Ferritin nano-composite based on synergistic effect of PROTAC and platinum drug and application of ferritin nano-composite

Abstract

The invention provides a ferritin nano-composite based on the synergistic effect of PROTAC and a platinum drug and application of the ferritin nano-composite, and belongs to the technical field of medicinal chemistry, biological medicine and nanometer.The ferritin nano-composite comprises a ferritin carrier, a platinum drug precursor and PROTAC molecules for targeted degradation of ERCC1 protein and / or XPF protein; the platinum drug precursor is loaded in an inner cavity of the ferritin carrier; the PROTAC molecule is modified on the surface of the ferritin carrier; the PROTAC molecule is formed by connecting an E3 ubiquitin ligase ligand and a ligand targeting ERCC1 protein and / or XPF protein through a connecting body. The nano-composite can degrade an ERCC1 / XPF protein complex in a targeted manner and release a Pt (IV) active drug, significantly inhibits an NER pathway and synergistically enhances the tumor toxicity of platinum drugs, provides an innovative strategy for overcoming the drug resistance of tumor chemotherapy, and has good application potential.

Description

Technical Field

[0001] The present invention belongs to the fields of medicinal chemistry, biopharmaceuticals and nanotechnology, and particularly relates to a ferritin nanocomplex based on the synergistic effect of PROTAC and platinum drugs and its application. Background Art

[0002] Platinum compounds (such as cisplatin and carboplatin) are commonly used clinical chemotherapy drugs, which inhibit DNA replication and transcription by forming adducts with DNA, thereby inducing apoptosis of tumor cells. However, due to the drug resistance of tumor cells to platinum drugs, the antitumor effect of platinum drugs is significantly reduced. Therefore, how to effectively overcome the drug resistance of platinum drugs has become an important research direction in the field of tumor chemotherapy. Summary of the Invention

[0003] In order to solve the problem of drug resistance of tumor tissues to platinum drugs, the present invention provides a ferritin nanocomplex based on the synergistic effect of PROTAC and platinum drugs. This nanocomplex can target and degrade the ERCC1 / XPF protein complex and release the Pt(IV) active drug, significantly inhibit the NER pathway and synergistically enhance the tumor toxicity of platinum drugs. The present invention proposes an innovative strategy to overcome tumor chemotherapy drug resistance and has good application potential.

[0004] The present invention also provides a preparation method and application of a ferritin nanocomplex based on the synergistic effect of PROTAC and platinum drugs.

[0005] The present invention is achieved by the following technical solutions:

[0006] The present invention provides a ferritin nanocomplex based on the synergistic effect of PROTAC and platinum drugs, and the nanocomplex includes a ferritin carrier, a platinum drug precursor, and a PROTAC molecule that targets and degrades ERCC1 protein and / or XPF protein;

[0007] The platinum drug precursor is loaded into the inner cavity of the ferritin carrier;

[0008] The PROTAC molecule is modified on the surface of the ferritin carrier;

[0009] The PROTAC molecule is formed by connecting an E3 ubiquitin ligase ligand and a ligand that targets ERCC1 protein and / or XPF protein through a linker.

[0010] Furthermore, in the PROTAC molecule, the ligand that targets ERCC1 protein and / or XPF protein includes the ligand NERi, and the structural formula of the ligand NERi is as follows:

[0011]

[0012] Further, the structural formula of the E3 ubiquitin ligase ligand is as follows:

[0013]

[0014] The PROTAC molecule includes at least one of C8-NERiP, C10-NERiP, PEG2-NERiP, PEG3-NERiP, PEG4-NERiP, and PEG5-NERiP;

[0015] The structural formulas of the C8-NERiP, C10-NERiP, PEG2-NERiP, PEG3-NERiP, PEG4-NERiP, and PEG5-NERiP are as follows respectively:

[0016]

[0017] Further, the platinum-based drug precursor includes platinum tetraiodide;

[0018] The ferritin carrier includes the ferritin carrier HFn.

