Nano material for targeted degradation of CXCR4 protein as well as preparation method and application of nano material

Abstract

The invention relates to the field of nano medicine, and particularly provides a nano material for targeted degradation of CXCR4 protein as well as a preparation method and application of the nano material. The nano material for targeted degradation of the CXCR4 protein comprises a nano sheet formed by a reaction of a manganese compound and polyphenol; and a binding oligopeptide which is connected to the surface of the nanosheet and targets CXCR4 protein. The nano material for targeted degradation of the CXCR4 protein provided by the invention can specifically recognize and combine the CXCR4 protein highly expressed on the surface of a tumor cell by utilizing the targeted oligopeptide connected to the surface of the nano material, and induce the CXCR4 protein to enter lysosome to be degraded, so that a CXCR4-mediated tumor promoting signal channel is blocked, and a tumor immune microenvironment is regulated and controlled; meanwhile, Mn < 2 + > released in an acid lysosome environment can effectively activate a cGAS-STING immune pathway, the anti-tumor immune response is enhanced, and dual synergistic treatment of target protein degradation and immune activation is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanomedicine, and specifically provides a kind of nanometer material for targeted degradation CXCR4 protein and its preparation method and application. BACKGROUND

[0002] The progression of tumor depends not only on the characteristics of malignant cells themselves, but also on the significant influence of tumor microenvironment. Although immunotherapy has changed the pattern of cancer treatment in the past decade, its overall efficacy is still limited. Studies have shown that at the early stage of tumorigenesis, macrophages are recruited to the tumor microenvironment under the action of chemokines, promoting the epithelial-mesenchymal transition and invasiveness of tumor cells, and inducing regulatory T cell (Treg) responses, thereby inhibiting adaptive anti-tumor immunity. At the same time, the physical barrier of tumor formation hinders T cell infiltration, further weakening the CD8 + T cell immune surveillance promotes tumor immune escape.

[0003] The chemokine network plays a key role in the regulation of tumor immunity, among which the CXCR4 signaling axis is particularly important. CXCR4 is highly expressed in Treg, myeloid-derived suppressor cells (MDSC) and various tumor cells infiltrated by tumors, which can mediate the recruitment of immunosuppressive cells to the tumor microenvironment and inhibit anti-tumor immune responses. At the same time, CXCR4 promotes the proliferation, migration and invasion of tumor cells by activating downstream pathways such as PI3K / AKT and MAPK, and is closely related to the poor prognosis of various solid tumors. Therefore, CXCR4 has become an important research target for the regulation and targeted therapy of tumor microenvironment, but its clinical translation is still restricted by factors such as normal tissue expression, drug resistance and tumor heterogeneity.

[0004] Currently, tumor immunotherapy mainly includes immune checkpoint inhibitors, adoptive cell transplantation and cancer vaccines. In recent years, with the advancement of metal immunology research, it has been found that a variety of transition metal ions have immunomodulatory effects, among which Mn 2+ has been confirmed to be used as an immune stimulator for tumor immunotherapy. Related studies have shown that Mn 2+ can activate the cGAS-STING signaling pathway and enhance the efficiency of cGAMP-mediated STING activation, thereby inducing the production of type I interferons. Based on the above mechanism, Mn-based nanomaterials have been used for tumor immunotherapy. However, free Mn 2+ in the prior art has problems such as high toxicity, low targeted delivery efficiency, narrow therapeutic dose window and fast in vivo metabolism, which leads to unstable efficacy and limits its further clinical application.

[0005] In view of the above technical problems, it is necessary to develop a multifunctional drug that has both Mn 2+ coordination ability and high-efficiency degradation ability of CXCR4 protein, so as to enhance the immune stimulation effect of Mn2+ To enhance the efficacy of Mn-mediated immunotherapy while reducing its toxic side effects, thereby overcoming the shortcomings of existing technologies and improving the safety and effectiveness of tumor immunotherapy. Given the above issues, there is an urgent need to develop a method that simultaneously possesses the ability to interact with Mn... 2+ Multifunctional drugs that coordinate to enhance immunotherapy efficacy while reducing toxic side effects and efficiently degrading CXCR4 protein are developed to overcome the shortcomings of existing technologies and provide more effective technical means for tumor immunotherapy and overcoming adverse reactions. Summary of the Invention

[0006] This invention aims to solve the aforementioned technical problems, namely, to address the low patient response rate in existing tumor immunotherapy, the insufficient targeting of existing CXCR4 targeting strategies (such as small molecule antagonists or antibodies), the ease of drug resistance, and the challenges of clinical translation, as well as the presence of free Mn in existing manganese-based immunotherapy nanoplatforms. 2+ It suffers from high toxicity, low targeted delivery efficiency, single function, and inability to synergistically intervene in the key immune escape target CXCR4.

[0007] In a first aspect, the present invention provides a nanomaterial for targeted degradation of CXCR4 protein, comprising: Nanosheets formed by reacting manganese compounds with polyphenols; and, A short peptide targeting the CXCR4 protein is attached to the surface of the nanosheet.

[0008] In the preferred embodiment of the above nanomaterial, the manganese compound is manganese chloride tetrahydrate, potassium permanganate, or manganese dioxide; and / or, the polyphenol is gallic acid, tannic acid, or catechin; and / or, the amino acid sequence of the bound short peptide is LGASWHRPDKC.

[0009] In the preferred embodiment of the above-mentioned nanomaterial, the mass ratio of the nanosheet to the bound short peptide is (1.0-10):1; and / or, in the nanosheet, the molar ratio of the manganese compound to the polyphenol is (1.0-2):1, wherein the number of moles of the manganese compound is calculated based on the number of moles of manganese element contained therein.

[0010] In a second aspect, the present invention provides a method for preparing the nanomaterials described in the first aspect, comprising the following steps: S1. Manganese compounds and polyphenols are mixed in an alkaline aqueous solution and the nanosheets are formed through a hydrothermal reaction. S2. The nanosheets are mixed with the binding short peptide targeting the CXCR4 protein and reacted to attach the binding short peptide to the surface of the nanosheets.

[0011] In the preferred embodiment of the above preparation method, in step S1, the manganese compound and the polyphenol are dissolved in DEPC water respectively; and / or, in step S1, the pH value of the alkaline aqueous solution is 8.0 to 10.0; and / or, in step S1, the temperature of the hydrothermal reaction is 120℃ to 180℃, and the reaction time is 1 to 5 h; and / or, in step S2, the reaction time is 12 h to 48 h.

[0012] In a third aspect, the present invention provides a nanomaterial for tumor imaging and treatment, comprising: Nanomaterials for targeted degradation of CXCR4 protein as described in the first aspect above; as well as, Near-infrared fluorescent probes attached to the nanomaterials.

[0013] In the preferred technical solutions of the above-mentioned nanomaterials for tumor imaging and treatment, the near-infrared fluorescent probe is Cy7-NHS, RhB-NHS, Cy3-NHS or Cy5.5-NHS.

[0014] In a fourth aspect, the present invention provides the application of the nanomaterials for targeted degradation of CXCR4 protein described in the first aspect above in the preparation of antitumor drugs.

[0015] In the preferred embodiment of the above application, the drug has the following synergistic anti-tumor effect: (i) Targeted degradation of CXCR4 protein to inhibit tumor cells; and (ii) Activate the cGAS-STING signaling pathway to promote anti-tumor immune response.

[0016] In a fifth aspect, the present invention provides the use of the nanomaterials for tumor imaging and treatment described in the third aspect above in the preparation of tumor diagnostic reagents and / or anti-tumor drugs.

