A method for preparing multifunctional nanocomposite materials and its application

Abstract

This invention relates to the field of biomedical technology and provides a method for preparing a multifunctional nanocomposite material and its applications. The material exhibits good biocompatibility and can be used as a safe agent for tumor treatment; its preparation is simple, green, and pollution-free; it expands the application of quercetin in anti-tumor immunotherapy; it possesses excellent fluorescence / computed tomography dual-mode imaging performance, which can be used to determine tumor boundaries and achieve precise treatment guidance; it has excellent photothermal and photothermally enhanced catalytic properties, possessing advantages such as minimally invasive and highly efficient treatment with few and controllable toxic side effects; it can effectively treat primary, metastatic, or recurrent head and neck squamous cell carcinoma by activating immunity; and by inducing tumor cells to release 2'3'-cGAMP and preventing its degradation, it efficiently activates the cGAS-STING signaling pathway, ultimately enhancing the anti-tumor immune effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a method for preparing a multifunctional nanocomposite material and its application. Background Technology

[0002] Head and neck squamous cell carcinoma (HNSCC), a common malignant tumor, is highly prone to local recurrence and / or distant metastasis. Recurrent or metastatic (R / M) HNSCC leads to a poor prognosis for patients. Currently, immunotherapy is one of the important means to improve the survival of R / M HNSCC patients. However, existing immunotherapies have problems such as limited efficacy, difficulty in building long-term immune memory, and frequent occurrence of immune-related adverse events, which greatly limit their clinical application. Therefore, developing long-acting immunotherapies with fewer side effects is of great significance for improving the current treatment status of R / M HNSCC.

[0003] Exonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) is highly expressed in relapsed / malignant neovascularization (R / M) chemoradioma (HNSCC) tissues, making it a potential therapeutic target. ENPP1 is also the main hydrolase of 2'3'-cGAMP and may drive tumor immune escape. 2'3'-cGAMP is a crucial agonist of the cGAS-STING signaling pathway, which can activate the immune system to achieve tumor therapy. Therefore, intervening in ENPP1 to increase 2'3'-cGAMP could enhance the immunotherapeutic effect in R / M HNSCC, and quercetin, a natural medicinal plant active ingredient, has been shown to inhibit ENPP1 enzyme activity.

[0004] Furthermore, multifunctional metal nanomaterials have attracted widespread attention due to their advantages in tumor diagnosis and treatment, as well as immune activation. Divalent manganese ions (Mn) 2+Gold-based nanomaterials possess excellent catalytic properties, enabling their application in chemokinetic therapy (CDT) for tumors and inducing mitochondrial damage in tumor cells. They can also act as metal adjuvants to directly promote the production of 2'3'-cGAMP, thereby activating the STING pathway. Furthermore, gold-based nanomaterials exhibit superior thermodynamic properties, making them suitable for photothermal therapy (PTT) and photothermally enhanced CDT for tumors. They also possess excellent fluorescence (FL) and computed tomography (CT) imaging capabilities, providing support for image-guided precision tumor treatment.

[0005] However, there is currently no combination of gold-based nanomaterials and Mn. 2+ This invention leverages the advantages of ions and utilizes quercetin as a raw material for the preparation of drugs related to the treatment of head and neck squamous cell carcinoma. Therefore, this invention proposes a method for preparing a multifunctional nanocomposite material and its applications. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing multifunctional nanocomposite materials and their applications, aiming to solve the problems mentioned in the background art.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A method for preparing a multifunctional nanocomposite material includes the following steps:

[0009] Synthesis of the ligand: 64 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 38 mg of N-hydroxysuccinimide were added to 40 mL of N,N-dimethylformamide and mixed. 400 μL of 3-mercaptopropionic acid was added and stirred. Then, an ethanol solution of polyethyleneimine was added dropwise. After stirring at room temperature under N2 atmosphere, the mixture was concentrated to 3 mL by vacuum distillation. The solution was washed three times with 27 mL of acetone and 9 mL of chloroform. The product was collected by centrifugation and dispersed in 3 mL of H2O to obtain the ligand, SH-PEI. The ethanol solution of polyethyleneimine was prepared by dissolving 0.6 g of polyethyleneimine in 2 mL of ethanol.

