Metal-polyamine nanocomposite, preparation method and application thereof

Abstract

The application belongs to the field of nanobiomaterials and tumor radioimmunotherapy, and particularly relates to a metal-polyamine nanocomposite as well as a preparation method and application thereof. The nanocomposite is a core-shell structure nanoparticle constructed by forming a hydrophobic core through coordination of divalent manganese ions and spermidine under mediation of oleic acid and coating the surface with DSPE-PEG. Through coordination assembly of divalent manganese ions and spermidine and surface modification with DSPE-PEG, the cGAS-STING pathway is simultaneously activated to promote maturation of DCs, and CD8 + T cell mitochondrial fatty acid oxidation function, and the anti-tumor effect induced by radiotherapy is enhanced.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of nanobiomaterials and radioimmunotherapy for tumors, and particularly relates to a metal-polyamine nanocomposite as well as a preparation method and application thereof. BACKGROUND

[0002] Radiotherapy is one of the core means for the clinical treatment of solid tumors, and its core mechanism is not only to directly induce DNA damage of tumor cells through ionizing radiation, but also to trigger immunogenic cell death (ICD). In this process, tumor cells release tumor-associated antigens (TAAs) and damage-associated molecular patterns (DAMPs), which provide key signals for the initiation of local anti-tumor immune response. However, the immune activation effect of simple radiotherapy has significant limitations: on the one hand, the intensity of ICD induced by radiotherapy is limited, and the number and activity of released TAAs and DAMPs are insufficient to effectively activate antigen-presenting cells (especially dendritic cells, DCs); on the other hand, even if radiotherapy can initially activate CD8 + T cells, the immunosuppressive properties of the tumor microenvironment, such as nutrient deficiency and oxidative stress, will severely hinder the tumor infiltration efficiency and functional exertion of CD8 + T cells, ultimately leading to low intensity and poor persistence of anti-tumor immune response, which significantly limits the long-term efficacy and clinical benefit of radiotherapy.

[0003] To break through the above bottleneck, various radiotherapy combined treatment strategies have been rapidly developed, including radiotherapy combined with immune checkpoint inhibitors, nano-drug mediated radiotherapy sensitization, and immune effector cell function regulation. Among them, the maturity of DCs directly determines the antigen presentation efficiency and subsequent specific T cell activation effect, and the activation of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is an important link for regulating the maturation of DCs. After the activation of the pathway, it can significantly promote the secretion of type I interferon, tumor necrosis factor and other pro-inflammatory factors, thereby strengthening the presentation ability of DCs to TAAs and laying a foundation for T cell activation. At present, the cGAS-STING pathway regulation strategies mainly include two categories of small molecule agonists (such as cGAMP analogs) and metal ions (such as Mn 2+ , Zn 2+ ). Mn 2+As a natural activator of cGAS, it can directly enhance the binding affinity of cGAS to double-stranded DNA (dsDNA) released by tumor cells, and efficiently activate the cGAS-STING pathway. Among them, manganese-based nanomaterials can specifically release Mn 2+ in response to the acidic conditions of the tumor microenvironment, achieving targeted activation of the pathway, and have the advantages of good biocompatibility, low toxicity and side effects, and have become a key research direction in this field.

