Protein degradation system based on polyamidoamine dendrimers and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and relates to a protein degradation system based on a polyamide-amine dendritic polymer, and a preparation method and application thereof. The protein degradation system is in a nanoparticle structure, the nanoparticle structure comprises a silica nanoparticle and a polyamide-amine dendritic polymer layer coated on the surface of the silica nanoparticle, and MDM2 protein ligands, GLUT1 protein ligands and E3 ubiquitin enzyme ligands are connected on the polyamide-amine dendritic polymer layer through chemical bonds. The protein degradation system provided by the application can synergistically degrade MDM2 protein and GLUT1 protein. This synergistic degradation strategy can not only effectively inhibit the proliferation and energy metabolism of tumor cells, but also significantly enhance the stability of p53 protein. By restoring the normal function of p53 protein, the growth of tumor cells is further inhibited, thereby providing a new strategy for tumor treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to a protein degradation system based on a polyamide-amine dendritic polymer as well as a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the general background of the application and does not necessarily constitute an admission or any kind of suggestion that this information forms the general prior art already known to a person of ordinary skill in the art.

[0003] The occurrence and development of malignant tumors involve abnormal expression and dysfunction of multiple key regulatory proteins, leading to uncontrolled cell proliferation and metabolic reprogramming. Among the numerous regulatory factors, the inactivation of certain key tumor suppressor proteins is an important driving factor for tumor development. For example, an important tumor suppressor protein (such as p53) often loses its function due to the overexpression of its negative regulatory factor, which significantly reduces the intracellular level or activity of the tumor suppressor protein by promoting its ubiquitination degradation pathway, thereby relieving the inhibition of tumor cell proliferation. Restoring the normal function of such key tumor suppressor proteins is one of the important strategies for tumor treatment.

[0004] However, for tumors, which are complex diseases driven by multiple signaling pathways, the protein degradation strategy of a single target often has limited effect, and it is difficult to completely inhibit tumor growth, adaptation and drug resistance; the multi-target synergistic degradation strategy faces severe challenges in practical application. SUMMARY

[0005] In order to solve the problems of the prior art, the present application aims to provide a protein degradation system based on a polyamide-amine dendritic polymer as well as a preparation method and application thereof. The protein degradation system provided by the present application can synergistically degrade MDM2 protein and GLUT1 protein, not only load ligands, but also achieve the co-delivery of MDM2 protein and GLUT1 protein degradation ligands. This synergistic degradation strategy can not only effectively inhibit the proliferation and energy metabolism of tumor cells, but also significantly enhance the stability of p53 protein. By restoring the normal function of p53 protein, the growth of tumor cells is further inhibited, providing a new strategy for tumor treatment.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0007] In a first aspect, a protein degradation system based on a polyamide-amine dendritic polymer is provided. The protein degradation system is in the form of a nanoparticle structure, which comprises a silica nanoparticle and a polyamide-amine dendritic polymer layer coated on the surface of the silica nanoparticle. The polyamide-amine dendritic polymer layer is chemically linked to an MDM2 protein ligand, a GLUT1 protein ligand and an E3 ubiquitin ligase ligand.

[0008] MDM2 protein is a key regulatory protein in tumor cells, and its overexpression can cause abnormal proliferation of tumor cells. At the same time, the overexpression of MDM2 protein can cause the down-regulation 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 the abnormal energy metabolism of tumor cells. The protein degradation system of the present application simultaneously connects MDM2 protein ligand and GLUT1 protein ligand, which can specifically bind the protein degradation system to MDM2 protein and GLUT1 protein. The E3 ubiquitin ligase ligand in the protein degradation system can specifically bind to the E3 ubiquitin ligase. At this time, the protein degradation system can make the E3 ubiquitin ligase close to the MDM2 protein and the GLUT1 protein through the binding of each ligand, so as to promote the E3 ubiquitin ligase to accurately recognize and interact with the MDM2 protein and the GLUT1 protein, thereby adding ubiquitin molecules to the target protein and gradually forming a polyubiquitin chain. The protease can degrade the MDM2 protein and the GLUT1 protein according to the formed polyubiquitin chain; by degrading the MDM2 protein, the normal function of the p53 protein is restored, the proliferation of tumor cells is inhibited, and the sensitivity of tumor cells to the system is enhanced; by degrading the GLUT1 protein, the energy metabolism of tumor cells is inhibited, and the survival ability of tumor cells is weakened. In addition, the E3 ubiquitin ligase ligand can also enhance the specificity of the entire degradation system, accurately guide the E3 ubiquitin ligase to the target protein, minimize the impact on non-target proteins, effectively reduce potential side effects, and significantly improve drug safety.

[0009] In order to exert the effects of MDM2 protein ligand, GLUT1 protein ligand and E3 ubiquitin ligase ligand, the present application selects silica nanoparticles coated with a layer of polyamide-amine dendrimer as a connecting carrier. The highly branched dendritic structure of PAMAM and the rich surface active groups (such as amino groups) in the 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 capacity, so as to efficiently load the ligand, and then realize the degradation of multiple targets.

[0010] On the other hand, a preparation method of a protein degradation system based on polyamide-amine dendrimer, comprising the following steps:

[0011] Silicon dioxide nanoparticles are provided, and the surface of the silicon dioxide nanoparticles is carboxylated and modified;

[0012] The silicon dioxide nanoparticles with carboxylated and modified surface are subjected to amidation reaction with polyamide-amine dendrimer (PAMAM), so as to coat the silicon dioxide nanoparticles with polyamide-amine dendrimer;

[0013] The silica nanoparticles coated with a polyamide-amine dendrimer layer are subjected to an amidation reaction with MDM2 protein ligands, GLUT1 protein ligands and E3 ubiquitinase ligands, so that the MDM2 protein ligands, GLUT1 protein ligands and E3 ubiquitinase ligands are connected to the polyamide-amine dendrimers on the surface of the silica nanoparticles.

