Protein degradation system based on polyamide-amine dendrimer and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and relates to a protein degradation system based on polyamide-amine dendrimers as well as a preparation method and application of the protein degradation system. The protein degradation system is of a nano-particle structure, and the nano-particle structure comprises silicon dioxide nano-particles and a polyamide-amine type dendritic polymer layer coating the surfaces of the silicon dioxide nano-particles. An MDM2 protein ligand, a GLUT1 protein ligand and an E3 ubiquitin enzyme ligand are connected to the polyamide-amine dendritic polymer layer through chemical bonds. The protein degradation system provided by the invention can be used for synergistically degrading the MDM2 protein and the GLUT1 protein. The synergistic degradation strategy not only can effectively inhibit proliferation and energy metabolism of tumor cells, but also can significantly enhance the stability of the p53 protein. By recovering the normal function of the p53 protein, the growth of tumor cells is further inhibited, and a new strategy is provided for tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to a protein degradation system based on polyamide-amine dendrimers, and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] The development and progression of malignant tumors involves the abnormal expression and dysfunction of multiple key regulatory proteins, leading to uncontrolled cell proliferation and metabolic reprogramming. Among these regulatory factors, the functional inactivation of certain key tumor suppressor proteins is an important driver of tumor development. For example, important tumor suppressor proteins (such as p53) often lose their function due to the overexpression of their negative regulators. These negative regulators promote their ubiquitination and degradation pathways, significantly reducing the intracellular levels or activity of the tumor suppressor proteins, thereby relieving the inhibition of tumor cell proliferation. Restoring the normal function of these key tumor suppressor proteins is an important strategy for tumor treatment.

[0004] However, for complex diseases such as tumors that are driven by multiple signaling pathways, single-target protein degradation strategies often have limited effects and are difficult to completely inhibit tumor growth, adaptation and drug resistance; multi-target synergistic degradation strategies face severe challenges in practical applications. Summary of the Invention

[0005] To address the deficiencies of the prior art, the present invention provides a protein degradation system based on polyamidoamine dendrimers, its preparation method, and its application. The protein degradation system provided by the present invention is capable of synergistically degrading MDM2 and GLUT1 proteins, not only loading ligands but also achieving co-delivery of MDM2 and GLUT1 degradation ligands. This synergistic degradation strategy not only effectively inhibits tumor cell proliferation and energy metabolism but also significantly enhances the stability of p53 protein. By restoring the normal function of p53 protein, tumor cell growth is further inhibited, providing a new strategy for tumor treatment.

[0006] In order to achieve the above object, the technical solution of the present invention is: In the first aspect, a protein degradation system based on polyamide-amine dendrimers is provided. The protein degradation system is a nanoparticle structure, which includes silica nanoparticles and a polyamide-amine dendrimer layer coated on the surface of the silica nanoparticles. The polyamide-amine dendrimer layer is chemically linked to an MDM2 protein ligand, a GLUT1 protein ligand, and an E3 ubiquitin enzyme ligand.

[0007] MDM2 protein is a key regulatory protein in tumor cells, and its overexpression can lead to abnormal proliferation of tumor cells. At the same time, overexpression of MDM2 protein can lead to downregulation of p53 protein expression, thereby relieving the inhibition of p53 protein on tumor cell proliferation. GLUT1 protein is a key transport protein for glucose uptake in tumor cells, and its overexpression is closely related to abnormal energy metabolism in tumor cells. In the protein degradation system of the present invention, MDM2 protein ligand and GLUT1 protein ligand are simultaneously connected, which can enable the protein degradation system to specifically bind to MDM2 protein and GLUT1 protein. In the protein degradation system, the E3 ubiquitin enzyme ligand connected can specifically bind to the E3 ubiquitin ligase. At this time, the protein degradation system can enable the E3 ubiquitin ligase to approach the MDM2 protein and GLUT1 protein through the binding of each ligand, thereby prompting the E3 ubiquitin ligase to accurately identify and interact with the MDM2 protein and GLUT1 protein, thereby adding ubiquitin molecules to the target protein and gradually forming a multi-ubiquitin chain. Proteases can degrade MDM2 and GLUT1 proteins based on the polyubiquitin chains they form. By degrading MDM2, they restore the normal function of p53, inhibiting tumor cell proliferation and increasing their sensitivity to this system. By degrading GLUT1, they inhibit tumor cell energy metabolism and weaken their viability. Furthermore, E3 ubiquitinase ligands enhance the specificity of the entire degradation system. By precisely directing the E3 ubiquitin ligase to the target protein, they minimize the impact on non-target proteins, effectively reducing potential side effects and significantly improving drug safety.

[0008] In order to bring into play the roles of MDM2 protein ligands, GLUT1 protein ligands and E3 ubiquitin enzyme ligands, the present invention selects silica nanoparticles with a surface-coated polyamide-amine dendritic polymer layer as a connection carrier. The highly branched tree structure and rich surface active groups (such as amino groups) of PAMAM in this carrier, combined with the high specific surface area, stable chemical properties and easy modification characteristics of SiO2, significantly improve the loading density and ligand binding ability, thereby enabling efficient loading of ligands and achieving multi-target degradation.

[0009] On the other hand, a method for preparing a protein degradation system based on polyamidoamine dendrimers comprises the following steps: Providing silica nanoparticles, and performing carboxyl modification on the surfaces of the silica nanoparticles; The silica nanoparticles with surface carboxyl modification are subjected to amidation reaction with polyamidoamine dendrimer (PAMAM), so that the polyamidoamine dendrimer coats the silica nanoparticles; The silica nanoparticles coated with polyamide-amine dendrimers are subjected to amidation reaction with MDM2 protein ligands, GLUT1 protein ligands, and E3 ubiquitin enzyme ligands, so that the MDM2 protein ligands, GLUT1 protein ligands, and E3 ubiquitin enzyme ligands are connected to the polyamide-amine dendrimers on the surface of the silica nanoparticles.