[0019] Further, the PROTAC molecule is connected to the COOH-SS-Mal group, and the maleimide in the COOH-SS-Mal group can react with the thiol group on the surface of the ferritin carrier to achieve the connection between the PROTAC molecule and the ferritin carrier;

[0020] The connection between the PROTAC molecule and the COOH-SS-Mal group is achieved by an esterification reaction between the carboxyl group in the COOH-SS-Mal group and the hydroxyl group on the E3 ubiquitin ligase ligand. The structural formula of the COOH-SS-Mal group is as follows:

[0021]

[0022] The platinum-based drug precursor is loaded into the inner cavity of the ferritin carrier through a temperature-controlled drug channel.

[0023] Further, in the nano-complex, the molar ratio of the ferritin carrier, the platinum-based drug precursor, and the PROTAC molecule is 1:18:58.

[0024] Based on the same inventive concept, the present invention provides an application of a ferritin nano-complex based on the synergistic effect of PROTAC and platinum drugs in the preparation of anti-tumor drugs.

[0025] Based on the same inventive concept, the present invention provides an anti-tumor drug, and the active ingredient of the drug includes the above-mentioned ferritin nano-complex based on the synergistic effect of PROTAC and platinum drugs.

[0026] Based on the same inventive concept, the present invention provides a method for preparing a ferritin nanocomplex based on the synergistic effect of PROTAC and platinum drugs, and the preparation method includes:

[0027] S1. Dissolve 2-Carboxybenzaldehyde (o-carboxybenzaldehyde) and 9-fluorenone hydrazone in an organic solvent, perform solid-liquid separation after reacting for a certain time, and wash and dry the obtained solid to obtain the ligand NERi;

[0028] S2. Dissolve 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecan-16-oic acid (1-tert-butyl 5,8,11-trioxa-2-azatridecane-1-carboxylate), HATU (2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate), and DIPEA (N,N-diisopropylethylamine) in an organic solvent, and then add (S,R,S)-AHPC-Me for reaction to obtain a mixed solution;

[0029] S3. Dilute, wash, dry, filter, concentrate under reduced pressure, and purify by silica gel column chromatography the mixed solution to obtain AHPC-PEG 4 -BOC;

[0030] S4. Dissolve the AHPC-PEG 4 -BOC in an organic solvent and react with DMAP and 4-nitrophenylcarbonochloridate. Dilute, wash, dry, filter, concentrate under reduced pressure, and purify by silica gel column chromatography the obtained reaction solution to obtain compound S5;

[0031] S5. Disperse 2,2′-Disulfanediylbis(ethan-1-ol) (2-hydroxyethyldisulfide) and DMAP in an organic solvent, and then add the compound S5 for reaction. Dilute, wash, dry, filter, concentrate under reduced pressure, and purify by silica gel column chromatography the obtained reaction solution to obtain compound S6;

[0032] S6. Dissolve the compound S6 in an organic solvent, and then add CF 3 COOH for reaction. After the reaction is completed, remove the solvent and purify by silica gel column chromatography to obtain compound S7;

[0033] S7. Dissolve the ligand NERi, HATU, and DIPEA together in an organic solvent, then add the compound S7 for reaction. The resulting reaction solution is diluted, washed, dried, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain compound S8;

[0034] S8. Dissolve the compound S8, succinic anhydride, and DMAP together in an organic solvent, react for a certain period of time, then remove the solvent, and purify by silica gel column chromatography to obtain compound S9;

[0035] S9. Dissolve the compound S9, EDC, Mal-PEG 4 -OH, and DMAP together in an organic solvent, react for a certain period of time, then remove the solvent, and purify by silica gel column chromatography to obtain compound S10, namely PEG4-NERiP-SS-Mal;

[0036] S10. Disperse the PEG4-NERiP-SS-Mal and the ferritin carrier HFn together in a buffer solution, react for a certain period of time and then purify to obtain HFn-NERiP;

[0037] S11. Using the platinum-based drug precursor solution and the HFn-NERiP solution as raw materials, prepare the HFn-NERiP-Pt(IV) nanocomposite by the method of heating to open the drug channel;

[0038] Among them, the structural formulas of the ligand NERi, the AHPC-PEG 4 -BOC, and the compounds S5 - S10 are as follows:

[0039]

[0040]