[0010] Synthesis of gold-manganese nanodots: 200 μL SH-PEI, 250 μL HAuCl4, 50 μL MnCl2 and 5 mL H2O were added to a round-bottom flask, and 300 μL hydrazine hydrate was added. After the reaction was completed, gold-manganese nanodots, namely AM, were obtained by dialysis.

[0011] Synthesis of PNIPAM-SS-AMPS nanospheres: 0.2586 g of 2-acrylamido-2-methylpropanesulfonic acid, 1.7415 g of N-isopropylacrylamide, 79.0 mg of benzyltriethylammonium chloride and 2 mg of sodium dodecyl sulfate were added to 185 mL of deionized water, ultrasonically dispersed and stirred under N2 atmosphere, stirred in a water bath until clear, and after reacting with 15 mL of (NH4)2S2O8 solution, the precipitate was removed and dialysis was performed to obtain PNIPAM-SS-AMPS nanospheres, i.e., NS;

[0012] Synthesis of multifunctional nanocomposite materials: 0.5 mg of quercetin was dissolved in 1 mL of ethanol to prepare a solution, and then the solution was added dropwise to 5 mL of 2 mg / mL NS solution. Then, 2 mL of 3 mg / mL AM solution was added, and the mixture was stirred in the dark and then dialyzed to obtain the multifunctional nanocomposite material, namely AM / Qu@NS.

[0013] A multifunctional nanocomposite material prepared according to the preparation method described above.

[0014] Furthermore, the multifunctional nanocomposite material exhibits photothermal properties under 808 nm laser irradiation.

[0015] Furthermore, the multifunctional nanocomposite material possesses dual-mode imaging capabilities of fluorescence and computed tomography.

[0016] Furthermore, the multifunctional nanocomposite material can induce tumor cells to release 2'3'-cGAMP and activate the cGAS-STING signaling pathway.

[0017] The application of the multifunctional nanocomposite material described above in the preparation of drugs for treating primary, metastatic, or recurrent head and neck squamous cell carcinoma.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] The multifunctional nanocomposite material prepared in this invention possesses numerous advantages and effects. It exhibits good biocompatibility, making it a safe formulation for tumor treatment; the preparation process is simple, green, and pollution-free; it expands the application of quercetin, a natural medicinal plant active ingredient, in anti-tumor immunotherapy; it possesses excellent fluorescence / computed tomography dual-mode imaging performance, potentially enabling precise clinical treatment; its superior imaging performance can be used to determine tumor boundaries, achieving image-guided precision treatment; it has excellent photothermal and photothermally enhanced catalytic properties, offering advantages such as minimally invasive and highly efficient treatment with few and controllable toxic side effects; it can effectively treat primary, metastatic, or recurrent head and neck squamous cell carcinoma by activating immunity; by inducing tumor cells to release 2'3'-cGAMP, the released quercetin inhibits ENPP1 to reduce 2'3'-cGAMP degradation, efficiently activating the cGAS-STING signaling pathway, ultimately enhancing the anti-tumor immune effect, demonstrating broad clinical application prospects. Attached Figure Description

[0020] Figure 1 The graph shows the photothermal and photothermally enhanced catalytic properties of the multifunctional nanocomposite material; where A is the photothermal performance curve of the multifunctional nanocomposite material under irradiation with different power densities of 808 nm laser; and B is the spectral curve of different systems.

[0021] Figure 2 The results show the FL / CT dual-mode imaging of multifunctional nanocomposite materials; where A represents the linear relationship of fluorescence imaging; B represents the linear relationship of CT imaging; C represents in vivo fluorescence imaging; and D represents in vivo CT imaging.

