PYT (at) Zn-MOF (at) siRNA nanoparticle, composite nanoparticle, and preparation method and application of PYT (at) Zn-MOF (at) siRNA nanoparticle and composite nanoparticle

Abstract

The invention belongs to the technical field of nano biomedicine, and particularly relates to PYT (at) Zn-MOF (at) siRNA nanoparticles, composite nanoparticles and a preparation method and application of the PYT (at) Zn-MOF (at) siRNA nanoparticles and the composite nanoparticles. The invention relates to a PYT (at) Zn-MOF (at) siRNA (small interfering Ribonucleic Acid) nano particle. In a tumor acid environment, Zn-MOF releases zinc ions, PYT serves as a zinc ion carrier to transfer zinc into cells, and zinc overload in the cells is caused; meanwhile, the loaded siRNA can recognize, cut and degrade target mRNA and block the expression of zinc ion transporter SLC30A1, so that zinc ion efflux is reduced, zinc overload is aggravated, active oxygen increase and oxidative stress are triggered, mitochondrial membrane potential is reduced, mitochondrial autophagy and immunogenic cell death are induced, and tumor cells are effectively removed. In addition, after the PYT is combined with the Zn-MOF and the siRNA, the antibacterial effect is remarkably improved. The nano particles show an excellent anti-tumor effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano biomedicine. More specifically, it relates to a PYT@Zn-MOF@siRNA nanoparticle, a composite nanoparticle, and their preparation methods and applications. Background Art

[0002] Oral squamous cell carcinoma (OSCC) is one of the most common head and neck malignancies. Approximately 64% of patients are in the advanced stage of the disease at the time of diagnosis, seriously affecting the quality of life of patients. Although chemotherapy and immunotherapy have made certain progress in the treatment of OSCC in recent years, while chemotherapy drugs effectively kill cancer cells, they inevitably cause damage to normal cells. Their specificity is limited and the side effects are large, and the overall efficacy still needs to be further improved. Therefore, it is particularly urgent to explore new and efficient strategies to combat OSCC and improve the prognosis of patients.

[0003] Dysregulation of zinc ion homeostasis has been identified as a key feature in the progression of OSCC. Zinc ions play multiple roles in cells, including regulating key biological processes such as gene expression, signal transduction, and enzymatic catalysis. Compared with normal cells, the zinc ion level in tumor cells is significantly increased, leading to zinc homeostasis imbalance. The disruption of zinc homeostasis not only disrupts intracellular signaling pathways, thereby promoting the proliferation and metastasis of tumor cells, but also affects immune cells in the tumor microenvironment, exacerbating the immune escape phenomenon. Therefore, metal ion interference therapy targeting the regulation of zinc ion levels in tumors is regarded as a potential new approach for the prevention and treatment of OSCC.

[0004] In addition to dysregulation of zinc ion homeostasis, the progression of OSCC is also affected by the oral microbiota. Imbalance of the oral microbial community may accelerate the process of OSCC through multiple mechanisms. Specifically, certain specific oral bacteria can promote tumor development by activating inflammatory responses, inducing immunosuppression, or directly acting on the biological characteristics of tumor cells. The presence of these bacteria not only exacerbates the inflammatory response in the tumor microenvironment, but may also interfere with the function of immune cells, further promoting tumor progression and enhancing the immune escape ability of tumor cells. Thus, the imbalance of the oral microbiota plays an important role in the occurrence and development of OSCC.

[0005] In view of the important roles of zinc ion homeostasis imbalance and the oral microbiota in the progression of OSCC, there is an urgent need to develop a treatment strategy that can simultaneously target zinc ion homeostasis dysregulation and oral microbiota imbalance, with the expectation of efficiently inhibiting the development of OSCC. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the poor treatment effect of existing oral squamous cell carcinoma, and there is an urgent need to develop a treatment strategy targeting both zinc ion homeostasis disorder and oral microbiota imbalance to efficiently inhibit the development of oral squamous cell carcinoma, and to provide a PYT@Zn-MOF@siRNA nanoparticle.

[0007] Another object of the present invention is to provide a preparation method of the above-mentioned PYT@Zn-MOF@siRNA nanoparticle.

[0008] Another object of the present invention is to provide a composite nanoparticle.

[0009] Another object of the present invention is to provide a preparation method of the above-mentioned composite nanoparticle.

[0010] Another object of the present invention is to provide the use of the above-mentioned PYT@Zn-MOF@siRNA nanoparticle or the above-mentioned composite nanoparticle in the preparation of anti-tumor drugs and / or antibacterial drugs.

[0011] The above objects of the present invention are achieved by the following technical solutions:

[0012] The present invention protects a PYT@Zn-MOF@siRNA nanoparticle, wherein the PYT@Zn-MOF@siRNA nanoparticle uses a zinc-based metal-organic framework (Zn-MOF) as a carrier to encapsulate drugs, and the drugs include pyrithione (PYT) and SLC30A1-siRNA.

[0013] The present invention provides a PYT@Zn-MOF@siRNA nanoparticle, which can induce zinc ion overload to promote tumor cell death, effectively kill oral tumorigenic microorganisms, and synergistically achieve the treatment of oral squamous cell carcinoma. In the acidic environment of the tumor, Zn-MOF, on the one hand, acts as a framework structure to load the antibacterial drug PYT and the nucleic acid drug SLC30A1-siRNA, and on the other hand, acts as a zinc source to continuously release zinc ions. PYT, as a zinc ion carrier, transports zinc into cells, causing zinc ion overload in the cells. In order to maintain zinc homeostasis, tumor cells upregulate the expression of SLC30A1, the only zinc ion efflux transporter on the cell membrane, to accelerate the efflux of zinc ions. In order to effectively inhibit this regulatory mechanism of tumor cells, the PYT@Zn-MOF@siRNA nanoparticles of the present application release small interfering RNA (siRNA) corresponding to the SLC30A1 protein. The siRNA recognizes the mRNA of SLC30A1 through the principle of base complementary pairing, and uses its endonuclease activity to cut messenger RNA (mRNA), resulting in mRNA degradation, thereby blocking the expression of SLC30A1 protein, and blocking the efflux of zinc ions in cells, further exacerbating the phenomenon of zinc ion overload. Zinc ion overload can cause an increase in reactive oxygen and oxidative stress, leading to a gradual decrease in mitochondrial membrane potential, thereby inducing mitochondrial autophagy and immunogenic cell death, and ultimately achieving the purpose of effectively removing tumor cells. In addition, when PYT is combined with Zn-MOF and SLC30A1-siRNA, its antibacterial effect is significantly improved. The nanoparticles work synergistically by regulating zinc homeostasis and exerting significant antibacterial efficacy, fully demonstrating excellent anti-tumor effects.

[0014] Preferably, the Zn-MOF includes at least one of ZIF8, MAF-6 and MAF-7.

[0015] More preferably, the Zn-MOF is ZIF8. As a typical zinc-based MOF material, it has multiple advantages such as excellent biocompatibility, responsive release, easy modification and efficient drug loading.

[0016] Furthermore, the ZIF8 can be prepared by itself or purchased from the market.

[0017] Further, as a preferred method, the preparation method of ZIF8 comprises the following steps:

[0018] Mix zinc salt and 2-methylimidazole in an organic solvent and allow to react fully to obtain ZIF8.