[0041] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0042] 1. A ferritin nanocomplex based on the synergistic effect of PROTAC and platinum drugs. This nanocomplex contains the ferritin carrier HFn, which is used for drug loading and targeted delivery, the PROTAC molecule NERiP, which can specifically target and degrade ERCC1 and / or XPF proteins, inhibit the NER (nucleotide excision repair) pathway, and the platinum drug prodrug Pt(IV), which enters the inner cavity of the ferritin shell through a temperature-controlled drug channel. This nanocomplex efficiently delivers NERiP and Pt(IV) to tumor cells through HFn mediation, specifically releases NERiP and Pt(IV) compounds in the tumor microenvironment, realizes an increase in intracellular platinum concentration, consumption of intracellular GSH, degradation of ERCC1 / XPF, inhibition of DNA repair, and enhancement of the tumor toxicity of platinum drugs, thereby achieving a synergistic anti-tumor effect. By targeting the degradation of the ERCC1 / XPF protein complex and releasing the Pt(IV) active drug, the present invention significantly inhibits the NER pathway and synergistically enhances the tumor toxicity of platinum drugs, providing a new strategy for overcoming tumor chemotherapy drug resistance and having good application potential.

[0043] 2. A ferritin nanocomplex based on the synergistic effect of PROTAC and platinum drugs. Utilizing the unique structure and targeting characteristics of ferritin HFn, a nanodelivery system based on HFn loaded with PROTAC and Pt(IV) prodrug is designed. After this system enters tumor cells through the CD71 targeting of ferritin, PROTAC and Pt(IV) compounds are specifically released in the tumor microenvironment. The PROTAC molecule can target and degrade the DNA repair protein ERCC1 / XPF, realize the inhibition of DNA repair in tumor cells, and utilize the loading and responsive release of the Pt(IV) prodrug to overcome the drug resistance of tumor cells to platinum drugs, significantly enhancing the chemotherapy effect.

[0044] 3. A preparation method of a ferritin nanocomplex based on the synergistic effect of PROTAC and platinum drugs. First, the HFn-NERiP complex is obtained by a chemical bonding method, and then the Pt(IV) prodrug enters the inner cavity of the HFn carrier through a temperature-controlled drug channel. Finally, the HFn-NERiP-Pt(IV) nanodelivery system is obtained. In in vitro and in vivo experiments, the HFn-NERiP-Pt(IV) prepared by the present invention demonstrates excellent anti-tumor effects and can effectively overcome the drug resistance of drug-resistant tumors such as esophageal squamous cell carcinoma to platinum drugs, providing an efficient and innovative solution for tumor drug resistance treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the accompanying drawings required for the description of the embodiments. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0046] Figure 1 For the differential expression of DNA repair-related proteins in parental and CDDP-resistant ESCC cells and the structure-guided design of NERiP.

[0047] Figure 2 For the in vitro targeted degradation of ERCC1 / XPF protein by NERiP.

[0048] Figure 3 For the design and characterization of the HFn-NERiP-Pt(IV) delivery system.

[0049] Figure 4 For the responsive drug release of the HFn-NERiP-Pt(IV) delivery system.

[0050] Figure 5 For the in vitro targeted degradation of ERCC1 / XPF protein by the HFn-NERiP-Pt(IV) delivery system.

[0051] Figure 6 For the anti-tumor efficacy and mechanism evaluation of HFn-NERiP-Pt(IV) in EC9706 / CDDP cells.

[0052] Figure 7 For the pharmacokinetics, biodistribution and biosafety evaluation of HFn-NERiP and HFn-NERiP-Pt(IV) in nude mice bearing EC9706 / CDDP tumors.

[0053] Figure 8 For the anti-tumor evaluation of HFn-NERiP-Pt(IV) in a CDDP-resistant esophageal squamous cell carcinoma CDX model. Specific embodiments

[0054] The following will specifically elaborate on the present invention in combination with specific embodiments. The advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific embodiments are used to illustrate the present invention, rather than limiting the present invention.

[0055] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains. In case of any contradiction, this specification shall prevail.

[0056] Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment, etc. used in the present invention can be obtained through market purchase or can be prepared by existing methods.