[0022] Figure 3 The results show the in vivo biocompatibility of the multifunctional nanocomposite material; A represents the changes in mouse body weight; B represents the histological analysis of major organs.

[0023] Figure 4 The results of the investigation into the generation of 2'3'-cGAMP and the activation of the cGAS-STING signaling pathway are shown; where A represents the amount of 2'3'-cGAMP released; and B represents proteins related to the cGAS-STING signaling pathway.

[0024] Figure 5 The results of in vivo treatment of different types of HNSCC (primary, metastatic, and recurrent) with multifunctional nanocomposite materials are shown; where A represents primary tumors; B represents metastatic tumors; and C represents recurrent tumors.

[0025] Figure 6 The morphological characteristics of gold / manganese nanodots (Au / MnNDs, abbreviated as AM) and multifunctional nanocomposite materials (AM / Qu@NS) are shown. Detailed Implementation

[0026] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0027] The specific implementation of the present invention will be described in detail below with reference to specific embodiments. The reagents used in the embodiments were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0028] Example 1: This example provides a method for preparing a multifunctional nanocomposite material, including the following steps:

[0029] (1) Synthesis of the ligand (SH-PEI, mercapto-coupled polyethyleneimine): First, 64 mg of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimine hydrochloride) and 38 mg of NHS (N-hydroxysuccinimide) were weighed and added to 40 mL of DMF (N,N-dimethylformamide), and mixed thoroughly. Then, 400 μL of MPA (3-mercaptopropionic acid) was added, and the mixture was stirred at room temperature for 30 min. Separately, 0.6 g of PEI (polyethyleneimine) was weighed, dissolved in 2 mL of ethanol, and slowly added dropwise to the above system. Subsequently, the mixture was stirred at room temperature under N2 atmosphere for 48 h, then concentrated to 3 mL by vacuum distillation, and washed three times with 27 mL of acetone and 9 mL of chloroform. The product was collected by centrifugation at 8800 rpm for 15 min, redispersed in 3 mL of H2O to obtain SH-PEI, which was stored in a -20 °C refrigerator for later use.

[0030] (2) Synthesis of gold / manganese nanodots (Au / MnNDs, abbreviated as AM): First, 200 μL of SH-PEI, 250 μL of HAuCl4 (tetrachloroauric acid, 50 mM), 50 μL of MnCl2 (50 mM) and 5 mL of H2O were added to a round-bottom flask, followed by the addition of 300 μL of hydrazine hydrate. The reaction was carried out continuously at 80 °C for 4 h. After the reaction was completed, the product was dialyzed in a dialysis bag (3500 Da Mw) for 8 h to obtain AM.

[0031] (3) Synthesis of PNIPAM-SS-AMPS nanospheres (NS): First, 0.2586 g AMPS (2-acrylamido-2-methylpropanesulfonic acid), 1.7415 g NIPAM (N-isopropylacrylamide), 79.0 mg BAC (benzyltriethylammonium chloride), and 2 mg SDS (sodium dodecyl sulfate) were added to 185 mL of deionized water. The mixture was ultrasonically vibrated for 30 min to ensure thorough dispersion of the solids, and then stirred for 30 min under a N2 atmosphere. Next, the mixture was stirred thoroughly in a 70 °C water bath until the system became clear. 15 mL of (NH4)2S2O8 solution (8.4 mg / mL) was slowly added, and the system exhibited a bluish-white opalescent appearance. After reacting in a 70 °C water bath for 12 h, a white turbid solution was obtained. The system was cooled to room temperature, and the white precipitate was removed. The resulting solution was dialyzed for 48 h to obtain NS, which was stored in a -20 °C refrigerator for later use.

[0032] (4) Synthesis of multifunctional nanocomposite material (AM / Qu@NS): 0.5 mg of quercetin (Qu) was dissolved in 1 mL of ethanol to prepare a solution with a mass concentration of 0.5 mg / mL. This solution was then added dropwise to 5 mL of 2 mg / mL NS solution. Subsequently, 2 mL of 3 mg / mL AM solution was added to the mixture, and the mixture was stirred for 12 h in the dark. The resulting solution was dialyzed for 12 h to obtain AM / Qu@NS, which was stored at -20 °C in the dark.

