Functional silylene compound, preparation method thereof and application of functional silylene compound in tumor mitochondrial targeted silicification treatment
By preparing functionalized silene complexes, and using their targeted destruction of tumor mitochondria membranes on tumor cells, the problems of limited efficacy and drug resistance of existing tumor treatment methods are solved, and efficient and accurate tumor treatment effects are achieved.
Patent Information
- Application Number
- CN202510431155.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-08
- Publication Date
- 2025-08-15
AI Technical Summary
The existing tumor treatment methods have problems such as limited efficacy, great toxic and side effects, and drug resistance. It is difficult for traditional drugs to efficiently target the mitochondria of tumor cells, resulting in poor treatment results.
By mixing natural polyphenol-modified silene nanosheets with mitochondrial targeting small molecules and polymer compounds, and ultrasonic reactions in alkaline buffer, functionalized silene complexes can be prepared, which can specifically target mitochondria of tumor cells, destroy the integrity of the tumor mitochondrial membrane and affect normal functions.
It achieves precise killing of tumor cells, reduces the therapeutic dose, solves the problem of drug resistance, and effectively inhibits tumor growth through drug-free methods and reduces damage to normal cells.
Smart Images
Figure CN120478659A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomaterials, and in particular relates to a functionalized silicene complex and a preparation method thereof, and application thereof in mitochondrial-targeted silicification therapy for tumors. Background Art
[0002] Cancer (tumors) has become the second leading cause of death worldwide, second only to cardiovascular disease. Every year, tens of millions of people die from cancer worldwide, and this number is on the rise as the global population ages and lifestyles change. Traditional tumor treatments such as surgery, radiotherapy, and chemotherapy have achieved efficacy to a certain extent, but still have problems such as limited efficacy, large toxic side effects, and drug resistance. With the deepening of tumor treatment research, new treatment strategies, such as targeted therapy and immunotherapy, have gradually been proposed and applied. These strategies face limitations such as high cost and individual differences in treatment. Therefore, there is a need to find more accurate and efficient tumor treatment strategies, especially those that can reduce damage to normal cells while effectively inhibiting tumor growth and preventing metastasis.
[0003] In cancer development, mitochondria play a key role in regulating energy metabolism, reactive oxygen species (ROS) production, and apoptosis. Previous studies have also shown that chemotherapeutic drugs can cause tumor cell death by inducing mitochondrial dysfunction; however, large drug doses are generally required to achieve this effect. Drug-free approaches have been introduced as an alternative to inducing mitochondrial dysfunction in cancer, potentially avoiding the development of drug resistance. These approaches involve the introduction or in situ formation of macromolecular systems in subcellular compartments, disrupting the integrity of tumor mitochondrial membranes, affecting normal function, and ultimately suppressing tumors.
[0004] Two-dimensional materials, due to their unique physicochemical properties and high specific surface area, have been widely used in drug delivery, tumor therapy, and tissue engineering. Silicene, a two-dimensional material similar to graphene, is a single-layer two-dimensional structure composed of silicon atoms. Currently, silicene is widely used for its photothermal tumor-killing function. Silicon compounds, as excellent mineralizing materials, also play a good role in inorganic-organic interactions in organisms. Some materials used to prepare biominerals are unstable in their natural state. For example, when exposed to water or extreme pH values, their chemical properties will change, causing structural damage. This requires ensuring that the mineralized materials are sufficiently stable during entry into the body, have selectivity for certain cell types, such as normal and tumor cells, and have subsequent tissue permeability, such as being able to penetrate cell membranes and enter subcellular interiors to exert their effects. Summary of the Invention
[0005] In view of the defects or shortcomings in the prior art, the primary purpose of the present invention is to provide a method for preparing a functionalized silicene composite.
[0006] Another object of the present invention is to provide a functionalized silicene complex obtained by the above preparation method.
[0007] Another object of the present invention is to provide an application of the functionalized silicene complex obtained by the above preparation method.
[0008] The purpose of the present invention is achieved through the following technical solutions:
[0009] A method for preparing a functionalized silicene complex comprises mixing natural polyphenol-modified silicene nanosheets with mitochondrial-targeting small molecules and high-molecular compounds, and subjecting the mixture to ultrasonic reaction in an alkaline buffer to obtain the functionalized silicene complex.
[0010] Furthermore, the natural polyphenol compound is any one of tannic acid (TA), catechin, gallic acid and coumarin; preferably tannic acid.
[0011] Furthermore, the mitochondrial targeting small molecule is any one of triphenylphosphine (TPP), mitochondrial targeting peptide, and anthocyanin dye; preferably triphenylphosphine.
