Preparation method and application of multifunctional nano composite material

Abstract

The invention is applicable to the technical field of biomedicine, and provides a preparation method and application of a multifunctional nano composite material. The material has good biocompatibility and can be used as a safe preparation for tumor treatment; the preparation is simple, green and pollution-free; the application of the quercetin in anti-tumor immunotherapy is expanded; the probe has excellent fluorescence / computed tomography dual-mode imaging performance, and can be used for determining tumor boundaries and realizing accurate treatment guidance; the nano-material has excellent photo-thermal and photo-thermal enhanced catalytic performance, has the advantages of being minimally invasive, efficient, small in toxic and side effect, controllable and the like, and 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 and preventing the 2 '3'-cGAMP from being degraded, a cGAS-STING signal channel is efficiently activated, and finally the anti-tumor immune effect is enhanced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a preparation method of a multifunctional nanocomposite material and application thereof. Background Art

[0002] Head and neck squamous cell carcinoma (HNSCC) is a common malignancy prone to local recurrence and / or distant metastasis. Recurrent or metastatic (R / M) HNSCC leads to a poor prognosis for patients. Currently, immunotherapy is an important approach to improve the survival of R / M HNSCC patients. However, existing immunotherapies have limited efficacy, difficulty in establishing long-term immune memory, and are often associated with immune-related adverse events, significantly limiting their clinical effectiveness. Therefore, the development of long-acting immunotherapies with minimal side effects is crucial for improving the current treatment of R / M HNSCC.

[0003] Ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) is highly expressed in R / M HNSCC tissues, making it a promising therapeutic target. ENPP1 is also the primary hydrolase of 2'3'-cGAMP and may drive tumor immune evasion. 2'3'-cGAMP is the most important agonist of the cGAS-STING signaling pathway, potentially promoting tumor therapy through immune activation. Therefore, increasing 2'3'-cGAMP by interfering with ENPP1 may enhance the immunotherapy efficacy of R / M HNSCC. Quercetin, a natural medicinal plant active ingredient, has been shown to inhibit ENPP1 enzymatic activity.

[0004] In addition, multifunctional metal nanomaterials have attracted extensive attention due to their advantages in tumor diagnosis and treatment and immune activation. 2+) possess excellent catalytic properties and can be used in chemokinetic therapy (CDT) for tumors, inducing mitochondrial damage in tumor cells. They can also serve as metallo-immune adjuvants to directly promote the production of 2'3'-cGAMP, thereby activating the STING pathway. Gold-based nanomaterials possess excellent thermodynamic properties and can be used in photothermal therapy (PTT) and photothermal-enhanced CDT for tumors. They also possess excellent fluorescence (FL) and computed tomography (CT) imaging capabilities, supporting imaging-guided precision tumor treatment.

[0005] However, there is no research on the integration of gold-based nanomaterials and Mn 2+ The advantages of ions are fully combined, and quercetin is used as a raw material to load an effective method for preparing drugs related to the treatment of head and neck squamous cell carcinoma. In view of this, the present invention proposes a preparation method and application of a multifunctional nanocomposite material. Summary of the Invention

[0006] The purpose of the present invention is to provide a preparation method of a multifunctional nanocomposite material and its application, aiming to solve the problems raised in the above background technology.

[0007] The purpose of the present invention is achieved through the following technical solutions: A method for preparing a multifunctional nanocomposite material comprises the following steps: Ligand synthesis: 64 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 38 mg of N-hydroxysuccinimide were added to 40 mL of N,N-dimethylformamide, mixed, and 400 μL of 3-mercaptopropionic acid was added and stirred. An ethanol solution of polyethyleneimine was then added dropwise. After stirring at room temperature under an N2 atmosphere, the mixture was concentrated to 3 mL by vacuum distillation. The mixture 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, namely 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 (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 15 mL of (NH4)2S2O8 solution was added for reaction. The precipitate was removed and dialyzed to obtain PNIPAM-SS-AMPS nanospheres, namely NS; Synthesis of multifunctional nanocomposites: 0.5 mg of quercetin was dissolved in 1 mL of ethanol to prepare a solution, which was then added dropwise to 5 mL of 2 mg / mL NS solution. Then, 2 mL of 3 mg / mL AM solution was added. The mixture was stirred in the dark and then dialyzed to obtain a multifunctional nanocomposite, namely AM / Qu@NS.

[0008] A multifunctional nanocomposite material prepared according to the above preparation method.

[0009] Furthermore, the multifunctional nanocomposite material has photothermal properties under 808 nm laser irradiation.