[0019] Furthermore, the zinc salt includes zinc nitrate, zinc chloride, zinc sulfate or a hydrate of any of the above zinc salts.

[0020] Preferably, the molar ratio of the zinc salt to 2-methylimidazole is 1:(5-10).

[0021] Further, the organic solvent is methanol and / or ethanol.

[0022] Preferably, the mixing is stirring.

[0023] Preferably, the time for sufficient reaction is 1-3 h.

[0024] Further, as a preferred method, the molar ratio of PYT to the zinc salt is 1:(5-7). When preparing PYT@Zn-MOF@siRNA nanoparticles by the "one-pot method", directly preparing ZIF8 in this preparation process can significantly improve the loading efficiency of PYT and siRNA, and make the two more uniformly and effectively integrated into the ZIF8 framework.

[0025] Preferably, the size of the Zn-MOF is 100-150 nm.

[0026] Further, the molar ratio of PYT to Zn-MOF is 1:(1-2).

[0027] Further, the mass ratio of PYT to SLC30A1-siRNA is (8-16):1.

[0028] The present invention protects the preparation method of the above-mentioned PYT@Zn-MOF@siRNA nanoparticles, which includes the following steps:

[0029] Mix PYT, Zn-MOF, and SLC30A1-siRNA sufficiently in an organic solvent, and centrifuge. The obtained precipitate is PYT@Zn-MOF@siRNA nanoparticles.

[0030] Further, the organic solvent is methanol and / or ethanol.

[0031] Further, the mass-volume ratio of PYT to the organic solvent is 1:(0.5-2) mg / mL.

[0032] Preferably, the mixing is stirring.

[0033] Preferably, the time for sufficient mixing is 40-90 min.

[0034] Preferably, the rotation speed of the centrifuge is 6000-10000 rpm.

[0035] More preferably, the centrifugation time is 3-6 min.

[0036] Further, the centrifugation also includes washing.

[0037] Further, the washing is to wash the precipitate obtained by centrifugation with methanol 3 to 6 times.

[0038] The present invention protects a composite nanoparticle, which is obtained by modifying the above-mentioned PYT@Zn-MOF@siRNA nanoparticle with polyethylene glycol or its derivative.

[0039] The present invention uses polyethylene glycol or its derivative to modify the surface of the PYT@Zn-MOF@siRNA nanoparticle by physical adsorption, significantly enhancing the stability and biocompatibility of the original nanoparticle. This improvement not only optimizes the drug delivery effect but also enables the anti-tumor efficacy of the nanoparticle to be more fully demonstrated.

[0040] Preferably, the derivative is folic acid-polyethylene glycol (FA-PEG) or polyoxyethylene polyoxypropylene ether (F127). FA-PEG is a functionalized PEG derivative, commonly used in targeted drug delivery systems. By connecting folic acid (FA) molecules to the PEG chain, it can utilize the specific targeting effect of FA on folate receptors on the surface of tumor cells to accurately deliver the drug to target cells, effectively reducing the off-target probability of nanoparticles; F127 is a non-ionic surfactant with an amphiphilic structure, also having good biocompatibility and biodegradability. It can self-assemble into micelles in aqueous solution to encapsulate nanoparticles, enhancing the stability and dispersibility of the particles. The above substances all have good biocompatibility, and through modification, the PYT@Zn-MOF@siRNA nanoparticle has better stability and biocompatibility.

[0041] More preferably, the polyethylene glycol or its derivative is folic acid-polyethylene glycol (FA-PEG).

[0042] More preferably, the molecular weight of the FA-PEG is 500 to 2000. FA-PEG within this molecular weight range helps to form nanoparticles with appropriate sizes, making it more convenient for cell uptake.

[0043] Further, the mass ratio of the PYT@Zn-MOF@siRNA nanoparticle to the polyethylene glycol or its derivative is 1:(3 to 10).

[0044] The present invention protects a method for preparing the above composite nanoparticle, including the following steps:

[0045] Disperse the aforementioned PYT@Zn-MOF@siRNA nanoparticle and polyethylene glycol or its derivative in a buffer solution to obtain a mixed solution, adjust the pH of the mixed solution to be alkaline, and after sufficient adsorption, centrifuge to obtain a precipitate, which is the composite nanoparticle.

[0046] Preferably, the buffer solution is PBS buffer solution.

[0047] Preferably, the sufficient dispersion is achieved by stirring dispersion.

[0048] More preferably, the time for the stirring dispersion is 40 - 90 min.

[0049] Furthermore, the alkalinity is 7 < pH ≤ 10.

[0050] Furthermore, the pH of the mixed solution can be adjusted by adding an alkaline reagent. Adjusting the pH of the solution to a weakly alkaline environment helps to promote the dissolution of polyethylene glycol or its derivatives, thereby more effectively surface - modifying the PYT@Zn - MOF@siRNA nanoparticles.

[0051] Even further, the alkaline reagent includes one or more of ammonia water, sodium bicarbonate, and sodium carbonate.

[0052] Preferably, the alkaline reagent is ammonia water. This is mainly because it is volatile and can avoid introducing impurity ions during the treatment process.

[0053] Preferably, the time for the sufficient adsorption is 12 - 36 h.

[0054] Preferably, the rotation speed of the centrifugation is 6000 - 10000 rpm.

[0055] More preferably, the time for the centrifugation is 3 - 6 min.

[0056] Furthermore, the centrifugation also includes washing.

[0057] Even further, the washing is to wash the precipitate obtained by centrifugation 3 - 6 times with methanol.

[0058] The present invention protects the application of the above - mentioned PYT@Zn - MOF@siRNA nanoparticles or the above - mentioned composite nanoparticles in the preparation of anti - tumor and / or antibacterial drugs.

[0059] Compared with the prior art, the present invention has the following beneficial effects:

[0060] The present invention prepared a PYT@Zn-MOF@siRNA nanoparticle. In the acidic environment of tumors, Zn-MOF releases zinc ions, and PYT, as a zinc ion carrier, transfers zinc into cells, resulting in intracellular zinc overload. At the same time, the loaded siRNA can recognize, cleave, and degrade the target mRNA, blocking the expression of the zinc ion transporter SLC30A1, thereby reducing zinc ion efflux, exacerbating zinc overload, triggering an increase in reactive oxygen species and oxidative stress, leading to a decrease in mitochondrial membrane potential, inducing mitophagy and immunogenic cell death, and effectively clearing tumor cells. In addition, when PYT binds to Zn-MOF and siRNA, the antibacterial effect is significantly enhanced. This nanoparticle exhibits excellent anti-tumor effects. Description of the Drawings

[0061] Figure 1 It is the TEM image of the composite nanoparticle in Example 1.

[0062] Figure 2 It is the hydrated particle size image (A) of the composite nanoparticle in Example 1 and the ZIF8 nanoparticle in Comparative Example 1, and the Zeta potential diagrams (B) of PYT and the ZIF8 nanoparticle in Comparative Example 1 and the composite nanoparticle in Example 1.

[0063] Figure 3 It is the X-ray powder diffraction pattern of the ZIF8 nanoparticle in Comparative Example 1, the PYT@ZIF8 composite nanoparticle in Comparative Example 2, and the composite nanoparticle in Example 1.