[0010] In a third aspect, a protein degradation system based on polyamidoamine dendrimers is used in the preparation of anti-tumor drugs.

[0011] In a fourth aspect, a method for treating tumors is provided, comprising administering to a subject a drug containing the above-mentioned protein degradation system based on polyamidoamine dendrimers.

[0012] The beneficial effects of the present invention are: The present invention uses silica nanoparticles coated with a polyamide-amine dendrimer layer to efficiently load MDM2 protein ligands, GLUT1 protein ligands and E3 ubiquitin enzyme ligands, so that the E3 ubiquitin ligase can approach the MDM2 protein and GLUT1 protein, thereby promoting the E3 ubiquitin ligase to accurately identify and interact with the MDM2 protein and GLUT1 protein, and then add ubiquitin molecules to the target protein, gradually forming polyubiquitin chains. By recognizing the polyubiquitin chains and achieving the degradation of the MDM2 protein and GLUT1 protein, the protease can not only restore the normal expression of the p53 protein, but also inhibit the energy metabolism of tumor cells, thereby synergistically regulating the key proteins of tumor cells at the molecular level. Through this synergistic degradation effect, the nanosystem more effectively inhibits the proliferation of tumor cells, providing a new strategy for tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0014] Figure 1 Transmission electron microscopy (TEM) images of SiO2 nanoparticles (A) and SG3TAC nanoparticles (B) prepared in Example 1 of the present invention, with a scale bar of 100 nm; Figure 2 Dynamic light scattering (DLS) data of SiO2 nanoparticles (A) and SG3TAC nanoparticles (B) prepared in Example 1 of the present invention; Figure 3 This is a graph showing the Zeta potential of the SG3 nanoparticles prepared in Example 1 of the present invention; Figure 4 This is a Fourier transform infrared (FT-IR) data result diagram of the SG3 nanoparticles prepared in Example 1 of the present invention; Figure 5 This is a graph showing the FT-IR data results of the SG3TAC nanoparticles prepared in Example 1 of the present invention; Figure 6 This is a graph showing the effects of different materials prepared in Example 1 of the present invention and Comparative Examples 1-2 on lung cancer cell toxicity; Figure 7 Live and dead cell fluorescence images of A549 cells incubated with different materials prepared in Example 1 and Comparative Examples 1-2 of the present invention. The fluorescent dyes used are Calcein-AM and Propidium Iodide (PI). Merge is a merged image. The scale bar is 100 μm. Figure 8 This is a Western blot analysis (WB) result of MDM2 protein in A549 cells treated with SG3-CM prepared in Comparative Example 1 at different concentrations in the present invention; Figure 9 This is a WB result diagram of MDM2 protein in A549 cells treated with SG3-CM prepared in Comparative Example 1 for different time periods; Figure 10 This is the WB result of MDM2 protein in A549 cells treated with different inhibitors in the present invention; Figure 11 This is the WB result of MDM2 protein in A549 cells treated with different materials in the present invention; Figure 12 The figure shows the immunofluorescence analysis results of the MDM2 protein expression in A549 cells treated with different materials in the present invention. The scale bar is 20 μm. Figure 13 This is a WB result of GLUT1 protein in A549 cells treated with SG3-CG prepared in Comparative Example 2 at different concentrations in the present invention; Figure 14 This is a WB result diagram of GLUT1 protein in A549 cells treated with SG3-CG prepared in Comparative Example 2 for different time periods; Figure 15 This is a graph showing the effects of different drug treatments on ATP levels in A549 cells; Figure 16 This is the WB result of GLUT1 protein in A549 cells treated with different inhibitors in the present invention; Figure 17 This is the WB result of GLUT1 protein in A549 cells treated with different materials in the present invention; Figure 18 The figure shows the immunofluorescence analysis results of the GLUT1 protein expression in A549 cells treated with different materials in the present invention. The scale bar is 20 μm. Figure 19The results of MDM2 protein and GLUT1 protein in A549 cells after treatment with SG3TAC at different concentrations for 12 hours in the present invention; Figure 20 This is the WB result of the p53 protein in A549 cells treated with different materials in the present invention; Figure 21 The immunofluorescence analysis results of the p53 protein expression in A549 cells treated with different materials in the present invention are shown in the figure. The scale bar is 20 μm. Figure 22 This is the WB result diagram of the effects of different treatments on the apoptosis proteins BAX, PUMA and NOXA in A549 cells. DETAILED DESCRIPTION

[0015] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0016] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0017] Given that the anti-cancer effect of single-target drug degradation is limited and the anti-cancer effect of multi-target drug synergistic degradation is difficult to achieve expected, the present invention proposes a protein degradation system based on polyamide-amine dendrimers, as well as its preparation method and application.

[0018] A typical embodiment of the present invention provides a protein degradation system based on polyamide-amine dendrimers. The protein degradation system is a nanoparticle structure, which includes silica nanoparticles and a polyamide-amine dendrimer layer coated on the surface of the silica nanoparticles. The polyamide-amine dendrimer layer is chemically linked to an MDM2 protein ligand, a GLUT1 protein ligand, and an E3 ubiquitin enzyme ligand.

[0019] In some embodiments, the nanoparticle structure has a particle size of 220-270 nm.

[0020] In some embodiments, the silica nanoparticles have a particle size of 200-250 nm.