[0010] Furthermore, the multifunctional nanocomposite material has fluorescence / computed tomography dual-mode imaging capability.

[0011] Furthermore, the multifunctional nanocomposite material can induce tumor cells to release 2'3'-cGAMP and activate the cGAS-STING signaling pathway.

[0012] A use of the multifunctional nanocomposite material described above in preparing a drug for treating primary, metastatic or recurrent head and neck squamous cell carcinoma.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The multifunctional nanocomposite prepared by the present invention possesses numerous advantages and effects. It has excellent biocompatibility and can be used as a safe preparation for tumor treatment. Its preparation is simple and environmentally friendly. It expands the application of quercetin, an active ingredient from a natural medicinal plant, in anti-tumor immunotherapy. Its excellent fluorescence / computed tomography dual-mode imaging performance enables clinical precision therapy, and its excellent imaging performance can be used to determine tumor boundaries and achieve imaging-guided precision therapy. Its excellent photothermal and photothermal-enhanced catalytic properties offer advantages such as minimally invasive and high efficacy, with minimal and controllable toxic side effects. It can effectively treat primary, metastatic, or recurrent head and neck squamous cell carcinoma by activating the immune system. By inducing tumor cells to release 2'3'-cGAMP, the released quercetin inhibits ENPP1, reducing 2'3'-cGAMP degradation, effectively activating the cGAS-STING signaling pathway, and ultimately enhancing anti-tumor immune effects, showing broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Figure 3 is a diagram of the photothermal and photothermal-enhanced catalytic performance of the multifunctional nanocomposite material; A is the photothermal performance curve of the multifunctional nanocomposite material under 808 nm laser irradiation with different power densities; B is the spectral curve of different systems.

[0015] Figure 2 FL / CT dual-mode imaging results of multifunctional nanocomposites; A is the linear relationship of fluorescence imaging; B is the linear relationship of CT imaging; C is in vivo fluorescence imaging; D is in vivo CT imaging.

[0016] Figure 3 The results of in vivo biocompatibility study of multifunctional nanocomposites; A represents the weight change of mice; B represents the histological analysis of major organs.

[0017] Figure 4 The results show the generation of 2'3'-cGAMP and the activation of the cGAS-STING signaling pathway; A represents the amount of 2'3'-cGAMP released; B represents proteins related to the cGAS-STING signaling pathway.

[0018] Figure 5 The in vivo treatment results of multifunctional nanocomposites on different types of HNSCC (primary, metastatic, and recurrent); A is the primary tumor; B is the metastatic tumor; and C is the recurrent tumor.

[0019] Figure 6 The morphological characteristics of gold-manganese nanodots (Au / MnNDs, AM for short) and multifunctional nanocomposites (AM / Qu@NS). DETAILED DESCRIPTION

[0020] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0021] The specific implementation of the present invention is described in detail below with reference to specific examples. The reagents used in the examples were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0022] Example 1: This example provides a method for preparing a multifunctional nanocomposite material, comprising the following steps: (1) Synthesis of ligand (SH-PEI, thiol-coupled polyethyleneimine): First, 64 mg of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide 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 stirred at room temperature for 30 min. 0.6 g of PEI (polyethyleneimine) was weighed and dissolved in 2 mL of ethanol. After dissolution, it was slowly added dropwise to the above system. Subsequently, the mixture was stirred at room temperature for 48 h under N2 atmosphere, and 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, and stored in a refrigerator at -20 °C for later use.

[0023] (2) Synthesis of gold-manganese nanodots (Au / MnNDs, AM): First, 200 μL of SH-PEI, 250 μL of HAuCl4 (50 mM tetrachloroauric acid), 50 μL of MnCl2 (50 mM), and 5 mL of H2O were added to a round-bottom flask. Then, 300 μL of hydrazine hydrate was added and the mixture was reacted at 80 °C for 4 h. After the reaction, the product was dialyzed in a dialysis bag (3500 Da Mw) for 8 h to obtain AM.

[0024] (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 deionized water, ultrasonically vibrated for 30 min to fully disperse the solids, and stirred for 30 min under N2 atmosphere. Then, the mixture was stirred in a 70 °C water bath until the system became clear, and 15 mL (NH4)2S2O8 solution (8.4 mg / mL) was slowly added. The system exhibited a blue-white opalescence. 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.

[0025] (4) Synthesis of multifunctional nanocomposite (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 a 2 mg / mL NS solution. Subsequently, 2 mL of a 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 in the dark at -20 °C.