[0064] Figure 4 It is the X-ray photoelectron spectroscopy (A) of the composite nanoparticle in Example 1, the enlarged characteristic peak diagram of zinc element (B), the enlarged characteristic peak diagram of carbon element (C), the enlarged characteristic peak diagram of oxygen element (D), the enlarged characteristic peak diagram of nitrogen element (E), and the enlarged characteristic peak diagram of sulfur element (F).

[0065] Figure 5 It is the high-resolution TEM image (A) of the composite nanoparticle in Example 1 and the distribution diagrams of zinc element (B), carbon element (C), nitrogen element (D), oxygen element (E), and sulfur element (F).

[0066] Figure 6 It is the bacterial blood agar plate counting experimental diagram of the blank control group (Control), the ZIF8 nanoparticle in Comparative Example 1, PYT, the PYT@ZIF8 composite nanoparticle in Comparative Example 2, and the composite nanoparticle in Example 1, and the data statistical chart (A) based on this bacterial blood agar plate counting, and the bacterial live / dead staining experimental diagram and the data statistical chart (B) based on this bacterial live / dead staining experimental diagram.

[0067] Figure 7Data statistical chart (A) of the gene knockdown efficiency of composite nanoparticles with different concentrations in Example 1 on the SLC30A1 gene, chelation zinc level diagrams of the blank control group (Control), ZIF8 nanoparticles in Comparative Example 1, PYT@ZIF8 composite nanoparticles in Comparative Example 2, and composite nanoparticles in Example 1, and the data statistical chart (B) based on the chelation zinc level diagram, co-localization diagram of zinc ions and mitochondria of the composite nanoparticles in Example 1, and the data statistical chart (C) based on the co-localization diagram.

[0068] Figure 8 Gene volcano plot of the RNA sequencing results of the composite nanoparticles in Example 1 on SCCI cells and the data statistical chart (A) and GSEA pathway enrichment analysis chart (B) based on the gene volcano plot.

[0069] Figure 9 Cell ROS level change diagram after treatment with composite nanoparticles with different concentrations in Example 1 and the data statistical chart (A) based on the ROS level change diagram, data statistical chart (B) of GSH change.

[0070] Figure 10 Cell morphology diagrams under transmission electron microscopy after treatment of the blank control group (Control) (A) and composite nanoparticles in Example 1 (B).

[0071] Figure 11 Flow cytometry diagram of mitochondrial membrane potential change after treatment with composite nanoparticles with different concentrations in Example 1 and the data statistical chart (B) based on the mitochondrial membrane potential change flow cytometry diagram (A).

[0072] Figure 12 Electrophoretic characterization diagrams of LC3, PINK1, and Parkin after treatment with composite nanoparticles with different concentrations in Example 1 and the data statistical chart (B) based on the electrophoretic characterization diagram (A).

[0073] Figure 13 Data statistical chart of ATP release amount after treatment with composite nanoparticles with different concentrations in Example 1.

[0074] Figure 14 Cell CRT expression amount change diagram after treatment with composite nanoparticles with different concentrations in Example 1 and the data statistical chart (A) based on the CRT expression amount change diagram, HMGB1 expression amount change diagram and the data statistical chart (B) based on the HMGB1 expression amount change diagram.

[0075] Figure 15 Electrophoretic characterization diagram of PD-L1 after treatment with composite nanoparticles with different concentrations in Example 1 and the data statistical chart (B) based on the electrophoretic characterization diagram (A).

[0076] Figure 16 Anti-tumor effect of composite nanoparticles on bacterial colonization in oral tumors and its synergistic enhancement with PD-1 inhibitor in Example 1; among them, (A) is the flow chart of in vivo experiments; (B) is the tumor volume growth curve during treatment; (C) is the statistical chart of tumor tissue map and weight analysis after treatment; (D) is the statistical chart of zinc content in tumor tissue after treatment; (E) is the experimental map of tumor HE pathological detection and IHC staining to verify bacterial content after treatment; (F) is the body weight curve of mice during treatment; (G) is the H&E staining map of organ tissues after treatment. Detailed implementation manners

[0077] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0078] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0079] Zinc nitrate hexahydrate was purchased from Guangzhou Chemical Reagent Factory;

[0080] 2-Methylimidazole was purchased from Bide Pharmatech;

[0081] ZIF8 was purchased from MedChemexpress Biotechnology Company;

[0082] Pyrithione was purchased from MedChemexpress Biotechnology Company;

[0083] FA-PEG (molecular weight 2000) was purchased from Yuanye Bio-Technology;

[0084] SLC30A1-siRNA was purchased from Ribobio;

[0085] PBS buffer was purchased from Solarbio's P1010;

[0086] Figure 2 A represents Figure 2 Figure A in Figure 2 B represents Figure 2 Figure B in , and so on for other orders;

[0087] Figures 1 - 16 ZIF8 and PYT@ZIF8 in respectively represent ZIF8 nanoparticles and PYT@ZIF8 composite nanoparticles.

[0088] Example 1 Preparation of a composite nanoparticle

[0089] S1. Co-doping of PYT, ZIF8, and siRNA

[0090] Zinc nitrate hexahydrate (297.5 mg, 1 mmol), 2-methylimidazole (MI) (660 mg, 8 mmol), pyrithione (PYT) (20 mg, 0.16 mmol), and SLC30A1-siRNA (2 mL, 1 mg·mL -1 ) were dissolved and dispersed in methanol solution (13 mL), and stirred thoroughly for 1 h by one-pot method. After centrifugation at 8000 rpm for 5 min and washing, PYT@Zn-MOF@siRNA nanoparticles were obtained;

[0091] S2. Surface modification with FA-PEG

[0092] The PYT@Zn-MOF@siRNA nanoparticles (10 mg) obtained in step S1 and FA-PEG (50 mg) were dispersed in 5 mL of PBS buffer, and about 1 mL of ammonia water was added to adjust the pH of the solution to 8. After stirring at room temperature for 24 h, centrifugation at 8000 rpm for 5 min and washing, composite nanoparticles were obtained.

[0093] Example 2 Preparation of PYT@Zn-MOF@siRNA nanoparticles

[0094] The difference from Example 1 is that the PYT@Zn-MOF@siRNA nanoparticles were not modified with FA-PEG, that is, step S2 was not carried out.

[0095] Zinc nitrate hexahydrate (297.5 mg, 1 mmol), 2-methylimidazole (MI) (660 mg, 8 mmol), pyrithione (PYT) (20 mg, 0.16 mmol), and SLC30A1-siRNA (2 mL, 1 mg·mL -1 ) were dissolved and dispersed in methanol solution (13 mL), and stirred thoroughly for 1 h by one-pot method. After centrifugation at 8000 rpm for 5 min and washing, PYT@Zn-MOF@siRNA nanoparticles were obtained.

[0096] Example 3 Preparation of composite nanoparticles

[0097] The difference from Example 1 is that in step S1, commercially available ZIF8 was used instead of self-made ZIF8 using zinc nitrate hexahydrate and 2-methylimidazole.