[0017] By adopting the above technical solution, the present invention can achieve the following beneficial effects: (1) A novel CXCR4-targeted degradation strategy is provided. Unlike existing small molecule antagonists or antibody therapies, the metal-polyphenol nanosheets (such as GAMnP) provided by this invention can bind to the CXCR4 protein through surface-modified specific short peptides and actively induce it to enter the lysosome for degradation, thereby clearing the target from the source and more thoroughly blocking its function, providing a new way to overcome the drug resistance problem of existing therapies; (2) Achieving efficient synergy between "signal blocking" and "immune activation". This invention combines the CXCR4 targeted degradation unit with Mn 2+Immunostimulants are integrated into a single nanoplatform (such as GAMnP). This system, on the one hand, effectively inhibits downstream pro-tumor signaling pathways such as ERK / AKT by degrading CXCR4 protein, thereby suppressing tumor cell proliferation, migration, and invasion, and reducing the proportion of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment, thus regulating the tumor microenvironment. On the other hand, it releases Mn2 in the acidic environment of lysosomes. 2+ It can efficiently activate the cGAS-STING innate immune pathway, promote dendritic cell maturation, and thus enhance the anti-tumor immune response. This dual mechanism works synergistically to achieve an organic combination of "precision strike" and "immune awakening" against tumors, producing a superimposed anti-tumor effect that transcends a single mechanism. (3) Excellent targeting and safety. This invention achieves specific binding to tumor cells highly expressing CXCR4 through targeting peptides, improving drug accumulation at the lesion site, enhancing treatment precision, and potentially reducing systemic toxicity. Simultaneously, it utilizes neurotoxic free Mn... 2+ Stable coordination within the nanostructure enables its controllable release, significantly improving Mn content. 2+ The delivery safety window overcomes the high toxicity of existing manganese-based formulations; (4) It has a significant inhibitory effect on the growth of in situ tumors. Based on the above-mentioned multi-level synergistic mechanism, the nanomaterials of the present invention significantly inhibited the growth of in situ breast tumors in animal models, verifying their great potential as a novel tumor immunotherapy drug. Attached Figure Description

[0018] The preferred embodiments of the present invention are described below with reference to the accompanying drawings, in which: Figure 1 Figure 1 shows the transmission electron microscopy (TEM) characterization images of GAMn and GAMnP; where Figure 2a is the TEM characterization image of GAMn and Figure 3b is the TEM characterization image of GAMnP. Figure 2 Particle size distribution diagrams for GAMn and GAMnP; Figure 3 The zeta potential diagrams for GAMn and GAMnP are shown. Figure 4 The ultraviolet spectra of GA, GAMn, and GAMnP are shown. Figure 5 Fourier transform infrared spectral characterization of GA, GAMn, and GAMnP; Figure 6 Figures show the stability characteristics of GAMn and GAMnP; where Figure a shows the stability characteristic of GAMn and Figure b shows the stability characteristic of GAMnP. Figure 7 A characterization diagram of the cellular uptake capacity of RhB@GAMnP; Figure 8 Fluorescence characterization of Cy3@GAMnP targeting lysosomes; Figure 9 Immunofluorescence images of GA, peptide, GAMn, and GAMnP degrading CXCR4 in 4T1 cells; Figure 10 A diagram showing the changes in CXCR4 mRNA in 4T1 cells after GAMnP treatment; Figure 11 Figure 1 shows the Western blot results of the effect of GAMnP on CXCR4 protein synthesis. Figure 12 To detect the dose-dependent degradation of CXCR4 protein by GAMnP in 4T1 cells using Western blot; Figure 13 Western blot analysis was performed to determine the time-dependent degradation of CXCR4 protein by GAMnP in 4T1 cells. Figure 14 To detect the dose-dependent degradation of CXCR4 protein by GAMnP in MC38 cells using Western blot; Figure 15 Western blot analysis was performed to determine the time-dependent degradation of CXCR4 protein by GAMnP in MC38 cells. Figure 16 To detect the degradation pathway of CXCR4 protein by GAMnP in 4T1 cells using Western blot. Figure 17 Figure showing the results of Western blot analysis of GAMnP activation of the cGAS / STING signaling pathway in 4T1 cells; Figure 18 Figure showing the effects of GA, peptide, GAMn, and GAMnP treatment on the secretory factor IFN-β in BMDCs cells; Figure 19 Figure showing the activation of dendritic cells by flow cytometry after treatment with GA, peptide, GAMn, and GAMnP. Figure 20 The figure shows the results of Western blot analysis of the effects of GA, peptide, GAMn, and GAMnP on the downstream signaling pathway ERK / AKT in 4T1 cells. Figure 21 The images show the cell viability of 4T1 and MC38 cells after GAMnP treatment; Figure a shows the cell viability of 4T1 cells after GAMnP treatment; Figure b shows the cell viability of MC38 cells after GAMnP treatment. Figure 22 The graph shows the results of detecting the proliferation capacity of 4T1 cells by GA, peptide, GAMn, and GAMnP. Figure 23 The graph shows the results of detecting the migration ability of GA, peptide, GAMn, and GAMnP in 4T1 cells. Figure 24 Image showing the tumor targeting results of Cy7@GAMnP in 4T1 tumor-bearing mice using in vivo imaging in small animals; Figure 25 Figure 1 shows the in vivo treatment experiment of GAMnP in 4T1 tumor-bearing mice; Figure 2a shows the weight change curve of mice in each group during the treatment period; Figure 3b shows the tumor volume change of mice in each group during the treatment period; Figure 4c shows the tumor mass of each group after the mice were sacrificed. Figure 26 The figure shows the results of Western blot detection of CXCR4 protein and downstream pathway ERK / AKT protein in tumors of different groups of 4T1 tumor-bearing mice. Figure 27 Figure 1 shows the immunomodulatory effects of GA, peptide, GAMn, and GAMnP on tumor tissues from 4T1 tumor-bearing mice, as detected by flow cytometry. Figure a shows the effect of CD11b on tumor tissues from 4T1 tumor-bearing mice. + GR-1 + Figure b shows the immune regulatory capacity of MDSCs cells; Figure b shows the CD3+ expression in tumor tissue from 4T1 tumor-bearing mice. + CD8 + Figure c shows the immune regulatory capacity of T cells; Figure c shows the CD11C expression in tumor tissue from 4T1 tumor-bearing mice. + MHCII + A diagram illustrating the immune regulatory capacity of DC cells; Figure 28 The effects of GA, Peptide, GAMn, and GAMnP on the secretory factor TNF-α in 4T1 tumor-bearing mice. Detailed Implementation

[0019] Preferred embodiments of the present invention will now be described. Those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0020] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0021] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, where a, b, and c can be single or multiple.

[0022] It should be understood that in the various embodiments of this application, the above-described order of steps does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. For example, step S1 may be executed at any node before steps S8, S9, and S10, or steps S8, S9, and S10 may be executed simultaneously or in any order. The execution order of each step should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0023] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0024] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a mass unit known in the chemical industry, such as μg, mg, g, or kg.

[0025] The terms "first" and "second" are used only to describe purpose, to distinguish purposes such as substances from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used in the following examples are commercially available unless otherwise specified.

[0027] The following explains the terminology used in this invention: GA: gallic acid; GAMn: Nanosheets formed by coordination of manganese chloride tetrahydrate and gallic acid; GAMnP: refers to nanomaterials composed of GAMn nanosheets linked to short peptides that target the CXCR4 protein; Cy7@GAMnP: refers to nanomaterials that are further linked to the fluorescent probe Cy7-NHS on the basis of GAMnP; ddH2O: Double-distilled water; DEPC: Diethyl pyrocarbonate, is a chemical reagent used to inactivate RNase (ribonuclease); DEPC water refers to ultrapure water that has been treated with DEPC to remove RNase, and then sterilized at high temperature to decompose any remaining DEPC, resulting in water that is free of RNase.

[0028] Based on the issues raised in the background section regarding low patient response rates in existing tumor immunotherapy, insufficient targeting of existing CXCR4-targeting strategies (such as small molecule antagonists or antibodies), drug resistance, and challenges in clinical translation, as well as the presence of free Mn in existing manganese-based immunotherapy nanoplatforms... 2+ The CXCR4 protein suffers from high toxicity, low targeted delivery efficiency, limited functionality, and inability to synergistically intervene in key immune escape targets. This invention provides a nanomaterial for targeted degradation of the CXCR4 protein, its preparation method, and its applications, aiming to simultaneously address these multiple technical bottlenecks through an integrated nanoplatform.