[0033] (5) Under the accelerating voltage of 200 kV, the morphological characteristics of AM and AM / Qu@NS were obtained by JEOL TECNAI F20 transmission electron microscope.

[0034] Example 2: Characterization and performance study of AM / Qu@NS;

[0035] I. Characterization methods for AM / Qu@NS: The fluorescence spectrum of the material was measured using a Shimadzu RF-5301 PC fluorescence spectrophotometer. The photothermal properties and photothermal stability of the material were evaluated using an 808 nm wavelength laser and an infrared thermal imaging camera. The ability of AM / Qu@NS to generate ·OH via a Fenton-like reaction was evaluated using a methylene blue probe. In vitro and in vivo detection was performed using a small animal in vivo imaging system and a small animal CT scanner system, respectively, employing dual-mode imaging.

[0036] II. In vitro activation of 2'3'-cGAMP and cGAS-STING signaling pathways: The generation of 2'3'-cGAMP was detected by enzyme-linked immunosorbent assay (ELISA), and the phosphorylation levels of key proteins (STING, TBK1, and IRF3) in the cGAS-STING signaling pathway were detected by Western blot.

[0037] III. In vivo treatment efficacy and safety testing: Constructing animal models of primary, metastatic, and postoperative recurrent HNSCC.

[0038] The process for establishing primary and metastatic HNSCC animal models was as follows: Each C57BL / 6J mouse underwent two tumor inoculations. First, 2×10⁻⁶ tumor cells were subcutaneously injected into the left back of the mouse. 6 A primary tumor was established using SCC7 cells (mouse head and neck squamous cell carcinoma carcinoma line). Six days later, 1.5 × 10⁻⁶ cells were subcutaneously injected into the right back. 6 Establish metastatic tumors using SCC7 cells.

[0039] The process of establishing an animal model of recurrent postoperative HNSCC was as follows: First, 2×10⁻⁶ mice were subcutaneously injected into the left back. 6 The primary tumor was established using SCC7 cells. The primary tumor tissue volume reached approximately 150 mm². 3 The tumor is fully exposed surgically, and then extensively removed under clear visualization. Any remaining tumor tissue (approximately 1 mm) is intentionally removed. 3 ), and carefully suture the surgical incision.

[0040] Mice were randomly divided into 6 groups: model control group (Control group), NS group, AM@NS group, AM@NS+NIR treatment group, AM / Qu@NS group, and AM / Qu@NS+NIR treatment group. Mouse body weight was measured during treatment, histological changes in major organs were observed after treatment, and tumor size was monitored.

[0041] IV. Experimental Results;

[0042] Figure 1 The photothermal and photothermally enhanced catalytic properties of AM / Qu@NS were demonstrated. For example... Figure 1 As shown in Figure A, 808 nm laser light was used at different laser power densities (1.0 W / cm²). 2 1.5 W / cm 2 2.0 W / cm 2 Under irradiation, the temperature changes corresponding to each power density all increased with time, and the higher the power density, the more significant the temperature rise within the same time period, demonstrating that AM / Qu@NS has good photothermal performance, and the higher the power density, the more prominent the photothermal heating effect. Figure 1As shown in Figure B, the absorbance of MB in the methylene blue (MB) + H₂O₂ system remained essentially unchanged after the addition of AuNDs. However, the absorbance of MB decreased upon the addition of AM, indicating that AM can react with H₂O₂ to produce ·OH. This demonstrates that AM possesses good catalytic performance. Importantly, the efficiency of the Fenton-like reaction is closely related to the reaction temperature. The photothermal properties of AM prompted us to investigate the effect of photothermal enhancement on the Fenton-like reaction. The results show that at an 808 nm laser (1.5 W / cm²), the absorbance of MB remained essentially unchanged after the addition of AuNDs. 2 After irradiation (MB+H2O2+AM+NIR group), the absorbance of MB decreased significantly, indicating a substantial increase in the production of ·OH. This result demonstrates that increased temperature promotes the catalytic efficiency of AM. Therefore, increasing temperature can effectively enhance the ability of AM to produce ·OH. This characteristic makes Au / MnNDs a promising candidate for photothermally enhanced CDT in tumor therapy.