[0098] S1. Co-doping of PYT, ZIF8, and siRNA

[0099] ZIF8 (50 mg, 0.22 mmol), pyrithione (PYT) (20 mg, 0.16 mmol), and SLC30A1-siRNA (2 mL, 1 mg·mL -1) Dissolve and disperse in methanol solution (13 mL), stir thoroughly for 1 h by one-pot method, centrifuge at 8000 rpm for 5 min, and wash to obtain PYT@Zn-MOF@siRNA nanoparticles;

[0100] S2. Surface modification with FA-PEG

[0101] Disperse the PYT@Zn-MOF@siRNA nanoparticles (10 mg) obtained in step S1 and FA-PEG (50 mg) in 5 mL of PBS buffer solution, add about 1 mL of ammonia water to make the pH of the solution 8, stir and react at room temperature for 24 h, centrifuge at 8000 rpm for 5 min, and wash to obtain composite nanoparticles.

[0102] Comparative Example 1 Preparation of a ZIF8 composite nanoparticle

[0103] The difference from Example 1 is that in step S1, PYT and siRNA were not added.

[0104] S1. Preparation of ZIF8

[0105] Dissolve zinc nitrate hexahydrate (297.5 mg, 1 mmol) and 2-methylimidazole (MI) (660 mg, 8 mmol) in methanol solution (13 mL), stir thoroughly for 1 h by one-pot method to obtain ZIF8 nanoparticles.

[0106] S2. Surface modification with FA-PEG

[0107] Disperse the ZIF8 nanoparticles (10 mg) obtained in step S1 and FA-PEG (50 mg) in 5 mL of pH buffer solution, add about 1 mL of ammonia water to make the pH of the solution 8, stir and react at room temperature for 24 h, centrifuge at 8000 rpm for 5 min, and wash to obtain ZIF8 composite nanoparticles.

[0108] Comparative Example 2 Preparation of a PYT@ZIF8 composite nanoparticle

[0109] The difference from Example 1 is that in step S1, siRNA was not added.

[0110] S1. Co-doping of PYT and ZIF8

[0111] Dissolve zinc nitrate hexahydrate (297.5 mg, 1 mmol), 2-methylimidazole (MI) (660 mg, 8 mmol), and pyrithione (PYT) (20 mg, 0.16 mmol) in methanol solution (13 mL), stir thoroughly for 1 h by one-pot method, centrifuge at 8000 rpm for 5 min, and wash to obtain PYT@ZIF8 composite nanoparticles;

[0112] S2. FA-PEG Surface Modification

[0113] Disperse the PYT@ZIF8 (10 mg) and FA-PEG (50 mg) obtained in step S1 in 5 mL of pH buffer solution, add about 1 mL of ammonia water to make the pH of the solution 8, stir at room temperature for 24 h, centrifuge at 8000 rpm for 5 min, and wash to obtain PYT@ZIF8 composite nanoparticles.

[0114] Experimental Example 1 Characterization of Composite Nanoparticles

[0115] 1. Detection by Transmission Electron Microscope (TEM)

[0116] (1) Experimental Method

[0117] Drop the PBS buffer solution containing the composite nanoparticles obtained in Example 1 on a 200-mesh copper grid, air-dry, and put it on the TEM (JEM-2010HR, JEOL, Japan).

[0118] (2) Experimental Results

[0119] According to Figure 1 the TEM images, it can be observed that the composite nanoparticles are cube-shaped, with uniform particle size and good dispersion in water.

[0120] 2. Detection of Dynamic Light Scattering (DLS) and zeta Potential

[0121] (1) Experimental Method

[0122] Use the dynamic light scattering method to measure the hydrodynamic diameter of the PYT@Zn-MOF@siRNA suspension (0.5 mg / mL); the measured scattered light intensity is displayed as the photon count rate, with the unit of thousands of counts per second (kcps); the scattering angle is set to 90°; for each sample suspension, three DLS measurements are performed at a fixed running time (60 s); use the Zetasizer Nanoinstrument DLS system to detect the zeta potential.

[0123] (2) Experimental Results

[0124] According to Figure 2 the DLS detection results of A, the hydrated particle size of the ZIF8 nanoparticles in Comparative Example 2 is about 150 nm, while the hydrated particle size of the composite nanoparticles in Example 1 is about 177 nm. Both have relatively small nanoparticle sizes, which is beneficial to enhancing the EPR effect. In addition, according to Figure 2Results of B. For the composite nanoparticles in Example 1, at pH 7.4, the potential was -6.27 ± 1.09 mV. This near-neutral potential characteristic is beneficial for enhancing its stability and biocompatibility.

[0125] 3. Detection of Powder X-ray Diffraction (PXRD)

[0126] (1) Experimental method

[0127] Load the composite nanoparticles (2 mg) in Example 1 onto a silicon wafer to make it flat without bumps or depressions, and then perform PXRD (D8, Bruker, Germany) on the machine.

[0128] (2) Experimental results

[0129] According to Figure 3 It can be obtained that after loading the drug PYT, the crystal structure of ZIF8 in Example 1 did not change.

[0130] 4. Detection of X-ray Photoelectron Spectroscopy (XPS)

[0131] (1) Experimental method

[0132] Load the composite nanoparticles (2 mg) in Example 1 onto a copper foil to make it flat without bumps or depressions, and then perform XPS (ThermoFisher Nexsa G2 electron spectrometer, USA) on the machine.

[0133] (2) Experimental results

[0134] According to Figure 4 A, it can be obtained that the composite nanoparticles in Example 1 exhibited characteristic peaks of elements such as zinc, sulfur, and nitrogen ( Figure 4 B~ Figure 4 F), which confirmed that PYT and ZIF had been successfully loaded onto the nanoparticles.

[0135] 5. Detection of High-resolution TEM (HR-TEM) and Elemental Distribution

[0136] (1) Experimental method

[0137] Drop the PBS buffer solution containing the composite nanoparticles in Example 1 onto a 200-mesh copper grid, air-dry it, and then perform tests on the machine (Tecnai G2 F30, Holland).

[0138] (2) Experimental results

[0139] According toFigure 5 The shown HR-TEM images and elemental distribution maps indicate that in Example 1, the composite nanoparticles exhibit a uniform spherical morphology ( Figure 5 A), and the distribution of elements such as zinc, sulfur, and nitrogen within the particles ( Figure 5 B~ Figure 5 F) is clearly visible, which further confirms that the drugs PYT and ZIF8 have been successfully loaded onto the nanoparticles. Experimental Example 2 Antibacterial properties of PYT, ZIF8 nanoparticles, PYT@ZIF8 composite nanoparticles, and composite nanoparticles

[0140] 1. Experimental method

[0141] (1) Blood agar plate counting method: Porphyromonas gingivalis was cultured to the mid-logarithmic phase, at which time the optical density was approximately 1.0 (OD = 1.0). PYT, ZIF8 nanoparticles (Comparative Example 1), PYT@ZIF8 composite nanoparticles (Comparative Example 2), and composite nanoparticles (Example 1) were added to the bacterial suspension (1 mL, 10 CFU / mL), with a final concentration of 10 μg / mL (based on the nanoparticle concentration), and incubated for 24 hours. The test concentration of PYT was 1.57 μM. Subsequently, the incubated suspension was diluted to an appropriate concentration. Then, 100 μL of the diluted solution was spread on a brain heart infusion (BHI) sheep blood agar plate and incubated under strict anaerobic conditions at 37°C for 48 h. Finally, the colonies on the solid medium were photographed and counted.