[0021] Another embodiment of the present invention provides a method for preparing a protein degradation system based on polyamidoamine dendrimers, comprising the following steps: Providing silica nanoparticles, and performing carboxyl modification on the surfaces of the silica nanoparticles; Carboxyl-modified silica nanoparticles are subjected to an amidation reaction with polyamide-amine dendrimers, so that the polyamide-amine dendrimers coat the silica nanoparticles; The silica nanoparticles coated with polyamide-amine dendrimers are subjected to amidation reaction with MDM2 protein ligands, GLUT1 protein ligands, and E3 ubiquitin enzyme ligands, so that the MDM2 protein ligands, GLUT1 protein ligands, and E3 ubiquitin enzyme ligands are connected to the polyamide-amine dendrimers on the surface of the silica nanoparticles.

[0022] In some embodiments, the surface of the silica nanoparticles is modified by carboxylation by first aminated the silica nanoparticles, and then converting the amino groups to carboxyl groups using a dianhydride. Specifically, the surface of the silica nanoparticles is aminated using 3-aminopropyltrimethoxysilane. Specifically, the temperature for the aminated modification is 110-130°C. Specifically, the duration of the aminated modification is 22-26 hours. The dianhydride described herein refers to a cyclic anhydride formed by removing a water molecule from an organic dian acid molecule containing two carboxyl groups, such as succinic anhydride and glutaric anhydride. The dianhydride reacts with the amino group to remove the amide bond and generate a free carboxyl group, thereby converting the amino group to a carboxyl group. Specifically, the mass ratio of dianhydride to aminated silica nanoparticles is 14-16:1.

[0023] The purpose of the present invention of subjecting silica nanoparticles with surface carboxyl modifications to an amidation reaction with PAMAM is to coat the surface of the silica nanoparticles with PAMAM by forming an amide bond. To increase the PAMAM, in some embodiments, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are used to activate the carboxyl groups on the surface of the silica nanoparticles, and then a polyamide-amine dendrimer is added to carry out an amidation reaction.

[0024] In some embodiments, the mass ratio of silica nanoparticles to polyamidoamine dendrimers is 100:20.0-21.0. This ratio allows for better coverage of the polyamidoamine dendrimer on the surface of the silica nanoparticles and facilitates subsequent efficient loading of various ligands.

[0025] The present invention involves subjecting silica nanoparticles coated with polyamidoamine dendrimers to an amidation reaction with MDM2 protein ligands, GLUT1 protein ligands, and E3 ubiquitinase ligands to form amide bonds between the ligands and the polyamidoamine dendrimers on the silica nanoparticles. To improve the connection of the various ligands, in some embodiments, the carboxyl groups of the MDM2 protein ligands, GLUT1 protein ligands, and E3 ubiquitinase ligands are activated using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), followed by addition of the polyamidoamine dendrimer-coated silica nanoparticles for the amidation reaction. Specifically, the amidation reaction temperature is 20-30°C, and the reaction time is 1-3 days.

[0026] In some embodiments, the mass ratio of the silica nanoparticles coated with polyamidoamine dendrimers, the MDM2 protein ligand, the GLUT1 protein ligand, and the E3 ubiquitin enzyme ligand is 10:2.3-2.4:1-2:1-2.

[0027] A third embodiment of the present invention provides a use of the above-mentioned protein degradation system based on polyamidoamine dendrimers in the preparation of anti-tumor drugs.

[0028] In some embodiments, the tumor is lung cancer. Specifically, the tumor is non-small cell lung cancer. Specifically, the tumor cell is A549 cell.

[0029] A fourth embodiment of the present invention provides a method for treating tumors, comprising administering to a subject a drug containing the above-mentioned protein degradation system based on polyamidoamine dendrimers.

[0030] The route of administration that the present invention uses to the experimenter can be intravenous, intraperitoneal, intramuscular, subcutaneous, spinal column or other parenteral administration routes. Parenteral administration refers to the mode of administration usually by injection rather than enteral and topical administration, including but not limited to intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardial, intradermal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, spinal column, epidural and intrasternal injection and infusion. Alternatively, can be used via non-parenteral route, such as local, epidermal or mucosal administration route, for example intranasal, oral, vaginal, rectal, sublingual or topical.

[0031] In some embodiments, the subject is a human or an animal. Specifically, the animal can be a mouse, rat, dog, rabbit, pig, etc.

[0032] In some embodiments, the tumor is lung cancer. Specifically, the tumor is non-small cell lung cancer. Specifically, the tumor cell is A549 cell.

[0033] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0034] The materials and reagents used in the following examples are: 3-Aminopropyltrimethoxysilane (APTS), anhydrous toluene, succinic anhydride (SA, 99%), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich. Acetone (AppliChem GmbH), anhydrous ethanol, ammonia, dimethyl sulfoxide (DMSO, 99%), methanol, and nonfat dry milk were purchased from MacLean Reagents. Trypsin-EDTA (0.25%) was purchased from Gibco (Thermo Fisher Scientific). 20× TBST buffer, fetal bovine serum (FBS), DMEM, double antibody (penicillin and streptomycin mixture), and GAPDH antibody were provided by Solebro. 4',6-diamidino-2-phenylindole (DAPI), phenylmethylsulfonyl fluoride (PMSF), 3-(4,5-dimethylthiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), calcein-AM, propidium iodide (PI), SDS lysis buffer, bovine serum albumin (BSA), BCA protein concentration assay kit, immunostaining permeabilization solution (Triton X-100), horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L), and horseradish peroxidase-conjugated goat anti-mouse IgG (H+L) were purchased from Beyotime. A PAGE gel rapid preparation kit, dual-color prestained protein marker, and polyvinylidene fluoride membrane (PVDF, 0.45 μm) were purchased from Yazyme. Polyvinylidene fluoride membrane (PVDF, 0.22 μm) was purchased from Millipore. The third-generation polyamidoamine dendrimer-amino group (PAMAM-G3-NH2) was purchased from Weihai Chenyuan Molecular New Materials Co., Ltd. The E3 ubiquitinase ligand (Thalidomide-NH-CH2-COOH, CAS No. 927670-97-1), the MDM2 protein ligand (Idasanutlin, CAS No. 1229705-06-9), the GLUT1 ligand (Lavendustin B, CAS No. 125697-91-8), MG132, and MLN4924 were all purchased from MCE (Shanghai, China). The MDM2 antibody was purchased from Wuhan Huamei Bioengineering Co., Ltd., and the GLUT1 antibody was purchased from Hangzhou Huaan Biotechnology Co., Ltd. p53 antibody was purchased from Wuhan Tri-Ting Biotechnology Co., Ltd., BAX and PUMA antibodies were purchased from Abcam, NOXA antibody was purchased from Shanghai Abibio Biotechnology Co., Ltd., and the ultrasensitive ECL chemiluminescence kit was purchased from Suzhou New Saimei Biotechnology Co., Ltd. AlexaFluor 555-labeled goat anti-rabbit IgG and AlexaFluor 488-labeled goat anti-mouse IgG were purchased from Sangon Biotechnology (Shanghai) Co., Ltd. A549 lung cancer cells were obtained from Wuhan Punosai Life Science Co., Ltd.ATP content determination kit was purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.