[0029] Specifically, in a first aspect, the present invention provides a nanomaterial for targeted degradation of CXCR4 protein, comprising: Nanosheets formed by the reaction of manganese compounds with polyphenols; as well as, A short peptide targeting the CXCR4 protein is attached to the surface of the nanosheet.

[0030] This material is a nanostructure based on the chemical interaction between metal and polyphenols, which combines the targeted degradation of CXCR4 and Mn. 2+ Synergistic function of immune activation. Specifically, its core is provided by manganese compounds (providing Mn). 2+ The nanosheets are formed by reacting manganese with polyphenols; these nanosheets are further modified with specific short peptides targeting the CXCR4 protein. This composite nanomaterial can be called a metal-polyphenol nanosheet. Taking manganese chloride tetrahydrate as the manganese compound and gallic acid as the polyphenol as an example, the resulting metal-polyphenol nanosheet can be denoted as GAMnP.

[0031] The nanomaterial (GAMnP) for targeting and degrading CXCR4 protein provided by this invention utilizes its surface-linked targeting short peptides to specifically recognize and bind to the highly expressed CXCR4 protein on the surface of tumor cells, inducing it to enter lysosomes for degradation. This blocks the CXCR4-mediated pro-tumor signaling pathway, inhibits tumor cell proliferation, migration, and invasion, and reduces the proportion of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment, thus regulating the tumor microenvironment. Simultaneously, Mn is released in the acidic lysosomal environment. 2+ It can effectively activate the cGAS-STING immune pathway, reshape the tumor immune microenvironment, and achieve dual synergistic therapy of "degrading target proteins" and "activating immunity".

[0032] In some specific embodiments, the manganese compound is manganese chloride tetrahydrate, potassium permanganate, or manganese dioxide.

[0033] In some specific embodiments, the polyphenol is gallic acid, tannic acid, or catechin.

[0034] In this invention, the nanosheets are formed by the chemical reaction of manganese compounds and polyphenols. The manganese compounds refer to compounds that can provide manganese to participate in the formation of the nanostructure, encompassing different valence states and forms, such as potassium permanganate (KMnO4), manganese dioxide (MnO2), and manganese chloride tetrahydrate (MnCl2·4H2O). The polyphenols refer to compounds containing at least two adjacent phenolic hydroxyl groups, such as gallic acid, tannic acid, and catechins.

[0035] The aforementioned manganese compounds interact with polyphenols under suitable conditions (e.g., in a hydrothermal environment) to form nanosheet structures with specific morphologies and functions. The formation mechanism varies depending on the valence state of the manganese source. (1) When manganese compounds are made using KMnO4 or MnO2, the process is a "reduction and coordination coupling" process. Polyphenols (such as catechol, gallic acid, tea polyphenols, etc.) contain ortho- or meta-phenolic hydroxyl structures, which donate electrons to be oxidized to quinones. Manganese is gradually reduced from +7 or +4 oxidation states to the thermodynamically stable Mn. 2+ Subsequently Mn 2+ It forms chelate coordination complexes with polyphenol oxygen / hydroxyl groups, thereby realizing the coupling process of "reduction + coordination" to form nanosheets; (2) When manganese compounds are used, MnCl2·4H2O, Mn 2+ It can directly coordinate with the phenolic hydroxyl groups of polyphenols to form nanosheets.

[0036] Regardless of which path is taken, the final product is formed from Mn 2+A stable nanosheet structure is formed with a polyphenol coordination network. This design allows the invention to be compatible with manganese compounds from different sources as starting materials. Those skilled in the art can optimize the specific reaction conditions through conventional experiments based on the selected raw materials.

[0037] In a preferred embodiment, the manganese compound is manganese chloride tetrahydrate, and the polyphenol is gallic acid.

[0038] In one specific embodiment, the amino acid sequence of the binding short peptide is LGASWHRPDKC.

[0039] In some specific embodiments, the mass ratio of the nanosheet to the bound short peptide is (1.0 to 10):1. For example, the mass ratio of the nanosheet to the bound short peptide is 1.0:1, 2.0:1, 3.0:1, 4.0:1, 5.0:1, 6.0:1, 7.0:1, 8.0:1, 9.0:1, 10:1, or any value within the range.

[0040] In some preferred embodiments, the mass ratio of the nanosheet to the bound short peptide is (1.0 to 8.0):1.

[0041] In a preferred embodiment, the mass ratio of the nanosheet to the bound short peptide is 5.0:1.

[0042] In some specific embodiments, the molar ratio of the manganese compound to the polyphenol in the nanosheet is (1.0–2):1. For example, the molar ratio of the manganese compound to the polyphenol can be 1.0:1, 1.2:1, 1.5:1, 1.8:1, 2.0:1, or any value within the range.

[0043] In some preferred embodiments, the molar ratio of the manganese compound to the polyphenol is (1.0 to 1.5):1.

[0044] In some specific embodiments, the molar ratio of the manganese compound to the polyphenol is 1:1.

[0045] The number of moles of the manganese compound is calculated based on the number of moles of manganese element (Mn) contained therein, and the number of moles of the polyphenol is calculated based on the number of moles of the polyphenol molecule.

[0046] Furthermore, the present invention provides a method for preparing the nanomaterials described in the first aspect in a second aspect, comprising the following steps: S1. Manganese compounds and polyphenols are mixed in an alkaline aqueous solution and the nanosheets are formed through a hydrothermal reaction. S2. The nanosheets are mixed with the binding short peptide targeting the CXCR4 protein and reacted to attach the binding short peptide to the surface of the nanosheets.

[0047] The method described above in this invention includes the preparation of nanosheets and their functionalization modification with targeting peptides. The process is simple, controllable, and easy to scale up.

[0048] In some specific embodiments, in step S1, the manganese compound and the polyphenol are dissolved in DEPC water respectively.

[0049] In some specific embodiments, in step S1, the pH value of the alkaline aqueous solution is 8.0 to 10.0. For example, the pH value can be 8.0, 8.5, 9.0, 9.5, 10, or any value within the range.

[0050] In some specific embodiments, in step S1, the temperature of the hydrothermal reaction is 120℃~180℃, and the reaction time is 1~5h. For example, the temperature of the hydrothermal reaction can be 120℃, 160℃, 180℃, or any value within the range; the reaction time can be 1h, 2h, 3h, 4h, 5h, or any value within the range.

[0051] In some specific embodiments, the process further includes separation, washing, and drying after the hydrothermal reaction. The washing may use water and anhydrous ethanol as detergents, and the washing may be performed at least three times to ensure thorough removal of impurities.

[0052] Specifically, step S1 can be broken down as follows: S11. Mix manganese compounds with polyphenols in an alkaline aqueous solution to form a mixture; S12. The mixture obtained in step S11 is subjected to a hydrothermal reaction; S13. The product after the hydrothermal reaction in step S12 is separated, washed and dried to obtain the nanosheets.

[0053] In some specific embodiments, the pH value of the alkaline aqueous solution can be adjusted using a buffer solution, which can be a pH buffer solution, preferably a sodium hydroxide (NaOH) solution.

[0054] In some specific embodiments, in step S1, the mixing is carried out by stirring at room temperature, with a stirring speed of 10,000 rpm to 15,000 rpm and a stirring time of 30 min to 90 min. As an example, the mixing can be carried out continuously at approximately 12,000 rpm for approximately 40 min. It should be understood that the above stirring speed and time parameters are intended to ensure thorough mixing of the reactants and are not intended to limit the invention; those skilled in the art can make adaptive adjustments according to actual circumstances.

[0055] In some specific embodiments, the reaction time in step S2 is 12h to 48h. For example, the reaction time can be 12h, 24h, 36h, 48h, or any value within the range.