[0142] (2) Live / Dead Bacterial Detection Kit: Live bacteria and dead bacteria were stained green and red, respectively. Bacteria were cultured routinely. PYT, ZIF8 nanoparticles (Comparative Example 1), PYT@ZIF8 composite nanoparticles (Comparative Example 2), and composite nanoparticles (Example 1) were incubated with the bacterial suspension (1 mL, 10 CFU / mL) at a final concentration of 50 μg / mL (based on the nanoparticle concentration). The test concentration of PYT was 7.86 μM. All groups were cultured at 37°C for 48 h. After incubation, the samples were washed three times with PBS and then stained with the Live / Dead Bacterial Staining Reagent for 30 min. After staining, the samples were rinsed three times with PBS and then imaged by a laser confocal microscope (Olympus Corporation, Japan), scale bar: 20 μm.

[0143] 2. Experimental results

[0144] From Figure 6On the left experimental figure of A, it can be observed that the more the number of white bacteria, the worse the antibacterial effect. On the solid media of PYT@ZIF8 composite nanoparticles (Comparative Example 2) and composite nanoparticles (Example 1), almost no bacteria can be seen, and the antibacterial effects of these two are significantly better than those of single ZIF8 (Comparative Example 1) and PYT. This indicates that the combination of PYT, ZIF8, and siRNA can significantly enhance their antibacterial effects. Figure 6 The data statistical chart on the right of A more intuitively demonstrates this result. Similarly, from Figure 6 From the live-dead staining figure and data statistical chart of B, it can be obtained that there are more dead bacteria (red) in PYT@ZIF8 composite nanoparticles (Comparative Example 2) and composite nanoparticles (Example 1), further verifying the above conclusion.

[0145] Experimental Example 3 Cytotoxicity Test of PYT, ZIF8 Nanoparticles, PYT@ZIF8 Composite Nanoparticles, and Composite Nanoparticles

[0146] 1. Experimental Method

[0147] CCK8 method: Inoculate the cell line into a 96-well plate, with 5×10 3 cells in each well, and place it in an incubator (37 °C, 5% CO2) for normal culture until the cells adhere to the wall. Subsequently, aspirate the original culture medium, and add drugs with different concentrations to the cells, 100 μL per well. Set 3 parallel wells, and at the same time set blank medium and cells without drugs as control wells. After culturing with drugs for 48 h, add 100 μL of CCK8 solution to each well and incubate at 37 °C for 1 h. Use a multifunctional microplate reader to measure the absorbance value (OD) of each well at 450 nm. The calculation formula for cell survival rate is: Cell survival rate % = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group).

[0148] The cytotoxicities of PYT, ZIF8 nanoparticles (Comparative Example 1), PYT@ZIF8 composite nanoparticles (Comparative Example 2), and composite nanoparticles (Example 1) were evaluated in vitro against human tongue squamous cell carcinoma (SCC1, HSC4, HN6), mouse squamous cell carcinoma (SCC7), and human oral mucosal precancerous cells (DOK). The results are shown in Table 1 (unit: μg / mL).

[0149] 2. Experimental Results

[0150] Table 1 Results of Cytotoxicity Test

[0151]

[0152] From the data in Table 1, it can be seen that the cytotoxicity from high to low is as follows: composite nanoparticles > PYT@ZIF8 composite nanoparticles > PYT > ZIF8 nanoparticles. It is worth noting that the toxicity of the composite nanoparticles to all cell lines is higher than that of cisplatin. Among the three human tongue squamous cell lines, the composite nanoparticles have the greatest cytotoxicity to the SCC1 cell line, and its IC 50 value is approximately 0.42 μg / mL. These results indicate that the composite nanoparticles have significant anti-tumor activity.

[0153] Experimental Example 4 Knockdown efficiency experiment of composite nanoparticles and analysis of zinc level changes and localization of PYT, ZIF8 nanoparticles, PYT@ZIF8 composite nanoparticles, and composite nanoparticles

[0154] 1. Experimental method

[0155] (1) Inoculate SCC1 cells (5×10 5 cells per well) into a 6-well plate and place it in an incubator at 37 °C for 24 h. Add composite nanoparticles (0.2, 0.4, 0.8, 1.6 μg / mL) and place it in an incubator at 37 °C for 24 h. Discard the cell culture medium, wash the cells 3 times with PBS, and according to the instructions, use a rapid RNA extraction kit to extract the total RNA of the sample. After reverse transcription, perform real-time quantitative polymerase chain reaction to detect the knockdown of the target gene SLC30A1.

[0156] (2) Detection of chelated zinc level by zinc ion probe TSQ: Inoculate SCC1 cells into a 35 mm confocal culture dish for 24 h, and then incubate them with the blank control group (Control), ZIF8 nanoparticles (Comparative Example 1), PYT@ZIF8 composite nanoparticles (Comparative Example 2), and composite nanoparticles (Example 1) at 37 °C for 24 h. Add TSQ DMSO solution (100 μM) 30 min before imaging. Wash the cells 3 times with PBS and observe under a laser confocal microscope (LSM980, Zeiss, Germany). Use ImageJ software to calculate the average fluorescence intensity. Scale bar: 20 μm.

[0157] (3) Determine the co-localization of zinc ions and mitochondria: Incubate SCC1 cells with the composite nanoparticles (4 μg / mL) in Example 1 for 2 h, add a mitochondrial red probe (MTDR, 200 nM) and incubate for 30 min, then take pictures using a confocal microscope (Olympus FV3000, TKY, Japan), and perform quantitative analysis of the Pearson correlation coefficient (PCC) using ImageJ software. Scale bar: 10 μm.

[0158] 2. Experimental results

[0159] FromFigure 7 The RNA results of A showed that the knockdown efficiency of the composite nanoparticles was positively correlated with the concentration, and the knockdown efficiency of all treatment groups was significantly higher than that of the untreated group. By utilizing the high porosity of Zn-MOF and the enhanced permeability and retention effect (EPR) of nanoparticles, the present invention can precisely deliver siRNA to tumor cells and effectively achieve efficient knockdown of the target gene SLC30A1. When the concentration of the composite nanoparticles was 1.6 μg / mL, the knockdown efficiency could reach 70%. In contrast, conventional siRNA transfection methods, such as calcium carbonate co-precipitation method, electroporation method, and cationic liposome transfection, are susceptible to various factors such as transfection reagents and cell status, resulting in low transfection efficiency and usually only about 50% knockdown efficiency; From Figure 7 It can be observed from the experimental graph of B that the blue fluorescence intensity of TSQ was positively correlated with its ability to chelate zinc, that is, the stronger the blue fluorescence, the higher the level of chelated zinc. Compared with the control group, ZIF8 nanoparticles (Comparative Example 1), and PYT@ZIF8 composite nanoparticles (Comparative Example 2), the composite nanoparticles (Example 1) group exhibited the strongest blue fluorescence, which clearly indicated that it had a higher level of chelated zinc. Figure 7 The data statistical graph of B more intuitively verified this conclusion. Further, from Figure 7 It can be seen from the experimental graph and data statistical graph of C that after the composite nanoparticles were co-incubated with cells, the green fluorescence of zinc and the red fluorescence of the mitochondrial probe MTDR showed a high degree of co-localization, and its Pearson correlation coefficient (PCC) was as high as 0.90, which fully demonstrated that zinc was mainly concentrated in mitochondria.