[0014] In a third aspect, the application provides a use of the polyamide-amine dendrimer-based protein degradation system in the preparation of an antitumor drug.

[0015] In a fourth aspect, the application provides a method for treating tumors, which comprises administering to a subject a drug containing the polyamide-amine dendrimer-based protein degradation system.

[0016] The application has the following advantages:

[0017] The silica nanoparticles coated with a polyamide-amine dendrimer layer are subjected to an amidation reaction with MDM2 protein ligands, GLUT1 protein ligands and E3 ubiquitinase ligands, so that the MDM2 protein ligands, GLUT1 protein ligands and E3 ubiquitinase ligands are connected to the polyamide-amine dendrimers on the surface of the silica nanoparticles. BRIEF DESCRIPTION OF DRAWINGS

[0018] The accompanying drawings, which form a part of the specification, are included to provide a further understanding of the application and are incorporated herein in conjunction with the description of the application. The drawings are not to be considered restrictive in scope and are included as a part of this specification only to explain certain embodiments of the application.

[0019] Figure 1 TEM images of the SiO2 nanoparticles (A) and SG3TAC nanoparticles (B) prepared in Example 1 of the application, with a scale bar of 100 nm;

[0020] Figure 2 DLS data graphs of the SiO2 nanoparticles (A) and SG3TAC nanoparticles (B) prepared in Example 1 of the application;

[0021] Figure 3 Zeta potential results of the SG3 nanoparticles prepared in Example 1 of the application;

[0022] Figure 4 FT-IR data result graph of SG3 nanoparticles prepared for Example 1 of the present application;

[0023] Figure 5 FT-IR data result graph of SG3TAC nanoparticles prepared for Example 1 of the present application;

[0024] Figure 6 Result graph of the influence of different materials prepared for Example 1 and Comparative Examples 1-2 of the present application on lung cancer cell toxicity;

[0025] Figure 7 Fluorescence image of A549 cell live and dead cell incubated with different materials prepared for Example 1 and Comparative Examples 1-2 of the present application, wherein the fluorescent dye used is Calcein-AM, PI (propidium iodide), Merge is a merged image, and the scale bar is 100 μm;

[0026] Figure 8 Western blot (WB) result graph of MDM2 protein of A549 cells treated with SG3-CM prepared by Comparative Example 1 in the present application at different concentrations;

[0027] Figure 9 WB result graph of MDM2 protein of A549 cells treated with SG3-CM prepared by Comparative Example 1 in the present application for different times;

[0028] Figure 10 WB result graph of MDM2 protein of A549 cells treated with different inhibitors in the present application;

[0029] Figure 11 WB result graph of MDM2 protein of A549 cells treated with different materials in the present application;

[0030] Figure 12 Result graph of immunofluorescence analysis of MDM2 protein expression of A549 cells treated with different materials in the present application, and the scale bar is 20 μm;

[0031] Figure 13 WB result graph of GLUT1 protein of A549 cells treated with SG3-CG prepared by Comparative Example 2 in the present application at different concentrations;

[0032] Figure 14 WB result graph of GLUT1 protein of A549 cells treated with SG3-CG prepared by Comparative Example 2 in the present application for different times;

[0033] Figure 15 Result graph of the influence of different drug treatments on ATP level of A549 cells in the present application;

[0034] Figure 16 This is a Western blot (WB) image of GLUT1 protein in A549 cells after treatment with different inhibitors in this invention.

[0035] Figure 17 This is a Western blot (WB) image of GLUT1 protein in A549 cells after treatment with different materials in this invention.

[0036] Figure 18 The image shows the immunofluorescence analysis results of the effects of different material treatments on GLUT1 protein expression in A549 cells in this invention. The scale bar is 20 μm.

[0037] Figure 19 The figure shows the results of MDM2 and GLUT1 protein in A549 cells after treatment with different concentrations of SG3TAC for 12 hours in this invention.

[0038] Figure 20 The image shows the Western blot results of different material treatments on p53 protein in A549 cells in this invention.

[0039] Figure 21 The image shows the immunofluorescence analysis results of the effects of different material treatments on the expression of p53 protein in A549 cells in this invention. The scale bar is 20 μm.

[0040] Figure 22 The image shows the Western blot results of different treatments on the apoptosis proteins BAX, PUMA, and NOXA in A549 cells in this invention. Detailed Implementation

[0041] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0042] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0043] Given that the anticancer effects of single-target drug degradation are limited and the anticancer effects of multi-target drug synergistic degradation are difficult to achieve as expected, this invention proposes a protein degradation system based on polyamide-amine dendritic polymers, its preparation method, and its application.

[0044] In an exemplary embodiment of the present application, a protein degradation system based on polyamide-amine dendrimers is provided, wherein the protein degradation system is in a nanoparticle structure, and the nanoparticle structure comprises silica nanoparticles and a polyamide-amine dendrimer layer coated on the surface of the silica nanoparticles, and the polyamide-amine dendrimer layer is chemically linked with MDM2 protein ligands, GLUT1 protein ligands and E3 ubiquitin ligase ligands.

[0045] In some embodiments, the particle size of the nanoparticle structure is 220-270 nm.

[0046] In some embodiments, the particle size of the silica nanoparticles is 200-250 nm.