[0035] The following instruments were used in the following examples: a Zeta potential and nanoparticle size analyzer (Zetasizer Nano, Malvern Instrument Ltd., UK); a transmission electron microscope (JEM2100, Thermo Fisher Scientific, Japan); a Fourier transform infrared spectrometer (FT-IR, TENSOR, Bruker, Germany); a chemiluminescence imager (ChemiScope 6200, Qinxiang, China); a fluorescence microscope (Ti2-E, Nikon, Japan); a microplate reader (K3 TOUCH, Fisher Scientific, China); an electrophoresis tank (Mini-PROTEAN® System, Bio-Rad, USA); and a protein transfer apparatus (Mini Trans-Blot cell, Bio-Rad, USA).

[0036] Example 1 A method for preparing a protein degradation system based on polyamidoamine dendrimers comprises the following steps: 1. Preparation of PAMAM-coated SiO2 nanoparticles: 1. Monodisperse spherical silica nanoparticles were prepared by the Stöber-Fink-Bohn method.

[0037] 500 μL of ammonia (25 w / w%), 14 mL of ethanol, and 2 mL of deionized water were stirred at 50°C for 20 minutes. Then, 500 μL of TEOS was added dropwise to the solution while stirring continuously at 50°C for 1.5 hours. After the reaction, the mixture was centrifuged (4000-14000 rpm, 10 minutes), washed three times with ethanol and deionized water, and dried at 70°C for 12 hours.

[0038] 2. PAMAM coated SiO2 nanoparticles 0.2 g of SiO2 was dispersed in 20 mL of anhydrous toluene containing 0.4 mL of APTS, stirred at 120°C for 24 h, centrifuged, washed sequentially with ultrapure water and methanol, and dried in vacuo at 50°C to obtain the product, SiO2-NH2. Subsequently, 0.2 g of the obtained SiO2-NH2 was dispersed in 20 mL of acetone, and 3 g of succinic anhydride was added to react for 24 h. After the reaction, the reaction was completed and dried in vacuo at 50°C to obtain the carboxylated product, SiO2-COOH. The obtained SiO2-COOH was then dispersed uniformly in 5 mL of DMSO. The mixture was activated sequentially with EDC (46 mg in 5 mL of DMSO) and NHS (27.6 mg in 5 mL of DMSO) and reacted with a solution of G3PAMAM dendrimers (41.6 mg in 5 mL of DMSO) under stirring for 1 day. The product was then centrifuged at 12,000 rpm for 10 min three times and freeze-dried to obtain PAMAM-coated SiO2 nanoparticles (SG3).

[0039] 2. Preparation of protein degradation system based on polyamidoamine dendrimers: NHS (45.0 mg, 5 mL DMSO), EDC (74.0 mg, 5 mL DMSO), E3 ubiquitinase ligand (11.7 mg, 5 mL DMSO), MDM2 protein ligand (10 mg, 5 mL DMSO), and GLUT1 protein ligand (10 mg, 5 mL DMSO) were mixed and incubated at 25°C for 2 h. SG3 particles (50.0 mg, 5 mL DMSO) were slowly added dropwise to the mixture. After stirring at 25°C for 2 days, the mixture was centrifuged at 12,000 rpm for 10 min. The resulting precipitate was washed three times with a water-anhydrous ethanol mixture and freeze-dried to obtain SG3TAC.

[0040] Example 2 This embodiment is the same as embodiment 1, except that: 2. Preparation of protein degradation system based on polyamidoamine dendrimers: NHS (45.0 mg, 5 mL DMSO), EDC (74.0 mg, 5 mL DMSO), E3 ubiquitinase ligand (11.7 mg, 5 mL DMSO), MDM2 protein ligand (10 mg, 5 mL DMSO), and GLUT1 protein ligand (10 mg, 5 mL DMSO) were mixed and incubated at 25°C for 3 h. SG3 particles (50.0 mg, 5 mL DMSO) were slowly added dropwise to the above mixture. After stirring at 25°C for 3 days, the mixture was centrifuged at 12,000 rpm for 10 min. The resulting precipitate was washed three times with a water-anhydrous ethanol mixture by centrifugation and freeze-dried to obtain the product.

[0041] Example 3 This embodiment is the same as embodiment 1, except that: 2. Preparation of protein degradation system based on polyamidoamine dendrimers: NHS (45.0 mg, 5 mL DMSO), EDC (74.0 mg, 5 mL DMSO), E3 ubiquitinase ligand (11.7 mg, 5 mL DMSO), MDM2 protein ligand (5 mg, 5 mL DMSO), and GLUT1 protein ligand (5 mg, 5 mL DMSO) were mixed and incubated at 25°C for 1 hour. SG3 particles (50.0 mg, 5 mL DMSO) were slowly added dropwise to the above mixture. After stirring at 25°C for 1 day, the mixture was centrifuged at 12,000 rpm for 10 minutes. The resulting precipitate was washed three times with a water-anhydrous ethanol mixture by centrifugation and freeze-dried to obtain the product.