[0160] Experimental Example 5 RNA Sequencing

[0161] The SCCI cells were treated with the drug (the composite nanoparticles in Example 1, 0.8 μg / mL) and cultured in an incubator at 37 °C for 24 h. Total RNA was extracted using a RNA rapid purification kit, and then RNA sequencing was performed.

[0162] 1. Experimental Method

[0163] The Qubit fluorescence quantifier and Qsep400 high-throughput biological fragment analyzer were used to perform quality inspection and quantitative analysis of the total RNA respectively to evaluate the integrity and concentration of RNA. Only samples with RNA integrity values not lower than 7 could be used for subsequent sequencing steps. The RNA sequencing work was completed by Wuhan Maiwei Metabolism Biotechnology Co., Ltd. After constructing the mRNA library, the Illumina HiSeq TM sequencing platform was used to sequence different libraries.

[0164] The gene expression levels were quantified using the featureCounts method, and the alignment statistics of the genes were calculated. Subsequently, based on the gene lengths, the fragments per kilobase of exon per million reads mapped (FPKM) and transcripts per million (TPM) values of each gene were calculated. In terms of data analysis, genes with an absolute value of Log2(fold change) ≥ 1 and a false discovery rate (FDR) significance score < 0.05 were defined as differentially expressed genes (DEGs) for subsequent analysis. Finally, the gene set enrichment analysis (GSEA) was performed using the clusterProfiler R package.

[0165] 2. Experimental results

[0166] As Figure 8 shown in the gene volcano plot of A, RNA sequencing (RNA-seq) analysis showed that compared with the control group, a total of 3695 significantly differentially expressed genes (DEGs) were detected. Among them, 2380 genes were up-regulated and 1315 genes were down-regulated. Among the down-regulated genes was SLC30A1. From Figure 8 the statistical chart of the data in A, it can be seen that after treatment with the composite nanoparticles in Example 1, the expression of SLC30A1 was down-regulated, and this down-regulation was significantly different compared with the control group. Further gene set enrichment analysis (GSEA) was performed, and the results were as Figure 8 shown in B. After treatment with the composite nanoparticles in Example 1, the genes related to oxidative phosphorylation were down-regulated, while the genes related to zinc ion response and regulation of the cell killing pathway were up-regulated. This indicates that the composite nanoparticles affected the mitochondrial function of tumor cells, inhibited mitochondrial respiration, and at the same time initiated the cell killing program, promoting tumor cell death.

[0167] Analysis of anti-tumor effect in Experimental Example 6

[0168] 1. Oxidative stress analysis

[0169] S1. Ability of composite nanoparticles to generate ROS

[0170] S1-1. Experimental method

[0171] First, take SCC1 cells in good growth condition, perform routine rinsing, digestion, and counting. Then, inoculate the cells into a 6-well plate and place it in an incubator (37°C, 5% CO2) for normal culture until the cells adhere to the wall. Next, add composite nanoparticles with certain concentrations (0.2, 0.4, 0.8, 1.6 μg / mL respectively), set 3 replicate wells for each group, and place it in a 37°C incubator for dark culture for 24 h. 30 min before imaging, add 10 μM DCFH-DA dye and incubate it at 37°C in the dark for 30 min. After that, wash the cells with PBS, then digest the cells with trypsin without EDTA for 2 min, add medium to terminate digestion, collect the cell suspension, centrifuge it at 1000 rpm for 3 min, discard the supernatant, resuspend the cells, centrifuge again, discard the supernatant, add 500 μL PBS to each tube to resuspend the cells, and then perform flow cytometry analysis.

[0172] S1-2. Experimental results

[0173] According to Figure 9 the experimental graph of A, it can be observed that in the cells treated with composite nanoparticles, the fluorescence intensity of 2',7'-dichlorofluorescein (DCF) increases in a concentration-dependent manner. This phenomenon indicates that the composite nanoparticles can induce the generation of reactive oxygen species (ROS) in cells. Specifically, when the concentration of the composite nanoparticles is 1.6 μg / mL, the intracellular ROS level induced by it is significantly higher than that of the control group (0 μg / mL), about 19 times that of the control group ( Figure 9 data statistical graph of A).

[0174] S2. Analyze the expression level of intracellular oxidative stress by detecting the level of glutathione (GSH)

[0175] S2-1. Experimental method

[0176] Culture SCC1 cells in a 12-well plate and perform reagent treatment according to the experimental requirements. Wash the cells with PBS, then centrifuge to collect the cells and count. Next, freeze-thaw the cells rapidly twice and centrifuge at 8000 g for 10 min. Finally, take the supernatant and place it in a GSH quantitative detection kit for measuring the total GSH content.

[0177] S2-2. Experimental results

[0178] As Figure 9 shown in B, GSH is a key antioxidant that can protect cells from oxidative stress damage. In SCC1 cells treated with composite nanoparticles, the content of GSH is significantly reduced. The test results show that the composite nanoparticles can effectively induce an oxidative stress response.

[0179] 2. Observe the morphological changes of cells after treatment with composite nanoparticles using transmission electron microscopy (TEM)

[0180] S1-1. Experimental method

[0181] Inoculate SCC1 cells in good growth condition into 6-cm culture dishes and culture them in an incubator at 37 °C until the cells adhere to the wall. Subsequently, culture the adherent SCC1 cells with media containing different concentrations of composite nanoparticles (0 and 0.8 μg / mL) in an incubator at 37 °C for 24 h. After the culture, digest and collect the cells using trypsin. The collected cells are washed 3 times with pre-cooled PBS, and after each wash, they are centrifuged at 1000 rpm for 5 min to remove the excess PBS. The obtained cell pellet is fixed overnight at 4 °C with glutaraldehyde. After that, the samples are treated with osmium tetroxide staining, and finally, the morphological changes of the cells are observed and recorded using transmission electron microscopy (TEM, Hitachi HT7800, Japan). Scale bars: 5 μm and 500 nm.

[0182] S1-1. Experimental results

[0183] As Figure 10 shown, compared with the intact cell organelles and plasma membranes in the control group cells, the cells treated with the composite nanoparticles in Example 1 showed obvious damage. Specifically, a large number of damaged mitochondria appeared in the treated cells, and these mitochondria were wrapped by a double-membrane structure. In addition, large areas of the plasma membrane of the cells were damaged, accompanied by the formation of numerous pores.

[0184] 3. Analysis of the effect on mitochondrial function

[0185] S1. Detect the change of mitochondrial membrane potential in cells

[0186] S1-1. Experimental method

[0187] SCC1 cells are inoculated in 6-well plates for culture and treated according to the instructions using a JC-1 mitochondrial membrane potential detection kit. Then, collect these cells and resuspend them in a staining working solution containing JC-1, and incubate them at 37 °C for 30 min. Subsequently, wash the cells 2 times with cold staining buffer. After the washing is completed, immediately perform the detection using flow cytometry, and use FlowJo software to analyze the obtained data in detail.

[0188] S1-2. Experimental results

[0189] According to Figure 11 the experimental results, it can be obtained that the proportion of cells with mitochondrial depolarization caused by the composite nanoparticles in Example 1 increased in a concentration-dependent manner, rising from 1.8% at 0 μg / mL to 97.7% at 1.6 μg / mL (Figure 11 A), and there were significant differences between the groups treated with different concentrations of composite nanoparticles and the untreated group ( Figure 11 B), indicating that the treatment with composite nanoparticles could lead to the loss of mitochondrial membrane potential.