[0047] In another exemplary embodiment of the present application, a preparation method of a protein degradation system based on polyamide-amine dendrimers is provided, comprising the following steps:

[0048] Silica nanoparticles are provided, and the surface of the silica nanoparticles is carboxylated and modified;

[0049] The silica nanoparticles with carboxylated and modified surface are subjected to an amidation reaction with polyamide-amine dendrimers, so that the polyamide-amine dendrimers coat the silica nanoparticles;

[0050] The silica nanoparticles with polyamide-amine dendrimers coated on the surface are subjected to an amidation reaction with MDM2 protein ligands, GLUT1 protein ligands and E3 ubiquitin ligase ligands, so that the MDM2 protein ligands, GLUT1 protein ligands and E3 ubiquitin ligase ligands are linked with the polyamide-amine dendrimers on the surface of the silica nanoparticles.

[0051] In some embodiments, the carboxylated and modified process of the surface of the silica nanoparticles is as follows: first, the surface of the silica nanoparticles is subjected to an amino modification, and then the amino group is modified to a carboxyl group by using a diacid anhydride. Specifically, the surface of the silica nanoparticles is subjected to an amino modification by using 3-aminopropyltrimethoxysilane. Specifically, the temperature of the amino modification is 110-130 ℃. Specifically, the time of the amino modification is 22-26 h. The diacid anhydride used in the present application refers to a cyclic acid anhydride formed by removing one molecule of water from an organic diacid molecule containing two carboxyl groups, such as succinic anhydride, glutaric anhydride, etc. The diacid anhydride can react with the amino group to delete the amide bond and generate a free carboxyl group, so as to modify the amino group to a carboxyl group. Specifically, the mass ratio of the diacid anhydride to the silica nanoparticles subjected to the amino modification is 14-16:1.

[0052] The purpose of the amide reaction of the silica nanoparticles with the carboxyl modification of the surface and PAMAM is to coat PAMAM on the surface of the silica nanoparticles by forming an amide bond, in order to improve PAMAM, in some embodiments, N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are used to activate the carboxyl group on the surface of the silica nanoparticles, and then polyamide-amine dendrimers are added for amide reaction.

[0053] In some embodiments, the mass ratio of the silica nanoparticles and the polyamide-amine dendrimers is 100:20.0-21.0. Under this ratio, the polyamide-amine dendrimers can be better covered on the surface of the silica nanoparticles, and it is more conducive to subsequent efficient loading of various ligands.

[0054] The purpose of the amide reaction of the silica nanoparticles with the carboxyl modification of the surface and PAMAM is to coat PAMAM on the surface of the silica nanoparticles by forming an amide bond, in order to improve PAMAM, in some embodiments, N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are used to activate the carboxyl group on the surface of the silica nanoparticles, and then polyamide-amine dendrimers are added for amide reaction.

[0055] In some embodiments, the mass ratio of the silica nanoparticles with the polyamide-amine dendrimers on the surface, the MDM2 protein ligand, the GLUT1 protein ligand, and the E3 ubiquitin ligase ligand is 10:2.3-2.4:1-2:1-2.

[0056] The third embodiment of the present application provides a use of the above-mentioned protein degradation system based on polyamide-amine dendrimers in the preparation of an antitumor drug.

[0057] In some embodiments, the tumor is lung cancer. Specifically, the tumor is non-small cell lung cancer. Specifically, the tumor cells are A549 cells.

[0058] The fourth embodiment of the present application provides a method for treating tumors, which comprises administering a drug containing the above-mentioned protein degradation system based on polyamide-amine dendrimers to a subject.

[0059] The administration route of the present application to the subject can be intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral administration routes. Parenteral administration refers to the administration mode of generally by injection rather than enterally and topically, including but not limited to intravenous, intraperitoneal, intramuscular, intra-arterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, trans-tracheal, subcutaneous, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and sternal injection and infusion. Alternatively, it can be administered via a non-parenteral route, such as a topical, epidermal or mucosal administration route, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

[0060] In some embodiments, the subject is a human or an animal. Specifically, the animal can be a mouse, a rat, a dog, a rabbit, a pig, etc.

[0061] In some embodiments, the tumor is lung cancer. Specifically, the tumor is non-small cell lung cancer. Specifically, the tumor cell is A549 cell.

[0062] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific examples and comparative examples.

[0063] The materials and reagents used in the following examples are as follows:

[0064] 3-aminopropyltrimethoxysilane (APTS), anhydrous toluene, succinic anhydride (SA, 99%), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich. Acetone (AppliChem GmbH), anhydrous ethanol, ammonia, dimethyl sulfoxide (DMSO, 99%), methanol and skimmed milk powder were purchased from Merck. Trypsin EDTA (0.25%) was purchased from Gibco (Thermo Fisher Scientific). 20x TBST buffer, fetal bovine serum (FBS), DMEM, double antibiotics (penicillin and streptomycin mixture) and GAPDH antibody were provided by Solabio. 4',6-diamidino-2-phenylindole (DAPI), phenylmethylsulfonyl fluoride (PMSF), 3-(4,5-dimethyl-2-thiazolyl)-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 labeled goat anti-rabbit IgG (H+L), horseradish peroxidase labeled goat anti-mouse IgG (H+L) were purchased from Biyun Tian. PAGE gel quick preparation kit, dual-color pre-stained protein marker and polyvinylidene fluoride membrane (PVDF, 0.45 μm) were purchased from Yaenzyme. Polyvinylidene fluoride membrane (PVDF, 0.22 μm) was purchased from Millipore. Third generation polyamidoamine dendrimer-amino (PAMAM-G3-NH2) was purchased from Weihai Chen Yuan Molecular New Material Co., Ltd. E3 ubiquitin ligase ligand (Thalidomide-NH-CH2-COOH, CAS No. 927670-97-1), MDM2 protein ligand (Idasanutlin, CAS No. 1229705-06-9), GLUT1 ligand (Lavendustin B, CAS No. 125697-91-8), MG132 and MLN4924 were purchased from MCE (Shanghai, China), MDM2 antibody was purchased from Wuhan Huamei Biological Engineering Co., Ltd., GLUT1 antibody was purchased from Hangzhou Huaan Biotechnology Co., Ltd. p53 antibody was purchased from Wuhan Sanying Biological Technology Co., Ltd., BAX antibody, PUMA antibody were purchased from Abeam, NOXA antibody was purchased from Shanghai Ebiwei Biological Technology Co., Ltd., Super sensitive ECL chemiluminescence kit was purchased from Suzhou Xinsaimai Biological Technology Co., Ltd., Alexa Fluor 555 labeled goat anti-rabbit IgG and Alexa Fluor 488 labeled goat anti-mouse IgG were purchased from Shengong Biological Engineering (Shanghai) Co., Ltd. A549 lung cancer cells were from Wuhan Punsai Life Science Co., Ltd.ATP content determination kit was purchased from Nanjing Jianshen Bioengineering Research Co., Ltd.

[0065] The instruments used in the following examples are as follows: Zeta potential and nanoparticle size analyzer (Zetasizer Nano, Malvern Instrument Ltd., UK). Transmission electron microscope (JEM2100, Thermo Fisher, Japan). Fourier infrared spectrometer (FT-IR, TENSOR, Bruker, Germany). Chemiluminescence imager (ChemiScope 6200, Kinxiang, China), fluorescence microscope (Ti2-E, Nikon, Japan). Microplate reader (K3 TOUCH, Fisher, China), electrophoresis tank (Mini-PROTEAN system, Bio-Rad, USA), protein transfer device (Mini Trans-Blot cell, Bio-Rad, USA).

[0066] Example 1

[0067] A preparation method of a protein degradation system based on a polyamide-amine dendrimer, comprising the following steps:

[0068] I. Preparation of PAMAM-coated SiO2 nanoparticles

[0069] 1. Monodisperse spherical silica nanoparticles were prepared by Stöber-Fink-Bohn method.

[0070] 500 μL of ammonia water (25 w / w%), 14 mL of ethanol and 2 mL of deionized water were stirred at 50 °C for 20 min. Then 500 μL of TEOS was added dropwise into the above solution, while continuously stirring at 50 °C for 1.5 h. After reaction, centrifugal separation (4000-14000 rpm, 10 min) was performed, and washing with ethanol and deionized water was repeated three times, and drying at 70 °C for 12 h.

[0071] 2. PAMAM-coated SiO2 nanoparticles

[0072] SiO2-NH2. Subsequently, 0.2 g of SiO2-NH2obtained was dispersed in 20 mL of acetone, 3 g of succinic anhydride was added and reacted for 24 h, and after the reaction, vacuum drying was performed at 50 °C to obtain a carboxylated product SiO2-COOH. Then, the obtained SiO2-COOH was dispersed in 5 mL of DMSO, and the mixture was activated with EDC (46 mg, 5 mL of DMSO) and NHS (27.6 mg, 5 mL of DMSO) in sequence, and reacted with a G3 PAMAM dendrimer solution (41.6 mg, 5 mL of DMSO) for 1 day while stirring. Then, centrifugation was performed at 12,000 rpm for 10 min three times, and freeze-drying was performed to obtain PAMAM-coated SiO2nanoparticles (SG3).

[0073] II. Preparation of a protein degradation system based on polyamidoamine dendrimers

[0074] NHS (45.0 mg, 5 mL of DMSO), EDC (74.0 mg, 5 mL of DMSO), an E3 ubiquitin enzyme ligand (11.7 mg, 5 mL of DMSO), an MDM2 protein ligand (10 mg, 5 mL of DMSO), and a GLUT1 protein ligand (10 mg, 5 mL of DMSO) were mixed and incubated at 25 °C for 2 h. SG3 particles (50.0 mg, 5 mL of DMSO) were slowly dropped into the above mixture, and after stirring at 25 °C for 2 days, centrifugation was performed at 12,000 rpm for 10 min, the obtained precipitate was washed with a "water-absolute ethanol" mixture three times, and freeze-drying was performed to obtain SG3TAC.

[0075] Example 2

[0076] This example is the same as Example 1, except that:

[0077] II. Preparation of a protein degradation system based on polyamidoamine dendrimers

[0078] NHS (45.0 mg, 5 mL DMSO), EDC (74.0 mg, 5 mL DMSO), E3 ubiquitin enzyme 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 into the above mixture, after stirring at 25 °C for 3 days, centrifuged at 12000 rpm for 10 min, the obtained precipitate was washed with a "water-absolute ethanol" mixture for three times, and freeze-dried to obtain.

[0079] Example 3

[0080] This example is the same as Example 1, except that:

[0081] II. Preparation of the protein degradation system based on polyamidoamine dendrimers:

[0082] NHS (45.0 mg, 5 mL DMSO), EDC (74.0 mg, 5 mL DMSO), E3 ubiquitin enzyme 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 h. SG3 particles (50.0 mg, 5 mL DMSO) were slowly added dropwise into the above mixture, after stirring at 25 °C for 1 day, centrifuged at 12000 rpm for 10 min, the obtained precipitate was washed with a "water-absolute ethanol" mixture for three times, and freeze-dried to obtain.