[0042] Comparative Example 1 This comparative example is the same as Example 1, except that: 2. Preparation of protein degradation system based on polyamidoamine dendrimers: NHS (45.0 mg, 5 mL DMSO), EDC (74.0 mg, 5 mL DMSO), E3 ubiquitinase ligand (11.7 mg, 5 mL DMSO), and MDM2 protein ligand (10 mg, 5 mL DMSO) were mixed and incubated at 25°C for 2 h. 50.0 mg of SG3 particles were added to 5 mL of DMSO and slowly added dropwise to the above mixture. After stirring at 25°C for 2 days, the mixture was centrifuged at 12,000 rpm for 10 min. The resulting precipitate was washed three times with a water-anhydrous ethanol mixture and freeze-dried to obtain SG3-CM.

[0043] Comparative Example 2 This comparative example is the same as Example 1, except that: 2. Preparation of protein degradation system based on polyamidoamine dendrimers: NHS (45.0 mg, 5 mL DMSO), EDC (74.0 mg, 5 mL DMSO), E3 ubiquitinase ligand (11.7 mg, 5 mL DMSO), and GLUT1 protein ligand (10 mg, 5 mL DMSO) were mixed and incubated at 25°C for 2 h. 50.0 mg of SG3 particles were added to 5 mL of DMSO and slowly added dropwise to the above mixture. After stirring at 25°C for 2 days, the mixture was centrifuged at 12,000 rpm for 10 min. The resulting precipitate was washed three times with a water-anhydrous ethanol mixture and freeze-dried to obtain SG3-CG.

[0044] Comparative Example 3 This comparative example is the same as Example 1, except that: 2. Preparation of protein degradation system based on polyamidoamine dendrimers: NHS (45.0 mg, 5 mL DMSO), EDC (74.0 mg, 5 mL DMSO), and E3 ubiquitinase ligand (11.7 mg, 5 mL DMSO) were mixed and incubated at 25°C for 2 h. 50.0 mg of SG3 particles were added to 5 mL of DMSO and slowly added dropwise to the mixture. After stirring at 25°C for 2 days, the mixture was centrifuged at 12,000 rpm for 10 min. The resulting precipitate was washed three times with a water-anhydrous ethanol mixture and freeze-dried to obtain SG3-C.

[0045] Comparative Example 4 This comparative example is the same as Example 1, except that: 2. Preparation of protein degradation system based on polyamidoamine dendrimers: NHS (45.0 mg, 5 mL DMSO), EDC (74.0 mg, 5 mL DMSO), and MDM2 protein ligand (10 mg, 5 mL DMSO) were mixed and incubated at 25°C for 2 h. SG3 particles (50.0 mg, 5 mL DMSO) were slowly added dropwise to the mixture. After stirring at 25°C for 2 days, the mixture was centrifuged at 12,000 rpm for 10 min. The resulting precipitate was washed three times with a water-anhydrous ethanol mixture and freeze-dried to obtain SG3-M.

[0046] Comparative Example 5 This comparative example is the same as Example 1, except that: 2. Preparation of protein degradation system based on polyamidoamine dendrimers: NHS (45.0 mg, 5 mL DMSO), EDC (74.0 mg, 5 mL DMSO), and GLUT1 protein ligand (10 mg, 5 mL DMSO) were mixed and incubated at 25°C for 2 h. SG3 particles (50.0 mg, 5 mL DMSO) were slowly added dropwise to the mixture. After stirring at 25°C for 2 days, the mixture was centrifuged at 12,000 rpm for 10 min. The resulting precipitate was washed three times with a water-anhydrous ethanol mixture and freeze-dried to obtain SG3-G.

[0047] Experimental Example 1 Study on the cytotoxicity of different materials to lung cancer cells Lung cancer A549 cells were seeded in a 96-well plate and incubated in a 37°C, 5% CO2 incubator for 12 hours to allow cell attachment. Subsequently, 20 μL of SG3, 20 μL of SG3-CM, 20 μL of SG3-CG, or 20 μL of SG3TAC at varying concentrations were added to each well and incubated for an additional 12 hours. MTT solution (100 μL, 5 mg / mL) was added to each well and incubated for an additional 4 hours. After removing the supernatant from the 96-well plate, 100 μL of DMSO was added to each well. The absorbance of the samples was recorded at 492 nm.

[0048] Test Example 2 Study on degradation conditions A549 cells were grown at 2 × 10 6 The cells were cultured in 6-well cell culture plates for 24 h.

[0049] In order to study the relationship between the concentration of SG3-CM, SG3-CG or SG3TAC and the protein degradation effect, SG3-CM, SG3-CG or SG3TAC were diluted with DMEM to 0-50 μg / mL, and 40 μL was incubated with cells for 12 h.

[0050] In order to study the effect of the incubation time of SG3-CM, SG3-CG or SG3TAC with cells on the protein degradation effect, 40 μL of 40 μg / mL SG3-CM solution, SG3-CG solution or SG3TAC solution was incubated with cells for 0-24 h.

[0051] To investigate the degradation pathway of SG3-CM or SG3-CG, cells were treated with MG132 or MLN4924 for 1 h, and then 40 μL of 40 μg / mL SG3-CM solution or 40 μL of 40 μg / mL SG3-CG solution were added to different wells and cultured at 37°C for 48 h.