[0012] Furthermore, the polymer compound is any one of polyethyleneimine (PEI), dendrimer polyamide, polyethyleneimine derivatives and polypropyleneimine (PPA); preferably polyethyleneimine.
[0013] Furthermore, the alkaline buffer refers to a Trizma-buffer buffer; preferably a Trizma-buffer buffer with a pH of 8.5±0.2.
[0014] Furthermore, the mass ratio of the natural polyphenol-modified silicene nanosheets to the mitochondrial-targeting small molecules and high molecular weight compounds is 1 to 8:1, preferably 1:1.
[0015] Furthermore, the preparation method comprises the following steps:
[0016] (1) Dispersing silicene nanosheets in an alkaline buffer solution, adding tannic acid (TA) during ultrasonication, and reacting to obtain tannic acid-modified silicene nanosheets, referred to as TA-Si;
[0017] (2) 3-(Carboxypropyl)-triphenylphosphine (TPP) is dissolved in an organic solvent, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) are added to react, and then polyethyleneimine (PEI) is added to continue the reaction; the reaction solution is dialyzed and freeze-dried to obtain a complex of 3-(carboxypropyl)-triphenylphosphine and polyethyleneimine, referred to as TPP-PEI;
[0018] (3) reacting the TA-Si obtained in step (1) with the TPP-PEI obtained in step (2) in a Trizma-buffer solution, and the reaction product is the functionalized silicene complex.
[0019] Furthermore, the silicene nanosheets described in step (1) are obtained by a method comprising the following steps:
[0020] Calcium silicide and iodine are added to an organic solvent for reaction. After the reaction is completed, the precipitate is collected by centrifugation, placed in N-methylpyrrolidone, and ultrasonically crushed to obtain silicene nanosheets.
[0021] Furthermore, the calcium silicide is micron calcium silicide with an average particle size of 1 to 10 μm; preferably micron calcium silicide with an average particle size of 3 to 5 μm.
[0022] Furthermore, the organic solvent is at least one of anhydrous acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide.
[0023] Furthermore, the molar ratio of calcium silicide to elemental iodine is 1-2:1-2, preferably 1:1.
[0024] Furthermore, the reaction conditions are: argon protection, rotation speed 400±100 rpm, and time 14±3 days.
[0025] Furthermore, the precipitate is washed with anhydrous acetonitrile and then with N-methylpyrrolidone before being placed in N-methylpyrrolidone.
[0026] Furthermore, the ultrasonic conditions are: ice bath, power 1200±200W, time 3±1 days.
[0027] Furthermore, the crushing conditions are: ice bath, time 8±2 hours.
[0028] Furthermore, the alkaline buffer described in step (1) refers to a Trizma-buffer buffer; preferably, it refers to a Trizma-buffer buffer having a pH of 8.5±0.2; more preferably, the Trizma-buffer buffer is obtained by a method comprising the following steps: dissolving Trizma@base in water and adjusting the pH to 8.5±0.2 with a 1M Trizma@hydrochloride aqueous solution.
[0029] Furthermore, the ultrasonic conditions in step (1) are: power 80W to 120W, time 30±5 minutes.
[0030] Furthermore, the mass ratio of the silicene nanosheets to tannic acid in step (1) is 1 to 3:1, preferably 1:1.
[0031] Furthermore, the reaction conditions in step (1) are: temperature 20-30° C. (room temperature) and time 16-24 hours (overnight).
[0032] Furthermore, the organic solvent in step (2) is at least one of dimethyl sulfoxide, N,N-dimethylformamide, and dichloromethane.
[0033] Furthermore, the polyethyleneimine described in step (2) is polyethyleneimine with a molecular weight of 1 to 10K; preferably polyethyleneimine with a molecular weight of 1 to 3K; more preferably polyethyleneimine with a molecular weight of 1.8K.
[0034] Furthermore, the molar ratio of 3-(carboxypropyl)-triphenylphosphine to polyethyleneimine in step (2) is 1 to 10:1, preferably 10:1.
[0035] Furthermore, the molar ratio of 3-(carboxypropyl)-triphenylphosphine (TPP), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide (NHS) in step (2) is 1:1-4:1-4; preferably 1:1.5:1.5.
[0036] Furthermore, the reaction time in step (2) is 4±1 hours, and the reaction time is continued for 48±10 hours.
[0037] Furthermore, the dialysis conditions in step (2) are: molecular weight cut-off 1 kD, time 48±10 hours.
[0038] Furthermore, the mass ratio of TA-Si to TPP-PEI in step (3) is 1 to 8:1, preferably 1:1.
[0039] A functionalized silicene nanocomposite for silicification of tumor mitochondria is obtained by the above preparation method. The particle size of the functionalized silicene nanocomposite is about 200 nm.