[0083] Comparative Example 1

[0084] This comparative example is the same as Example 1, except that:

[0085] II. Preparation of the protein degradation system based on polyamidoamine dendrimers:

[0086] NHS (45.0 mg, 5 mL DMSO), EDC (74.0 mg, 5 mL DMSO), E3 ubiquitin enzyme ligand (11.7 mg, 5 mL DMSO), 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 into the above mixture, after stirring at 25 °C for 2 days, centrifuged at 12000 rpm for 10 min, the obtained precipitate was washed with a "water-absolute ethanol" mixture for three times, and freeze-dried to obtain SG3-CM.

[0087] Comparative Example 2

[0088] This comparative example is identical to Example 1, except that:

[0089] II. Preparation of the protein degradation system based on polyamidoamine dendrimers:

[0090] NHS (45.0 mg, 5 mL DMSO), EDC (74.0 mg, 5 mL DMSO), E3 ubiquitin enzyme ligand (11.7 mg, 5 mL DMSO), 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 dropped into the above mixture. After stirring at 25 °C for 2 days, centrifugation was performed at 12000 rpm for 10 min, and the obtained precipitate was washed three times with a "water-absolute ethanol" mixture and freeze-dried to obtain SG3-CG.

[0091] Comparative Example 3

[0092] This comparative example is identical to Example 1, except that:

[0093] II. Preparation of the protein degradation system based on polyamidoamine dendrimers:

[0094] NHS (45.0 mg, 5 mL DMSO), EDC (74.0 mg, 5 mL DMSO), E3 ubiquitin enzyme 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 dropped into the above mixture. After stirring at 25 °C for 2 days, centrifugation was performed at 12000 rpm for 10 min, and the obtained precipitate was washed three times with a "water-absolute ethanol" mixture and freeze-dried to obtain SG3-C.

[0095] Comparative Example 4

[0096] This comparative example is identical to Example 1, except that:

[0097] II. Preparation of the protein degradation system based on polyamidoamine dendrimers:

[0098] NHS (45.0 mg, 5 mL DMSO), EDC (74.0 mg, 5 mL DMSO), 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 into the above mixture, after stirring at 25 °C for 2 days, centrifuged at 12000 rpm for 10 min, the obtained precipitate was washed with "water-absolute ethanol" mixture for three times, and freeze-dried to obtain SG3-M.

[0099] Comparative Example 5

[0100] This comparative example is the same as Example 1, except that:

[0101] II. Preparation of protein degradation system based on polyamidoamine dendrimers:

[0102] NHS (45.0 mg, 5 mL DMSO), EDC (74.0 mg, 5 mL DMSO), 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 into the above mixture, after stirring at 25 °C for 2 days, centrifuged at 12000 rpm for 10 min, the obtained precipitate was washed with "water-absolute ethanol" mixture for three times, and freeze-dried to obtain SG3-G.

[0103] Test Example 1 Study on the influence of different materials on the toxicity of lung cancer cells

[0104] Lung cancer cells A549 cells were inoculated in 96-well plates and incubated at 37 °C, 5% CO2 in an incubator for 12 h to adhere the cells. Then, 20 μL SG3 or 20 μL SG3-CM or 20 μL SG3-CG or 20 μL SG3TAC of different concentrations were added to each well, respectively, and incubated for 12 h. MTT solution (100 µL, 5 mg / mL) was added to each well, respectively, and incubated for another 4 h. After removing the supernatant in the 96-well plate, 100 μL DMSO was added to each well. Finally, the absorbance of the samples at 492 nm was recorded.

[0105] Test Example 2 Study on degradation conditions

[0106] A549 cells were cultured in 6-well cell culture plates at a density of 2×10 6 cells per well for 24 h.

[0107] To investigate the relationship between the concentration of SG3-CM or SG3-CG or SG3TAC and the effect of degrading proteins, SG3-CM or SG3-CG or SG3TAC was diluted with DMEM to 0-50 μg / mL, and 40 μL was used to incubate with cells for 12 h.

[0108] To investigate the effect of the incubation time of SG3-CM or SG3-CG or SG3TAC with cells on the degradation of proteins, 40 μL of SG3-CM solution or SG3-CG solution or SG3TAC solution with a concentration of 40 μg / mL was used to incubate with cells for 0-24 h.

[0109] 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 SG3-CM solution with a concentration of 40 μg / mL or 40 μL of SG3-CG solution with a concentration of 40 μg / mL was added to different wells, and the cells were further cultured at 37 ℃ for 48 h.

[0110] The cells obtained in the above three paragraphs were washed twice with ice-cold PBS, and 150-200 μL of SDS lysis buffer (containing 1 mM protease inhibitor cocktail) was added. The protein concentration was determined using a BCA protein concentration determination kit. Then the protein sample and SDS-PAGE sample loading buffer (5x) were boiled together for 30 min, and 20-40 μg of each sample was loaded onto a 10% or 12% SDS-PAGE gel according to the calculation, and electrophoresis was performed using a protein electrophoresis instrument (80 V) for 1-2 h. The proteins in the gel were transferred to a PVDF membrane (0.45 μm or 0.22 μm) using a protein transfer device (120 mA, 1 h). After blocking the membrane with 25 mL of 5% skim milk in TBST at room temperature for 1 h, the membrane was washed with TBST three times, and then incubated with MDM2 antibody (1:2000) or GLUT1 antibody (1:50000) or GAPDH antibody (1:50000) or p53 antibody (1:25000) or BAX antibody (1:5000) or PUMA antibody (1:3000) or NOXA antibody (1:2000) at 4 ℃ for 12 h. Then, the membrane was washed with TBST buffer three times (10 min each time). The membrane was incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) (1:1000) and anti-mouse IgG antibody (1:1000) at room temperature for 1 h. Finally, the membrane was washed with TBST buffer three times (10 min each time), and developed using electrochemiluminescence (ECL) exposure solution, and the Western blot bands were detected on a chemiluminescence imager.