[0043] Figure 2 The results of FL / CT dual-mode imaging of AM / Qu@NS are presented. Figure 2 As shown in Figure A, the fluorescence intensity increased linearly with increasing material concentration (0.4-2.0 μg / mL) (RA). 2 =0.905), the brightness of the actual fluorescent spot increased synchronously, reflecting a positive correlation between fluorescence signal and concentration; Figure 2 The B-mode image shows that CT intensity increases linearly with increasing concentration (R0). 2 =0.979), and the brightness of the CT spot also increased, indicating a positive correlation between CT signal and concentration. After establishing a tumor-bearing mouse model and administering the drug via tail vein, Figure 2 The C-ray image shows that the tumor site (indicated by the arrow) in tumor-bearing mice exhibits prominent fluorescence signal, showing good in vivo fluorescence imaging. Figure 2 The mid-D diagram shows that the tumor is clearly identifiable in the CT image (indicated by the arrow), demonstrating high-quality in vivo CT imaging. In summary, both the AM / Qu@NS fluorescence signal and CT signal increase with concentration, and the in vivo and in vitro imaging effects are good, exhibiting FL / CT dual-mode imaging capabilities.

[0044] Figure 3 The results of the in vivo biocompatibility study of AM / Qu@NS are presented. Figure 3 The results showed that the body weight of mice changed over time (0-10 days). The body weight of mice in the Control group and the groups treated with NS, AM@NS, AM@NS+NIR, AM / Qu@NS, and AM / Qu@NS+NIR fluctuated within the normal range (17-19 g), with no obvious abnormal decrease or disorder, indicating that the materials and related treatments did not cause adverse changes in the body weight of mice. Figure 3In our study, HE staining was used to observe the major organs of mice: heart, liver, spleen, lung, and kidney. Compared with the control group, no obvious pathological damage (such as necrosis or inflammation) was observed in the cell morphology and tissue structure of the tissue sections of each organ in the different treatment groups, and normal histological characteristics were maintained. Combined with weight monitoring and organ histological analysis, it is shown that AM / Qu@NS does not have significant adverse effects on mouse weight or major organs when administered in vivo, demonstrating good in vivo biocompatibility.

[0045] Figure 4 The results of the study on the generation of 2'3'-cGAMP and the activation of the cGAS-STING signaling pathway were presented. Figure 4 The results showed that, compared with the Control, NS, AM@NS, AM@NS+NIR, and AM / Qu@NS groups, the amount of 2'3'-cGAMP released by tumor cells was significantly increased after AM / Qu@NS+NIR treatment, indicating that AM / Qu@NS combined with NIR can effectively induce tumor cells to generate and release more 2'3'-cGAMP. Figure 4 Western blotting analysis of cGAS-STING signaling pathway-related proteins revealed that, compared to other groups, the AM / Qu@NS + NIR group showed the most significant upregulation of Phospho-TBK1, Phospho-IRF3, and Phospho-STING (phosphorylated forms of key pathway proteins, representing protein activation). Meanwhile, the total protein (TBK1, IRF3, STING) bands remained relatively stable across all groups. This indicates that AM / Qu@NS can activate the cGAS-STING signaling pathway in dendritic cells, participating in signal transduction by regulating the phosphorylation of key pathway proteins. In conclusion, AM / Qu@NS can induce tumor cells to release more 2'3'-cGAMP and activate the cGAS-STING signaling pathway in dendritic cells.