[0026] (5) Under an accelerating voltage of 200 kV, the morphological characteristics of AM and AM / Qu@NS were obtained using a JEOL TECNAI F20 transmission electron microscope.

[0027] Example 2: Characterization and performance study of AM / Qu@NS; 1. Characterization of AM / Qu@NS: The fluorescence spectrum of the material was measured using a Shimadzu RF-5301 PC fluorescence spectrophotometer. The photothermal performance and stability of the material were evaluated using an 808 nm 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. Dual-mode imaging was performed in vivo and in vitro using a small animal in vivo imaging system and a small animal CT scanner.

[0028] 2. In vitro activation of 2'3'-cGAMP and cGAS-STING signaling pathways: The production of 2'3'-cGAMP was detected by enzyme-linked immunosorbent assay, and changes in the phosphorylation levels of key proteins in the cGAS-STING signaling pathway (STING, TBK1, and IRF3) were detected by Western blot.

[0029] 3. In vivo therapeutic efficacy and safety testing: Construction of primary, metastatic and postoperative recurrent HNSCC animal models.

[0030] The process of establishing primary and metastatic HNSCC animal models was as follows: each C57BL / 6J mouse was inoculated with tumors twice. First, 2×10 6 SCC7 cells (mouse head and neck squamous cell carcinoma cell line) were used to establish primary tumors. Six days later, 1.5×10 6 SCC7 cells established metastatic tumors.

[0031] The process of establishing the postoperative recurrent HNSCC animal model was as follows: first, 2×10 6 When the primary tumor tissue volume reached approximately 150 mm 3 The tumor was completely exposed by surgery, and then the tumor was extensively removed under clear vision. 3 ) and carefully suture the surgical incision.

[0032] Mice were randomly divided into six 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. Body weights were measured during treatment, and histological changes in major organs and tumor size were observed after treatment.

[0033] 4. Experimental results; Figure 1 The photothermal and photothermal-enhanced catalytic performance of AM / Qu@NS was demonstrated. Figure 1 As shown in A, the laser power density of 808 nm was 1.0 W / cm 2 , 1.5 W / cm 2 , 2.0 W / cm 2 ) irradiation, as time increases, the temperature changes corresponding to each power density increase, and the greater the power density, the more significant the temperature rise in the same time, reflecting 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 remains basically unchanged after the addition of AuNDs to the methylene blue (MB) + H2O2 system. However, when AM is added, the absorbance of MB decreases, indicating that AM can react with H2O2 to produce ·OH. This shows that AM has good catalytic performance. Importantly, the efficiency of Fenton-like reactions is closely related to the reaction temperature. The photothermal properties of AM prompted us to explore the effect of photothermal enhancement of Fenton-like reactions. The results showed that under the conditions of 808nm laser (1.5 W / cm 2 After irradiation (MB+H₂O₂+AM+NIR group), the absorbance of MB decreased significantly, indicating a significant increase in ·OH production. This result suggests that increasing temperature promotes the catalytic efficiency of AM. This indicates that increasing temperature can effectively enhance AM's ability to generate ·OH. This property makes Au / MnNDs promising for photothermally enhanced CDT in tumor therapy.

[0034] Figure 2 The FL / CT dual-mode imaging results of AM / Qu@NS were demonstrated. Figure 2 A in the middle shows that as the concentration of the material (0.4-2.0 μg / mL) increases, the fluorescence intensity increases linearly (R 2 =0.905), the brightness of the fluorescent spot of the object increased synchronously, indicating that the fluorescence signal is positively correlated with the concentration; Figure 2 Middle B shows that the CT intensity increases linearly with increasing concentration (R 2 =0.979), the brightness of the physical CT spot also increased, indicating that the CT signal is positively correlated with the concentration. After establishing a tumor-bearing mouse model and administering the drug through the tail vein, Figure 2 Middle C shows that the fluorescence signal at the tumor site (indicated by the arrow) of the tumor-bearing mouse is prominent, showing good in vivo fluorescence imaging; Figure 2 Center D shows that the tumor is clearly discernible in the CT image (arrowed), demonstrating high-quality in vivo CT imaging. In summary, AM / Qu@NS demonstrates that both fluorescence and CT signals increase with concentration, and offers excellent in vitro and in vivo imaging, demonstrating dual-mode FL / CT imaging capabilities.