[0056] In some specific embodiments, after the reaction in step S2, the reactants are further centrifuged and washed to remove unreacted bound short peptides, thereby obtaining purified nanomaterials that target and degrade CXCR4 protein.

[0057] In some specific embodiments, the centrifugation is performed at 12,000–15,000 rpm for 5–20 minutes. As an example, the centrifugation is carried out continuously at 12,000 rpm for 15 minutes. It should be understood that the above centrifugation speed and time parameters are intended to achieve sufficient separation of the product and solution and are not intended to limit the invention. Those skilled in the art can make adaptive adjustments according to actual circumstances.

[0058] In some specific embodiments, the detergent used for washing after centrifugation is DEPC water, and the washing is performed more than three times.

[0059] In some specific embodiments, the water used to dissolve reactants or wash products is DEPC-treated RNase-free water (hereinafter referred to as "DEPC water"). The inventors have found that using DEPC water in the preparation of nanomaterials helps to further reduce the risk of non-specific immune responses caused by nucleases or microbial-derived impurities that may remain in the water, thereby potentially improving the safety and purity of the nanomaterials in biological applications. It is understood that conventional ultrapure water or water for injection is also applicable to this invention.

[0060] Furthermore, in a third aspect, the present invention provides a nanomaterial for tumor imaging and treatment, comprising: Nanomaterials for targeted degradation of CXCR4 protein as described in the first aspect above; as well as, Near-infrared fluorescent probes attached to the nanomaterials.

[0061] This material is composed of the nanomaterials mentioned in the first aspect, further connected with near-infrared fluorescent probes (such as Cy7-NHS). While retaining the synergistic therapeutic function, it also has the ability to trace tumor fluorescence imaging, providing a visualization tool for real-time monitoring of the treatment process and efficacy evaluation.

[0062] In some preferred embodiments, the near-infrared fluorescent probe is Cy7-NHS, RhB-NHS, Cy3-NHS, or Cy5.5-NHS.

[0063] It is understood that the selection of the above probes is mainly based on the following two points: first, their emission wavelength is located in the near-infrared region, which is suitable for in vivo imaging; second, their molecular structure contains reactive groups (such as NHS esters), which facilitates stable coupling with nanomaterials. Those skilled in the art will know that other fluorescent probes with similar optical properties and reactive groups are also applicable to this invention. Therefore, the probes listed above are merely examples, and the scope of protection of this invention is not limited thereto.

[0064] It should be noted that the nanomaterials used for tumor imaging and treatment can be prepared using conventional methods in the art for conjugating fluorescent probes to nanomaterials. For example, the fluorescent probe can be mixed with nanomaterials that target and degrade CXCR4 protein and reacted to attach the fluorescent probe to the nanomaterials that target and degrade CXCR4 protein. This invention does not limit the specific conjugation method.

[0065] Furthermore, in a fourth aspect, the present invention provides the application of the nanomaterials for targeting and degrading CXCR4 protein as described in the first aspect in the preparation of antitumor drugs.

[0066] Specifically, the drug exerts its anti-tumor effect through the synergistic effect of the following two mechanisms: (i) Targeted degradation of CXCR4 protein to inhibit tumor cells; and (ii) Activate the cGAS-STING signaling pathway to promote anti-tumor immune response.

[0067] The nanomaterials for targeted degradation of CXCR4 protein provided by this invention utilize surface-linked targeting short peptides to specifically recognize and bind to the highly expressed CXCR4 protein on the surface of tumor cells, inducing its entry into lysosomes for degradation. This blocks the CXCR4-mediated pro-tumor signaling pathway, inhibits tumor cell proliferation, migration, and invasion, and reduces the proportion of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment, thus regulating the tumor microenvironment. Simultaneously, Mn is released in the acidic lysosomal environment. 2+ It can effectively activate the cGAS-STING immune pathway, reshape the tumor immune microenvironment, and achieve dual synergistic therapy of "degrading target proteins" and "activating immunity".

[0068] Based on the aforementioned synergistic effect, the drug is particularly suitable for treating tumors with high CXCR4 expression, such as breast cancer, bladder cancer, and lung cancer.

[0069] Furthermore, in a fifth aspect, the present invention provides the use of the nanomaterials for tumor imaging and treatment described in the third aspect above in the preparation of tumor diagnostic reagents and / or anti-tumor drugs.

[0070] The nanomaterial (GAMnP) of this invention can achieve targeted degradation of CXCR4 and Mn. 2+ This technology enables synergistic therapy through immune activation; further loading with fluorescent probes (such as Cy7@GAMnP, Cy3@GAMnP, or RhB@GAMnP) also provides tumor optical imaging capabilities. This system overcomes the shortcomings of existing technologies, such as poor targeting, high toxicity, and limited functionality, offering a new solution for developing highly effective anti-tumor immunotherapies with efficacy monitoring capabilities.

[0071] The following detailed description of the nanomaterials for targeted degradation of CXCR4 protein, their preparation methods, and applications are provided through several specific examples.

[0072] Example 1-1, Preparation of GAMn This embodiment provides a method for preparing GAMn, including the following steps: S11. Weigh 0.5 mmol of manganese chloride tetrahydrate (MnCl2·4H2O) and dissolve it in 15 mL of DEPC aqueous solution. Stir for 5 minutes to ensure complete dissolution. Then add 0.5 mmol of gallic acid (GA) dissolved in DEPC water and stir at room temperature for 30 minutes until GA is completely dissolved. During the entire reaction process, continuously add 2 M sodium hydroxide (NaOH) aqueous solution to adjust the pH of the reaction system to 8.5. S12. After the reaction is complete, transfer the mixed solution to a 20ml reaction vessel and place it in a vacuum drying oven to slowly heat to 150℃ and react for 3 hours. S13. After the reaction is complete, transfer the crude product to a centrifuge tube, centrifuge and discard the supernatant. Wash the product three times with DEPC water and anhydrous ethanol respectively, and then freeze-dry it under vacuum to obtain GAMn nanosheets. Store at 4°C for later use.

[0073] Example 1-2, Preparation of GAMn This embodiment provides a method for preparing GAMn, referring to Example 1-1. The difference from Example 1-1 is that in step S11, the amount of MnCl2·4H2O used is 1 mmol.

[0074] Example 1-3, Preparation of GAMn This embodiment provides a method for preparing GAMn. Referring to Example 1-1, the difference from Example 1-1 is that in step S12, the heating temperature is 120°C and the reaction time is 5 hours.

[0075] Example 1-4, Preparation of GAMn This embodiment provides a method for preparing GAMn. Referring to Example 1-1, the difference from Example 1-1 is that in step S12, the heating temperature is 180°C and the reaction time is 1 hour.

[0076] Example 1-5, Preparation of GAMn This embodiment provides a method for preparing GAMn. Referring to Example 1-1, the difference from Example 1-1 is that in step S11, the pH value of the reaction system is adjusted to 8.0.

[0077] Example 1-6, Preparation of GAMn This embodiment provides a method for preparing GAMn, referring to Example 1-1. The difference from Example 1-1 is that in step S11, the pH value of the reaction system is adjusted to 10.0.

[0078] Example 1-7, Preparation of GAMn This embodiment provides a method for preparing GAMn. Referring to Example 1-1, the difference from Example 1-1 is that in step S11, MnCl2·4H2O is replaced with KMnO4.

[0079] Example 1-8, Preparation of GAMn This embodiment provides a method for preparing GAMn. Referring to Example 1-1, the difference from Example 1-1 is that in step S11, MnCl2·4H2O is replaced with MnO2.

[0080] Example 1-9, Preparation of metal-polyphenol nanosheets This embodiment provides a method for preparing metal-polyphenol nanosheets. Referring to Example 1-1, the difference from Example 1-1 is that in step S11, gallic acid is replaced with tannic acid.