[0052] Wash the cells obtained from the above three sections twice with ice-cold PBS, add 150-200 μL of SDS lysis buffer (containing 1 mM protease inhibitor cocktail), and quantify protein using a BCA protein assay kit. Then, boil the protein sample and SDS-PAGE sample loading buffer (5×) for 30 minutes. Load 20 μg to 40 μg of sample per well onto a 10% or 12% SDS-PAGE gel and run the gel on an 80 V electrophoresis apparatus for 1-2 hours. Transfer the proteins from the gel to a 0.45 μm or 0.22 μm PVDF membrane using a protein transfer apparatus (120 mA, 1 hour). The membrane was blocked with 25 mL of 5% nonfat dry milk in TBST at room temperature for 1 hour, then washed three times with TBST and incubated with antibodies against MDM2 (1:2000), GLUT1 (1:50,000), GAPDH (1:50,000), p53 (1:25,000), BAX (1:5000), PUMA (1:3000), or NOXA (1:2000) for 12 hours at 4°C. The membrane was then washed three times with TBST buffer (10 minutes each). The membrane was then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L) (1:1000) and anti-mouse IgG (1:1000) for 1 hour at room temperature. Finally, the membrane was washed three times with TBST buffer (10 minutes each) and developed using electrochemiluminescence (ECL) developer. Western blot bands were detected on a chemiluminescence imager.

[0053] Experimental Example 3 Study on the feasibility of SG3-CM and SG3-CG to degrade proteins A549 cells were cultured in confocal microplates and incubated in a 37°C, 5% CO2 incubator for 12 hours to allow attachment. PBS, SG3, SG3-CM, SG3-CG, and SG3TAC were added to different culture dishes and incubated for an additional 12 hours. The cells were then rinsed three times with PBS. A549 cells were then fixed with anhydrous methanol for 15 minutes. 0.5% Triton X-100 was then added and incubated at room temperature for 10 minutes. After washing with PBS, 3% BSA solution was added to the samples and incubated at room temperature for 2 hours. After washing with PBS, antibodies against MDM2, GLUT1, or p53 were incubated overnight at 4°C. After washing with PBS, AlexaFluor 488-conjugated goat anti-mouse IgG or AlexaFluor 555-conjugated goat anti-rabbit IgG was added and incubated at room temperature for 1 hour. After washing with PBS, the cells were stained with DAPI for 15 minutes. After washing with PBS, images were captured using a fluorescence microscope.

[0054] Results and Discussion 1. Characterization of SiO2, SG3, SG3-CM, SG3-CG, and SG3TAC Nanoparticles The morphologies of SiO2 and SG3TAC nanoparticles were characterized by TEM. Figure 1 As shown. The prepared SiO2 and SG3TAC have uniform morphology and good dispersion. Compared with SiO2 nanoparticles, Figure 1 As shown in Figure A, the surface of SG3TAC nanoparticles is rougher. Figure 1 As shown in Figure 3B, this is because the coverage of PAMAM reduces the surface flatness.

[0055] The particle size of SiO2 nanoparticles is approximately 230 nm, which is consistent with the dynamic light scattering (DLS) data, e.g. Figure 2 As shown in Figure A. The particle size of SG3TAC nanoparticles is about 250 nm, which is also consistent with the DLS data, as shown in Figure 4. Figure 2 As shown in B.

[0056] By surface potential analysis, such as Figure 3 As shown in Figure 3, the surface potential of carboxylated SiO2 nanoparticles was negative (-24.8 ± 0.62 mV). After loading with PAMAM, the surface potential of the particles was positive (3.34 ± 0.27 mV), which was due to the potential reversal caused by the abundant amino groups on PAMAM.

[0057] The groups on the surface of nanoparticles were analyzed by FT-IR, such as Figure 4 As shown in the infrared absorption spectrum of carboxylated SiO2 nanoparticles, at 960 cm -1 , 1020 - 1110 cm -1 、1640 cm -1 There are obvious absorption peaks at 1560-1640 cm-1. These three absorption peaks are caused by the asymmetric bending and stretching vibration of Si-OH, the asymmetric stretching vibration of Si-O-Si, and the vibration of -COOH. -1 There is an absorption peak at 960 cm, which is caused by NH bending vibration. -1 , 1020 - 1110 cm -1 、1560-1640 cm -1 There were three obvious absorption peaks, which confirmed that SG3 was SiO2 nanoparticles with rich amino groups on the surface.

[0058] Observe the infrared absorption peak of the product (SG3-C) after the reaction of SG3 and E3 ubiquitin enzyme ligand, such as Figure 5As shown, it was found that 1700-2000 cm -1 The product of the reaction between SG3-C and GLUT1 ligand (SG3-CG) has an absorption peak at 3200-3500 cm -1 The absorption peak appears at 2210-2260 cm, which is the -OH stretching vibration peak on the GLUT1 ligand. The product (SG3-CM) after the reaction of SG3-C and MDM2 ligand is at 2210-2260 cm -1 An absorption peak appears at 595 cm, which is the characteristic peak of the C≡N triple bond on the MDM2 ligand. SG3-C combines with the GLUT1 ligand and the MDM2 ligand to form SG3TAC, which is at 595 cm -1 、3200-3500 cm -1 It has the same absorption peak as SG3-CG. SG3TAC is at 2210-2260 cm -1 It has the same absorption peak as SG3-CM.

[0059] 2. Evaluation of cytotoxicity of SG3, SG3-CM, SG3-CG, and SG3TAC nanoparticles The MTT assay was used to detect the viability of A549 cells treated with SG3, SG3-CG, SG3-CM, and SG3TAC. Figure 6 As shown, SG3 had no significant effect on A549 cell viability within the 0-40 μg / mL concentration range, demonstrating good compatibility. A549 cell viability gradually decreased with increasing concentrations of SG3-CG, SG3-CM, and SG3TAC (0-40 μg / mL). SG3TAC had the most pronounced inhibitory effect on A549 cells, demonstrating its stronger anti-tumor activity.