[0004] Many studies have confirmed that manganese-based biomaterials can enhance the synergistic anti-tumor immune effect of radiotherapy by activating the cGAS-STING pathway. For example, the prior art (Deng Zheng, et al. "Biomineralized MnO2 Nanoplatforms Mediated Delivery of Immune Checkpoint Inhibitors with STING Pathway Activation to Potentiate Cancer Radio-Immunotherapy.." ACS nano 17.5 (2023): doi:10.1021 / ACSNANO.2C10352.) proposed a biomineralization method, synthesized αPDL1 encapsulated MnO2 (αPDL1@MnO2) nanoparticles, and through Mn 2+ mediated cGAS-STING pathway activation and immune suppression microenvironment regulation, enhanced anti-tumor immune response after radiotherapy. The prior art (Shuai Zhang, et al. "DNA-Capturing Manganese-Coordinated Chitosan Microparticles Potentiate Radiotherapy via Activating the cGAS-STING Pathway and Maintaining Tumor-Infiltrating CD8 + T-Cell Stemness.." Advanced materials (Deerfield Beach, Fla.) 37.12 (2025): e2418583. doi:10.1002 / ADMA.202418583.) constructed manganese-coordinated chitosan microparticles (CS-Mn), which can specifically capture DNA fragments released by radiotherapy, and synergize with Mn 2+Activate the cGAS-STING pathway, promote DCs maturation and enhance the effect of radiotherapy. The existing technology (Bo Chen, et al. "Boosting Peroxidase-Mimetic Activity of FeMn-NCe Dual-Atom Radiosensitizing Nanozymes for Augmented Radiodynamic Immunotherapy.." ACS nano (2025): doi:10.1021 / ACSNANO.4C17148.) developed FeMn-NCe dual-atom nanozyme, through the modification of ultra-small gold nanoparticles to enhance X-ray absorption, catalyze H2O2 to generate •OH, and simultaneously load STING agonist diABZI, multi-mechanism synergistic radiotherapy activates the cGAS-STING pathway. But the above-mentioned manganese-based materials generally have the core limitation of single function - only focus on the activation of upstream cGAS-STING pathway to regulate DCs maturation, and cannot synergistically intervene in the metabolic function and exhausted state of downstream effector T cells, resulting in that the immune response is difficult to form a complete chain of "DCs activation-antigen presentation-T cell effect amplification", and finally cannot realize the sustained anti-tumor immune response after radiotherapy. SUMMARY

[0005] In view of the shortcomings of the prior art, the present application provides a metal-polyamine nanocomposite and a preparation method and application thereof, which realizes the synchronous activation of the cGAS-STING pathway to promote DCs maturation and enhances the CD8 + T cell mitochondrial fatty acid oxidation function, enhances the anti-tumor effect induced by radiotherapy.

[0006] The technical scheme provided by the present application is as follows:

[0007] The present application provides a metal-polyamine nanocomposite, which is a core-shell structure nanoparticle formed by coordination of divalent manganese ions and spermidine under the mediation of oleic acid to form a hydrophobic core, and surface coated with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol DSPE-PEG.

[0008] Further, the divalent manganese ions are selected from one or more of manganese chloride, manganese sulfate and manganese acetate; the particle size of the nanocomposite is 100-200 nm, and the Zeta potential is -20 to -10 mV.

[0009] The present application also provides a preparation method of the above-mentioned metal-polyamine nanocomposite, comprising the following steps:

[0010] The oleic acid dispersion, the manganese salt solution and the spermidine solution are respectively prepared by using a first solvent, and the pH of the spermidine solution is adjusted;

[0011] The manganese salt solution is added dropwise into the oleic acid dispersion under stirring to obtain a mixed solution; the spermidine solution is added dropwise into the mixed solution under ultrasonic condition, and the mixed solution is treated by ultrasonic dispersion and stirring to form a milky white suspension;

[0012] The light yellow solid obtained after centrifugal purification of the suspension is dissolved in a second solvent, and DSPE-PEG is added after stirring; the stirring is continued until the DSPE-PEG is completely dissolved, and a transparent light yellow liquid is obtained;

[0013] The second solvent is added to the transparent light yellow liquid, and the second solvent is removed by rotary evaporation after ultrasonic treatment, so that the metal-polyamine nanocomposite is obtained.

[0014] Further, the manganese salt is selected from one or more of manganese chloride, manganese sulfate and manganese acetate; the concentration of the manganese salt in the manganese salt solution is 0.1-0.5 mol / L.

[0015] Preferably, the manganese salt is manganese chloride.

[0016] Further, the concentration of spermidine in the spermidine solution is 0.05-0.5 mol / L, and the pH of the spermidine solution is adjusted to 4.0-5.5 by using 0.6-2 mmol / L formic acid.

[0017] Further, the mass ratio of DSPE-PEG to light yellow solid is 1:2-1:10.

[0018] Further, the first solvent is anhydrous ethanol, and the mass-volume ratio of the oleic acid to the first solvent is 0.5-10 g:1-5 mL; the second solvent is anhydrous chloroform.

[0019] Further, the power of ultrasonic dispersion is 300-500 W, the frequency is 20-40 kHz, and the time is 10-20 min; the dropwise adding speed of the manganese salt solution and the spermidine solution is 1-2 mL / min and 0.5-1 mL / min, respectively.