[0081] Example 1-10, Preparation of metal-polyphenol nanosheets This embodiment provides a method for preparing metal-polyphenol nanosheets. Referring to Example 1-1, the difference from Example 1-1 is that in step S11, gallic acid is replaced with catechin.

[0082] Example 2-1, Preparation of GAMnP This embodiment provides a GAMnP, comprising: a nanosheet (GAMn) formed from manganese chloride tetrahydrate and gallic acid; and a short peptide targeting the CXCR4 protein attached to the surface of the nanosheet; the sequence of the short peptide is LGASWHRPDKC.

[0083] The preparation method of GAMnP is as follows: 5 mg of GAMn nanosheets (obtained in Example 1-1) and 1 mg of short peptide (sequence LGASWHRPDKC) are weighed and placed in a 10 mL glass bottle. The mixture is stirred at room temperature for 24 h to allow the GAMn nanosheets and short peptide to fully bind. Then, the mixture is centrifuged (12000 rpm, 15 min) and washed three times with ddH2O solvent to remove excess unbound short peptides. The resulting GAMnP nanomaterial is dispersed and stored in sterile water for later use.

[0084] Example 2-2, Preparation of GAMnP This embodiment provides a GAMnP and its preparation method, referring to Example 2-1. The difference from Example 2-1 is that the amount of GAMn nanosheets used in the preparation method is 1 mg.

[0085] Example 2-3, Preparation of GAMnP This embodiment provides a GAMnP and its preparation method, referring to Example 2-1. The difference from Example 2-1 is that the amount of GAMn nanosheets used in the preparation method is 10 mg.

[0086] Example 2-4, Preparation of GAMnP This embodiment provides a GAMnP and its preparation method, referring to Example 2-1. The difference from Example 2-1 is that the stirring time at room temperature in the preparation method is 12 hours.

[0087] Example 2-5, Preparation of GAMnP This embodiment provides a GAMnP and its preparation method, referring to Example 2-1. The difference from Example 2-1 is that the stirring time at room temperature in the preparation method is 48 hours.

[0088] Example 3-1, Preparation of Cy7@GAMnP This embodiment provides a nanomaterial (Cy7@GAMnP) for tumor imaging and treatment, comprising: a nanomaterial for targeting and degrading CXCR4 protein; and a fluorescent probe attached to the nanomaterial.

[0089] The preparation method of the Cy7@GAMnP nanomaterial is as follows: 10 mg of pre-prepared GAMnP nanomaterial (obtained in Example 2-1) was dispersed in water, and 1 mg of Cy7-NHS (purchased from Ruixibio) was added at room temperature. The mixture was stirred at room temperature for 24 h, and then the mixture was transferred to a dialysis bag (molecular weight of 1 kDa) and dialyzed in 2 L of ddH2O solution to remove unreacted Cy7-NHS. After dialysis, Cy7@GAMnP nanomaterials with coupled Cy7-NHS fluorescence were obtained.

[0090] Example 3-2, Preparation of Cy3@GAMnP Similar to the preparation of Cy7@GAMnP in Example 3-1, except that Cy7-NHS is replaced with Cy3-NHS.

[0091] Example 3-3, Preparation of RhB@GAMnP Similar to the preparation of Cy7@GAMnP in Example 3-1, except that Cy7-NHS is replaced with RhB-NHS.

[0092] Example 4, Transmission electron microscopy characterization of GAMn and GAMnP GAMn nanosheets (prepared in Example 1-1) and GAMnP nanomaterials (prepared in Example 2-1) were added to ddH2O solution at a concentration of 0.2 mg / mL and dispersed uniformly using an ultrasonic homogenizer. 10 μL of each solution was dropped onto a carbon-supported copper grid. The copper grid was dried at room temperature in a dry and clean place. The particle morphology was then observed and photographed under a transmission electron microscope.

[0093] The results are as follows Figure 1 As shown, both GAMn nanosheets and GAMnP nanomaterials exhibit a sheet-like morphology under electron microscopy, with a size of approximately 140 nm.

[0094] Example 5, Particle size determination of GAMn and GAMnP GAMn nanosheets (prepared in Example 1-1) and GAMnP nanomaterials (prepared in Example 2-1) were dispersed in ddH2O at a concentration of 1 mg / mL. The dispersed materials were then placed in an ultrasonic homogenizer for ultrasonication and then placed in a particle size measurement cell. The particle size was measured using a particle size potential analyzer.

[0095] The results are as follows Figure 2 As shown. From Figure 2It can be seen that the hydrated particle size of GAMn is about 127.1 nm, and the PDI value is 0.1661; while the hydrated particle size of GAMnP is slightly larger than that of GAMn, with a size of about 142 nm and a PDI value of 0.1817. This may be due to the successful adsorption of CXCR4 peptide onto GAMn, resulting in its larger particle size.

[0096] Example 6, Potential Measurement of GAMn and GAMnP GAMn nanosheets (prepared in Example 1-1) and GAMnP nanomaterials (prepared in Example 2-1) were diluted to a concentration of 0.2 mg / mL and dispersed in ddH2O. Then, they were placed in a potential measurement cell and their potential was measured using a potential particle size analyzer.

[0097] The results are as follows Figure 3 As shown in the figure, GAMn is negatively charged with a zeta potential of -20.9 mV; while the potential of GAMnP nanomaterials adsorbed with positively charged short peptides is much smaller than that of GAMn, with a potential of approximately -9.65 mV. This is because positively charged short peptides are adsorbed on the surface of the nanomaterials.

[0098] Example 7, UV spectral characterization of GAMn and GAMnP Gallic acid (GA), GAMn nanosheets (prepared in Example 1-1), and GAMnP nanomaterials (prepared in Example 2-1) were added to ddH2O solution at a concentration of 0.2 mg / mL. The dispersed samples were then added to a clean quartz cuvette. The UV-Vis spectrophotometer was set to scan wavelengths between 200-400 nm, absorbance measurement mode was selected, baseline scanning was performed using ddH2O and zeroing was performed, and then the UV absorption wavelengths of GA, GAMn, and GAMnP nanomaterials were measured.

[0099] The results are as follows Figure 4 As shown. From Figure 4 It can be seen that the characteristic ultraviolet absorption peak of GA is at 213 nm, while that of GAMn is at 207 nm. This significant shift in ultraviolet absorption wavelength proves the difference between GA and Mn. 2+ The successful complexation of gallic acid and manganese ions, and the stabilization of the UV absorption of GAMn nanosheets after 263 nm, indicate that the coordination between gallic acid and manganese ions has reached equilibrium, further proving the successful coordination of gallic acid and manganese ions. The lower UV absorption intensity of GAMnP compared to GAMn may be due to the aggregation of short peptides on the surface of GAMn nanosheets when the short peptides are adsorbed.

[0100] Example 8, Infrared spectral characterization of GA, GAMn and GAMnP Gallic acid (GA), GAMn nanosheets (prepared in Example 1-1), and GAMnP nanomaterials (prepared in Example 2-1) were freeze-dried into solid powders using a vacuum freeze dryer. An appropriate amount of powder was then thoroughly mixed with potassium bromide and pressed into transparent discs using a tablet press. Finally, infrared spectroscopy was used for detection.

[0101] The results are as follows Figure 5 As shown. From Figure 5 It can be seen that gallic acid (GA) is at 3264 cm⁻¹ -1 A broad absorption peak appeared at 593 cm⁻¹, which was caused by the stretching vibration of the phenolic hydroxyl group in gallic acid. However, the intensity of this absorption peak was significantly reduced in GAMn nanosheets, which is likely due to the coordination of gallic acid with manganese ions, indicating successful coordination between gallic acid and manganese ions. Meanwhile, GAMn showed a peak intensity at 593 cm⁻¹. -1 The absorption peak in the far-infrared region is due to the characteristic stretching vibration of Mn-O, while GA shows no obvious absorption peak in this region, further indicating the successful coordination of gallic acid with manganese ions. An amide I band peak (1580 cm⁻¹) appears in the infrared spectrum of GAMnP. -1 The reason for this is the stretching vibration of the peptide bond C=O, which indicates the existence of the peptide bond structure, confirms the existence of its polypeptide backbone, and proves the successful adsorption of short peptides on GAMn nanomaterials.