[0111] Experimental Example 3: Feasibility Study of SG3-CM and SG3-CG Degradation Proteins

[0112] A549 cells were cultured in confocal culture dishes and incubated at 37 °C with 5% CO2 for 12 h to allow cell adhesion. PBS, SG3, SG3-CM, SG3-CG, and SG3TAC were added to different culture dishes, and the cells were cultured for another 12 h. The cells were then washed three times with PBS buffer. A549 cells were then fixed with anhydrous methanol for 15 min. Subsequently, 0.5% Triton X-100 was added, and the cells were incubated at room temperature for 10 min. After washing with PBS buffer, 3% BSA solution was added to the sample, and the cells were incubated at room temperature for 2 h. After washing with PBS buffer, MDM2 antibody, GLUT1 antibody, or p53 antibody was incubated overnight at 4 °C. After washing with PBS buffer, Alexa Fluor 488-labeled goat anti-mouse IgG or Alexa Fluor 555-labeled goat anti-rabbit IgG was added, and the cells were incubated at room temperature for 1 h. After washing with PBS buffer, the cells were stained with DAPI for 15 min. After washing with PBS buffer, images were captured using a fluorescence microscope.

[0113] Results and Discussion

[0114] 1. Characterization of SiO2, SG3, SG3-CM, SG3-CG and SG3TAC nanoparticles

[0115] The morphology of SiO2 and SG3TAC nanoparticles was characterized by TEM, such as Figure 1 As shown, the prepared SiO2 and SG3TAC exhibit uniform morphology and good dispersibility. Compared to SiO2 nanoparticles, such as... Figure 1 As shown in A, the surface of SG3TAC nanoparticles is rougher, such as... Figure 1 As shown in B, this is because the coverage of PAMAM reduces surface smoothness.

[0116] The particle size of the SiO2 nanoparticles is approximately 230 nm, consistent with dynamic light scattering (DLS) data, such as... Figure 2 As shown in A, the particle size of the SG3TAC nanoparticles is approximately 250 nm, which is consistent with the DLS data. Figure 2 As shown in B in the diagram.

[0117] Through surface potential analysis, such as Figure 3 As shown, the surface of carboxylated SiO2 nanoparticles exhibits a negative potential (-24.8 ± 0.62 mV). After loading PAMAM, the particle surface displays a positive potential (3.34 ± 0.27 mV), which is attributed to the potential reversal caused by the abundant amino groups on PAMAM.

[0118] FT-IR analysis of functional groups on the surface of nanoparticles, such as... Figure 4 As shown. In the infrared absorption spectrum of carboxylated SiO2 nanoparticles, at 960 cm⁻¹... -1 1020 - 1110 cm -1 1640 cm -1 A distinct absorption peak appears at [location missing]. These three absorption peaks are caused by the asymmetric bending and stretching vibrations of Si-OH, the asymmetric stretching vibrations of Si-O-Si, and the vibrations of -COOH, respectively. In the PAMAM infrared absorption spectrum, the peaks are located at 1560-1640 cm⁻¹. -1 An absorption peak appears at 960 cm⁻¹, which is caused by the bending vibration of NH₃. In the infrared absorption spectrum of SG₃, an absorption peak appears at 960 cm⁻¹. -1 1020 - 1110 cm -1 1560-1640 cm -1 The presence of three distinct absorption peaks confirms that SG3 is a SiO2 nanoparticle with an amino-rich surface.

[0119] Observe the infrared absorption peak of the product (SG3-C) after the reaction of SG3 with E3 ubiquitinase ligand, such as... Figure 5 As shown, 1700-2000 cm were found. -1 An absorption peak appears at 3200-3500 cm⁻¹, which is a characteristic triplet of benzene. The product of the reaction between SG3-C and the GLUT1 ligand (SG3-CG) shows an absorption peak at 3200-3500 cm⁻¹. -1 An absorption peak appears at 2210-2260 cm⁻¹, which is the stretching vibration peak of the -OH group on the GLUT1 ligand. The product of the reaction between SG3-C and the MDM2 ligand (SG3-CM) shows an absorption peak at 2210-2260 cm⁻¹. -1 An absorption peak appears at 595 cm⁻¹, which is a characteristic peak of the C≡N triple bond on the MDM2 ligand. SG3-C binds to the GLUT1 and MDM2 ligands to form SG3TAC, which has an absorption peak at 595 cm⁻¹. -1 3200-3500 cm -1 It possesses the same absorption peaks as SG3-CG. SG3TAC has absorption peaks in the 2210-2260 cm⁻¹ range. -1 It has the same absorption peak as SG3-CM.

[0120] 2. Evaluation of the cytotoxicity of SG3, SG3-CM, SG3-CG, and SG3TAC nanoparticles

[0121] The viability of A549 cells treated with SG3, SG3-CG, SG3-CM, and SG3TAC was detected using the MTT assay. Results are as follows: Figure 6As shown, SG3 had no significant effect on A549 cell viability in the concentration range of 0-40 μg / mL, showing good compatibility. SG3-CG, SG3-CM and SG3TAC gradually decreased the viability of A549 cells with increasing concentration (0-40 μg / mL). Among them, SG3TAC had the most prominent inhibitory effect on A549 cells, indicating that it had stronger anti-tumor activity.