[0020] Further, the centrifugal speed in the centrifugal purification is 8000-12000 rpm for 10-15 min; and the temperature of rotary evaporation is 35 ± 2℃.

[0021] The application also provides a use of the metal-polyamine nanocomposite prepared by the above method in preparation of a drug for enhancing tumor radiotherapy.

[0022] Advantages

[0023] The nanocomposite constructed in the application adopts manganese ions, spermidine and other easily obtained raw materials, the preparation method is simple, the conditions are mild, complex equipment is not needed, the repeatability is good and the scale production is facilitated. The obtained nanocomposite has uniform particle size and high stability; the stability of spermidine is significantly improved through coordination assembly and DSPE-PEG surface coating, and the bioavailability of functional molecules is ensured.

[0024] The nanocomposite constructed in the application integrates the cGAS-STING pathway activator (Mn 2+ ) and spermidine, realizes the synergistic regulation of “DCs activation-T cell function enhancement”, overcomes the limitation of single functional material, and has an important application prospect in tumor radioimmunotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is a preparation flowchart of the metal-polyamine nanocomposite in the embodiment of the application;

[0026] Figure 2 It is a transmission electron microscope image of the MnFSP nanocomposite (the left side is the MnFSP nanocomposite prepared in example 1, and the right side is the MnFSP nanocomposite prepared in example 2) in the embodiment of the application;

[0027] Figure 3 It is a Fourier transform infrared spectrum and an X-ray diffraction pattern of the MnFSP nanocomposite in the embodiment of the application, wherein a is the Fourier transform infrared spectrum, and b is the X-ray diffraction pattern;

[0028] Figure 4 It is a stability and particle size distribution diagram of the MnFSP nanocomposite in the embodiment of the application, wherein a is the dispersion of the MnFSP nanocomposite in different solution systems, and b is the particle size change of the MnFSP nanocomposite in different solution systems;

[0029] Figure 5 It is a flow cytometry detection and quantitative analysis result of the MnFSP nanocomposite promoting dendritic cell maturation (CD80 and CD86 are key costimulatory molecules expressed on the surface of antigen-presenting cells) in the embodiment of the application, wherein a is the flow cytometry result, and b is the flow cytometry result statistics;

[0030] Figure 6 It is a detection result of the influence of the MnFSP nanocomposite on tumor necrosis factor-α secretion in the embodiment of the application;

[0031] Figure 7 It is a detection result of the influence of the MnFSP nanocomposite on CD8 +Flow cytometry detection and quantitative analysis results of T cell proliferation influence (CFSE is a fluorescent dye used to track cell division and proliferation), wherein a is flow cytometry results, and b is flow cytometry results statistics;

[0032] Figure 8 Tumor volume and body weight change results of the MnFSP nanocomposite in the tumor-bearing mouse model in the embodiments of the present application, wherein a is a tumor volume growth curve, and b is a mouse weight curve. DETAILED DESCRIPTION

[0033] The present application will be further described in conjunction with specific embodiments, and the following examples are not used to limit the present application, but only to illustrate the present application. Unless otherwise specified, the experimental methods used in the following examples are generally performed under conventional conditions, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0034] The model numbers and manufacturers of some reagents used in this embodiment are as follows:

[0035] Spermidine, brand: Aladdin, product number: S416439-1g;

[0036] Manganese chloride, brand: Macklin, product number: M799397-100g;

[0037] Manganese sulfate, brand: Macklin, product number: M75911-100ML;

[0038] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol DSPE-PEG, brand: Macklin, product number: N864074-10mg.

[0039] Example 1: Preparation of metal-polyamine nanocomposite (MnFSP)

[0040] As shown in Figure 1 , the embodiments of the present application provide a preparation method of a metal-polyamine nanocomposite, and the specific steps are as follows:

[0041] Take oleic acid (12 mmol) into a 15 mL centrifuge tube, add anhydrous ethanol to a total volume of 2.5 mL, and place the centrifuge tube in an ultrasonic cleaner (power: 400 W, frequency: 30 kHz) for 15 minutes to disperse into a transparent homogeneous oleic acid dispersion solution; take manganese chloride (0.2 mmol) into a 2 mL anhydrous ethanol solution, and then magnetically stir (400 rpm) for 15 minutes to obtain a manganese chloride solution; take spermidine (0.15 mmol) into 3 mL anhydrous ethanol, and then magnetically stir (400 rpm) for 10 minutes, dropwise add formic acid (1.2 mmol / L), and continue to stir (400 rpm) for 10 minutes, and finally adjust the pH to 4.0-5.5 to obtain a spermidine solution.