[0040] Application of the functionalized silicene nanocomplex for tumor mitochondrial silicification in the preparation of anti-tumor drugs.
[0041] Furthermore, the tumor includes but is not limited to at least one of colon cancer, melanoma, and pancreatic cancer.
[0042] The technical solution provided by the present invention has the following beneficial effects:
[0043] (1) The silicene nanocomplex provided by the present invention and the addition of TPP small molecules can specifically target mitochondria in tumor cells, drive them to accumulate in the mitochondrial matrix through the potential gradient, and introduce or form a macromolecular system in the mitochondria, thereby destroying the integrity of the tumor mitochondrial membrane, affecting normal function and thus inhibiting the tumor to induce tumor death;
[0044] (2) The functionalized silicene nanocomplex provided by the present invention can not only improve the shortcomings of small molecules such as poor targeting and large side effects, but also, as a method for treating tumors without drugs, can solve drug resistance and reduce the dosage of therapeutic agents; at the same time, it can precisely mineralize mitochondria, the energy supply center in subcellular organelles, and achieve more accurate and efficient treatment of tumors through the controllable regulation of inorganic elements on organisms. BRIEF DESCRIPTION OF THE DRAWINGS
[0045] Figure 1 Transmission (TEM) images of unfunctionalized and functionalized silicene complexes;
[0046] Figure 2 Figure 1 is the particle size data of unfunctionalized and functionalized silicene composites;
[0047] Figure 3 Scanning electron microscopy (SEM) images showing the affinity verification of unfunctionalized and functionalized silicene complexes for bacterial membranes;
[0048] Figure 4 Scanning electron microscopy (SEM) images of the mitochondrial affinity verification of unfunctionalized and functionalized silicene complexes;
[0049] Figure 5 This is the MTT toxicity test diagram of unfunctionalized and functionalized silicene complexes on breast cancer cells;
[0050] Figure 6 Live / dead cell double-staining fluorescence microscopy images of breast cancer cells treated with unfunctionalized and functionalized silicene complexes;
[0051] Figure 7 Fluorescence microscopy images verifying the targeting effect of functionalized silicene complexes on mitochondria;
[0052] Figure 8 Fluorescence microscopy images of the damage to mitochondrial membrane potential caused by unfunctionalized and functionalized silicene complexes;
[0053] Figure 9 Transmission (TEM) images of different functionalized silicene complexes. DETAILED DESCRIPTION
[0054] In order to better understand the technical solutions of the present invention, the present invention is further described below with reference to the following embodiments, but the present invention is not limited to the following embodiments.
[0055] Unless otherwise specified, the reagents and raw materials used in this application can be obtained commercially.
[0056] Example 1
[0057] (1) Synthesis of Silicene
[0058] Weigh 1.15g of micron calcium silicide (3-5μm) and 3.03g of I2, add them to 160mL of anhydrous acetonitrile in a molar ratio of 1:1, and stir at room temperature at 400rpm under argon protection for 2 weeks. After the reaction, collect the precipitate by centrifugation, wash it with anhydrous acetonitrile 3 times, and wash it with methylpyrrolidone (NMP) once. The obtained silicane precipitate is dispersed in 50mL of NMP, and ultrasonicated in a water bath for 3 days under ice bath conditions with a power of 1200W. After the ultrasonication, crush it with a crusher for 8h, centrifuge it at 1000rpm for 30min, and collect the supernatant to obtain silicane nanosheets.
[0059] (2) Synthesis of tannic acid-modified silicene (TA-Si)
[0060] ① Preparation of 1M Trizma@hydrochloride: Dissolve 3.152g Trizma@hydrochloride solid in 20mL deionized water;
[0061] ② Preparation of 0.1M Trizma-buffer solution: Dissolve 8.72g Trizma@base in 1L deionized water and adjust the pH of the solution to 8.5 with 1M Trizma@hydrochloride;
[0062] ③ The silicane nanosheets obtained in step (1) were washed three times with anhydrous ethanol, washed once with Trizma-buffer, and then redispersed in Trizma-buffer solution to a concentration of 1 mg / mL. TA dissolved in Trizma-buffer solution was added during ultrasound (120W) at a concentration of 1 mg / mL. The mass ratio of silicane to TA was 1:1. After ultrasound for 30 minutes, the reaction was allowed to proceed at room temperature overnight. The nanosheets were washed three times with anhydrous ethanol and stored in anhydrous ethanol to obtain TA-Si.