[0190] S2. Analyze the expression levels of mitophagy-related proteins in cells by Western Blot

[0191] S2-1. Experimental method

[0192] SCC1 cells were co-cultured with different concentrations of composite nanoparticles (0.2, 0.4, 0.8, 1.6 μg / mL) in an incubator at 37 °C for 24 h. After that, these cells were collected and lysed on ice using RIPA buffer containing 1% phosphatase inhibitor and 1% protease inhibitor for 30 min. Next, the protein concentration was measured by the BCA method, followed by electrophoresis loading, membrane transfer, and blocking steps. After blocking, the membrane was incubated with the primary antibody overnight at 4 °C. The next day, after washing the membrane, the corresponding secondary antibody was incubated. Finally, color development and exposure were performed using a gel imager to collect images, and the images were further analyzed using ImageJ software.

[0193] S2-2. Experimental results

[0194] According to Figure 12 the experimental results of A: After treatment with composite nanoparticles, the expressions of the key mitophagy proteins PTEN-induced kinase 1 (PINK1) and Parkin were significantly upregulated with the increase in concentration. Specifically, as the concentration of composite nanoparticles increased from 0 μg / mL to 1.6 μg / mL, the relative expression levels of PINK1 and Parkin both showed a significant increasing trend. Statistical analysis of the data showed ( Figure 12 B) that this change was significant at each concentration (P < 0.0001 or P < 0.0002). In addition, the conversion of LC3-I to its lipidated form LC3-II was also enhanced with the increase in nanoparticle concentration. These results indicate that composite nanoparticles can effectively induce the expression of mitophagy-related proteins and promote the conversion of LC3-I to LC3-II.

[0195] 4. Immunogenic death effect analysis

[0196] During typical immunogenic cell death, tumor cells express damage-associated molecular patterns (DAMPs), which are characterized by the release of ATP, exposure of calreticulin (CRT), and release of high-mobility group box 1 (HMGB1). DAMPs promote the activation of antigen-presenting cells, migration to lymph nodes, and activation of T cells, thereby achieving anti-tumor immunotherapy; while PD-L1 is a protein expressed on the surface of tumor cells and immune cells. After binding to programmed death protein 1 (PD-1), it inhibits the immune activity of T cells. A decrease in its expression means enhanced activation of the immune system, and T cells can better recognize and kill tumor cells.

[0197] S1. Effect of composite nanoparticles on intracellular ATP level

[0198] S1-1. Experimental method

[0199] SCC1 cells in good growth state were seeded in 6-well plates and cultured in an incubator at 37 °C until the cells adhered to the wall. Then, the cells were washed with PBS and then treated with composite nanoparticles at three concentrations of 0.4, 0.8, and 1.6 μg / mL, with 3 replicates in each group to ensure the reliability of the data. The treated cells were placed back in the incubator at 37 °C for 24 h. After the culture, the cells were collected and lysed. The protein concentration in the lysate was measured using a BCA protein quantification detection kit. Subsequently, the supernatant containing the protein was transferred to a 96-well plate with a black wall and clear bottom, and an ATP detection reagent was added to each well. Finally, the ATP level in each well was detected by chemiluminescence method on an enzyme-linked immunosorbent assay (ELISA) reader.

[0200] S1-2. Experimental results

[0201] According to Figure 13 the results, it can be obtained that: the chemiluminescence method detection shows that the intracellular ATP content shows a dose-dependent decreasing trend with the increase in the treatment concentration of composite nanoparticles. Specifically, compared with the untreated control group (0 μg / mL), the ATP content of the cells treated with 0.4 μg / mL, 0.8 μg / mL, and 1.6 μg / mL concentrations all decreased. Among them, the P value of the decrease in the ATP content of the cells treated with 0.8 μg / mL concentration was 0.0023, and the P value of the decrease in the ATP content of the cells treated with 1.6 μg / mL concentration was 0.0007, indicating that this decrease was statistically significant. This further confirmed that composite nanoparticles can effectively reduce the intracellular ATP content.

[0202] S2. Effect of composite nanoparticles on the expression of cell surface CRT and intracellular HMGB1

[0203] S2-1. Experimental method

[0204] Inoculate well-growing SCC1 cells into a 6-well plate and place them in an incubator at 37 °C until the cells adhere to the wall. Subsequently, wash the cells with PBS, and then add composite nanoparticles at three concentrations of 0.4, 0.8, and 1.6 μg / mL respectively, with 3 replicate wells set in each group. Put the treated cells back into the incubator at 37 °C for 24 h. After the culture ends, collect the cells and wash them 3 times with PBS. Then, fix the cells with 4% paraformaldehyde (PFA) at room temperature for 15 min. To analyze the exposure of calreticulin (CRT) on the cell surface (i.e., ecto-CRT), the fixed cells (without permeabilization) were incubated with Calreticulin (D3E6) Rabbit mAb (Alexa 488 Conjugate) at 4 °C for 12 h to specifically label the CRT on the cell surface. To analyze the intracellular HMGB1 level, first permeabilize the cells with 0.1% Triton X-100 at room temperature for 10 min to enable the antibody to enter the cells. Then, incubate the permeabilized cells with PE anti-HMGB1 Antibody at 4 °C for 12 h to specifically label the intracellular HMGB1. Finally, analyze the labeled cells using a flow cytometer to detect the exposure of cell CRT and the intracellular HMGB1 level.

[0205] S2-2. Experimental results

[0206] According to Figure 14 The experimental graph results of A flow cytometry show that after treatment with composite nanoparticles, the exposure of calreticulin (CRT) on the cell surface increased significantly, and this increasing trend was positively correlated with the treatment concentration. Specifically, compared with the untreated control group (0 μg / mL), the exposure of cell surface CRT in cells treated with 0.4 μg / mL, 0.8 μg / mL, and 1.6 μg / mL concentrations all showed a significant increase ( Figure 14 The data statistical graph of A), and this increase was statistically significant (P < 0.0001). At the same time, Figure 14 The experimental graph results of B show that the content of HMGB1 in the cell nucleus decreased significantly with the increase in the treatment concentration of composite nanoparticles. In cells treated with 0.8 μg / mL and 1.6 μg / mL concentrations, the decrease in HMGB1 content was particularly obvious, with P values of 0.001 and P < 0.0001 respectively. In cells treated with 0.4 μg / mL concentration, although the decrease in HMGB1 content was not as significant as the former two, it also reached statistical significance (P = 0.0478)( Figure 14Statistical graph of data of B). These results together indicate that the composite nanoparticles can effectively regulate the expression of CRT on the cell surface and the content of HMGB1 in the cell nucleus.

[0207] S3. Analyze the expression level of PD-L1 in cells by Western Blot

[0208] S3-1. Experimental method

[0209] Co-culture SCC1 cells with composite nanoparticles at different concentrations (0.2, 0.4, 0.8, 1.6 μg / mL) in an incubator at 37 °C for 24 h. Subsequently, collect these cells and lyse them on ice using RIPA buffer containing 1% phosphatase inhibitor and 1% protease inhibitor for 30 min. Quantitatively detect the protein concentration after lysis using the Bicinchoninic Acid (BCA) method. Then, electrophoretically separate the protein samples and transfer them to a membrane. After that, block the membrane and incubate it with the primary antibody overnight at 4 °C. The next day, wash the membrane and incubate it with the corresponding secondary antibody. Finally, use a gel imager for color development and exposure to collect images, and use ImageJ software for data analysis of the images.