[0042] Slowly drop the manganese chloride solution (1 mL / min) into the oleic acid dispersion solution, and magnetically stir (500 rpm) for 5 minutes; then, under the condition of continuous ultrasonic environment (power: 400 W, frequency: 30 kHz), slowly drop the spermidine solution (0.5 mL / min) into the oleic acid dispersion solution, and ultrasonic for 20 minutes; after completion, continue to magnetically stir (500 rpm) for 30 minutes to obtain a milky white suspension.

[0043] Then, centrifuge the milky white suspension for 10 minutes (10000 rpm), resuspend the precipitate with anhydrous ethanol (15 mL) after discarding the supernatant, repeat the centrifugal washing for 3 times to obtain a light yellow solid. Further, add the light yellow solid into anhydrous chloroform (5 mL), magnetically stir (400 rpm) for 20 minutes, add DSPE-PEG (the mass ratio of DSPE-PEG to the light yellow solid is 1:5) and continue to stir for 30 minutes until completely dissolved into a transparent light yellow liquid.

[0044] Supplement anhydrous chloroform (5 mL) and further ultrasonic (power: 400 W, frequency: 30 kHz) for 10 minutes, and then transfer to a rotary evaporator (35°C) to remove anhydrous chloroform, thereby obtaining the MnFSP nanocomposite.

[0045] Example 2: Preparation of metal-polyamine nanocomposite (MnFSP)

[0046] The embodiment of the present application provides a preparation method of a metal-polyamine nanocomposite, and the specific steps are as follows:

[0047] Take oleic acid (12 mmol) into a 15 mL centrifuge tube, add anhydrous ethanol to a total volume of 2.5 mL, and place the centrifuge tube in an ultrasonic cleaner (power: 400 W, frequency: 30 kHz) for 15 minutes to disperse into a transparent homogeneous oleic acid dispersion solution; take manganese sulfate (0.2 mmol) into a 2 mL anhydrous ethanol solution, and then magnetically stir (400 rpm) for 15 minutes to obtain a manganese sulfate solution; take spermidine (0.15 mmol) into 3 mL anhydrous ethanol, and then magnetically stir (400 rpm) for 10 minutes, dropwise add formic acid (1.2 mmol / L), and continue to stir (400 rpm) for 10 minutes, and finally adjust the pH to 4.0-5.5 to obtain a spermidine solution.

[0048] Slowly drop the manganese sulfate solution (1 mL / min) into the oleic acid dispersion solution, and magnetically stir (500 rpm) for 5 minutes; then, under the condition of continuous ultrasonic environment (power: 400 W, frequency: 30 kHz), slowly drop the spermidine solution (0.5 mL / min) into the oleic acid dispersion solution, and ultrasonic for 20 minutes; after completion, continue to magnetically stir (500 rpm) for 30 minutes to obtain a milky white suspension.

[0049] Then, centrifuge the milky white suspension for 10 minutes (10000 rpm), resuspend the precipitate with anhydrous ethanol (15 mL) after discarding the supernatant, repeat the centrifugal washing for 3 times to obtain a light yellow solid. Further, add the light yellow solid into anhydrous chloroform (5 mL), magnetically stir (400 rpm) for 20 minutes, add DSPE-PEG (the mass ratio of DSPE-PEG to the light yellow solid is 1:5) and continue to stir for 30 minutes until completely dissolved into a transparent light yellow liquid.

[0050] Supplement anhydrous chloroform (5 mL) and further ultrasonic (power: 400 W, frequency: 30 kHz) for 10 minutes, and then transfer to a rotary evaporator (35°C) to remove anhydrous chloroform, thereby obtaining the MnFSP nanocomposite.