[0063] (3) Synthesis of TPP-PEI
[0064] 215 mg of 3-(carboxypropyl)-triphenylphosphine (TPP) was added to 3 mL of dimethyl sulfoxide (DMSO), and then 144 mg of N-ethyl N-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDCI) and 87 mg of N-hydroxysuccinimide (NHS) were added to the system. After reacting at room temperature for 4 hours, 88 μL of polyethyleneimine (PEI) with a molecular weight of 1.8K was added. The molar ratio of TPP to PEI was 10:1. At this time, the system was reacted for another 48 hours. After the reaction, it was dialyzed using a 1 kD molecular weight cutoff (MWCO) dialysis bag for 48 hours and lyophilized to obtain TPP-PEI.
[0065] (4) Synthesis of TPTS
[0066] The TA-Si obtained in the above steps (2) and (3) was reacted with TPP-PEI in a 0.1M Trizma-buffer (pH 8.5) solution at a feed mass ratio of 1:1 for 24 hours. After the reaction, the mixture was washed three times with anhydrous ethanol to obtain a functionalized silicene complex TPTS.
[0067] Example 2
[0068] (1) Synthesis of Silicene
[0069] 1.15 g of calcium silicide and 3.03 g of I2 were weighed and added to 160 mL of anhydrous acetonitrile at a molar ratio of 1:1. The mixture was stirred at room temperature for 2 weeks at 400 rpm under argon protection. After the reaction, the mixture was washed with anhydrous acetonitrile 3 times and NMP once. The obtained silicane precipitate was dispersed in 50 mL of NMP and ultrasonicated in a water bath for 3 days under ice bath conditions. After the ultrasonication, the mixture was crushed with a crusher for 8 hours. After the sonication, the mixture was centrifuged at 1000 rpm for 30 minutes and the supernatant was collected to obtain silicane nanosheets.
[0070] (2) Synthesis of TA-Si
[0071] ① Preparation of 1M Trizma@hydrochloride: Dissolve 3.152g Trizma@hydrochloride solid in 20mL deionized water;
[0072] ② Preparation of 0.1M Trizma-buffer solution: Dissolve 8.72g Trizma@base in 1L deionized water and adjust the pH of the solution to 8.5 with 1M Trizma@hydrochloride;
[0073] ③ The silicane nanosheets obtained in step (1) were washed three times with ethanol, washed once with Trizma-buffer, and then redispersed in Trizma-buffer solution to a concentration of 1 mg / mL. TA dissolved in Trizma-buffer solution was added during the ultrasonic process to a concentration of 1 mg / mL. The mass ratio of silicane to TA was 1:1. After ultrasonication for 30 minutes, the reaction was allowed to react at room temperature overnight. The nanosheets were washed three times with ethanol and stored in anhydrous ethanol to obtain TA-silicene.
[0074] (3) Synthesis of TPP-PEI
[0075] 215 mg of 3-(carboxypropyl)-triphenylphosphine (TPP) was added to 3 mL of dimethyl sulfoxide, and then 144 mg of EDCI and 87 mg of NHS were added to the system. After reacting at room temperature for 4 hours, 88 μL of PEI with a molecular weight of 1.8K was added. The molar ratio of TPP to PEI was 5:1. At this time, the system was reacted for another 48 hours. After the reaction, it was dialyzed with a 1kD MWCO dialysis bag for 48 hours and lyophilized to obtain TPP-PEI.
[0076] (4) Synthesis of TPTS
[0077] The TA-Si obtained in the above steps (2) and (3) was reacted with TPP-PEI in a 0.1M Trizma-buffer (PH 8.5) solution at a feed mass ratio of 1:1 for 24 hours. After the reaction, the mixture was washed three times with anhydrous ethanol to obtain a functionalized silicene complex TPTS.
[0078] Example 3
[0079] (1) Synthesis of Silicene
[0080] 1.15 g of calcium silicide and 3.03 g of I2 were weighed and added to 160 mL of anhydrous acetonitrile at a molar ratio of 1:1. The mixture was stirred at room temperature for 2 weeks at 400 rpm under argon protection. After the reaction, the mixture was washed 3 times with anhydrous acetonitrile and once with NMP. The obtained silicane precipitate was dispersed in 50 mL of NMP and ultrasonicated in a water bath for 3 days under ice bath conditions. After the ultrasonication, the mixture was crushed with a crusher for 8 hours. After the sonication, the mixture was centrifuged at 1000 rpm for 30 minutes and the supernatant was collected to obtain silicane nanosheets.
