis. Through combined immunotherapy, the anti-tumor function of cytotoxic immune cells (CD8+ T cells) can be further activated, significantly enhancing the ferroptosis treatment effect. Multimodal synergistic therapy overcomes the limitations of single treatment modes, effectively improves the treatment effect and reduces systemic side effects.

[0067] 2) By forming a stable connection by coating linoleic acid outside the PDA-coated Fe nanoparticles, multiple advantages can be achieved. The nanoparticles can utilize the characteristics of PDA to extend the circulation time in vivo and improve the stability of linoleic acid; at the same time, with the help of the acidic microenvironment of tumor cells, the nanoparticles can specifically release iron ions to play a role, reducing damage to normal tissues; the Fe nanoparticles can not only act as carriers, but also participate in the ferroptosis process, and synergistically with linoleic acid to promote ferroptosis of tumor cells, further strengthening the anti-tumor effect.

[0068] 3) In the nanomaterial prepared by the present invention, the Fe nanoparticles exist in zero-valent and divalent states. Compared with traditional divalent or trivalent iron ion materials (such as Fe2O3, Fe3O4, FeOOH), zero-valent iron has higher stimulus-responsive characteristics in the tumor microenvironment. Zero-valent iron can release divalent iron ions on demand in an acidic microenvironment, inducing local Fenton reaction, thereby achieving a more efficient ferroptosis effect.

[0069] 4) In the nanomaterial prepared by the present invention, polydopamine not only endows the material with good biocompatibility and photothermal properties, but also protects the internal zero-valent iron through its reducibility to prevent its oxidation, and reduces a small amount of oxidized trivalent iron to divalent iron, thereby ensuring the continuous and on-demand release of iron ions, significantly enhancing the ferroptosis induction ability of the material.

[0070] 5) The present invention uses linoleic acid as an immune adjuvant, which not only enhances the anti-tumor effect of ferroptosis through peroxidation reaction, but also improves the mitochondrial function of CD8+ T cells through metabolic reprogramming, reduces their exhaustion and enhances the anti-tumor immune response, significantly improving not only the ferroptosis and photothermal therapy effects, but also greatly enhancing the anti-tumor immune response.

[0071] 6) The present invention provides a nano-material LA-Fe@PDA based on Fe@PDA. Its nano-size and surface properties enable it to be specifically taken up by 4T1 tumor cells and can be used as a drug carrier to accurately deliver chemotherapeutic drugs, targeted drugs, etc. to tumor tissues, which not only significantly increases the concentration of drugs at the tumor site, but also reduces the toxic and side effects on normal tissues. LA-Fe@PDA has photothermal properties and can achieve controlled release of drugs after irradiation with near-infrared light when it reaches the tumor site. It has high efficient ferroptosis induction ability, excellent photothermal conversion performance and significant immune activation characteristics, and can be used as a core material for multimodal combined treatment of tumors. This material integrates photothermal effect, high efficient iron ion release and stimulus-responsive characteristics to construct a highly efficient and controllable drug delivery system. Through the on-demand release of iron ions, the photothermal properties of polydopamine, and the characteristics of high hydrogen peroxide concentration and low pH in the tumor microenvironment, this material realizes the controllability and synergy of the treatment effect.

[0072] 7) Compared with the traditional complex drug delivery system design, the multimodal synergistic effect of LA-Fe@PDA shows extremely excellent effects in tumor treatment. Its preparation method is simple, the conditions are mild, and the repeatability is good. The prepared products have obvious anti-tumor effects, providing an innovative path for the clinical transformation of multimodal combined treatment. Description of the Drawings

[0073] Other features, purposes and advantages of the present invention will become more obvious by reading the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0074] Figure 1 It is the SEM morphology diagram of nano iron powder and the Fe@PDA nano-material prepared in Example 1 in the present invention;

[0075] Figure 2 It is the EDS element distribution diagram of nano iron powder and the Fe@PDA nano-material prepared in Example 1 in the present invention;

[0076] Figure 3 It is the XPS image of nano iron powder and the Fe@PDA nano-material prepared in Example 1 in the present invention. Among them, A is the full spectrum diagram of nano iron powder, B is the full spectrum diagram of Fe@PDA, C is the iron valence composition of Fe@PDA, and D is the iron valence composition of nano iron powder;

[0077] Figure 4 Zeta potential of nano iron powder and Fe@PDA nanomaterial prepared in Example 1 in the present invention;

[0078] Figure 5 XRD patterns of nano iron powder and Fe@PDA nanomaterial prepared in Example 1 in the present invention;

[0079] Figure 6 Fe@PDA nanomaterial prepared in Example 1, configured into solutions with different concentrations in ultrapure water, photo-thermal imaging in EP tubes (left figure), and temperature-time change curve of materials with different concentrations (right figure);

[0080] Figure 7 4T1 cell uptake of LA-Fe@PDA nanoparticles, where DAPI corresponds to nuclear fluorescence, RB-LA@PDA corresponds to fluorescence of the nanodrug, Lysosome corresponds to fluorescence of lysosome probe, and Merge corresponds to the superposition of the above three fluorescence images;

[0081] Figure 8 Cytotoxicity analysis of nanodrugs with different concentrations on 4T1 cells, with the vertical axis being the survival rate of 4T1 cells (n = 3);

[0082] Figure 9 Fluorescence images of ROS signals generated by treating 4T1 cells with different drug treatment groups, where NC is the negative control, PC is the positive control, Bright field is the bright field image, DCFH-DA corresponds to ROS distribution, and Merged corresponds to the superposition of the above two fluorescence images;

[0083] Figure 10 JC-1 staining of 4T1 cells in different drug treatment groups;

[0084] Figure 11 Graph of the change in relative tumor volume of tumor-bearing mice in different drug treatment groups over the treatment time (n = 3);

[0085] Figure 12 Tumor photos (left figure) and quantitative analysis of tumor weight (n = 5) (right figure) of tumor-bearing mice in different drug treatment groups;

[0086] Figure 13 Blood routine test of tumor-bearing mice in different drug treatment groups (n = 3);

[0087] Figure 14 H&E staining results of tumor tissues of tumor-bearing mice in different drug treatment groups;

[0088] Figure 15H&E staining results of major organs of tumor-bearing mice in different drug treatment groups;

[0089] Figure 16 TUNEL fluorescence staining (left figure) and quantitative analysis results (n = 3) (right figure) of tumor-bearing mice in different drug treatment groups;

[0090] Figure 17 Immunohistochemistry images (left figure) and quantitative analysis results (n = 3) (right figure) of tumor-bearing mice in different drug treatment groups. Detailed implementation manners

[0091] The present invention will be described in detail below with reference to the embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those of ordinary skill in the art can make several adjustments and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0092] For easy understanding, the abbreviations or nouns mentioned in the following text are first explained:

[0093] Nano iron powder: Purchased from Macklin, product number: I812029, average particle size 50 nm, purity 99.9%;

[0094] Culture medium: RPMI 1640;

[0095] 4T1 breast cancer cells: Mouse breast cancer cells, purchased from Shanghai Institute of Life Sciences, Chinese Academy of Sciences;

[0096] TUNEL kit: Purchased from Servicebio, Shanghai, China.