[0051] Example 3: Preparation of metal-polyamine nanocomposite (MnFSP)

[0052] The embodiment of the present application provides a preparation method of a metal-polyamine nanocomposite, and the specific steps are as follows:

[0053] Take oleic acid (12 mmol) into a 15 mL centrifuge tube, add anhydrous ethanol to a total volume of 2.5 mL, and place the centrifuge tube in an ultrasonic cleaner (power: 400 W, frequency: 30 kHz) for 20 minutes to disperse into a transparent homogeneous oleic acid dispersion solution; take manganese chloride (0.3 mmol) into a 2 mL anhydrous ethanol solution, and then magnetically stir (400 rpm) for 15 minutes to obtain a manganese chloride solution; take spermidine (1.5 mmol) into 3 mL anhydrous ethanol, and then magnetically stir (400 rpm) for 10 minutes, take formic acid (0.6 mmol / L) dropwise, and continue to stir (400 rpm) for 10 minutes, and finally adjust the pH to 4.0-5.5 to obtain a spermidine solution.

[0054] Slowly drop the manganese chloride solution (1 mL / min) into the oleic acid dispersion solution, and magnetically stir (500 rpm) for 5 minutes; then, under the condition of continuous ultrasonic environment (power: 400 W, frequency: 30 kHz), slowly drop the spermidine solution (0.5 mL / min) into the oleic acid dispersion solution, and ultrasonic for 20 minutes; after completion, continue to magnetically stir (500 rpm) for 30 minutes to obtain a milky white suspension.

[0055] Then, centrifuge the milky white suspension for 10 minutes (10000 rpm), resuspend the precipitate with anhydrous ethanol (15 mL) after discarding the supernatant, repeat the centrifugal washing for 3 times to obtain a light yellow solid. Further, add the light yellow solid into anhydrous chloroform (5 mL), magnetically stir (400 rpm) for 20 minutes, add DSPE-PEG (the mass ratio of DSPE-PEG to the light yellow solid is 1:2) and continue to stir for 30 minutes until completely dissolved into a transparent light yellow liquid.

[0056] Supplement anhydrous chloroform (5 mL) and further ultrasonic (power: 400 W, frequency: 30 kHz) for 10 minutes, and then transfer to a rotary evaporator (35°C) to remove anhydrous chloroform, thereby obtaining the MnFSP nanocomposite.

[0057] Example 4: Preparation of metal-polyamine nanocomposite (MnFSP)

[0058] The embodiment of the present application provides a preparation method of a metal-polyamine nanocomposite, and the specific steps are as follows:

[0059] Take oleic acid (12 mmol) into a 15 mL centrifuge tube, add anhydrous ethanol to a total volume of 2.5 mL, place the centrifuge tube in an ultrasonic cleaner (power: 400 W, frequency: 30 kHz) for 20 minutes, and disperse to form a transparent homogeneous oleic acid dispersion solution for standby; take manganese chloride (0.5 mmol) into a 2 mL anhydrous ethanol solution, then magnetically stir (400 rpm) for 15 minutes to obtain a manganese chloride solution; take spermidine (0.5 mmol) into 3 mL anhydrous ethanol, then magnetically stir (400 rpm) for 10 minutes, take formic acid (2 mmol / L) dropwise, and continue to stir (400 rpm) for 10 minutes, and finally adjust the pH to 4.0-5.5 to obtain a spermidine solution.

[0060] Slowly drop the manganese chloride solution (2 mL / min) into the oleic acid dispersion solution, and magnetically stir (500 rpm) for 5 minutes; then, under the condition of continuous ultrasonic environment (power: 400 W, frequency: 30 kHz), slowly drop the spermidine solution (1 mL / min) into the oleic acid dispersion solution, and ultrasonic for 20 minutes; after completion, continue to magnetically stir (500 rpm) for 30 minutes to obtain a milky white suspension.

[0061] Then, centrifuge the milky white suspension for 10 minutes (10000 rpm), resuspend the precipitate with anhydrous ethanol (15 mL) after discarding the supernatant, repeat the centrifugal washing for 3 times to obtain a light yellow solid. Further, add the light yellow solid into anhydrous chloroform (5 mL), magnetically stir (400 rpm) for 20 minutes, add DSPE-PEG (the mass ratio of DSPE-PEG to light yellow solid is 1:10) and continue to stir for 30 minutes until completely dissolved into a transparent light yellow liquid.

[0062] Supplement anhydrous chloroform (5 mL) and further ultrasonic (power: 400 W, frequency: 30 kHz) for 10 minutes, then transfer to a rotary evaporator (35°C), and remove the anhydrous chloroform by rotary evaporation to obtain the MnFSP nanocomposite.