[0081] (2) Synthesis of TA-Si
[0082] ① Preparation of 1M Trizma@hydrochloride: Dissolve 3.152g Trizma@hydrochloride solid in 20mL deionized water;
[0083] ② Preparation of 0.1M Trizma-buffer solution: Dissolve 8.72g Trizma@base in 1L deionized water and adjust the pH of the solution to 8.5 with 1M Trizma@hydrochloride;
[0084] ③ The silicane nanosheets obtained in step (1) were washed three times with ethanol, washed once with Trizma-buffer, and then redispersed in Trizma-buffer solution to a concentration of 1 mg / mL. TA dissolved in Trizma-buffer solution was added during the ultrasonic process to a concentration of 1 mg / mL. The mass ratio of silicane to TA was 1:1. After ultrasonication for 30 minutes, the reaction was allowed to react at room temperature overnight. The nanosheets were washed three times with ethanol and stored in anhydrous ethanol to obtain TA-silicene.
[0085] (3) Synthesis of TPP-PEI
[0086] 215 mg of 3-(carboxypropyl)-triphenylphosphine (TPP) was added to 3 mL of dimethyl sulfoxide, and then 144 mg of EDCI and 87 mg of NHS were added to the system. After reacting at room temperature for 4 hours, 88 μL of PEI with a molecular weight of 1.8K was added. The molar ratio of TPP to PEI was 2:1. At this time, the system was reacted for another 48 hours. After the reaction, it was dialyzed using a 1KD MWCO dialysis bag for 48 hours and freeze-dried to obtain TPP-PEI.
[0087] (4) Synthesis of TPTS
[0088] The TA-Si obtained in the above steps (2) and (3) was reacted with TPP-PEI in a 0.1M Trizma-buffer (PH 8.5) solution at a feed mass ratio of 1:1 for 24 hours. After the reaction, the mixture was washed three times with anhydrous ethanol to obtain a functionalized silicene complex TPTS.
[0089] Comparative Example 1: Silicene without any modification
[0090] (1) Synthesis of Silicene
[0091] 1.15 g of calcium silicide and 3.03 g of I2 were weighed and added to 160 mL of anhydrous acetonitrile at a molar ratio of 1:1. The mixture was stirred at room temperature for 2 weeks at 400 rpm under argon protection. After the reaction, the mixture was washed 3 times with anhydrous acetonitrile and once with NMP. The obtained silicane precipitate was dispersed in 50 mL of NMP and ultrasonicated in a water bath for 3 days under ice bath conditions. After the ultrasonication, the mixture was crushed with a crusher for 8 hours. After the sonication, the mixture was centrifuged at 1000 rpm for 30 minutes and the supernatant was collected to obtain silicane nanosheets.
[0092] Comparative Example 2: TA-Si without mitochondrial targeting modification
[0093] (1) Synthesis of Silicene
[0094] 1.15 g of calcium silicide and 3.03 g of I2 were weighed and added to 160 mL of anhydrous acetonitrile at a molar ratio of 1:1. The mixture was stirred at room temperature for 2 weeks at 400 rpm under argon protection. After the reaction, the mixture was washed 3 times with anhydrous acetonitrile and once with NMP. The obtained silicane precipitate was dispersed in 50 mL of NMP and ultrasonicated in a water bath for 3 days under ice bath conditions. After the ultrasonication, the mixture was crushed with a crusher for 8 hours. After the sonication, the mixture was centrifuged at 1000 rpm for 30 minutes and the supernatant was collected to obtain silicane nanosheets.
[0095] (2) Synthesis of TA-Si
[0096] ① Preparation of 1M Trizma@hydrochloride: Dissolve 3.152g Trizma@hydrochloride solid in 20mL deionized water;
[0097] ② Preparation of 0.1M Trizma-buffer solution: Dissolve 8.72g Trizma@base in 1L deionized water and adjust the pH of the solution to 8.5 with 1M Trizma@hydrochloride;
[0098] ③ The silicane nanosheets obtained in step (1) were washed three times with ethanol, washed once with Trizma-buffer, and then redispersed in Trizma-buffer solution to a concentration of 1 mg / mL. TA dissolved in Trizma-buffer solution was added during the ultrasonic process to a concentration of 1 mg / mL. The mass ratio of silicane to TA was 1:1. After ultrasonication for 30 minutes, the reaction was allowed to react at room temperature overnight. The nanosheets were washed three times with ethanol and stored in anhydrous ethanol to obtain TA-silicene.
[0099] Comparative Example 3: Different feed mass ratios of TA-Si and TPP-PEI
[0100] (1) Synthesis of Silicene
[0101] 1.15 g of calcium silicide and 3.03 g of I2 were weighed and added to 160 mL of anhydrous acetonitrile at a molar ratio of 1:1. The mixture was stirred at room temperature for 2 weeks at 400 rpm under argon protection. After the reaction, the mixture was washed with anhydrous acetonitrile 3 times and NMP once. The obtained silicane precipitate was dispersed in 50 mL of NMP and ultrasonicated in a water bath for 3 days under ice bath conditions. After the ultrasonication, the mixture was crushed with a crusher for 8 hours. After the sonication, the mixture was centrifuged at 1000 rpm for 30 minutes and the supernatant was collected to obtain silicane nanosheets.