[0122] Further through A549 cell live and dead experiment, it was found that compared with the PBS group, the number of live cells (showing green fluorescence) in the cells treated by SG3TAC decreased significantly, and the number of dead cells (showing red fluorescence) increased significantly; and the proportion of dead cells in the SG3TAC treatment group was significantly higher than that in the SG3-CM group and the SG3-CG group, as shown in Figure 7 This result is consistent with the conclusion of the MTT experiment, which together confirms the cytotoxicity and anti-proliferation effect of SG3TAC.

[0123] 3. Evaluation of SG3-CM on MDM2 protein degradation

[0124] The degradation of MDM2 protein in A549 cells was analyzed by Western blot. 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 8 As the concentration of SG3-CM increases, the expression of MDM2 protein gradually decreases, showing a concentration-dependent degradation trend. Especially 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.

[0125] To evaluate the effect of exposure time on the expression of MDM2 protein, A549 cells were exposed to 40 μg / mL SG3-CM for 0-24 h. As shown in Figure 9 After 9 h, the MDM2 protein band gradually lightened, showing the strong degradation ability of SG3-CM.

[0126] 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 for experiments. The results are shown in Figure 10As shown, compared to A549 cells directly exposed to SG3-CM, A549 cells pre-treated with MG132 and then exposed to SG3-CM significantly inhibited the degradation of MDM2 protein, which revealed that the degradation of MDM2 protein by SG3-CM was related to the proteasome pathway. A549 cells pre-treated with MLN4924 and then exposed to SG3-CM significantly inhibited the degradation of MDM2 protein, which revealed that the degradation of MDM2 protein by SG3-CM was also related to the ubiquitination pathway.

[0127] Meanwhile, the degradation of MDM2 protein in A549 cells treated with SG3-C, SG3-M (without E3 ubiquitin ligase ligand) and their mixture was investigated. As shown, compared to SG3-CM, there was almost no degradation of MDM2 protein in the SG3-C group, the SG3-M group and the mixture group, which fully demonstrated that only SG3-CM could effectively induce the degradation of MDM2 protein. At the same time, it explained the mechanism of action of MDM2 protein degradation by forming a ternary complex of PAMAM, MDM2 protein and E3 ubiquitin ligase in A549 cells. Figure 11

[0128] In addition, immunofluorescence imaging analysis of MDM2 protein in cells was carried out. Here, Alexa Fluor 488 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 12 , which demonstrated the high efficiency of SG3-CM and SG3TAC in regulating the expression of MDM2 protein.

[0129] 4. Evaluation of GLUT1 protein degradation by SG3-CG

[0130] The degradation of GLUT1 protein in A549 cells was analyzed by Western blotting. A549 cells were exposed to different concentrations of SG3-CG nanoparticles (0-50 μg / mL) for 12 h. The results of Western blotting analysis are shown in Figure 13 , with the increase of SG3-CG concentration, the expression of GLUT1 protein gradually decreased, showing a concentration-dependent degradation trend. Especially at higher concentrations (40 μg / mL and 50 μg / mL), the brightness of 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.

[0131] To evaluate the effect of exposure time on the expression of GLUT1 protein, A549 cells were exposed to 40 μg / mL SG3-CG for 0-24 h. As shown in Figure 14 ​As shown, the GLUT1 protein band gradually became lighter after 6 h, showing the strong degradation ability of SG3-CG.

[0132] Meanwhile, the detection of intracellular ATP content found that the ATP level of SG3-CG treated cells decreased significantly, as shown in Figure 15 As shown, this indicates that SG3-CG effectively inhibits the function of GLUT1 protein, reduces the input amount of glucose, and thus affects the generation of ATP, suggesting that it exerts an anti-tumor effect by interfering with cellular energy metabolism.

[0133] On this basis, in order to further explore the degradation pathway of SG3-CG, proteasome inhibitor MG132 and NEDD8 activating enzyme (NAE) inhibitor MLN4924 were used for experiments, and the results are shown in Figure 16 As shown, compared with A549 cells directly exposed to SG3-CG, A549 cells pretreated with MG132 and then exposed to SG3-CG significantly inhibited the degradation of GLUT1 protein, revealing that the degradation of GLUT1 protein by SG3-CG is related to the proteasome pathway. A549 cells pretreated with MLN4924 and then exposed to SG3-CG significantly inhibited the degradation of GLUT1 protein, revealing that the degradation of GLUT1 protein by SG3-CG is also related to the ubiquitination pathway.

[0134] Meanwhile, the degradation of GLUT1 protein in A549 cells treated with SG3-C, SG3-G (without E3 ubiquitin ligase ligand) and their mixture was investigated. Compared with SG3-CG, there was almost no degradation of GLUT1 protein in the SG3-C group, the SG3-G group and the mixture of the two groups, as shown in Figure 17 This result fully shows that only SG3-CG can effectively induce the degradation of GLUT1 protein. At the same time, it explains the mechanism of action of GLUT1 protein degradation by forming a ternary complex of PAMAM, GLUT1 protein and E3 ubiquitin ligase in A549 cells.

[0135] In addition, immunofluorescence imaging analysis of GLUT1 protein in cells was carried out. Here, Alexa Fluor 555 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 18 This indicates the high efficiency of SG3-CG and SG3TAC in regulating the expression of GLUT1 protein.