[0097] First, a preparation method of a nanomaterial with nano iron powder coated with PDA is introduced through Examples 1 to 4.

[0098] Example 1

[0099] A preparation method of a nanomaterial with nano iron coated with PDA includes the following steps:

[0100] S1. Add 100 mg of nano iron powder (Fe) to 50 mL of Tris-HCl buffer solution (concentration 10 mM, pH value 8.5), and ultrasonically treat the mixture for 25 min under nitrogen protection to obtain a mixed solution containing Fe;

[0101] S2. Add 50 mg of dopamine hydrochloride to the mixed solution containing Fe obtained in step S1, and perform electric stirring at room temperature. The rotation speed of the electric stirrer is 800 rpm, and stir for 6 h to promote the self-assembly reaction between dopamine hydrochloride and the Fe surface, forming an ordered complex Fe@PDA to obtain a mixed solution containing Fe@PDA;

[0102] S3. Centrifuge the mixed solution containing Fe@PDA obtained in step S2 to separate the solid from the liquid, collect the grayish-black precipitate, with a centrifuge speed of 12,000 rpm and a centrifuge time of 10 min, and wash the precipitate with ultrapure water repeatedly for 3 - 5 times to remove impurities to obtain a clean precipitate;

[0103] S4. Freeze-dry the clean precipitate obtained in step S3 at -50 °C and 2 Pa for 48 h to obtain dry Fe@PDA, and encapsulate it in a brown bottle filled with nitrogen for storage.

[0104] Example 2

[0105] A method for preparing a nanomaterial with nano-iron coated with PDA, the steps are basically the same as those in Example 1, and the differences are as follows:

[0106] In step S1, the dosage of nano-iron powder is 50 mg, and in step S2, the dosage of dopamine hydrochloride is 50 mg.

[0107] Example 3

[0108] A method for preparing a nanomaterial with nano-iron coated with PDA, the steps are basically the same as those in Example 1, and the differences are as follows:

[0109] In step S1, the dosage of nano-iron powder is 150 mg, and in step S2, the dosage of dopamine hydrochloride is 50 mg.

[0110] Example 4

[0111] A method for preparing a nanomaterial with nano-iron coated with PDA, the steps are basically the same as those in Example 1, and the differences are as follows:

[0112] In step S1, the dosage of nano-iron powder is 25 mg, and in step S2, the dosage of dopamine hydrochloride is 50 mg.

[0113] It should be noted that in addition to the purchased method, nano-iron powder can also be prepared by oneself. The steps for preparing nano-iron powder by oneself are as follows:

[0114] Dissolve (0.1 g of ferric chloride (FeCl3) in 100 mL of deionized water to obtain a precursor solution;

[0115] Under the stirring condition of 500 rpm, 10 mL of 0.1 M sodium borohydride (NaBH4) solution was added dropwise to the precursor solution, the dropping rate was controlled at 1 mL / min, and the reaction was carried out at room temperature for 30 min until the solution changed from yellow to black. During this period, 0.1 g of polyvinylpyrrolidone (PVP) or 0.05 g of citric acid was added as a stabilizer;

[0116] After the reaction, the solution was centrifuged at 10000 rpm for 10 min, the precipitate was washed 3 times with deionized water, and dried in a vacuum drying oven at 40 °C for 6 - 12 h.

[0117] The dried iron powder was stored in a sealed container filled with nitrogen.

[0118] Example 5

[0119] A preparation method of a nanomaterial with nano - copper coated with PDA. The Cu@PDA nanoparticles were synthesized by the pre - chelation polymerization method. The preparation steps were basically the same as those in Example 1 (Fe@PDA), except that:

[0120] Hydrated copper chloride was used instead of nano - iron powder as the metal precursor. 50 mg of hydrated copper chloride and 50 mg of dopamine hydrochloride were directly dissolved in 100 mL of Tris - HCl buffer solution (concentration: 10 mM, pH value: 8.5), centrifuged and freeze - dried to obtain the Cu@PDA nanomaterial.

[0121] Example 6

[0122] A preparation method of a nanomaterial with nano - copper coated with PDA. The preparation steps were basically the same as those in Example 5, except that:

[0123] The amount of hydrated copper chloride used was 100 mg, and the amount of dopamine hydrochloride used in step S2 was 50 mg.

[0124] Example 7

[0125] A preparation method of a nanomaterial with nano - copper coated with PDA. The preparation steps were basically the same as those in Example 5, except that:

[0126] The amount of hydrated copper chloride used was 25 mg, and the amount of dopamine hydrochloride used in step S2 was 50 mg.

[0127] Detection and analysis

[0128] (1) Relationship between the mass ratio of dopamine hydrochloride to iron nanoparticles or copper salt and the particle size of Fe@PDA nanomaterial

[0129] Dynamic light scattering (DLS) was used to measure the particle size at different mass ratios. As shown in Table 1, the particle size of the iron nanoparticles changes significantly with the mass ratio of dopamine hydrochloride to iron nanoparticles. When the mass ratio of dopamine hydrochloride to iron nanoparticles is 1:2, the average particle size of the iron nanoparticles is the smallest, approximately 80 nm. Conversely, when the mass ratio is 2:1, the particle size of the iron nanoparticles reaches its maximum, approximately 800 nm. A dopamine hydrochloride:iron ratio of 1:2 is most preferred.

[0130] This phenomenon may be closely related to the polymerization behavior of dopamine and its surface modification effect on iron nanoparticles. When the dopamine hydrochloride content is high (such as a 2:1 mass ratio), dopamine molecules may form a thicker polymer layer on the surface of the iron nanoparticles through chemical reactions, thereby increasing the adhesion of the material, causing aggregation between the iron nanoparticles and increasing the particle size. When the mass ratio of dopamine hydrochloride to iron nanoparticles is 1:2, the lower dopamine concentration may not form an overly thick polymer layer, thereby effectively avoiding the aggregation of particles and ultimately minimizing the particle size.