[0102] (2) Synthesis of TA-Si
[0103] ① Preparation of 1M Trizma@hydrochloride: Dissolve 3.152g Trizma@hydrochloride solid in 20mL deionized water;
[0104] ② Preparation of 0.1M Trizma-buffer solution: Dissolve 8.72g Trizma@base in 1L deionized water and adjust the pH of the solution to 8.5 with 1M Trizma@hydrochloride;
[0105] ③ The silicane nanosheets obtained in step (1) were washed with ethanol three times, washed with Trizma-buffer once, and then redispersed in Trizma-buffer solution to a concentration of 1 mg / mL. TA dissolved in Trizma-buffer solution was added during the ultrasonic process to a concentration of 1 mg / mL. The mass ratio of silicane to TA was 1:1. After ultrasonication for 30 minutes, the reaction was allowed to proceed at room temperature overnight. The nanosheets were washed with ethanol three times and stored in anhydrous ethanol to obtain TA-Si.
[0106] (3) Synthesis of TPP-PEI
[0107] 215 mg of 3-(carboxypropyl)-triphenylphosphine (TPP) was added to 3 mL of dimethyl sulfoxide, and then 144 mg of EDCI and 87 mg of NHS were added to the system. After reacting at room temperature for 4 hours, 88 μL of PEI with a molecular weight of 1.8K was added. The molar ratio of TPP to PEI was 10:1. At this time, the system was reacted for another 48 hours. After the reaction, it was dialyzed with a 1kD MWCO dialysis bag for 48 hours and lyophilized to obtain TPP-PEI.
[0108] (4) Synthesis of TPTS
[0109] The TA-Si obtained in the above steps (2) and (3) was reacted with TPP-PEI in a 0.1M Trizma-buffer (pH 8.5) solution at a feed mass ratio of 2:1 for 24 hours. After the reaction, the mixture was washed three times with anhydrous ethanol to obtain a functionalized silene complex TPTS.
[0110] Figure 1 Transmission electron microscopy (TEM) images of unfunctionalized and functionalized silicene composites show the morphological changes before and after modification. The left image shows an unmodified single-layer 2D nanomaterial, which is thin and has a relatively uniform surface. The right image shows the material modified with tannic acid (TA), polyethyleneimine (PEI), and sodium tripolyphosphate (TPP). The modification significantly increases the thickness and enhances the surface structure, indicating that the modification was successful and effectively altered the material's physical and chemical properties.
[0111] Figure 2 Figures 2 and 3 show the particle size data for unfunctionalized and functionalized silicene complexes. The figures show the hydrated particle size distribution before and after modification. The left image shows unmodified silicene, which exhibits a smaller hydrated particle size and a narrower distribution. The right image shows functionalized silicene, which exhibits a significant increase in the hydrated particle size of TPTS and a higher polydispersity index (PDI), indicating a wider particle size distribution. This is likely due to increased aggregation or surface interactions after the introduction of the modifying molecules.
[0112] Figure 3 Scanning electron microscopy (SEM) images show affinity verification of unfunctionalized and functionalized silicene complexes for bacterial membranes. Comparing the mineralization results of lactic acid cocci treated with unfunctionalized and functionalized silicene reveals that the bacterial surface of the silicene-treated group is smoother due to the lack of targeting, while the surface of the targeted group becomes thicker. EDS spectra show an increase in silicon content and the presence of phosphorus signals, demonstrating that targeted silicene can better target the bacterial surface.
[0113] Figure 4 Scanning electron micrographs (SEM) show affinity verification of mitochondria with unfunctionalized and functionalized silicene complexes. Comparing the mineralization results of mitochondria extracted from 4T1 tumor cells with those of silicene before and after modification reveals that fully functionalized silicene exhibits significantly more surface mineralized deposits, a coarser particle structure, and increased volume, indicating that modification promotes mineralization. This result demonstrates that functionalization enhances silicene's mineralization within the mitochondrial microenvironment.
[0114] The functionalized silicene complexes prepared in Example 1 and Comparative Examples 1, 2, and 3 were used for relevant experimental tests.