[0063] Comparative Example 1: Preparation of metal-polyamine nanocomposite (MnFSP)

[0064] The present application provides a preparation method of a metal-polyamine nanocomposite, and the specific steps are as follows:

[0065] Take oleic acid (12 mmol) into a 15 mL centrifuge tube, add anhydrous ethanol to a total volume of 2.5 mL, and place the centrifuge tube in an ultrasonic cleaner (power: 400 W, frequency: 30 kHz) for 20 minutes to form a transparent homogeneous oleic acid dispersion solution; take manganese chloride (0.004 mmol) into a 2 mL anhydrous ethanol solution, then magnetically stir (400 rpm) for 15 minutes to obtain a manganese chloride solution; take spermidine (0.01 mmol) into 3 mL anhydrous ethanol, then magnetically stir (400 rpm) for 10 minutes, take formic acid (0.2 mmol / L) dropwise, and continue to stir (400 rpm) for 10 minutes, and finally adjust the pH to 4.0-5.5 to obtain a spermidine solution.

[0066] Slowly add the manganese chloride solution (0.2 mL / min) to the oleic acid dispersion solution, and magnetically stir (500 rpm) for 5 minutes; then, under continuous ultrasonic environment (power: 400 W, frequency: 30 kHz), slowly add the spermidine solution (0.1 mL / min) to the oleic acid dispersion solution, and ultrasonically for 20 minutes; after completion, continue to magnetically stir (500 rpm) for 30 minutes, and a milky white suspension cannot be obtained.

[0067] Structural characterization and performance test

[0068] 1. Transmission electron microscope (TEM) characterization of MnFSP nanocomposites

[0069] Take the MnFSP nanocomposites prepared in Examples 1 and 2, dilute to an appropriate concentration, and ultrasonically disperse; take a small amount of the diluted solution and drop it onto a clean copper mesh to dry naturally. Place the dried copper mesh under a transmission electron microscope, select multiple fields of view, and take images. As shown in Figure 2 , the MnFSP nanocomposites prepared in Example 1 exhibit clear spherical structure, with smooth and regular particle edges, uniform electron density in the core region, and no obvious agglomeration (left image in Figure 2 ); the MnFSP prepared in Example 2 has poorer size uniformity and regularity than the MnFSP prepared in Example 1 (right image in Figure 2 ), and the MnFSP prepared in Example 1 is more suitable for subsequent in vitro and in vivo experiments.

[0070] 2. Zeta potential characterization of MnFSP nanocomposites

[0071] The MnFSP nanocomposite prepared in Example 1 was diluted to a suitable concentration and then ultrasonically dispersed. A small amount of the dispersion was added to a DLS-specific cuvette and placed in a dynamic light scattering (DLS) instrument for particle size and polydispersity index (PDI) measurement. Subsequently, the same dispersion was added to a Zeta potential-specific sample cell, and the Zeta potential was measured using a laser particle size analyzer under the same conditions as before. The results showed that the particle size of the nanocomposite was 100–200 nm (see Example 1). Figure 4 The Zeta potential is -20 to -10 mV.

[0072] 3. Fourier transform infrared (FT-IR) characterization of MnFSP nanocomposites

[0073] The MnFSP nanocomposite prepared in Example 1 was mixed with potassium bromide, ground until homogeneous, and then pressed into transparent sheets. Simultaneously, potassium bromide pellets of MnFSP and DSPE-PEG were prepared as control samples. The samples were sequentially placed in a Fourier transform infrared spectrometer at 4000-400 cm⁻¹. -1 The scan was performed within the specified range. The results are as follows: Figure 3 As shown in Figure a, the characteristic absorption peaks of both MnFS and DSPE-PEG were simultaneously observed in the spectrum of the MnFSP sample, indicating that the two were successfully recombinated; furthermore, characteristic absorption peaks were observed at 1284, 1112, and 854 cm⁻¹. -1 The presence of characteristic absorptions for CN stretching, COC stretching, and Mn-N stretching indicates that the components form a stable composite structure through chemical interactions.

[0074] 4. X-ray diffraction (XRD) characterization of MnFSP nanocomposites

[0075] The MnFSP nanocomposite prepared in Example 1 was vacuum dried to constant weight, then ground into a fine powder, and subjected to X-ray diffraction analysis. The results are as follows: Figure 3 As shown in Figure b, the sample exhibits multiple sharp diffraction peaks at a specific angle, with clear peak shapes and stable baselines, indicating that MnFSP is a crystalline material.