[0131] Further analysis showed that when the mass ratio of dopamine hydrochloride to iron nanoparticles was 1:3, the particle size increased to 400 nm. This may be because the concentration of dopamine was too low and failed to fully coat the surface of the iron nanoparticles, causing the particle aggregation phenomenon to occur again, but the degree of aggregation was slightly lower than that in the 2:1 case.

[0132] For Cu@PDA nanomaterials, when the mass ratio of dopamine hydrochloride to copper salt is 1:1, the resulting Cu@PDA nanoparticles exhibit the most uniform particle size distribution, with an average particle size of approximately 150 nm. As the mass ratio increases, the average particle size of the nanoparticles shows a clear increasing trend: when the mass ratio increases to 2:1, the average particle size increases to approximately 320 nm; and when the mass ratio decreases to 1:2, the particle size is approximately 200 nm. Based on the particle size distribution and stability test results, the optimal synthesis condition is a 1:1 mass ratio of dopamine hydrochloride to copper salt. This phenomenon may be related to the synergistic effect of the polymerization rate of dopamine under different ratios and the copper ion chelation efficiency.

[0133] In summary, the mass ratio of dopamine hydrochloride to iron nanoparticles (or copper salt) has a significant effect on the particle size distribution of Fe@PDA (@PDA) nanomaterials, and the optimal mass ratio is 1:2, which can effectively reduce the particle size of iron nanoparticles and avoid aggregation.

[0134] Table 1

[0135]

[0136] (2) Morphology and element distribution

[0137] The morphology, size and element content of the nano-iron powder and the Fe@PDA nanomaterial prepared in Example 1 were measured using a Carl Zeiss Gemini 300 cryo-high resolution scanning electron microscope (SEM). Figure 1 and Figure 2 shown. Figure 1 In the figure, the Fe@PDA nanomaterial is spherical with a size of about 80 nm. Compared with the morphology of nano-iron powder, it can be found that there is an irregular sticky coating layer on the surface of Fe@PDA, indicating the presence of surface PDA. Figure 2 Elemental analysis revealed that N elements accumulated on the surface of Fe@PDA, further proving the successful preparation of Fe@PDA.

[0138] It should be noted that in order to ensure the quality of SEM testing, nano-iron powder and Fe@PDA were dispersed on the conductive glue respectively, and then gold spraying was performed before SEM testing.

[0139] (3) Analysis of surface chemical properties

[0140] The nano-iron powder and the Fe@PDA nanomaterial obtained in Example 1 were pressed into powder, and the surface chemical properties of Fe and Fe@PDA were analyzed by X-ray photoelectron spectroscopy (AXIS UltraDLD, Shimadzu). Figure 3 As shown in the figure, through the comparison of N elements, it can be seen that PDA is successfully wrapped on the Fe nanoparticles. Further analysis such as peak separation processing shows that the valence state of iron is mainly divalent, with a small amount of zero valence.

[0141] (4) Electrochemical performance analysis

[0142] Nano iron powder and Fe@PDA nanomaterial obtained in Example 1 were dispersed evenly with ultrapure water to form different dispersion systems with concentrations of 1 to 100 mg / L. In the present invention, a concentration of 50 mg / L was used for testing and analysis. The Zeta potential of the different dispersion systems was measured on a nanoparticle size analyzer (Zetasizer Nano series). The results are shown in Figure 2. Figure 4 After coating with PDA, the surface potential of Fe nanoparticles shifted from positive to negative, demonstrating successful PDA coating of the Fe nanoparticles. Because PDA is a material with numerous functional groups, such as phenolic hydroxyl and amine groups, these groups readily dissociate in aqueous solution to form negative charges, significantly altering the surface potential of the Fe nanoparticles. This further demonstrates the successful preparation of Fe@PDA.

[0143] (5) Crystal structure analysis

[0144] The crystal structures of nano iron powder and the Fe@PDA nanomaterial obtained in Example 1 were analyzed by XRD using a Bruker multi-functional X-ray diffractometer, with a scanning rate of 0.5° / min -1 , a working voltage of 40 kV, a working current of 40 mA, and the diffraction patterns are as shown in Figure 5 . It can be seen that the diffraction peaks of Fe@PDA are very consistent with those of nano iron powder, and there are no diffraction peaks of other phases, indicating that the original Fe crystal structure was not damaged during the preparation process, nor was there an iron crystal phase transformation

[0145] (6) In vitro photothermal effect analysis

[0146] The Fe@PDA nanomaterial obtained in Example 1 was fully dispersed in ultrapure water, and a series of solutions with concentrations of 0 μg / ml (pure water), 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, and 400 μg / ml were prepared in sequence. NIR irradiation was carried out for 5 min, and the NIR irradiation conditions were a power density of 1 W·cm -2 , a wavelength of 808 nm, and an irradiation distance of 2 cm. The results are as shown in Figure 6 , indicating that the prepared Fe@PDA has an obvious temperature dependence and effective photothermal properties

[0147] In summary, Fe@PDA was systematically analyzed by characterization techniques such as XPS, SEM, XRD, and Zetasizer Nano, which proved the successful preparation of Fe@PDA. The XPS results showed that the material surface contains elements such as Fe, C, and N, and the valence states of iron are mainly divalent and zero-valent. The SEM images showed that the prepared Fe@PDA presented an irregular spherical structure on the surface, with a particle size of about 80 nm. XRD analysis further confirmed the crystal structure characteristics of the material, and Zetasizer Nano showed that the surface potential of Fe changed from positive to negative after being coated with PDA. This is because polydopamine (PDA) contains abundant functional groups such as phenolic hydroxyl (-OH) and amino (-NH2). These functional groups will ionize in the solution, carrying negative charges, thus masking the original positive charges of Fe nanoparticles and making the overall surface potential show a negative value

[0148] The temperature of Fe@PDA was monitored by 808 nm near-infrared light to evaluate its photothermal properties. The results showed that the photothermal effect of Fe@PDA exhibited a high concentration dependence (0 - 0.4 mg / mL). When the laser power density was 1 W·cm -2 , the temperature of 0.2 mg / mL Fe@PDA increased by 19.3 °C compared to room temperature within 5 min, while the water hardly increased, indicating that due to the coating of PDA, Fe@PDA exhibited excellent photothermal properties

[0149] Next, in this specific embodiment, a preparation method of a metal nanomaterial for anti-tumor is introduced through Examples 5 to 6.

[0150] Example 5

[0151] A preparation method of a metal nanomaterial for anti-tumor uses the dried Fe@PDA obtained in step S4 of Example 1 as a raw material, and includes the following steps:

[0152] S5. Disperse 0.2 mg of the dried Fe@PDA obtained in step S4 in 1 mL of linoleic acid (LA) solution, and ultrasonically treat it at room temperature for 20 min to obtain a linoleic acid-modified nanoparticle LA-Fe@PDA solution.