[0115] (1) Cytotoxicity test
[0116] The cytotoxicity of unmodified Silicene nanosheets and functionalized Silicene complexes on 4T1 mouse breast cancer cells was analyzed by MTT kit. 4T1 cell suspension was seeded in a 96-well cell culture plate, and 100 μL of a 1×10 4 cells / mL cell suspension. After culturing for 24 hours in a 5% CO2 incubator at 37°C, DMEM prepared in Example 1, Comparative Example 1, or Comparative Example 2 was added according to the experimental grouping, with five replicate wells per group. The 96-well plate was then incubated in a 37°C 5% CO2 cell culture incubator for 24 hours. Cell viability was calculated by measuring OD570 wavelength according to the MTT kit instructions. Figure 5 The results showed that the modified silicene (TPTS) had a significant effect on cell survival. Compared with the unmodified group, the cell survival rate of the TPTS group decreased, indicating that the modified silicene enhanced the cytotoxic effect. This suggests that TPTS kills tumor cells through mitochondrial damage, mineralization induction, or other cell death mechanisms.
[0117] (2) Calcein-AM / PI live cell / dead cell double staining test
[0118] 4T1 cells were cultured at a rate of 1×10 5 The cells were seeded at a density of 10 cells per well in a 24-well cell culture plate and cultured until the cells were fully adhered to the wall. The culture medium was then aspirated and the TA-Si without mitochondrial targeting modification and the functionalized silicene complex were added at a concentration of 25 μg / mL. After 8 h, Calcein AM / PI staining was performed and the cells were observed under a fluorescence microscope. Figure 6 The results showed that after silicene functionalization, more red fluorescence was exhibited, proving that more cells died, while the cells in the unfunctionalized group TA-Si and the group with nothing added had fewer dead cells than the functionalized group. In contrast, both the TA-Silicene and TPTS treatment groups induced varying degrees of cytotoxicity. Among them, the cytotoxicity of the TPTS treatment group was more significant, as evidenced by a significant decrease in the number of live cells and a significant increase in the number of dead cells. Further analysis showed that TPTS triggered the cell death process more accurately by inducing mitochondrial mineralization, indicating that TPTS has a stronger specific killing effect on tumor cells.
[0119] (3) Verification of the mitochondrial targeting effect of functionalized silicene complexes
[0120] 4T1 cells were cultured at a rate of 1×10 5Cells were seeded at a density of 100 cells per well in a 24-well cell culture plate and cultured until the cells were fully adhered to the wall. The culture medium was then aspirated and the mitochondrial-targeted modified silicene complex Cy3-TPTS labeled with a fluorescent dye was added. The control group was added with nothing. After incubation with the cells for 8 hours, the material was washed away and the cells were stained with the mitochondrial dye Mitotracker and the nuclear dye Hochest for mitochondrial and nuclei staining and observed under a fluorescence microscope. Figure 7 The results showed that in the Cy3-TPTS group, the red fluorescence of TPTS and the green fluorescence of mitochondria had obvious overlap, proving that TPTS can effectively target the mitochondria in tumor cells and play the next function.
[0121] (4) Mitochondrial membrane potential test:
[0122] The effects of silicane, TA-Si and the functionalized silicane complex TPTS on the mitochondrial membrane potential of 4T1 mouse breast cancer cells were analyzed by JC-1 kit. 4T1 cells were plated at 1×10 5 The cells were seeded at a density of 10 cells per well in a 24-well cell culture plate and cultured until the cells were fully adhered and expanded. The culture medium was then aspirated and the unmodified target and modified target (i.e., functionalized silicene complex) were added at a concentration of 25 μg / mL. After 8 h, the cells were incubated with a JC-1 probe and observed under a fluorescence microscope. Figure 8 The results showed that the mitochondrial membrane potential of the targeted group decreased more significantly than that of the other groups, and TPTS could cause greater damage to the mitochondrial membrane potential.
[0123] (5) Thickness comparison:
[0124] The targeted silicene materials in Example 1 and Comparative Example 3 were dispersed in anhydrous ethanol, ultrasonicated for 10 minutes, and then dropped onto a copper mesh. After natural air drying, the materials were observed under a transmission electron microscope. Figure 9 The results show that due to the different feed mass ratios of TA-Si and TPP-PEI, the silicene in Comparative Example 3 (left) is thicker and unevenly distributed, while the targeted silicene prepared by the method of Example 1 (right) is thinner.
[0125] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.
Claims
1. A method for preparing a functionalized silicene complex, characterized by: The natural polyphenol-modified silicene nanosheets were mixed with mitochondrial-targeting small molecules and polymer compounds, and ultrasonically reacted in an alkaline buffer to obtain a functionalized silicene complex. The natural polyphenol compound is any one of tannic acid, catechin, gallic acid and coumarin; The mitochondrial targeting small molecule is any one of triphenylphosphine, mitochondrial targeting peptide and anthocyanin dye; The polymer compound is any one of polyethyleneimine, dendrimer polyamide, polyethyleneimine derivatives and polypropyleneimine.