[0057] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0058] The overall concept of the present invention is as follows:

[0059] Platinum-based compounds (such as cisplatin and carboplatin) are commonly used chemotherapeutic drugs in clinics. They inhibit DNA replication and transcription by forming adducts with DNA, thereby inducing apoptosis. However, due to the drug resistance of tumor cells to platinum-based drugs, the anti-tumor effect of platinum-based drugs is significantly reduced. Therefore, how to effectively overcome the drug resistance of platinum-based drugs has become an important research direction in the field of tumor chemotherapy. The applicant found that the high activity of the nucleotide excision repair (NER) pathway in tumor cells, such as the overexpression of the ERCC1 / XPF complex, is an important factor leading to the drug resistance of tumor cells to platinum-based drugs. The ERCC1 protein and the XPF protein are the core components of the nucleotide excision repair (NER) pathway and are responsible for repairing DNA crosslinks and damage.

[0060] In recent years, the proteolysis targeting chimera (PROTAC) technology, as a novel drug design strategy, has shown great potential in specifically inhibiting key tumor proteins by inducing the ubiquitination and degradation of target proteins.

[0061] Based on this, the present invention proposes a ferritin nanocomposite based on the synergistic effect of PROTAC and platinum drugs and its application. Using the unique structure and targeting characteristics of heavy-chain ferritin (HFn), a nanodelivery system based on HFn loaded with PROTAC and Pt(IV) prodrug is designed. By targeting the degradation of the ERCC1 / XPF protein complex and releasing the Pt(IV) active drug, it significantly inhibits the NER pathway and synergistically enhances the tumor toxicity of platinum-based drugs. The present invention proposes an innovative strategy for overcoming tumor chemotherapy drug resistance and has good application potential.

[0062] First, the present invention designs a PROTAC molecule NERiP based on the specific target of ERCC1 / XPF. NERiP has a ligand NERi that can target and bind to the ERCC1 / XPF protein, and at the same time has an E3 ubiquitin ligase ligand (Von Hippel-Lindau disease, VHL), which can bind to the E3 ubiquitin ligase complex in cells and promote the ubiquitination and degradation of the target protein ERCC1 / XPF. Then, NERiP is chemically modified with functional groups such as GSH-responsive disulfide bonds and maleimide, and NERiP is bound to the outer surface of HFn through the reaction of maleimide and thiol to form a HFn-NERiP complex with GSH-responsive release. Finally, Pt(IV) is loaded into the inner cavity of the ferritin shell through a temperature-controlled drug channel to obtain a HFn-NERiP-Pt(IV) nanodelivery system.

[0063] The HFn-NERiP-Pt(IV) nanodelivery system prepared by the present invention comprises the following components:

[0064] 1. Ferritin shell structure (HFn)

[0065] HFn is a natural protein shell with good biocompatibility and stability, and can achieve targeted delivery by binding to tumor cell surface receptors (such as CD71 receptor). HFn also has a unique cavity structure and can be used as an ideal carrier for drugs.

[0066] 2. PROTAC molecule (NERiP)

[0067] The PROTAC molecule NERiP is designed to specifically target and degrade the key proteins ERCC1 and / or XPF in the nucleotide excision repair (NER) pathway, thereby inhibiting the DNA damage repair mechanism and enhancing the killing effect of platinum drugs on tumor cells. Through chemical bonding, the NERiP molecule is modified on the surface of HFn to ensure its efficient delivery into cells.

[0068] 3. Platinum prodrug (Pt(IV))

[0069] Pt(IV) is an inactive precursor of platinum drugs and can be reduced to active platinum ions (Pt(II)) through intracellular glutathione (GSH) responsiveness, thereby enhancing the toxicity of the drug in tumor cells. Pt(IV) is loaded into the inner cavity of HFn by a temperature-controlled method to ensure the stability of delivery and the controllability of drug release.

[0070] Through the synergistic effect, this system achieves the effects of targeted delivery, tumor microenvironment-responsive release, and combined anti-tumor therapy.