[0153] In this example, the concentration of the LA solution used is 100 μM, and the solvent is RPMI1640 medium.

[0154] Example 6

[0155] A preparation method of a metal nanomaterial for anti-tumor uses the dried Fe@PDA obtained in step S4 of Example 1 as a raw material, and includes the following steps:

[0156] S5. Disperse 1 mg of the dried Fe@PDA obtained in step S4 in 1 mL of linoleic acid (LA) solution, and ultrasonically treat it at room temperature for 20 min to obtain a linoleic acid-modified nanoparticle LA-Fe@PDA solution.

[0157] In this example, the concentration of the LA solution used is 1 mM, and the solvent is PBS buffer (pH 7.4).

[0158] Detection and analysis

[0159] The Fe@PDA used in (7) below refers to the Fe@PDA nanoparticles obtained in Example 1, and the LA-Fe@PDA in the detections of (8) to (10) refers to the LA-Fe@PDA solution obtained in Example 5; the solvents in the Fe@PDA solution, Fe solution and PDA solution are media;

[0160] The LA-Fe@PDA in the detections of (11) and (12) refers to the LA-Fe@PDA solution obtained in Example 6; the solvents in the Fe@PDA solution, Fe solution and PDA solution are PBS buffer.

[0161] (7) Cell uptake analysis

[0162] S71. Add 2 mg of Fe@PDA to 10 mL of ultrapure water, then add 5 μmol of Rhodamine B (RB). Sonicate under dark conditions at room temperature for 4 h, wash several times with ultrapure water, and then perform centrifugation at 12,000 rpm to obtain particles labeled with Rhodamine B, RB-Fe@PDA;

[0163] S72. Drop a small amount of medium into a 24-well plate. The medium used is RPMI-1640; add 500 μL of 4T1 cell dilution to each well. The cell concentration of the cell dilution is 5×10 4 cells / well. Then, culture the cells at 37 °C for 24 h, aspirate the medium in the well plate, and rinse the cells 4 times with PBS buffer;

[0164] S73. Using the medium as a solvent, prepare an RB-Fe@PDA solution with a concentration of 200 μg / mL, and add this solution to the well plate for an uptake experiment. Culture at 37 °C in an incubator. After culturing for 1 h, 2 h, and 4 h respectively, aspirate the mixture in the well plate, and rinse 3 times with PBS to ensure that the residual medium and drug are completely removed, terminating the uptake;

[0165] S74. Dilute the Lyso Tracker Green lysosome fluorescence probe (Lysosome) with the medium to a staining reagent with a concentration of 500 nM, and add 500 μL of the staining reagent to each well plate. After co-culturing at 37 °C for 30 min, aspirate the liquid in each well plate, and rinse the cells three times with PBS. Then fix the cells with a 4% (w / v) paraformaldehyde solution (prepared with PBS) for 15 min. After fixation, add 200 μL of DAPI working solution with a concentration of 10 μg / mL to each well, place it in the incubator and let it stand for 10 min, then aspirate the DAPI working solution, and rinse 3 times with PBS again. Finally, add 200 μL of fresh medium to each well to keep the cells moist. Among them, the DAPI working solution is: the initial concentration of DAPI is 5 mg / mL, restore the reagent to room temperature, use ultrapure water as the diluent, and dilute 500 times to obtain a 10 μg / mL DAPI working solution.

[0166] S75. Use an Olympus IX73 research-grade inverted fluorescence microscope to observe the endocytosis of RB-Fe@PDA by cells, take and record fluorescence images for further analysis of cell uptake behavior. The results are as Figure 7 . It can be seen that in the 4T1 cells cultured through a 4-h uptake experiment, the green fluorescence signals of RB-LA-Fe@PDA and the lysosome probe Lyso Tracker Green overlap well, proving that the prepared Fe@PDA can be effectively taken up by cancer cells, which is also the key for this metal nanoparticle to exert its anti-tumor effect.

[0167] (8) Cytotoxicity analysis

[0168] S81. Take the 4T1 cells in good growth state in step S72, add them to the culture medium to prepare a cell suspension, and then inoculate them into a 96-well plate. Add 100 μL of the cell suspension to each well, and the cell concentration is 10,000 cells / well. After culturing the cells at 37 °C for 24 h, discard the culture medium in the well plate and rinse the cells with PBS.

[0169] S82. Add 100 μL of the experimental treatment solution to each well in the 96-well plate. Then continue to culture the cells at 37 °C for 24 h, discard the drug treatment solution in the well plate, and rinse the cells with PBS. Among them, the addition of the experimental treatment solution adopts the method of a four-factor three-level orthogonal experiment. Set the LA-Fe@PDA solution, Fe@PDA solution, Fe solution, and PDA solution to three concentration gradients of 50 μg / mL, 100 μg / mL, and 200 μg / mL respectively, and allocate 3 replicate wells (n = 3) for each concentration gradient to verify the data reliability; and use RPMI 1640 culture medium as a blank control.

[0170] S83. Add 110 μL of CCK-8 working solution to each well, and incubate the cells added with the working solution in an incubator at 37 °C for 3 hours. Among them, the CCK-8 working solution is a mixed solution of 10 μL of CCK-8 reagent and 100 μL of culture medium.

[0171] S84. After the incubation in step S83 is completed, use a Bio-Tek microplate reader to read the absorbance data of the 96-well plate at an absorption wavelength of 450 nm to evaluate the toxicity of the nanodrugs to cells under different treatment conditions. The results are as Figure 8 shown. LA-Fe@PDA and Fe@PDA have obvious killing effects on tumor cells, and with the increase of the concentration, the killing effect of the material on tumor cells is enhanced. Within a certain concentration range, single Fe shows good biocompatibility. However, low-concentration Fe@PDA can show obvious killing effects on tumor cells, reflecting that the enhanced endocytosis may localize to the acidic environment of lysosomes, enhancing the accumulation of Fe ions and enhancing the killing effect. The stronger killing effect of LA-Fe@PDA also indicates the promoting effect of linoleic acid on ferroptosis.

[0172] (9) Analysis of ROS generation level

[0173] S91. Add 500 μL of different treatment solutions to each cell well plate rinsed in step S72 and culture them at 37 °C for 6 h as the experimental group, as shown in Table 2 specifically:

[0174] Table 2

[0175]

[0176]

[0177] A positive control group was set up. 500 μL of CCCP solution was added to the cell culture plate after rinsing in step S72, and cultured at 37 °C for 30 min. Before staining, the cell culture plate with CCCP solution as the treatment solution was further processed. The culture medium was aspirated, 500 μL of ROSup working solution was added, and the culture was continued at 37 °C for 30 min to induce the production of reactive oxygen species. Among them, the ROSup working solution was: the initial concentration of the stock solution was 50 mg / mL, and it was diluted with the culture medium solvent at a dilution factor of 1:1000 as the ROSup working solution.