[0136] 5. Effect of SG3TAC nanoparticles on p53 protein expression

[0137] Figure 19The results showed that the expression levels of GLUT1 protein and MDM2 protein in A549 cells treated with SG3TAC nanoparticles at different concentrations (10-40 pg / mL) for 12 h exhibited significant concentration-dependent changes. Specifically, the expression level of GLUT1 protein gradually decreased with the increase of SG3TAC concentration, and was almost completely inhibited at a concentration of 30 pg / mL, indicating that SG3TAC had a significant degradation effect on GLUT1 protein. Similarly, the expression of MDM2 protein also showed a concentration-dependent decreasing trend after SG3TAC treatment, reaching the lowest level at the highest concentration (40.0 pg / mL).

[0138] The expression level of p53 protein in A549 cells treated with SG3, SG3-CM, SG3-CG and SG3TAC was changed compared with the PBS group, as shown in Figure 20 . Among them, the SG3 treatment group had no obvious effect on the expression of p53 protein, while the SG3-CM and SG3-CG treatment groups increased the expression of p53 protein, especially the SG3TAC treatment group, which reached the highest level of p53 protein expression, indicating that SG3TAC could more effectively up-regulate the expression of p53 protein.

[0139] In addition, immunofluorescence imaging analysis of p53 protein in cells was carried out. Here, Alexa Fluor 555 labeled goat anti-rabbit IgG was used, and the red fluorescence signal of p53 protein in A549 cells treated with SG3-CM, SG3-CG and SG3TAC was significantly enhanced, as shown in Figure 21 , indicating that these two treatments could effectively promote the expression of p53 protein. Most obviously, the SG3TAC treatment group had the strongest red fluorescence signal of p53 protein in cells, showing its high efficiency in inducing the expression of p53 protein.

[0140] The expression of apoptosis proteins (BAX protein, PUMA protein and NOXA protein) in cells is shown in Figure 22 . Compared with the control group, the expression levels of BAX protein, PUMA protein and NOXA protein in A549 cells treated with SG3-CM, SG3-CG and SG3TAC were all up-regulated. Specifically, SG3-CM and SG3-CG treatment could increase the expression of these proteins, while the expression of BAX protein, PUMA protein and NOXA protein in the SG3TAC treatment group was further significantly increased, reaching the highest level. This indicates that SG3TAC has stronger efficiency in inducing the expression of these pro-apoptotic proteins. This result suggests that SG3TAC can more effectively activate the p53 signaling pathway and up-regulate the expression of pro-apoptotic proteins, thereby enhancing the process of cell apoptosis.

[0141] In summary, SG3TAC nanoparticles significantly reduced the expression levels of MDM2 and GLUT1 proteins in a concentration-dependent manner, while effectively enhanced the expression of p53 protein, showing its potential in regulating the expression of key proteins in tumor cells.

[0142] The above only the preferred embodiments of the present application, and is not intended to limit the present application, for those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application, should be included in the scope of protection of the present application.

Claims

1. A protein degradation system based on polyamidoamine dendrimers, characterized in that, The protein degradation system is a nanoparticle structure, the nanoparticle structure comprises silica nanoparticles and a polyamide-amine dendrimer layer coated on the surface of the silica nanoparticles, and an MDM2 protein ligand, a GLUT1 protein ligand and an E3 ubiquitin ligase ligand are connected on the polyamide-amine dendrimer layer by chemical bonds, so that the E3 ubiquitin ligase is close to the MDM2 protein and the GLUT1 protein, and the E3 ubiquitin ligase is prompted to accurately recognize and interact with the MDM2 protein and the GLUT1 protein.

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

3. The protein degradation system of claim 1, wherein, The particle size of the silica nanoparticles is 200-250 nm.

4. A method for preparing a protein degradation system based on polyamidoamine dendrimers, characterized by, The method comprises the following steps: Silica nanoparticles are provided, and the surface of the silica nanoparticles is carboxylated and modified; The silica nanoparticles with the carboxylated and modified surface are subjected to amidation reaction with polyamide-amine dendrimers, so that the polyamide-amine dendrimers coat the silica nanoparticles; The silica nanoparticles with the polyamide-amine dendrimers on the surface are subjected to amidation reaction with an MDM2 protein ligand, a GLUT1 protein ligand and an E3 ubiquitin ligase ligand, so that the MDM2 protein ligand, the GLUT1 protein ligand and the E3 ubiquitin ligase ligand are connected with the polyamide-amine dendrimers on the surface of the silica nanoparticles.

5. The production method according to claim 4, wherein The carboxylated and modified surface of the silica nanoparticles is obtained by first modifying the surface of the silica nanoparticles with amino groups, and then modifying the amino groups into carboxyl groups by using a diacid anhydride.

6. The production method according to claim 4, wherein The carboxyl groups on the surface of the silica nanoparticles are activated by using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and then polyamide-amine dendrimers are added to perform amidation reaction.

7. The production method according to claim 4, wherein The mass ratio of the silica nanoparticles to the polyamide-amine dendrimers is 100:20.0-21.

0.

8. The production method according to claim 4, wherein The mass ratio of the silica nanoparticles with the polyamide-amine dendrimers on the surface to the MDM2 protein ligand, the GLUT1 protein ligand and the E3 ubiquitin ligase ligand is 10:2.3-2.4:1-2:1-2.

9. Use of the protein degradation system of any one of claims 1-3 or the protein degradation system obtained by the preparation method of any one of claims 4-8 in the preparation of an antitumor drug.

10. The use according to claim 9, wherein the compound is ###00002### or a pharmaceutically acceptable salt thereof. The tumor is lung cancer.

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