[0060] Further A549 cell live-death experiments revealed that compared with the PBS group, the number of live cells (showing green fluorescence) in the SG3TAC-treated cells was significantly reduced, while the number of dead cells (showing red fluorescence) was significantly increased. Moreover, the proportion of dead cells in the SG3TAC-treated group was significantly higher than that in the SG3-CM and SG3-CG groups. Figure 7 This result is consistent with the conclusion of the MTT experiment, confirming the cytotoxicity and anti-proliferative effects of SG3TAC.

[0061] 3. Evaluation of MDM2 protein degradation by SG3-CM Western blot analysis was performed to analyze the degradation of MDM2 protein in A549 cells. A549 cells were exposed to different concentrations of SG3-CM nanoparticles (0-50 μg / mL) for 12 h. The results of Western blot analysis are shown in Figure 2. Figure 8As shown in the figure, as the concentration of SG3-CM increases, the expression level of MDM2 protein gradually decreases, showing a concentration-dependent degradation trend. In particular, at higher concentrations (40 μg / mL and 50 μg / mL), the brightness of the MDM2 protein band is significantly lower than that of the control group (0 μg / mL), indicating that SG3-CM effectively promotes the degradation of MDM2 protein at these concentrations.

[0062] To evaluate the effect of exposure time on MDM2 protein expression, A549 cells were exposed to 40 μg / mL SG3-CM for 0-24 h. Figure 9 As shown in the figure, the MDM2 protein band gradually became lighter after 9 h, indicating the powerful degradation ability of SG3-CM.

[0063] On this basis, in order to further explore the degradation pathway of SG3-CM, the proteasome inhibitor MG132 and the NEDD8 activating enzyme (NAE) inhibitor MLN4924 were used to conduct experiments. Figure 10 As shown in the figure, compared with A549 cells directly exposed to SG3-CM, A549 cells pretreated with MG132 and then exposed to SG3-CM significantly inhibited MDM2 protein degradation, indicating that SG3-CM degradation of MDM2 protein is related to the proteasome pathway. A549 cells pretreated with MLN4924 and then exposed to SG3-CM also significantly inhibited MDM2 protein degradation, indicating that SG3-CM degradation of MDM2 protein is also related to the ubiquitination pathway.

[0064] At the same time, the degradation of MDM2 protein in A549 cells by SG3-C, SG3-M (without E3 ubiquitinase ligand) and their mixture was investigated. Compared with SG3-CM, MDM2 protein was hardly degraded in the SG3-C group, SG3-M group and their mixture. Figure 11 This result clearly demonstrates that only SG3-CM can effectively induce MDM2 protein degradation. It also indirectly suggests that the mechanism of MDM2 protein degradation in A549 cells is through the formation of a ternary complex between PAMAM, MDM2 protein, and E3 ubiquitinase.

[0065] In addition, immunofluorescence imaging analysis of MDM2 protein in cells was performed. Here, AlexaFluor488-labeled goat anti-mouse IgG was used. In A549 cells treated with SG3-CM and SG3TAC, the green fluorescence signal was significantly weakened, as shown in Figure 2. Figure 12 As shown, it illustrates the high efficiency of SG3-CM and SG3TAC in regulating MDM2 protein expression.

[0066] 4. Evaluation of SG3-CG on GLUT1 protein degradation Western blot analysis was performed to analyze the degradation of GLUT1 protein in A549 cells. A549 cells were exposed to different concentrations of SG3-CG nanoparticles (0-50 μg / mL) for 12 h. The results of Western blot analysis are shown in Figure 2. Figure 13 As shown in the figure, as the concentration of SG3-CG increased, the expression of GLUT1 protein gradually decreased, showing a concentration-dependent degradation trend. In particular, at higher concentrations (40 μg / mL and 50 μg / mL), the brightness of the GLUT1 protein band was significantly lower than that of the control group (0 μg / mL), indicating that SG3-CG effectively promoted the degradation of GLUT1 protein at these concentrations.

[0067] To evaluate the effect of exposure time on GLUT1 protein expression, A549 cells were exposed to 40 μg / mL SG3-CG for 0-24 h. Figure 14 As shown in the figure, the GLUT1 protein band gradually became lighter after 6 h, indicating the strong degradation ability of SG3-CG.

[0068] At the same time, the detection of intracellular ATP content found that the ATP level of cells treated with SG3-CG decreased significantly, such as Figure 15 As shown, this indicates that SG3-CG effectively inhibits the function of GLUT1 protein, reduces the input of glucose, and thus affects the generation of ATP, suggesting that it exerts its anti-tumor effect by interfering with cellular energy metabolism.

[0069] On this basis, in order to further explore the degradation pathway of SG3-CG, the proteasome inhibitor MG132 and the NEDD8 activating enzyme (NAE) inhibitor MLN4924 were used to carry out experiments. The results are as follows Figure 16 As shown in the figure, compared with direct exposure of A549 cells to SG3-CG, MG132 pre-treatment of A549 cells and subsequent exposure to SG3-CG significantly inhibited GLUT1 protein degradation, suggesting that SG3-CG degradation of GLUT1 protein is related to the proteasome pathway. MLN4924 pre-treatment of A549 cells and subsequent exposure to SG3-CG significantly inhibited GLUT1 protein degradation, suggesting that SG3-CG degradation of GLUT1 protein is also related to the ubiquitination pathway.

[0070] At the same time, the degradation of GLUT1 protein in A549 cells by SG3-C, SG3-G (without E3 ubiquitinase ligand) and their mixture was investigated. Compared with SG3-CG, GLUT1 protein was almost not degraded in the SG3-C group, SG3-G group and their mixture group. Figure 17This result clearly demonstrates that only SG3-CG can effectively induce GLUT1 protein degradation. It also indirectly suggests that in A549 cells, PAMAM forms a ternary complex with GLUT1 protein and E3 ubiquitinase, leading to GLUT1 protein degradation.