[0178] S92. After the treatments of each experimental group and the positive control group were completed, the culture solution in the well plate was aspirated, and 100 μL of 2’,7’-dichlorofluorescein diacetate (DCFH-DA) reactive oxygen species fluorescent probe working solution with a concentration of 10 mM was added to each well for staining. After the samples were incubated in an incubator at 37 °C for 20 min under light-proof conditions, the cells were rinsed 3 times with PBS to remove the excess staining solution.

[0179] S93. The fluorescence intensity of each treatment group was observed through a confocal high-content imaging system (Molecular Devices, USA) to analyze the ROS generation level, and the experimental results were recorded by taking pictures. The results were as Figure 9 . After treating 4T1 cells with LA-Fe@PDA and Fe@PDA, obvious green fluorescence was produced. In addition, the strongest fluorescence signal was detected in NIR+LA-Fe@PDA, which reflected that photothermal promoted the release of Fe ions, and due to the participation of linoleic acid in lipid peroxidation reaction, ferroptosis was aggravated, and more ROS were produced.

[0180] (10) Mitochondrial damage analysis

[0181] S101. 500 μL of different experimental treatment solutions (as shown in Table 3) were added to each cell culture plate after rinsing in step S72, incubated in an incubator at 37 °C for 6 hours, then the culture medium was discarded, and the cells were rinsed once with PBS.

[0182] Table 3

[0183]

[0184] Among them, the CCCP working solution was a 10 mM CCCP stock solution diluted with the culture medium as the solvent at a ratio of 1:1000.

[0185] S102. Add 150 μL of JC-1 dye with a concentration of 10 μg / mL and 150 μL of DAPI dye working solution with a concentration of 10 μg / mL into each well to label mitochondria and nuclei respectively. Place the cells added with the dyes in an incubator and incubate at 37 °C for 30 minutes.

[0186] S103. After staining, rinse the cells once with PBS, then add 4% (w / v) formaldehyde solution to fix the cells, and let them stand at room temperature for 5 min.

[0187] S104. Observe the mitochondrial damage using an inverted fluorescence microscope, and record the fluorescence signals of JC-1-labeled mitochondria and DAPI-labeled nuclei. The results are as Figure 10 . It can be seen that Fe@PDA induced the transformation of J aggregates (red fluorescence, healthy mitochondria) to J monomers (green fluorescence, unhealthy depolarized mitochondria), indicating the loss of mitochondrial membrane potential during apoptosis; while the green fluorescence in the NIR+LA-Fe@PDA group was the most obvious, suggesting that the accumulation of iron ions in cells may produce a large amount of reactive oxygen species (ROS) through the Fenton reaction. These ROS can directly damage the mitochondrial membrane, leading to its depolarization, thus triggering cell death, which is highly consistent with the ROS results.

[0188] (11) Experiment on tumor-bearing mice

[0189] S111. Select healthy Balb / c female nude mice aged 6 - 8 weeks, and place them in a sterile environment for one week to adapt to the experimental conditions. Before the experiment, the mice are fasted for 6 h. Subsequently, using aseptic operation, collect the suspended cells from the 4T1 cells in the logarithmic growth phase cultured in step S72, wash them 3 times with PBS, and adjust the cell concentration to 1×10 7 cells / mL. Slowly subcutaneously inject 100 μL of 4T1 cell suspension (1×10 6 cells) into the right abdominal site (near the mammary gland) of the mice to establish a tumor-bearing mouse model.

[0190] Weigh the mice every two days and measure the tumor volume using a vernier caliper until the tumor size reaches about 80 mm 3 (with a diameter of about 5 mm), and then start drug administration.

[0191] S112. Randomly divide the tumor-bearing mice into five groups for drug intervention, as shown in Table 4.

[0192] Table 4

[0193] Figure 13 Middle marker Fe-LA@PDA Fe@PDA PDA Fe Control Drug Fe-LA@PDA Solution Fe@PDA Solution PDA Solution Fe Solution Blank Drug Dosage 5mg / kg 5mg / kg 5mg / kg 5mg / kg /

[0194] Peritumoral subcutaneous injection was adopted, and the drug was administered once every two days for a total of 14 days. NIR (808 nm, 1 W·cm -2 ) irradiation was performed for 3 min 4 h after each drug administration.

[0195] S113. Tumor volume measurement: The long diameter (L) and short diameter (W) of the tumor were measured using an electronic vernier caliper every two days, and the tumor volume (V) was calculated. The formula was: The results were as Figure 11 . It can be seen that over time, the tumor volumes of all groups increased. Among them, the tumors in the Control group grew the fastest, and the tumor volume reached a maximum of approximately 12 V / V% at 14 days. The tumor growth trends in the PDA group, Fe group, and Fe@PDA group were similar, and the tumor volumes at 14 days were approximately 9 V / V%, 7 V / V%, and 6 V / V% respectively. The tumors in the LA-Fe@PDA group grew the slowest, and the tumor volume was approximately 4 V / V% at 14 days. LA-Fe@PDA significantly improved the anti-tumor efficiency through the synergistic effect of photothermal-ferroptosis and targeted delivery design.

[0196] S114. After 14 days of drug administration intervention, blood was collected from the orbits of the mice. The collected blood was placed in an anticoagulation tube, and the blood was detected using a blood cell analyzer. The main indicators included: red blood cell count (RBC), white blood cell count (WBC), platelet count (PLT), mean corpuscular volume (MCV), mean corpuscular hemoglobin content (MCH), and mean corpuscular hemoglobin concentration (MCHC). The results were as Figure 13 . There were no significant differences in the detected indicators between the experimental group and the Control control group, indicating that Fe@PDA and LA-Fe@PDA had little impact on the physiological state of the mice.

[0197] (12) Tumor pathology analysis

[0198] First, sample collection was carried out. According to the ethical standards of experimental animals, all mice were humanely euthanized, and the main organs (heart, liver, spleen, lung, kidney) and tumor tissues were quickly collected after dissection.