2. The method for preparing a functionalized silicene complex according to claim 1, wherein: The natural polyphenol compound is tannic acid; The mitochondrial targeting small molecule is triphenylphosphine; The polymer compound is polyethyleneimine; The alkaline buffer refers to Trizma-buffer buffer; The mass ratio of the natural polyphenol-modified silicene nanosheets to the mitochondria-targeted small molecules and high-molecular compounds is 1 to 8:
1.
3. The method for preparing a functionalized silicene complex according to claim 1, wherein: The preparation method comprises the following steps (1) Dispersing silicene nanosheets in an alkaline buffer solution, adding tannic acid during ultrasonication, and reacting to obtain tannic acid-modified silicene nanosheets, referred to as TA-Si; (2) 3-(Carboxypropyl)-triphenylphosphine is dissolved in an organic solvent, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide are added to react, and then polyethyleneimine is added to continue the reaction; the reaction solution is dialyzed and freeze-dried to obtain a complex of 3-(carboxypropyl)-triphenylphosphine and polyethyleneimine, referred to as TPP-PEI; (3) reacting the TA-Si obtained in step (1) with the TPP-PEI obtained in step (2) in a Trizma-buffer solution, and the reaction product is the functionalized silicene complex.
4. The method for preparing a functionalized silicene complex according to claim 3, wherein: The silicene nanosheets described in step (1) are obtained by a method comprising the following steps: adding calcium silicide and iodine to an organic solvent, reacting, collecting a precipitate by centrifugation after the reaction, placing the precipitate in N-methylpyrrolidone, and ultrasonically crushing the silicene nanosheets; The calcium silicide is micron calcium silicide with an average particle size of 1 to 10 μm; The organic solvent is at least one of anhydrous acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide; The molar ratio of the calcium silicide to the iodine element is 1-2:1-2.
5. The method for preparing a functionalized silicene complex according to claim 4, wherein: The calcium silicide is micron calcium silicide with an average particle size of 3 to 5 μm; The organic solvent is anhydrous acetonitrile; The molar ratio of calcium silicide to iodine is 1:1; The reaction conditions are: argon protection, rotation speed 400±100 rpm, time 14±3 days; Before placing the precipitate in N-methylpyrrolidone, it was washed with anhydrous acetonitrile and then with N-methylpyrrolidone; The ultrasonic conditions are as follows: ice bath, power 1200±200W, time 3±1 days; The crushing conditions are: time 8±2 hours.
6. The method for preparing a functionalized silicene complex according to any one of claims 3 to 5, wherein: The alkaline buffer described in step (1) refers to a Trizma-buffer buffer having a pH of 8.5±0.2; The mass ratio of the silicene nanosheets to tannic acid in step (1) is 1 to 3:
1. The organic solvent in step (2) is at least one of dimethyl sulfoxide, N,N-dimethylformamide, and dichloromethane; The polyethyleneimine described in step (2) is polyethyleneimine with a molecular weight of 1 to 10K; The molar ratio of 3-(carboxypropyl)-triphenylphosphine to polyethyleneimine in step (2) is 1 to 10:1; The molar ratio of 3-(carboxypropyl)-triphenylphosphine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide in step (2) is 1:1-4:1-4; The mass ratio of TA-Si to TPP-PEI in step (3) is 1 to 8:
1.
7. The method for preparing a functionalized silicene complex according to any one of claims 3 to 5, wherein: The mass ratio of the silicene nanosheets to tannic acid in step (1) is 1:1; The organic solvent described in step (2) is dimethyl sulfoxide; The polyethyleneimine described in step (2) is polyethyleneimine with a molecular weight of 1.8K; The molar ratio of 3-(carboxypropyl)-triphenylphosphine to polyethyleneimine in step (2) is 10:1; The molar ratio of 3-(carboxypropyl)-triphenylphosphine (TPP), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide (NHS) in step (2) is 1:1.5:1.
5. The mass ratio of TA-Si to TPP-PEI in step (3) is 1:1; The ultrasonic conditions described in step (1) are: power 80-120W, time 30±5 minutes; The reaction conditions in step (1) are: temperature 20-30° C., time 8-16 hours; The reaction time in step (2) is 4±1 hours, and the reaction time is continued for 48±10 hours; The dialysis conditions described in step (2) are: molecular weight cut-off 1 kD, time 48±10 hours.
8. A functionalized silicene complex for silicification of tumor mitochondria, characterized by: The method is obtained by the preparation method described in any one of claims 1 to 7.
9. Use of the method for preparing the functionalized silicene complex for silicification of tumor mitochondria as claimed in claim 8 in anti-tumor drugs.
10. The use according to claim 9, characterized in that: The tumor is at least one of colon cancer, melanoma, and pancreatic cancer.