[0071] The specific mechanism of action of the HFn-NERiP-Pt(IV) nanodelivery system prepared by the present invention in anti-tumor therapy is as follows:

[0072] 1. Combination therapy

[0073] Mediated by HFn, the PROTAC molecule and Pt(IV) are delivered to tumor cells, and the active drugs are specifically released in the tumor microenvironment. The PROTAC molecule can specifically degrade ERCC1 / XPF, block the NER pathway, and enhance the level of DNA damage; after Pt(IV) is reduced to Pt(II), it further induces DNA cross-linking and breakage, thus achieving a combined killing effect on tumor cells.

[0074] 2. Therapeutic mechanism

[0075] Internalization and GSH consumption: The HFn shell structure targets and binds to the receptors on the surface of tumor cells, delivering the nanosystem into the cells; Pt(IV) is reduced and released in the tumor microenvironment with a high GSH concentration, consuming the GSH in tumor cells and enhancing the activity of platinum drugs.

[0076] Inhibition of DNA repair: The PROTAC molecule degrades the ERCC1 / XPF protein, inhibits the DNA damage repair ability, and makes tumor cells more vulnerable to DNA damage induced by platinum drugs.

[0077] Synergistic apoptosis: The combination therapy enhances the level of DNA damage in tumor cells, activates the apoptosis signaling pathway, and thus significantly improves the anti-tumor activity of platinum drugs.

[0078] 3. Therapeutic effect

[0079] In in vitro and in vivo experiments, this system significantly inhibited the proliferation of tumor cells, especially showing excellent therapeutic effects in cell lines with high platinum drug resistance.

[0080] Next, a ferritin nanocomposite based on the synergistic effect of PROTAC and platinum drugs and its application of the present invention will be described in detail in combination with examples and experimental data.

[0081] Example 1

[0082] This example provides a preparation method for the PROTAC molecule NERiP, which is as follows:

[0083] 1. Western blot

[0084] EC9706, EC109, and TE1 parental and drug-resistant cells were seeded in 10-cm cell culture dishes. Cells were collected using a cell scraper, lysed in frozen RIPA lysis buffer containing protease inhibitors, and the protein concentrations of ERCC1 and XPF were quantified by the BCA protein assay. Proteins were separated on a 15% SDS-PAGE gel and transferred to a PVDF membrane, which was then blocked with 5% non-fat milk (room temperature, 2 h). After incubation with primary and secondary antibodies, the membrane was exposed and imaged using an ECL chemiluminescence solution and an automatic chemiluminescence image analysis system (MiniChemi 610).

[0085] 2. Synthesis of S1 (NERi)

[0086] 600 mg of 2-Carboxybenzaldehyde was dissolved in 50 mL of ethanol, and 776 mg of 9-fluorenone hydrazone was added. The reaction mixture was stirred overnight at room temperature. The precipitate was collected by filtration and washed twice with 50 mL of diethyl ether. The final product was obtained by vacuum drying to give the ligand NERi.

[0087] 3. Synthesis of S2 (AHPC-PEG 4 -BOC)

[0088] 0.71 g of 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecan-16-oic acid (1-tert-butyl 5,8,11-trioxa-2-azatridecane-1,13-dioate), 1.7 g of HATU, and 0.75 g of DIPEA were dissolved in 10 mL of DMF, and 1.0 g of (S,R,S)-AHPC-Me was added slowly. The reaction mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was diluted with ethyl acetate (10 mL × 3) and washed with water (30 mL) and saturated NaCl solution (10 mL) respectively. The organic layer was dried over anhydrous Na 2 SO 4 dried, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to give AHPC-PEG 4 -BOC (the structural formula is shown in Figure 1 F).

[0089] 4. Synthesis of S3 (AHPC-PEG 4 -NH 2 )

[0090] 500 mg of AHPC-PEG 4 -BOC was dissolved in 10 mL of CH 2 Cl 2Slowly add 3.0 mL of CF 3 COOH and stir for 12 h at room temperature. After the reaction, the solvent was evaporated under reduced pressure and purified by silica gel column chromatography to obtain AHPC-PEG 4 -NH 2 .