[0210] S3-2. Experimental results

[0211] According to Figure 15 As shown, in SCC1 cells treated with composite nanoparticles, the expression level of programmed death ligand 1 (PD-L1) decreases significantly with the increase of the treatment concentration ( Figure 15 A). Specifically, compared with the untreated control group (0 μg / mL), the relative expression levels of PD-L1 in cells treated with 0.2, 0.4, 0.8, and 1.6 μg / mL concentrations all showed significant decreases, and this decrease was statistically significant. Among them, the P value of the relative expression level of PD-L1 in cells treated with 0.2 μg / mL concentration was 0.0089, the P value in cells treated with 0.4 μg / mL concentration was 0.0012, the P value in cells treated with 0.8 μg / mL concentration was 0.0006, and the P value in cells treated with 1.6 μg / mL concentration was less than 0.0001 ( Figure 15 B). These results indicate that the composite nanoparticles can effectively reduce the expression level of PD-L1 in a concentration-dependent manner.

[0212] In summary, the above test results further indicate that the treatment with composite nanoparticles can effectively induce immunogenic death of tumor cells and significantly reduce the expression of PD-L1, thereby playing an immunomodulatory role.

[0213] S4. Evaluate the anti-tumor effect and immunomodulatory ability of composite nanoparticles using a CDX infection model

[0214] Four-week-old female C3H-HeN mice were housed in the SPF-class barrier system of South China Agricultural University. Feed and drinking water were sterilized by the animal house and provided for the animals to eat and drink freely.

[0215] S4-1. Experimental method

[0216] Inject 100 μL (containing 1×10 6 cells) of a live cell suspension of SCC7 (squamous cell carcinoma cell line) pretreated with Porphyromonas gingivalis (MOI = 100) subcutaneously into the right axilla of C3H-HeN mice to establish a CDX infection model ( Figure 16 A). When the tumor volume reached approximately 200 mm 3 , the mice were randomly divided into the following 4 groups, with 6 mice in each group: (1) normal saline control group; (2) PD-1 monoclonal antibody treatment group (10 mg / kg); (3) composite nanoparticle treatment group (10 mg / kg); (4) PD-1 monoclonal antibody + composite nanoparticle treatment group (10 mg / kg). The composite nanoparticles were injected intratumorally once every 3 days for a total of 6 injections. The PD-1 monoclonal antibody was injected intraperitoneally once every 3 days for a total of 6 times. Starting from the day of administration, the tumor volume and body weight of the mice were recorded every other day. Seventeen days after the end of treatment, the mice were sacrificed and the tumors were excised and weighed. Part of the tumor tissue was fixed with 10% buffered formalin, embedded in paraffin, and stained with H&E and IHC (immunohistochemistry), with scale bars of 50 μm (H&E) and 20 μm (IHC), respectively. Another part of the tumor tissue was used for inductively coupled plasma mass spectrometry, flow cytometry, and cytokine detection. At the same time, the main organs of the mice, such as the heart, liver, spleen, lungs, and kidneys, were embedded in paraffin and subjected to histopathological examination and bacterial analysis, with a scale bar of 100 μm.

[0217] S4-2. Experimental results

[0218] According to the tumor volume growth curve graph ( Figure 16 B), the gross tumor tissue graph, and the tumor tissue weight bar graph ( Figure 16 C), both the composite nanoparticle group and the PD-1 monoclonal antibody significantly inhibited tumor growth, and their combined effect further significantly promoted tumor regression. The results of inductively coupled plasma mass spectrometry (ICP-MS) showed that both the composite nanoparticle group and the PD-1 monoclonal antibody had obvious accumulation in the tumor, and the accumulation of zinc content in the tumor was more significant when used in combination ( Figure 16D). Meanwhile, HE staining showed that tumor necrosis was most obvious in the combined treatment group; IHC bacterial staining showed that the bacterial content was the lowest in the combined treatment group, indicating the highest bacterial clearance rate ( Figure 16 E). Meanwhile, the body weights of mice in all groups remained stable during the treatment process, and there was no sharp change in body weight in the short term. Figure 16 F). In addition, no obvious toxic side effects were observed in the H&E staining sections of the main organs (heart, liver, spleen, lung, and kidney) of the mice. Figure 16 G), suggesting that the composite nanoparticles have good administration safety.

[0219] In summary, the above test results confirm that the combined treatment strategy of composite nanoparticles and anti-PD-1 monoclonal antibody has a powerful anti-tumor effect, can effectively inhibit tumor growth, relieve the immunosuppressive tumor microenvironment, and also reveals its significant effect in bacterial clearance. At the same time, this combined treatment synergistically enhances anti-tumor adaptive immunity, shows good feasibility, and has high biosafety.

[0220] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A PYT@Zn-MOF@siRNA nanoparticle, characterized in that, The PYT@Zn-MOF@siRNA nanoparticles use zinc-based metal organic framework materials as carriers to encapsulate drugs, and the drugs include pyrithione and SLC30A1-siRNA.

2. The PYT@Zn-MOF@siRNA nanoparticles according to claim 1, wherein The molar ratio of the pyrithione to the zinc-based metal organic framework material is 1:(1-2).

3. The PYT@Zn-MOF@siRNA nanoparticles according to claim 1, characterized in that, The mass ratio of pyrithione to SLC30A1-siRNA is (8-16):

1.

4. The preparation method of the PYT@Zn-MOF@siRNA nanoparticles according to any one of claims 1 to 3, characterized in that, The following steps are involved: Pyridinethione, zinc-based metal organic framework material, and SLC30A1-siRNA are fully mixed in an organic solvent and centrifuged to obtain PYT@Zn-MOF@siRNA nanoparticles.

5. The preparation method according to claim 4, wherein The organic solvent is methanol and / or ethanol.

6. A composite nanoparticle, characterized in that, The PYT@Zn-MOF@siRNA nanoparticles according to any one of claims 1 to 3 are obtained by modifying the nanoparticles with polyethylene glycol or its derivatives.

7. The composite nanoparticle according to claim 6, wherein The derivative is folic acid-polyethylene glycol or polyoxyethylene-polyoxypropylene ether.

8. The composite nanoparticle according to claim 6, wherein The mass ratio of the PYT@Zn-MOF@siRNA nanoparticles to polyethylene glycol or its derivatives is 1:(3-10).

9. The preparation method of the composite nanoparticle according to any one of claims 6 to 8, characterized in that, The following steps are involved: The PYT@Zn-MOF@siRNA nanoparticles according to any one of claims 1 to 3 and polyethylene glycol or its derivatives are fully dispersed in a buffer to obtain a mixed solution, the pH of the mixed solution is adjusted to be alkaline, and after sufficient adsorption, centrifugation is performed to obtain a precipitate as a composite nanoparticle.

10. Use of the PYT@Zn-MOF@siRNA nanoparticles according to any one of claims 1 to 3 or the composite nanoparticles according to any one of claims 6 to 8 in the preparation of anti-tumor and / or antibacterial drugs.

Images