[0199] S121. Tumor weight analysis

[0200] The collected tumors were arranged by group, photographed and recorded, and the tumor tissues of different treatments were weighed, and the tumor weight data were quantitatively analyzed to obtain Figure 12It can be seen that the tumor tissues in the blank control group were relatively large, indicating that tumor growth was more obvious without treatment; the tumor tissues in the PDA administration group and the Fe administration group were smaller than those in the blank control group, but the effect was not as obvious as that of other treatment groups. This indicates that the use of PDA or Fe alone has a certain inhibitory effect on tumor growth, but the effect is limited; the tumor tissues in the Fe@PDA group were significantly smaller than those in the first three groups, and the average tumor weight was 44% of that in the blank control group, indicating that the Fe@PDA administration treatment had a more significant inhibitory effect on tumors; the tumor tissues in the LA-Fe@PDA group were the smallest, and the average tumor weight was only 23% of that in the control group, indicating that the LA-Fe@PDA administration treatment had the most obvious inhibitory effect on tumors, and the introduction of linoleic acid LA further enhanced the tumor inhibition effect.

[0201] S122, Tissue Fixation and Embedding

[0202] The collected tumor tissue samples were immediately placed in a 4% (w / v) paraformaldehyde solution pre-cooled to 4°C for fixation for 24 h, and direct light was avoided at room temperature to ensure the integrity of the tissue structure. The fixed tissue samples were subjected to gradient dehydration treatment, and then transparentized with xylene to ensure that there was no residual water in the samples. The transparentized samples were immersed in molten paraffin to form paraffin blocks.

[0203] Among them, the gradient dehydration treatment was as follows: ethanol solutions with concentrations gradually increasing from 70% to 100%, and the gradient concentrations were set as 70% → 80% → 90% → 95% → 100% in sequence. The samples were soaked in each concentration for 120 min, and during the soaking process, the volume of ethanol was 5 - 10 times the volume of the tissue samples (in this invention, 8 times the volume of ethanol was used for soaking treatment).

[0204] S123, Tissue Sectioning

[0205] The paraffin-embedded tissues were cut into continuous sections with a thickness of 4 - 6 μm using a paraffin slicer, and the sections were flattened on glass slides.

[0206] S124, H&E Staining and Analysis

[0207] The sections were stained with hematoxylin and eosin in sequence, where: the nuclei were stained with hematoxylin for 5 min, and the excess stain was removed by rinsing with running water; then the sections were transferred to eosin stain and soaked for 2 min, and the excess stain was removed by rinsing with running water; then the sections were dehydrated (refer to the gradient dehydration treatment in step S122); finally, they were mounted and preserved for microscopic observation, as Figure 14 and Figure 15 .

[0208] Figure 14In this study, due to the synergistic anti-tumor effects of ferroptosis, photothermal therapy, and immunity, the cell morphology of both the Fe@PDA group and the LA-Fe@PDA group changed, with the nuclei becoming smaller. Further, larger nuclear fragmentation and karyolysis were observed in the tumors of mice treated with LA-Fe@PDA compared to those treated with other drugs.

[0209] Figure 15 In this study, H&E staining of the major organs showed that the negative impacts of these treatments on the health of these mice were negligible, which was consistent with the results of the in vivo biosafety study.

[0210] S125, TUNEL Staining and Analysis

[0211] Tumor sections were stained using a TUNEL kit (according to the kit instructions). After staining, the sections were imaged using a fluorescence microscope (model: Olympus), and the positive cells in each group were quantitatively analyzed using ImageJ software. As Figure 16 shown, histologically, compared with the blank control group, the Fe@PDA administration group and the LA-Fe@PDA administration group had a significant effect on promoting tumor cell apoptosis in 4T1 breast cancer tumors. Among them, the LA-Fe@PDA induced the most obvious apoptosis, with a 19.8% increase in TUNEL-positive cells.

[0212] S126, Immunohistochemical Staining and Analysis

[0213] The tumor tissue sections were successively subjected to antigen retrieval, blocking of endogenous peroxidase, and blocking treatment, and then primary antibody, secondary antibody, color development, counterstaining, dehydration, and mounting were added. The specific steps were as follows:

[0214] Antigen retrieval: After dewaxing and rehydrating the tissue sections, microwave antigen retrieval was used. Among them, the dewaxing and rehydrating process was as follows: The tissue sections were successively placed in environmentally friendly dewaxing solution I for 10 min, environmentally friendly dewaxing solution II for 10 min, and environmentally friendly dewaxing solution III for 10 min, then placed in absolute ethanol I for 5 min, absolute ethanol II for 5 min, and absolute ethanol III for 5 min, and finally rinsed with distilled water to complete the entire dewaxing and rehydrating process; Microwave antigen retrieval method: The sections were placed on a glass slide and then placed in a container containing citrate buffer (pH 6.0) antigen retrieval solution, and heated in a microwave oven at 92 - 98 °C for 10 min. During this process, the evaporation of the buffer should be prevented, and the slides should not be dried. Then, they were allowed to cool naturally, the glass slides were removed from the retrieval solution, placed in a container containing PBS buffer, and shaken on a decolorizing shaker for 3 times, 5 min each time, to remove the residual retrieval solution and impurities on the surface of the sections through washing.

[0215] Blocking endogenous peroxidase: Place the glass slides in 3% (w / w) hydrogen peroxide solution and incubate in the dark at room temperature for 25 min. Then place the glass slides in PBS (pH 7.4) and wash them on a shaker for 3 times, 5 min each time.

[0216] Blocking treatment: Use a histochemical pen to draw a circle around the tissue section on the glass slide to form a relatively enclosed area. Drop the blocking solution (3% (m / v) BSA) into the histochemical circle to evenly cover the tissue section and block at room temperature for 30 min. During this period, the proteins in the blocking solution will bind to the non-specific binding sites in the tissue section, blocking the possible non-specific binding of the subsequent primary antibody, thereby reducing the interference of background staining.

[0217] Three primary antibodies are involved in the primary antibody incubation, all purchased from Servicebio (Shanghai, China). PBS is selected as the diluent. ① Anti-iNOS antibody (mouse monoclonal anti-iNOS antibody, dilution ratio 1:1000); ② Anti-CD206 antibody (recombinant human CD206 protein, dilution ratio 1:1000); ③ Anti-CD8 antibody (mouse monoclonal anti-CD8 antibody, dilution ratio 1:800).

[0218] Adding primary antibody: Gently shake off the blocking solution on the glass slide, drop the diluted primary antibody on the section, and place the glass slide flat in a wet box and incubate overnight at 4°C.

[0219] Adding secondary antibody: Place the glass slides in PBS buffer and wash them on a shaker for 3 times, 5 min each time, to wash away the unbound primary antibody; after slightly drying the glass slides, drop the secondary antibody (HRP-labeled) corresponding to the primary antibody in the circle to cover the tissue and incubate at room temperature for 50 min.