[0091] 5. Synthesis of S4(PEG 4 -NERiP)

[0092] Dissolve 90 mg of NERi, 210 mg of HATU and 90 mg of DIPEA in 10 mL of DMF, and slowly add 350 mg of AHPC-PEG 4 -NH2. The reaction mixture was stirred at room temperature for 24 h. After the reaction was completed, the mixture was diluted with ethyl acetate (10 mL×3) and washed with water (30 mL) and saturated NaCl solution (10 mL) respectively. The organic layer was dried with anhydrous Na 2 SO 4 , filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain PEG 4 -NERiP.

[0093] 6. Synthesis of P-NERiP or C-NERiP

[0094] This method is the same as the synthesis method of PEG 4 -NERiP, except that 1-tert-butyl 5,8,11-trioxa-2-azatridecanedioate is replaced by COOH-PEG 2 -BOC, COOH-PEG 3 -BOC, COOH-PEG 5 -BOC, COOH-C 8 -BOC or COOH-C 10 -BOC.

[0095] 7. Transcriptome sequencing and data analysis

[0096] Inoculate EC9706 parental and drug-resistant cells in a 10 cm cell culture dish. After the cells adhered, incubate for 48 h, and collect the cells using a cell scraper. Total RNA was isolated from the cells using TRIzol reagent (Solarbio, R1100), and then quickly frozen in liquid nitrogen. RNA sequencing analysis was completed by Biomarker Technologies (Beijing).

[0097] Results:

[0098] To investigate the drug resistance mechanisms related to DNA repair, we selected esophageal squamous cell carcinoma EC9706 as the parental cell model and induced cisplatin-resistant cells EC9706 / CDDP. Transcriptome sequencing was performed to analyze the differential gene expression between EC9706 and EC9706 / CDDP cells. The results are as Figure 1 shown: Among them, A. Volcano plot comparing the mRNA expression levels between parental and drug-resistant ESCC cells. B. KEGG enrichment analysis of upregulated genes in CDDP-resistant ESCC cells compared with parental cells. C. Western blot analysis of the protein levels of XPF and ERCC1 in ESCC cell lines. D-E. Molecular docking of PROTAC with ERCC1 and XPF proteins (D), and details of the NERiP binding sites in ERCC1 and XPF (E). F. Synthetic roadmap of NERiP. G. 1H NMR spectra of C8-NERiP, C10-NERiP, PEG2-NERiP, PEG3-NERiP, PEG4-NERiP, and PEG5-NERiP.

[0099] Figure 1 Figure A shows the volcano plot of the mRNA expression profiles of EC9706 and EC9706 / CDDP cells. The results showed that 3636 genes were upregulated and 3525 genes were downregulated, indicating significant transcriptional changes in drug-resistant cells. KEGG analysis ( Figure 1 Figure B) found that multiple DNA repair pathways, including mismatch repair, NER, base excision repair, and homologous recombination, were upregulated, suggesting that these pathways contribute to the ability of drug-resistant cells to cope with CDDP-induced DNA damage. To verify the transcriptome results, Figure 1 Figure C shows the expression of the key proteins XPF and ERCC1 of the NER pathway detected by Western blotting. The expressions of XPF and ERCC1 in CDDP-resistant cells EC109 / CDDP, TE1 / CDDP, and EC9706 / CDDP were significantly increased, indicating that they play important roles in cisplatin resistance. These results suggest that drug-resistant cells enhance their tolerance to cisplatin by upregulating the key proteins XPF and ERCC1 of the NER pathway. Targeted degradation of these proteins can disrupt the DNA repair mechanism and increase the sensitivity of drug-resistant cells to cisplatin. Based on this, we designed PROTAC molecules aiming to selectively degrade XPF and ERCC1 to overcome drug resistance.

[0100] PROTACs promote protein degradation by linking target proteins and E3 ligases. The key to designing effective PROTACs lies in selecting appropriate target protein ligands. In this invention, NERi (a small molecule NER inhibitor) was used as a candidate ligand targeting XPF. To verify this, molecular docking simulations were performed to predict the interaction between NERi and XPF. The results showed that NERi formed hydrogen bonds with ASN834 and LYS860 of XPF and had hydrophobic interactions with residues such as LYS832, TYR833, PRO837, GLN838, MET856, and ASN861. In addition, NERi also formed π-π interactions with TYR833, further supporting the binding potential of NERi to XPF ( Figure 1 D and 1E).