[0046] Figure 5 The study demonstrated the in vivo treatment results of AM / Qu@NS for different types of HNSCC (primary, metastatic, and recurrent). Figure 5 The results showed that, compared with the Control, NS, AM@NS, AM@NS+NIR, and AM / Qu@NS groups, the volume of primary tumor samples was significantly smaller after treatment with AM / Qu@NS+NIR, indicating that AM / Qu@NS combined with NIR can effectively inhibit the growth of primary tumors. Figure 5The results showed that, although no photothermal treatment was applied to the metastatic tumors, the volume of metastatic tumor samples in the AM / Qu@NS+NIR group was significantly smaller than that in other groups, demonstrating the good therapeutic effect of immune activation on metastatic tumors under this approach. Figure 5 The results showed that recurrent tumors almost disappeared after AM / Qu@NS+NIR treatment, indicating that AM / Qu@NS combined with NIR can effectively treat recurrent tumors. In summary, AM / Qu@NS combined with near-infrared NIR can effectively treat primary, metastatic and recurrent HNSCC, and has a significant inhibitory effect on the growth of different types of tumors.

[0047] Figure 6 The morphological features of AM and AM / Qu@NS are shown. Figure 6 As shown in Figure A, the synthesized AM has a spherical morphology with a particle size of less than 5 nm. Figure 6 Figure B shows that the prepared AM / Qu@NS is uniformly dispersed with a diameter of approximately 50 nm.

[0048] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. The application of a multifunctional nanocomposite material in the preparation of drugs for treating primary, metastatic, or recurrent head and neck squamous cell carcinoma, characterized in that, The multifunctional nanocomposite material is prepared by the following steps: Synthesis of the ligand: 64 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 38 mg of N-hydroxysuccinimide were added to 40 mL of N,N-dimethylformamide and mixed. 400 μL of 3-mercaptopropionic acid was added and stirred. Then, an ethanol solution of polyethyleneimine was added dropwise. After stirring at room temperature under N2 atmosphere, the mixture was concentrated to 3 mL by vacuum distillation and washed three times with 27 mL of acetone and 9 mL of chloroform. The product was collected by centrifugation and dispersed in 3 mL of H2O to obtain the ligand, i.e., SH-PEI. The ethanol solution of polyethyleneimine was prepared by dissolving 0.6 g of polyethyleneimine in 2 mL of ethanol. Synthesis of gold-manganese nanodots: 200 μL SH-PEI, 250 μL HAuCl4, 50 μL MnCl2 and 5 mL H2O were added to a round-bottom flask, and 300 μL hydrazine hydrate was added. After the reaction was completed, gold-manganese nanodots, namely AM, were obtained by dialysis. Synthesis of PNIPAM-SS-AMPS nanospheres: 0.2586 g of 2-acrylamido-2-methylpropanesulfonic acid, 1.7415 g of N-isopropylacrylamide, 79.0 mg of benzyltriethylammonium chloride and 2 mg of sodium dodecyl sulfate were added to 185 mL of deionized water, ultrasonically dispersed and stirred under N2 atmosphere, stirred in a water bath until clear, and then reacted with 15 mL of (NH4)2S2O8 solution. After removing the precipitate and dialysis, PNIPAM-SS-AMPS nanospheres, i.e., NS, were obtained. Synthesis of multifunctional nanocomposite materials: 0.5 mg of quercetin was dissolved in 1 mL of ethanol to prepare a solution, and then the solution was added dropwise to 5 mL of 2 mg / mL NS solution. Then, 2 mL of 3 mg / mL AM solution was added, and after stirring in the dark, the mixture was dialyzed to obtain the multifunctional nanocomposite material, namely AM / Qu@NS. The multifunctional nanocomposite material exhibits photothermal properties under 808 nm laser irradiation, possesses dual-mode imaging capabilities of fluorescence / computed tomography, and can induce tumor cells to release 2'3'-cGAMP and activate the cGAS-STING signaling pathway.

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