[0220] DAB color development: Prepare the DAB color development solution according to the instructions of the DAB kit. Place the glass slides in PBS and wash them on a shaker for 3 times, 5 min each time; after slightly drying the section, drop the DAB color development solution in the circle and observe the color development degree under the microscope. The positive color is brownish yellow, and then rinse the section with running water to terminate the color development.

[0221] Counterstaining the cell nucleus: Counterstain with hematoxylin staining solution (HE staining kit) at room temperature for 3 min, and rinse with running water to remove the excess stain; then use 1 vol.% hydrochloric acid alcohol to differentiate for a few seconds and rinse with running water; finally, use hematoxylin blueing solution to blue and rinse with running water.

[0222] Dehydration and mounting: Immerse the sections in 75% alcohol for 5 min → 85% alcohol for 5 min → absolute ethanol I for 5 min → absolute ethanol II for 5 min → n-butanol for 5 min → xylene for 5 min in sequence for dehydration treatment. Take out the sections from xylene, let them dry slightly, and mount them with mounting medium.

[0223] The different sections were imaged using a fluorescence microscope, and the distribution and intensity of positive markers were recorded. The ImageJ software was used to quantitatively analyze the positive cells in each group. The results were as Figure 17 follows:

[0224] CD8: The anti-tumor immune cells (such as M1-type TAMs and CD8+ T cells) in the Fe@PDA group and the LA-Fe@PDA group were significantly increased, while the pro-tumor immune cells (such as M2-type TAMs) were significantly decreased. Notably, in the tumors treated with LA-Fe@PDA, the positive rate of CD8+ T cells reached 9.88 times that of the control group, showing a significant difference compared with the simple Fe@PDA group. This significant difference is closely related to the immunomodulatory effect of the immune adjuvant linoleic acid (LA);

[0225] CD206: Compared with the control group, the positive rates of CD206 in the PDA group and the Fe group were slightly lower than those in the control group, but the differences were not significant. The positive rates of CD206 in the LA-Fe@PDA group and the Fe@PDA group were further decreased, being 40% and 35% of the control group, respectively;

[0226] iNOS: For iNOS representing M1-type macrophages with anti-tumor effects, the positive rate of iNOS in the Fe@PDA group was significantly increased compared with the control group, and the positive rate of iNOS in the LA-Fe@PDA group was the highest, being significantly higher than all other groups.

[0227] It can be seen that peritumoral injection of LA-Fe@PDA can not only achieve the synergistic anti-tumor effect of ferroptosis and photothermal therapy to inhibit tumor growth, but also enhance the anti-tumor immune response by remodeling the tumor microenvironment (TME) and enhancing the CD8 killing effect, thus showing excellent therapeutic effects.

[0228] In summary, in order to analyze the anti-tumor effects of the prepared nanomaterials in the present invention, various experimental analyses were carried out, mainly including:

[0229] Through the cell uptake experiment, it was demonstrated that the nanoparticles could enter cells through endocytosis to play a role. Given that effective cell uptake is the key to the entire treatment process, Fe@PDA was labeled with rhodamine B and co-incubated with mouse breast cancer 4T1 cells to study cell uptake. Microscopic imaging showed that the green fluorescence signal of rhodamine B-labeled Fe@PDA overlapped well with the commercially available lysosome probe LysoTracker Green, indicating that Fe@PDA could be captured in lysosomes through endocytosis. The acidic environment in lysosomes promoted the biodegradation of Fe@PDA, releasing Fe 2+ .

[0230] The killing ability of the material against mouse breast cancer cells (4T1 cells) was detected by the Cell Counting Kit-8 (CCK-8) method, and the results showed that it had concentration-dependent cytotoxicity. The principle of CCK-8 for detecting cytotoxicity is based on the reduction of water-soluble tetrazolium salt (WST-8) by cell metabolic activities. WST-8 is catalyzed by dehydrogenase in living cells to generate formazan with yellow light absorption characteristics, and its absorbance (usually at 450 nm) is proportional to the number of living cells. By measuring the change in absorbance, the effect of the material on cell viability can be evaluated, thus indirectly reflecting its cytotoxicity. It can be seen from the experimental results that within a certain concentration range, Fe alone showed good biocompatibility. A higher concentration of Fe is required for Fe alone to achieve an ideal therapeutic effect, while a high concentration of Fe will have toxic side effects on healthy organs. In addition, this small molecule drug is easily metabolized and eliminated from the body. In contrast, Fe encapsulated with PDA showed good killing effects at lower concentrations. At the same incubation concentration, the survival rates of 4T1 cells incubated with 200 μg / ml LA-Fe@PDA and Fe@PDA were reduced to 46.7% and 58.92% respectively. Compared with Fe@PDA, LA-Fe@PDA had higher cytotoxicity to tumor cells. This is mainly because LA promoted the accumulation of lipid peroxide (LPO) through peroxidation reaction, enhancing the ferroptosis anti-tumor effect.

[0231] To deeply understand the anti-tumor mechanism through reactive oxygen species verification, DCFH-DA was used to detect the intracellular ROS level. DCFH-DA is a classic reactive oxygen species detection probe, and its principle is based on its lipophilic non-fluorescent state. It can enter cells by passive diffusion through the cell membrane and then be hydrolyzed by intracellular esterase to DCFH. Deacylated DCFH is a sensitive substrate for ROS and can be oxidized by intracellular ROS to generate DCF with green fluorescence. The fluorescence intensity of DCF is proportional to the level of ROS, so the accumulation of intracellular ROS can be quantitatively reflected by the fluorescence signal.

[0232] After treating 4T1 cells with LA-Fe@PDA and Fe@PDA, due to the Fenton reaction, a large amount of ROS was generated in the cells, manifested as an obvious increase in green fluorescence. In addition, the strongest fluorescence signal was detected in the NIR+LA-Fe@PDA group, indicating that the photothermal effect promoted the release of Fe ions, and linoleic acid participated in the lipid peroxidation reaction, exacerbating the ferroptosis process, thus generating more ROS. This result further verified the importance of the synergistic effect of photothermal effect and ferroptosis in the mechanism of ROS generation and cell death.

[0233] Through the mitochondrial membrane potential JC-1 staining experiment, it was observed that after treatment with LA-Fe@PDA, the mitochondrial membrane potential in cells decreased significantly, manifested as the transformation of JC-1 dye from mainly J aggregates (red fluorescence, representing healthy mitochondria) to mainly J monomers (green fluorescence, unhealthy depolarized mitochondria). JC-1 is a sensitive fluorescent probe for detecting mitochondrial membrane potential, and its principle is that the fluorescence signal changes according to different states of mitochondrial membrane potential: when the mitochondrial membrane potential is high, JC-1 aggregates in mitochondria to form J aggregates and emits red fluorescence; when the mitochondrial membrane potential decreases, JC-1 exists in monomer form and emits green fluorescence. The experimental results showed that NIR+LA-Fe@PDA induced the most obvious loss of mitochondrial membrane potential, suggesting that a large amount of reactive oxygen species (ROS) may be generated through the Fenton reaction due to the accumulation of iron ions in cells. These ROS can directly damage the mitochondrial membrane, leading to its depolarization, thus triggering cell death.