[0101] Since XPF and ERCC1 form a functional complex in the NER pathway, we further investigated whether NERi could also interact with ERCC1. Docking analysis showed that NERi formed hydrogen bonds with Ser259, Leu260, and Glu261 of ERCC1 and had hydrophobic interactions with residues such as Leu227, AsPEG230, and Phe231 ( Figure 1 D and 1E). These results indicate that NERi can bind to both XPF and ERCC1 simultaneously, thus potentially disrupting the function of this key repair complex, providing a basis for its selection as a PROTAC ligand.

[0102] Based on this, we synthesized six variants, linking NERi with the E3 ligase ligand through different alkane chains and PEG chains. The synthetic route is as Figure 1 shown in F. The final obtained PROTACs, including C8-NERiP, C10-NERiP, PEG2-NERiP, PEG3-NERiP, PEG4-NERiP, and PEG5-NERiP ( Figure 1 G), were characterized and identified by nuclear magnetic resonance hydrogen spectrum 1 1H NMR.

[0103] Example 2

[0104] This example provides an experiment on the in vitro targeted degradation of ERCC1 / XPF proteins by NERiP.

[0105] Experimental method:

[0106] Western blot: The parental EC9706 and drug-resistant cells were respectively seeded in 10-cm cell culture dishes. After continued culturing for 24 hours to allow them to adhere, the old culture medium was discarded, and the cells were further cultured for 48 hours in the culture medium containing different concentrations (0, 0.1, 1, 5, 10 μM) of PROTACs. The cells were collected using a cell scraper, lysed in a frozen RIPA lysis buffer containing protease inhibitors, and the protein concentration was quantified by the BCA protein assay. The proteins were separated on a 15% SDS-PAGE gel and transferred to a PVDF membrane, which was then blocked with 5% skim milk (at room temperature, 2 h). After incubation with the primary and secondary antibodies, the membrane was exposed and imaged using an ECL chemiluminescence solution and an automatic chemiluminescence image analysis system (MiniChemi 610).

[0107] Experimental results:

[0108] We further evaluated the protein degradation effects of these PROTACs in tumor cells, and the results are as Figure 2 shown: Among them, A - B. Western blot was used to detect the degradation of ERCC1 and XPF mediated by NERiP in parental and CDDP-resistant ESCC cells, and the fold change of ERCC1 and XPF compared with the control group was quantitatively analyzed (n = 3).

[0109] Figure 2 A and 2B show the degradation of ERCC1 and XPF proteins by NERiP in EC9706 and EC9706 / CDDP cells. Dose-dependent Western blot analysis showed that the degradation activities of C8-NERiP and C10-NERiP were weak, probably because the rigid structure of the saturated alkane chain hindered the approach of the target protein to the E3 ligase. PEG2-NERiP, PEG3-NERiP, and PEG5-NERiP showed limited degradation efficiency, indicating that the chain length is a key factor affecting the cellular activity of PROTACs. Notably, PEG4-NERiP significantly reduced the protein levels of XPF and ERCC1 in drug-resistant cells, with DC 50 values of 4.22 μM (XPF) and 5.10 μM (ERCC1), respectively. These results indicate that PEG4-NERiP is worthy of further investigation as a degrader of XPF and ERCC1.

[0110] Example 3

[0111] This example provides the synthesis method and characterization results of the HFn-NERiP-Pt(IV) delivery system.

[0112] 1. Synthesis of S5

[0113] As Figure 3As shown in B, 500 mg of S2 was added to anhydrous DCM (10 mL), and 417 mg of DMAP and 687 mg of 4-nitrophenyl carbonochloridate were slowly added at room temperature. The reaction mixture was stirred at room temperature for 24 hours. After the reaction was completed, DCM was added to dilute the solution to 40 mL, and then it was washed three times with 10% aqueous citric acid solution (20 mL), three times with saturated aqueous sodium carbonate solution (20 mL), and once with saturated brine (20 mL). The organic phase was dried over anhydrous Na 2 SO 4 dried, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain S5.