[0071] In addition, immunofluorescence imaging analysis of GLUT1 protein in cells was performed. Here, AlexaFluor555-labeled goat anti-rabbit IgG was used. In A549 cells treated with SG3-CG and SG3TAC, the red fluorescence signal was significantly weakened, as shown in Figure 2. Figure 18 As shown, it illustrates the high efficiency of SG3-CG and SG3TAC in regulating GLUT1 protein expression.

[0072] 5. Effect of SG3TAC Nanoparticles on p53 Protein Expression Figure 19 Results showed that after 12 hours of treatment with different concentrations of SG3TAC nanoparticles (10-40 µg / mL), the expression levels of both GLUT1 and MDM2 proteins exhibited significant concentration-dependent changes. Specifically, GLUT1 protein expression gradually decreased with increasing SG3TAC concentration, being almost completely inhibited at 30 µg / mL, indicating that SG3TAC has a significant degradation effect on GLUT1 protein. Similarly, MDM2 protein expression also showed a concentration-dependent decrease after SG3TAC treatment, reaching its lowest level at the highest concentration (40.0 µg / mL).

[0073] The results of the expression level study in A549 cells are as follows Figure 20 As shown in the figure, compared with the PBS group, p53 protein expression levels in A549 cells treated with SG3, SG3-CM, SG3-CG, and SG3TAC also changed. SG3 treatment had no significant effect on p53 protein expression, while SG3-CM and SG3-CG treatment increased p53 protein expression. The SG3TAC treatment group, in particular, achieved the highest p53 protein expression, indicating that SG3TAC is a more effective upregulator of p53 protein expression.

[0074] In addition, immunofluorescence imaging analysis of p53 protein in cells was performed. Here, AlexaFluor555-labeled goat anti-rabbit IgG was used. In A549 cells treated with SG3-CM, SG3-CG, and SG3TAC, SG3-CM and SG3-CG treatments significantly enhanced the red fluorescence signal. Figure 21As shown, these two treatments can effectively promote the expression of p53 protein. The most obvious is the SG3TAC treatment group, where the red fluorescence signal of p53 protein in cells is the strongest, demonstrating its high efficiency in inducing p53 protein expression.

[0075] The expression of intracellular apoptosis proteins (BAX protein, PUMA protein and NOXA protein) is as follows Figure 22 As shown in the figure, compared with the control group, the expression levels of BAX, PUMA, and NOXA proteins were all upregulated in A549 cells treated with SG3-CM, SG3-CG, and SG3TAC. Specifically, treatment with SG3-CM and SG3-CG increased the expression of these proteins, while the expression of BAX, PUMA, and NOXA proteins in the SG3TAC-treated group further increased significantly, reaching the highest levels. This indicates that SG3TAC is more potent in inducing the expression of these pro-apoptotic proteins. This result suggests that SG3TAC can more effectively activate the p53 signaling pathway, upregulate the expression of pro-apoptotic proteins, and thus enhance the apoptotic process.

[0076] In summary, SG3TAC nanoparticles significantly reduced the expression levels of MDM2 protein and GLUT1 protein in a concentration-dependent manner, while effectively enhancing the expression of p53 protein, demonstrating its potential in regulating the expression of key proteins in tumor cells.

[0077] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A protein degradation system based on polyamidoamine dendrimers, characterized in that: The protein degradation system is a nanoparticle structure, which includes silica nanoparticles and a polyamide-amine dendrimer layer coated on the surface of the silica nanoparticles. The polyamide-amine dendrimer layer is connected to the MDM2 protein ligand, the GLUT1 protein ligand and the E3 ubiquitin enzyme ligand through chemical bonds.

2. The protein degradation system according to claim 1, wherein: The particle size of the nanoparticle structure is 220~270 nm.

3. The protein degradation system according to claim 1, wherein: The particle size of silica nanoparticles is 200~250nm.

4. A method for preparing a protein degradation system based on polyamidoamine dendrimers, characterized in that: The steps include: Providing silica nanoparticles, and performing carboxyl modification on the surfaces of the silica nanoparticles; Carboxyl-modified silica nanoparticles are subjected to an amidation reaction with polyamide-amine dendrimers, so that the polyamide-amine dendrimers coat the silica nanoparticles; The silica nanoparticles coated with polyamide-amine dendrimers are subjected to amidation reaction with MDM2 protein ligands, GLUT1 protein ligands, and E3 ubiquitin enzyme ligands, so that the MDM2 protein ligands, GLUT1 protein ligands, and E3 ubiquitin enzyme ligands are connected to the polyamide-amine dendrimers on the surface of the silica nanoparticles.

5. The preparation method according to claim 4, wherein: The process of carboxyl modification of the surface of the silicon dioxide nanoparticles is as follows: firstly, the surface of the silicon dioxide nanoparticles is amino modified, and then the amino groups are modified into carboxyl groups by dianhydride.

6. The preparation method according to claim 4, wherein: The carboxyl groups on the surface of silica nanoparticles were activated by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and then polyamidoamine dendrimer was added to carry out amidation reaction.

7. The preparation method according to claim 4, characterized in that: The mass ratio of silica nanoparticles to polyamide-amine dendrimer is 100:20.0~21.

0.

8. The preparation method according to claim 4, wherein: The mass ratio of silica nanoparticles coated with polyamide-amine dendrimers, MDM2 protein ligand, GLUT1 protein ligand, and E3 ubiquitin enzyme ligand is 10:2.3~2.4:1~2:1~2.

9. Use of the protein degradation system according to any one of claims 1 to 3 or the protein degradation system obtained by the preparation method according to any one of claims 4 to 8 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that: The tumor is lung cancer.

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