[0234] The excellent anti-tumor effect of the material in vivo was verified through animal experiments. According to the results of relative tumor volume, Fe@PDA and LA-Fe@PDA had obvious effects on inhibiting tumor growth. Further studying their biosafety, H&E staining of major organs showed that compared with the control group, the experimental group did not cause significant changes in the major organs of the heart, liver, spleen, lungs, and kidneys, indicating that the negative impacts on the health of these mice caused by these treatments were negligible, which was consistent with the results of in vitro biosafety studies.

[0235] To further analyze the therapeutic effect of LA-Fe@PDA, H&E and TUNEL staining analyses were performed on tumor tissues after different treatments. Due to the synergistic anti-tumor effects of ferroptosis, photothermal effect, and immunity, the cell morphology in both the Fe@PDA group and the LA-Fe@PDA group changed, and the cell nuclei became smaller. It was found that larger karyorrhexis and karyolysis appeared in the tumors of mice treated with LA-Fe@PDA than in the tumors of mice treated with other drugs. In the TUNEL staining results, compared with the control group, LA-Fe@PDA significantly inhibited cell proliferation and induced apoptosis in 4T1 breast cancer tumors (the number of TUNEL-positive cells increased by 19.8%), indicating that lipid peroxidation and excessive ROS caused by ferroptosis may trigger DNA damage and induce secondary apoptotic responses, thus increasing the positive rate of TUNEL staining.

[0236] Due to the immunomodulatory potential of Fe@PDA and LA-Fe@PDA, the infiltration of immune cells in tumor tissues treated with different treatments was observed by immunohistochemical staining. Immunohistochemical staining utilizes the principle of antigen-antibody reaction and combines a label (such as an enzyme or a fluorescent dye) to visualize the antigen. In the immunohistochemical staining experiment, after the tumor tissue sections were subjected to antigen retrieval and blocking treatments, the primary antibody was added for staining. In the blank control group, the expression of CD8 was low, slightly increased in the PDA administration group and the Fe administration group, while in the two administration groups of Fe@PDA and LA-Fe@PDA, the expression of CD8 increased significantly; for CD206, the expression in the blank control group was high, slightly decreased in the two administration groups of Fe and PDA, while the expression in the two administration groups of Fe@PDA and LA-Fe@PDA decreased significantly; for iNOS, the expression in the blank control group was high, decreased in the two administration groups of PDA and Fe, the expression in the Fe@PDA administration group increased significantly, the expression in the LA-Fe@PDA administration group was the highest, and the positive rate was significantly higher than that of all other administration groups, reaching the highest level. It shows that LA-Fe@PDA can not only synergistically promote the anti-tumor effects of ferroptosis and photothermal therapy and inhibit tumor growth, but also enhance the killing effect of CD8 + T cells by remodeling the tumor microenvironment (TME), thereby significantly improving the anti-tumor immune response and ultimately showing excellent therapeutic effects.

[0237] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A preparation method of a nano-metal nanomaterial coated with linoleic acid-modified polydopamine, characterized in that, It includes the following steps: Prepare a pre-coated material with a nano-metal coated with polydopamine (PDA). The average particle size of the pre-coated material is not more than 200 nm. The nano-metal is nano-iron (Fe) or nano-copper (Cu), and the pre-coated material is Fe@PDA or Cu@PDA; Disperse the pre-coated material in a linoleic acid solution, and perform ultrasonic treatment to disperse it evenly to obtain a mixed solution containing nanoparticles modified with linoleic acid. The dosage ratio of the pre-coated material to the linoleic acid solution is (0.05 - 1) mg:(1 - 2) mL, and the concentration of the linoleic acid solution is 100 - 1000 μM.

2. The preparation method according to claim 1, wherein The solvent of the linoleic acid solution is RPMI1640 medium or PBS buffer.

3. The preparation method according to claim 1, characterized in that The pre-coated material is spherical. The valence states of iron in the Fe@PDA material are divalent and zero-valent; the valence states of copper in the Cu@PDA material are zero-valent and monovalent.

4. The preparation method according to claim 1, characterized in that, It also includes one or more of the following technical features: A. The preparation method of the pre-coated material is: Add the nano-metal into Tris-HCl buffer solution, and perform ultrasonic treatment on the mixture under nitrogen protection for mixing to obtain a suspension containing the nano-metal; Add dopamine hydrochloride into the suspension containing the nano-metal, and perform electric stirring to promote the self-assembly reaction between dopamine hydrochloride and the surface of the nano-metal to form a suspension containing the pre-coated material; Perform solid-liquid separation on the suspension containing the pre-coated material, collect the precipitate and wash it, and perform freeze-drying on the precipitate to obtain the dry pre-coated material; When the nano-metal is Fe, the obtained pre-coated material is Fe@PDA; When the nano-metal is Cu, the obtained pre-coated material is Cu@PDA; B. The preparation method of the pre-coated material is: Dissolve the metal salt and dopamine hydrochloride in Tris-HCl buffer solution, perform electric stirring and mixing reaction, and after the reaction is completed, perform centrifugation and freeze-drying to obtain the dry pre-coated material; When the metal salt is a soluble iron salt, the obtained pre-coated material is Fe@PDA; When the metal salt is a soluble copper salt, the obtained pre-coated material is Cu@PDA.

5. The preparation method according to claim 4, wherein The mass ratio of the nano-metal to dopamine hydrochloride is 1:(1.8 - 2.2).

6. The preparation method according to claim 4, wherein The mass ratio of the metal salt to dopamine hydrochloride is 1:(0.8 - 1.2).

7. The preparation method according to claim 4, characterized in that, The time of the ultrasonic treatment is 15 - 30 min.

8. The preparation method according to claim 4, wherein The parameters of the freeze-drying are -45 - -50 °C, 1 - 5 Pa, 48 - 72 h.

9. A linoleic acid-modified polydopamine-coated nano-iron or copper metal nano-material is prepared by the method according to any one of claims 1 to 8.

10. Use of a linoleic acid-modified polydopamine-coated nano-iron or copper metal nano-material as described in claim 9 in the preparation of an anti-tumor preparation.

Images