[0035] Figure 3 The results of in vivo biocompatibility investigation of AM / Qu@NS are presented. Figure 3 Figure A shows that the weight of mice changed over time (0-10 days). The weight of mice in the control group and the NS, AM@NS, AM@NS+NIR, AM / Qu@NS, and AM / Qu@NS+NIR-treated groups 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 weight of mice. Figure 3Figure B shows HE staining of the heart, liver, spleen, lung, and kidney of mice. Comparison of the different treatment groups with the control group revealed no significant pathological damage (such as necrosis or inflammation) in the cellular morphology and tissue structure of the tissue sections of each organ, maintaining normal histological characteristics. Combined weight monitoring and organ histological analysis demonstrated that AM / Qu@NS did not significantly adversely affect mouse body weight or major organs when used in vivo, demonstrating good in vivo biocompatibility.

[0036] Figure 4 The results of the investigation into the generation of 2'3'-cGAMP and activation of the cGAS-STING signaling pathway are presented. Figure 4 Figure A shows 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 cell generation and release more 2'3'-cGAMP. Figure 4 Figure B shows Western Blot analysis of proteins involved in the cGAS-STING signaling pathway. The results showed that, compared with the 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) after treatment. The total protein (TBK1, IRF3, STING) bands in each group were relatively stable, indicating that AM / Qu@NS can activate the cGAS-STING signaling pathway in dendritic cells and participate in the signal transduction process by regulating the phosphorylation of key pathway proteins. In summary, AM / Qu@NS can induce tumor cells to release more 2'3'-cGAMP and activate the cGAS-STING signaling pathway in dendritic cells.

[0037] Figure 5 The in vivo therapeutic results of AM / Qu@NS on different types of HNSCC (primary, metastatic, and recurrent) were demonstrated. Figure 5 Figure A shows that compared with the Control, NS, AM@NS, AM@NS+NIR, and AM / Qu@NS groups, the volume of the primary tumor sample 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 5Figure B shows that although the metastatic tumor was not directly treated with photothermal or other treatments, the volume of metastatic tumor samples in the AM / Qu@NS+NIR group was significantly smaller than that in other groups, reflecting the good therapeutic effect of immune activation on metastatic tumors under this method. Figure 5 Figure C shows that the recurrent tumor almost disappeared after treatment with AM / Qu@NS+NIR, indicating that AM / Qu@NS combined with NIR is effective in treating recurrent tumors. In summary, AM / Qu@NS combined with near-infrared NIR is effective in treating primary, metastatic, and recurrent HNSCC, significantly inhibiting the growth of different tumor types.

[0038] Figure 6 The morphological characteristics of AM and AM / Qu@NS are demonstrated. Figure 6 As shown in Figure A, the synthesized AM has a spherical morphology and the particle size is within 5 nm. Figure 6 Figure B shows that the prepared AM / Qu@NS is uniformly dispersed and has a diameter of about 50 nm.

[0039] The above are only preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the concept of the present invention. These should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A method for preparing a multifunctional nanocomposite material, characterized in that: The following steps are involved: Ligand synthesis: 64 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 38 mg of N-hydroxysuccinimide were added to 40 mL of N,N-dimethylformamide, mixed, and 400 μL of 3-mercaptopropionic acid was added and stirred. An ethanol solution of polyethyleneimine was then added dropwise. After stirring at room temperature under an N2 atmosphere, the mixture was concentrated to 3 mL by vacuum distillation. The mixture 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, namely 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 (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 15 mL of (NH4)2S2O8 solution was added for reaction. The precipitate was removed and dialyzed to obtain PNIPAM-SS-AMPS nanospheres, namely NS; Synthesis of multifunctional nanocomposites: 0.5 mg of quercetin was dissolved in 1 mL of ethanol to prepare a solution, which was then added dropwise to 5 mL of 2 mg / mL NS solution. Then, 2 mL of 3 mg / mL AM solution was added. The mixture was stirred in the dark and then dialyzed to obtain a multifunctional nanocomposite, namely AM / Qu@NS.

2. A multifunctional nanocomposite material obtained according to the preparation method of claim 1.

3. The multifunctional nanocomposite material according to claim 2, characterized in that The multifunctional nanocomposite material has photothermal properties under 808 nm laser irradiation.

4. The multifunctional nanocomposite material according to claim 2, characterized in that The multifunctional nanocomposite material has fluorescence / computed tomography dual-mode imaging capability.

5. The multifunctional nanocomposite material according to claim 2, characterized in that The multifunctional nanocomposite material can induce tumor cells to release 2'3'-cGAMP and activate the cGAS-STING signaling pathway.

6. Use of the multifunctional nanocomposite material according to any one of claims 2 to 5 in the preparation of a drug for treating primary, metastatic or recurrent head and neck squamous cell carcinoma.

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