[0102] Example 9, Stability characterization of GAMn and GAMnP GAMn nanosheets (prepared in Example 1-1) and GAMnP nanomaterials (prepared in Example 2-1) were dispersed at a concentration of 100 μg / mL in ddH2O, PBS, and DMEM containing 10% fetal bovine serum (FBS), respectively. The hydrated particle size of the nanomaterials was then immediately measured in a particle size analyzer, and this was recorded as day 0. Subsequently, the hydrated particle size of both nanomaterials was measured again using a nanoparticle size analyzer on days 1, 2, 3, 4, 5, 6, and 7.

[0103] The results are as follows Figure 6 As shown. From Figure 6 It can be seen that the hydrated particle size of GAMn nanosheets and GAMnP nanomaterials fluctuated within a small range in H2O, PBS, and DMEM containing 10% FBS within 7 days, indicating that they all have good stability.

[0104] Example 10, RhB@GAMnP's cellular uptake capacity 4T1 cells (purchased from ATCC) were seeded in 24-well cell culture plates at a density of approximately 5 × 10⁶ cells / well. 4Cells were cultured overnight in fresh DMEM medium (purchased from Gibco) and 100 μg / mL of RhB@GAMnP was added (the RhB@GAMnP prepared in Example 3-3 was first dissolved in DMEM medium to a concentration of 100 μg / mL before being added to the cells; the same applies below). The blank control was an equal volume of PBS buffer. After culturing the cells for 1, 2, and 4 hours, the fluorescence of the cells at a wavelength of 527 nm was detected using a fluorescence microscope (Nikon).

[0105] The results are as follows Figure 7 As shown. Figure 7 The results showed that after treatment with RhB@GAMnP, the fluorescence in 4T1 cells gradually increased over time, proving that RhB@GAMnP can effectively enter tumor cells.

[0106] Example 11, Cy3@GAMnP's ability to target lysosomes 4T1 cells were seeded into 24-well cell culture plates pre-placed with circular spreaders, at a density of approximately 5 × 10⁶ cells / well. 4 Cells were cultured overnight in wells of fresh DMEM medium before loading. Before loading, the cells were transferred to fresh DMEM medium and 100 μg / mL of Cy3@GAMnP (prepared in Example 3-2) was added. An equal volume of PBS buffer (0 h) was used as a blank control. After 6 h of cell culture, the lysosomes were stained using a Lysosensor (purchased from Invitrogen), followed by confocal microscopy.

[0107] The results are as follows Figure 8 As shown. From Figure 8 As can be seen, compared with the 0h group, the red Cy3@GAMnP nanosheets and the green lysosomes in the 4T1 cells treated with Cy3@GAMnP nanosheets showed obvious co-localization, proving that Cy3@GAMnP can target and enter the lysosomes.

[0108] Example 12, Assessment of GAMnP's ability to clear CXCR4 protein 4T1 cells were seeded in 24-well cell culture plates at a density of approximately 5 × 10⁶ cells / well. 4 Cells were cultured overnight at 1 cell / well for later use. Before adding samples, the cells were replaced with fresh DMEM medium, and PBS (as control group Con) was added, along with peptide (25 μg / mL), GA (25 μg / mL), GAMn (50 μg / mL, prepared in Example 1-1) and GAMnP (50 μg / mL, prepared in Example 2-1). After culturing the cells for 12 hours, immunofluorescence detection was performed.

[0109] The results are as follows Figure 9 As shown. From Figure 9 It can be seen that, compared with other groups, GAMnP can effectively reduce the level of CXCR4 protein in 4T1 cells.

[0110] Example 13, Assessing the effect of GAMnP on CXCR4 transcriptional levels 4T1 cells were seeded in 24-well cell culture plates at a density of approximately 5 × 10⁶ cells / well. 4 Cells were cultured overnight at 1 cell / well for later use. Before adding samples, the cells were transferred to fresh DMEM medium and GAMnP (prepared in Example 2-1) at a concentration of 100 μg / mL was added. The blank control was an equal volume of PBS buffer. After culturing the cells for 12 h, the cells were washed twice with PBS buffer, and then RNA was extracted using RNAexPro Regent (purchased from Aikerui Biotechnology). The mRNA level of CXCR4 was detected by RT-PCR.

[0111] The results are as follows Figure 10 As shown. From Figure 10 As can be seen, compared with the control group, the mRNA level of CXCR4 protein did not change significantly after GAMnP treatment.

[0112] Example 14, Assess whether GAMnP affects CXCR4 protein synthesis. 4T1 cells were seeded in 24-well cell culture plates at a density of approximately 5 × 10⁶ cells / well. 4 Cells per well were cultured overnight and ready for use. Before adding samples, the cells were replaced with fresh DMEM medium and then treated with 20 μM cyclohexylimide (CHX, purchased from Selleck) at specified times (0, 4, 8, and 12 h) with or without the addition of GAMnP (50 µg / mL, prepared in Example 2-1), followed by Western blot analysis.

[0113] The results are as follows Figure 11 As shown. From Figure 11 It can be seen that, compared with CHX alone, the addition of GAMnP promotes the reduction of CXCR4 protein level, indicating that the reduction of CXCR4 protein level induced by GAMnP is mainly due to degradation.

[0114] Example 15, Assess the dose-dependent degradation of CXCR4 protein by GAMnP in 4T1 cells. 4T1 cells were seeded in 24-well cell culture plates at a density of approximately 5 × 10⁶ cells / well. 4Cells were cultured overnight in fresh DMEM medium before use. Before adding samples, the cells were replaced with GAMnP (prepared in Example 2-1) at concentrations of 12.5, 25, 50, 100, 150 and 200 µg / mL. The control group was PBS buffer alone. After 12 h of cell treatment, Western blot was used for detection.

[0115] The results are as follows Figure 12 As shown. From Figure 12 It can be seen that as the concentration of GAMnP increases, the level of CXCR4 protein decreases, indicating that the degradation of CXCR4 protein by GAMnP is dose-dependent.

[0116] Example 16, Assess the time-dependent degradation of CXCR4 protein by GAMnP in 4T1 cells. 4T1 cells were seeded in 24-well cell culture plates at a density of approximately 5 × 10⁶ cells / well. 4 Cells were cultured overnight at 100 µg / well for use. Before adding samples, the cells were replaced with fresh DMEM medium, and then 100 µg / mL GAMnP (prepared in Example 2-1) was added at different times (0, 4, 8 and 12 h). The control group was treated with PBS buffer alone. After 12 h of cell treatment, Western blot was used for detection.

[0117] The results are as follows Figure 13 As shown. From Figure 13 It can be seen that the level of CXCR4 protein decreased with increasing GAMnP treatment time, indicating that the degradation of CXCR4 protein by GAMnP in 4T1 cells is time-dependent.

[0118] Example 17, Assess the dose-dependent degradation of CXCR4 protein by GAMnP in MC38 cells. MC38 cells were seeded in 24-well cell culture plates at a density of approximately 5 × 10⁶ cells / well. 4 Cells were cultured overnight in fresh DMEM medium before loading. GAMnP (prepared in Example 2-1) at concentrations of 0, 25, 50, 100, 150, and 200 µg / mL was added to each well. The control group consisted of PBS buffer alone. After 12 hours of cell treatment, Western blot analysis was performed.

[0119] The results are as follows Figure 14 As shown. From Figure 14 It can be seen that the level of CXCR4 protein decreases with increasing GAMnP concentration, indicating that the degradation of CXCR4 protein by GAMnP in MC38 cells is dose-dependent.