[0076] 5. Stability testing of MnFSP nanocomposites

[0077] The MnFSP nanocomposite prepared in Example 1 was dispersed in ultrapure water, physiological saline (pH 7.4), and DMEM medium containing 10% fetal bovine serum, respectively, maintaining a consistent concentration. The nanocomposite was incubated at 37°C, and samples were taken at 0 h, 24 h, 48 h, and 72 h for observation and particle size determination using dynamic light scattering. The results are as follows: Figure 4 As shown, no visible precipitation was observed in any of the three media within 72 h, and the particle size did not change significantly, indicating that MnFSP has good stability in physiological media.

[0078] Example 5: In vitro experiment

[0079] MnFSP nanocomposites prepared in Example 1 were used for in vitro experiment.

[0080] 1. In vitro experiment of MnFSP nanocomposites promoting DCs maturation

[0081] Immature BMDCs were inoculated in 6-well plates, and MnFSP experimental groups (5, 10, 20, 40 µM), negative control group (PBS) and positive control group (LPS) were set. After 24 h of incubation, cells were collected and detected by flow cytometry, taking CD11c + as the gate, and analyzing the proportion of CD80 + CD86 + double positive cells (DCs maturation marker). As shown in Figure 5 , with the increase of MnFSP nanocomposite concentration, the proportion of CD80 + CD86 + double positive cells increased in a concentration-dependent manner, and the proportion of 40 µM MnFSP group was significantly higher than that of the negative control group, indicating that MnFSP could effectively promote the maturation of dendritic cells.

[0082] 2. Experiment of MnFSP nanocomposites promoting DCs to secrete proinflammatory cytokines

[0083] The culture supernatant of each group of cells was collected, centrifuged and stored at -20℃. The absorbance was measured at 450 nm using a mouse TNF-α ELISA kit, and the TNF-α concentration was calculated according to the standard curve. As shown in Figure 6 , the secretion amount of TNF-α in the supernatant of 40 µM MnFSP group was significantly higher than that of the negative control group, indicating that MnFSP could effectively activate the cGAS-STING signaling pathway and promote the release of downstream proinflammatory cytokines, thereby enhancing the immune response.

[0084] 3. Experiment of MnFSP nanocomposites mediating spermidine regulating CD8 + T cell proliferation

[0085] CD8 + T cells were sorted from mouse spleen, pre-activated by anti-CD3 / CD28 magnetic beads for 24 h, and then stained with CFSE for labeling. Cells were inoculated in 6-well plates, and MnFSP experimental groups (2, 5, 10 µM) and PBS control group were set. After 72 h of continuous culture, cells were collected, and CFSE fluorescence intensity was detected by flow cytometry to analyze cell proliferation index. As shown in Figure 7 , with the increase of MnFSP nanocomposite concentration, the proliferation index of CD8 +The T cell proliferation index gradually increased, and the proportion of the 10 mM MnFSP group was significantly higher than that of the PBS group, indicating that spermidine released by MnFSP can effectively promote CD8 + T cell proliferation Figure 7 ).

[0086] Example 6: In vivo experiment

[0087] Evaluation of the anti-tumor effect of MnFSP nanocomposites combined with radiotherapy

[0088] The in vivo anti-tumor effect of the MnFSP nanocomposites prepared in Example 1 was evaluated by constructing a CT26 mouse subcutaneous tumor model. The mice were randomly divided into four groups: PBS group, X-ray group, MnFSP group, and MnFSP combined with X-ray group (MnFSP + X-ray). After the start of treatment, the tumor volume was recorded every 2 days and the body weight of the mice was monitored, and the experiment lasted for 14 days.

[0089] The results are shown in Figure 8 The average tumor volume of the PBS group exceeded 1500 mm 3 ; the tumor growth of the MnFSP group was partially inhibited; the MnFSP + X-ray group showed the strongest tumor growth inhibition effect, and its tumor volume was significantly lower than that of the X-ray group. In addition, there was no significant change in the body weight of the mice in each group during the treatment, indicating that the MnFSP nanocomposites have good safety. The above results show that MnFSP nanocomposites combined with radiotherapy can significantly inhibit tumor growth.