[0120] Example 18, Assess the time-dependent degradation of CXCR4 protein by GAMnP in MC38 cells. MC38 cells were seeded in 24-well cell culture plates at a density of approximately 5 × 10⁶ cells / well. 4 Cells were cultured overnight at 100 µg / well for later use. Before adding samples, the cells were replaced with fresh DMEM medium, and then 100 µg / mL GAMnP (prepared in Example 2-1) was added at different times (0, 2, 4, 6, 8 and 12 h). The control group was treated with PBS buffer alone. After 12 h of cell treatment, Western blot was used for detection.

[0121] The results are as follows Figure 15 As shown. From Figure 15 It can be seen that the level of CXCR4 protein decreases with increasing GAMnP treatment time, indicating that the degradation of CXCR4 protein by GAMnP is time-dependent. Example 19, Assessment of the degradation pathway of CXCR4 protein by GAMnP 4T1 cells were seeded in 24-well cell culture plates at a density of approximately 5 × 10⁶ cells / well. 4 Cells were cultured overnight at 100 μg / well for later use. Before adding samples, the cells were replaced with fresh DMEM medium, and then PBS, 100 μg / mL GAMnP (prepared in Example 2-1), 10 μM proteasome inhibitor MG-132 (purchased from Selleck), 100 μg / mL GAMnP + 10 μM proteasome inhibitor MG-132 (purchased from Selleck), 5 μM autophagy-lysosome inhibitor chloroquine (abbreviated as CQ, purchased from Selleck), and 100 μg / mL GAMnP + autophagy-lysosome inhibitor CQ were added respectively. After 12 h of cell culture, Western blot analysis was performed.

[0122] The results are as follows Figure 16 As shown. From Figure 16 It can be seen that the autophagy-lysosome inhibitor CQ can effectively inhibit the ability of GAMnP to degrade CXCR4 protein; while the proteasome inhibitor MG-132 cannot, indicating that the degradation of CXCR4 protein induced by GAMnP is through the lysosomal pathway.

[0123] Example 20, Evaluate the impact of GAMnP on the cGAS / STING signaling pathway 4T1 cells were seeded in 24-well cell culture plates at a density of approximately 5 × 10⁶ cells / well. 4Cells were cultured overnight at 1 cell / well for later use. Before adding samples, the cells were replaced with fresh DMEM medium, and PBS (as control group Con) was added, along with peptide (25 μg / mL), GA (25 μg / mL), GAMn (50 μg / mL, prepared in Example 1-1) and GAMnP (50 μg / mL, prepared in Example 2-1). After culturing the cells for 12 hours, Western blot analysis was performed.

[0124] The results are as follows Figure 17 As shown. From Figure 17 It can be seen that, compared with other groups, GAMnP can effectively enhance the levels of phosphorylated IRF3 (p-IRF3), phosphorylated STING (p-STING), and phosphorylated TBK1 (p-TBK1) proteins, proving that GAMnP significantly activates the cGAS / STING signaling pathway.

[0125] Example 21, Assess the effect of GAMnP on the secretory factor IFN-β Immature bone marrow-derived dendritic cells (BMDCs) were isolated from the femur and tibia of 12-week-old C57BL / 6J mice and cultured in RPMI 1640 medium containing M-CSF (20 ng / mL) and IL-4 (10 ng / mL). After 6 days, BMDCs were added to PBS (as control group Con), peptide (25 μg / mL), GA (25 μg / mL), GAMn (50 μg / mL, prepared in Example 1-1), and GAMnP (50 μg / mL, prepared in Example 2-1), respectively. After 12 hours of cell culture, the culture supernatant was collected, and the level of secreted IFN-β factor in the cell supernatant was detected using an IFN-β kit (purchased from Abcam).

[0126] The results are as follows Figure 18 As shown. From Figure 18 It can be seen that, compared with other groups, GAMnP can effectively promote the level of IFN-β factor in the supernatant of BMDCs, further proving that GAMnP activates the cGAS / STING signaling pathway.

[0127] Example 22, Assess the effect of GAMnP on dendritic cell activation Immature bone marrow-derived dendritic cells (BMDCs) were isolated from the femur and tibia of 12-week-old C57BL / 6J mice and cultured in RPMI 1640 medium containing M-CSF (20 ng / mL) and IL-4 (10 ng / mL). After 6 days, BMDCs were incubated with 4T1 cells, which were treated for 12 hours with PBS (as control group Con), peptide (25 μg / mL), GA (25 μg / mL), GAMn (50 μg / mL, prepared in Example 1-1), and GAMnP (50 μg / mL, prepared in Example 2-1), respectively. After 24 hours, the DCs were collected and analyzed by flow cytometry.

[0128] The results are as follows Figure 19 As shown. From Figure 19 It can be seen that, compared with the control group, GAMnP treatment enhanced the expression of co-stimulatory molecules (CD80, CD86, CD40, and MHC-II) in BMDCs. These results demonstrate that GAMnP can stimulate DC maturation, thereby triggering an anti-tumor immune response.

[0129] Example 23, Assess the impact of GAMnP on downstream ERK / AKT signaling pathways 4T1 cells were seeded in 24-well cell culture plates at a density of approximately 5 × 10⁶ cells / well. 4 Cells were cultured overnight in wells of fresh DMEM medium before use. Before adding samples, PBS (as control group Con), peptide (25 μg / mL), GA (25 μg / mL), GAMn (50 μg / mL, prepared in Example 1-1), and GAMnP (50 μg / mL, prepared in Example 2-1) were added. After culturing the cells for 12 hours, Western blot analysis was performed.

[0130] The results are as follows Figure 20 As shown. From Figure 20 It can be seen that, compared with other groups, the protein levels of phosphorylated AKT (p-AKT) and phosphorylated ERK (p-ERK) were significantly reduced after GAMnP treatment, while having no effect on the baseline AKT and ERK protein levels, proving that GAMnP inhibits the downstream ERK / AKT signaling pathway.

[0131] Example 24, Assess the killing ability of GAMnP against 4T1 and MC38 cells. 4T1 and MC38 cells (both from ATCC) were seeded in 96-well cell culture plates at a density of approximately 1 × 10⁻⁶ cells / well. 4 / well, cultured overnight, ready for use. Add PBS and different concentration gradients (0, 25, 50, 100, 200, 400 and 600 μg / mL) of GAMnP (prepared in Example 2-1), and perform MTT assay after 24 h of cell culture.

[0132] The results are as follows Figure 21 As shown. From Figure 21 It can be seen that GAMnP has a significant killing effect on both 4T1 and MC38 cells at the degradation concentration, and the killing effect gradually increases with further increase in concentration.

[0133] Example 25, Assess the effect of GAMnP on the proliferation capacity of 4T1 cells 4T1 cells were seeded in 6-well cell culture plates at a density of approximately 5 × 10⁶ cells / well. 2 Cells were cultured overnight at 1 cell / well for later use. Before adding samples, the cells were replaced with fresh DMEM medium, and PBS (as control group Con) was added, along with peptide (25 μg / mL), GA (25 μg / mL), GAMn (50 μg / mL, prepared in Example 1-1) and GAMnP (50 μg / mL, prepared in Example 2-1). After culturing the cells for 12 hours, the medium was removed and replaced with fresh medium. The medium was then changed every 2 days. After 7 days, the medium was removed, the cells were washed three times with PBS, fixed with methanol, and stained with 0.1% crystal violet. After washing three times with PBS, the cells were imaged under a microscope.

[0134] The results are as follows Figure 22 As shown. From Figure 22 It can be seen that, compared with other treatment groups, the number of clones formed in 4T1 cells treated with GAMnP was significantly reduced, indicating that GAMnP can effectively inhibit the proliferation of 4T1 cells.

[0135] Example 26, Assess the effect of GAMnP on the migration ability of 4T1 cells 4T1 cells were seeded in 24-well cell culture plates at a density of approximately 5 × 10⁶ cells / well. 4 Cells per well, incubated overnight, ready for use. Using a 200 μL pipette tip and a ruler, draw a vertical line in the cell culture plate. After washing the cells with PBS, add serum-free culture medium and photograph under a microscope. Then add PBS (as control group Con), peptide (25 μg / mL), GA (25 μg / mL), GAMn (50 μg / mL, prepared in Example 1-1), and GAMnP (50 μg / mL, prepared in Example 2-1). Incubate for 12 h and 24 h respectively, and photograph again.

[0136] The results are as follows Figure 23 As shown. FromFigure 23 It can be seen that, compared with other treatment groups, the distance between cells in the 4T1 cells in the GAMnP treatment group was significantly greater, indicating that GAMnP can effectively inhibit the migration ability of 4T1 cells.

[0137] Example 27, Evaluation of Cy7@GAMnP's tumor-targeting ability in a 4T1 mouse model 1×10 6 4T1 cells were injected orally into the mammary pads of BALB / c mice (purchased from Vital River Pharmaceuticals Beijing, weighing approximately 20g) to construct an orthotopic breast cancer mouse model. When the tumor volume in the mice reached approximately 200 mm², the tumors were targeted. 3 At approximately 2:00 AM, 2 mg of Cy7@GAMnP (prepared in Example 3-1) was injected intravenously (iv), and imaging was performed at 2, 4, 8, 24, and 48 hours using the Xenogen IVIS Lumina system.

[0138] like Figure 24 As shown, in mouse tissues injected with Cy7@GAMnP, compared with the heart, liver, spleen, lungs, and kidneys, there was more obvious Cy7 fluorescence enrichment in the tumor sites, indicating that Cy7@GAMnP has good tumor targeting ability.

[0139] Example 28, Evaluation of the antitumor effect of GAMnP in the 4T1 mouse model 1×10 6 4T1 cells were injected orally into the mammary pads of BALB / c mice (purchased from Vital River Pharmaceuticals Beijing, weighing approximately 20g) to construct an orthotopic breast cancer mouse model. When the tumor volume in the mice reached approximately 100mm... 3 Around the 13th day of treatment, mice were randomly divided into 5 groups of 7 mice each: PBS group (Con), peptide group, GA group, GAMn group, and GAMnP group. Administered the medication every other day at a dose of 10 mg / kg, with a volume of 100 μL per mouse. GAMn was prepared in Example 1-1, and GAMnP was prepared in Example 2-1. Tumor volume, tumor weight, and body weight were recorded. On day 13 of treatment, the mice were sacrificed, the tumor tissue was removed, and the mice were weighed.

[0140] The results are as follows Figure 25 As shown. Among them, Figure 25 As shown in (a), no significant changes in mouse body weight were observed throughout the treatment process, demonstrating that the components in each experimental group did not cause significant systemic toxicity to the mice, and also reflecting the good biocompatibility of GAMnP; Figure 25(b) The mouse tumor growth curves show that GAMnP effectively inhibited tumor growth compared to the PBS group, peptide group, GA group, and GAMn. This is consistent with the tumor volume data. Figure 25 (c) The tumor weight of mice in each group showed that the tumor weight of the GAMn group decreased the most significantly compared with other treatment groups, indicating that GAMnP has a good anti-tumor effect.

[0141] Example 29, Evaluate the effect of GAMnP on inhibiting CXCR4 in a 4T1 mouse model. In Example 28, the 4T1 mouse model was sacrificed after 13 days of treatment. The tumor was removed, chopped, thoroughly mixed using a tissue homogenizer, centrifuged, and the supernatant was collected to prepare a tumor tissue sample, which was then subjected to Western blot detection.

[0142] The results are as follows Figure 26 (The numbers in the diagram indicate the number of mice taken from each group.) From... Figure 26 It can be seen that the levels of CXCR4 protein and its downstream signaling proteins p-AKT and p-ERK were significantly reduced in the tumor tissues of mice treated with GAMnP, proving that GAMnP can also degrade CXCR4 protein and inhibit its downstream signaling pathways in the tumor tissues of 4T1 tumor-bearing mice.

[0143] Example 30, Assess the immunomodulatory capacity of GAMnP in the 4T1 mouse model In Example 28, the 4T1 mouse model was sacrificed after 13 days of treatment. The tumor was removed, weighed, chopped, digested into a single-cell suspension, stained with antibodies for the target marker, and then detected by flow cytometry.

[0144] The results are as follows Figure 27 As shown. From Figure 27 It can be seen that after GAMnP processing, CD11b + GR-1 + The proportion of MDSCs cells was significantly reduced, and CD3... + CD8 + T cells and CD11C + MHCII + The significantly increased proportion of dendritic cells (DCs) indicates that GAMnP treatment significantly reduced the number of MDSCs and CD8+ in mouse tumor tissues. + The number of T cells and DC cells increased significantly.

[0145] Example 31, Evaluate the effect of GAMnP on the secretory factor TNF-α in a 4T1 mouse model. In the 4T1 mouse model of Example 28, after 13 days of treatment, the mice were sacrificed, blood was collected from the eyeballs, and the serum was collected by centrifugation (3000g, 20min) after resting. The level of TNF-α in the mouse serum was detected using a TNF-α kit (purchased from Abcam).

[0146] The results are as follows Figure 28 As shown. From Figure 28 It can be seen that, compared with other groups, the level of TNF-α in the serum of mice treated with GAMnP was significantly increased, indicating that GAMnP treatment promoted tumor immune regulation in the mouse model.

[0147] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after such changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. A nanomaterial for targeted degradation of CXCR4 protein, characterized in that, include: Nanosheets formed by the reaction of manganese compounds with polyphenols; as well as, A short peptide targeting the CXCR4 protein is attached to the surface of the nanosheet.

2. The nanomaterial according to claim 1, characterized in that, The manganese compound is manganese chloride tetrahydrate, potassium permanganate, or manganese dioxide. And / or, the polyphenols are gallic acid, tannic acid or catechin; And / or, the amino acid sequence of the binding short peptide is LGASWHRPDKC.

3. The nanomaterial according to claim 1 or 2, characterized in that, The mass ratio of the nanosheet to the bound short peptide is (1.0-10):1; And / or, in the nanosheet, the molar ratio of the manganese compound to the polyphenol is (1.0 to 2):1, wherein the number of moles of the manganese compound is calculated based on the number of moles of manganese element contained therein.

4. A method for preparing the nanomaterial according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Manganese compounds and polyphenols are mixed in an alkaline aqueous solution and the nanosheets are formed through a hydrothermal reaction. S2. The nanosheets are mixed with the binding short peptide targeting the CXCR4 protein and reacted to attach the binding short peptide to the surface of the nanosheets.

5. The preparation method according to claim 4, characterized in that, In step S1, the manganese compounds and polyphenols are dissolved separately using DEPC water; And / or, in step S1, the pH value of the alkaline aqueous solution is 8.0 to 10.0; And / or, in step S1, the temperature of the hydrothermal reaction is 120℃~180℃, and the reaction time is 1~5h; And / or, in step S2, the reaction time is 12h to 48h.

6. A nanomaterial for tumor imaging and treatment, characterized in that, include: Nanomaterials for targeted degradation of CXCR4 protein as described in any one of claims 1-3; as well as, Near-infrared fluorescent probes attached to the nanomaterials.

7. The nanomaterial for tumor imaging and treatment according to claim 6, characterized in that, The near-infrared fluorescent probe is Cy7-NHS, RhB-NHS, Cy3-NHS, or Cy5.5-NHS.

8. The use of a nanomaterial that targets and degrades CXCR4 protein as described in any one of claims 1-3 in the preparation of antitumor drugs.

9. The application according to claim 8, characterized in that, The drug has the following synergistic anti-tumor effects: (i) Targeted degradation of CXCR4 protein to inhibit tumor cells; and (ii) Activate the cGAS-STING signaling pathway to promote anti-tumor immune response.

10. The use of a nanomaterial for tumor imaging and treatment as described in claim 6 or 7 in the preparation of tumor diagnostic reagents and / or antitumor drugs.

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