[0090] The specific embodiments of the present application are described in detail above, but the scope of the present application is not limited to the above embodiments. For those skilled in the art, various adjustments, modifications or replacements can be made to the described embodiments without departing from the core principles and spirit of the present application, but these changes still fall within the protection scope of the present application.

Claims

1. A metal-polyamine nanocomposite, characterized in that, The nanocomposite is a core-shell structured nanoparticle constructed by coordinating divalent manganese ions and spermidine to form a hydrophobic core under oleic acid-mediated coordination, and then coating the surface with DSPE-PEG. The preparation method of the nanocomposite includes the following steps: Oleic acid dispersion, manganese salt solution and spermidine solution were prepared using the first solvent, and the pH of the spermidine solution was adjusted. Manganese salt solution was added dropwise to oleic acid dispersion and stirred to obtain a mixture; under ultrasonic conditions, spermidine solution was added dropwise to the mixture, and after ultrasonic dispersion and stirring, a milky white suspension was formed. The pale yellow solid obtained after centrifugation and purification of the suspension was dissolved in a second solvent, stirred, and then DSPE-PEG was added. Stirring was continued until completely dissolved to obtain a transparent pale yellow liquid. A second solvent was added to the transparent pale yellow liquid, and after ultrasonic treatment, the second solvent was removed by rotary evaporation to obtain the metal-polyamine nanocomposite.

2. The metal-polyamine nanocomposite according to claim 1, characterized in that, The divalent manganese ions are selected from one or more of manganese chloride, manganese sulfate, and manganese acetate; the particle size of the nanocomposite is 100~200 nm, and the zeta potential is -20~-10 mV.

3. A method for preparing the metal-polyamine nanocomposite according to any one of claims 1-2, characterized in that, Includes the following steps: Oleic acid dispersion, manganese salt solution and spermidine solution were prepared using the first solvent, and the pH of the spermidine solution was adjusted. Manganese salt solution was added dropwise to oleic acid dispersion and stirred to obtain a mixture; under ultrasonic conditions, spermidine solution was added dropwise to the mixture, and after ultrasonic dispersion and stirring, a milky white suspension was formed. The pale yellow solid obtained after centrifugation and purification of the suspension was dissolved in a second solvent, stirred, and then DSPE-PEG was added. Stirring was continued until completely dissolved to obtain a transparent pale yellow liquid. A second solvent was added to the transparent pale yellow liquid, and after ultrasonic treatment, the second solvent was removed by rotary evaporation to obtain the metal-polyamine nanocomposite.

4. The method for preparing the metal-polyamine nanocomposite according to claim 3, characterized in that, The manganese salt is selected from one or more of manganese chloride, manganese sulfate, and manganese acetate; the concentration of the manganese salt in the manganese salt solution is 0.1~0.5 mol / L.

5. The method for preparing the metal-polyamine nanocomposite according to claim 3, characterized in that, The concentration of spermidine in the spermidine solution is 0.05~0.5 mol / L, and the pH of the spermidine solution is adjusted to 4.0~5.5 with 0.6~2 mmol / L formic acid.

6. The method for preparing the metal-polyamine nanocomposite according to claim 3, characterized in that, The mass ratio of DSPE-PEG to the pale yellow solid is 1:2 to 1:

10.

7. The method for preparing the metal-polyamine nanocomposite according to claim 3, characterized in that, The first solvent is anhydrous ethanol, and the mass-to-volume ratio of oleic acid to the first solvent is 0.5~10g:1~5mL; the second solvent is anhydrous chloroform.

8. The method for preparing the metal-polyamine nanocomposite according to claim 3, characterized in that, The ultrasonic dispersion has a power of 300-500 W, a frequency of 20-40 kHz, and a duration of 10-20 min; the dropping rates of the manganese salt solution and the spermidine solution are 1-2 mL / min and 0.5-1 mL / min, respectively.

9. The method for preparing the metal-polyamine nanocomposite according to claim 3, characterized in that, The centrifugation purification process involves centrifuging at 8000~12000 rpm for 10~15 min; the rotary evaporation temperature is 35 ± 2℃.

10. The use of a metal-polyamine nanocomposite according to any one of claims 1-2 or a metal-polyamine nanocomposite prepared by the method according to any one of claims 3-9 in the preparation of a medicament for enhancing tumor radiotherapy.

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