[0114] 2. Synthesis of S6

[0115] 860 mg of 2,2′-Disulfanediylbis(ethan-1-ol) and 275 mg of DMAP were added to 5 mL of DCM. 1 g of S5 was dissolved in 5 mL of DCM and added. The reaction mixture was stirred at room temperature for 24 hours. After the reaction was completed, DCM was added to dilute the solution to 40 mL, and then it was washed three times with 10% aqueous citric acid solution (20 mL), three times with saturated aqueous sodium carbonate solution (20 mL), and once with saturated brine (20 mL). The organic phase was dried over anhydrous Na 2 SO 4 dried, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain S6.

[0116] 3. Synthesis of S7

[0117] 500 mg of S6 was dissolved in 10 mL of CH 2 Cl 2 and 3.0 mL of CF 3 COOH was slowly added. The mixture was stirred at room temperature for 12 hours. After the reaction, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain S7.

[0118] 4. Synthesis of S8

[0119] 214 mg of NERi, 500 mg of HATU, and 212 mg of DIPEA were dissolved in 10 mL of DMF, and 640 mg of S7 was slowly added. The reaction mixture was stirred at room temperature for 24 hours. After the reaction was completed, the mixture was diluted with ethyl acetate (10 mL × 3) and washed with water (30 mL) and saturated NaCl solution (10 mL) respectively. The organic layer was dried over anhydrous Na 2 SO 4It was dried, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S8.

[0120] 5. Synthesis of S9

[0121] 300 mg of intermediate S8, 120 mg of succinic anhydride and 29 mg of DMAP were added to 10 mL of DCM, and the reaction mixture was stirred at room temperature for 12 hours. After the reaction was completed, the solvent was removed, and it was purified by silica gel column chromatography to obtain S9.

[0122] 6. Synthesis of S10 (NERiP-SS-Mal)

[0123] 80 mg of intermediate S9, 125 mg of EDC, 36 mg of Mal-PEG 4 -OH (maleimide-tetraethylene glycol) and 1.6 mg of DMAP were added to 10 mL of DCM, and the reaction mixture was stirred at room temperature for 12 hours. After the reaction was completed, the solvent was removed, and it was purified by silica gel column chromatography to obtain NERiP-SS-Mal.

[0124] 7. Preparation of HFn-NERiP-Pt(IV) nanodrug

[0125] First, Mal-SS-NERiP (i.e., NERiP-SS-Mal) was mixed with HFn solution through the reaction of maleimide in Mal-SS-NERiP with the thiol groups on the HFn surface, and HFn-NERiP was prepared by purification through size exclusion chromatography on HiLoad 16 / 600 Superdex 200 pg. The HFn-NERiP-Pt(IV) nanodrug was prepared by the method of heating to open the drug channel. First, the Pt(IV) prodrug solution (1 mL, 2 mg / mL in DMSO) was vortexed with 1 mL of glycerol, then 2 mL of 20 mM Tris-HCl (pH 8.0) was added and mixed evenly, and then it was dropped into the HFn-NERiP solution (1 mL, 20 mg / mL in 20 mM Tris-HCl). It was heated at 55 °C for 2 h, centrifuged at 10000 rpm for 10 min to remove impurities, and the free Pt(IV) prodrug was removed using a dialysis bag (molecular weight cut-off 12000 - 14000 DA) and the buffer was replaced with PBS, pH 7.4. Then it was purified by size exclusion chromatography on HiLoad 16 / 600 Superdex 200 pg.

[0126] 8. Transmission electron microscopy imaging

[0127] 10 μL of a 1 mg / mL sample was dropped onto the front of the copper grid, left to stand for 10 min, then the excess liquid was blotted off with filter paper, a phosphotungstic acid negative staining solution was dropped, stained for 100 s, the negative staining solution was blotted off with filter paper, and after drying, it was observed using an electron microscope.

[0128] 9. High performance liquid chromatography size exclusion (SEC):

[0129] Samples with the same protein concentration (0.5 mg / ml) were taken respectively, and size exclusion analysis by high performance liquid chromatography was performed using a TSKgel G4000SWXL Column. The mobile phase was 200 mM Tris-HCl, pH 8.0, and the ultraviolet absorption at 280 nm was detected. The sample injection volume was 40 μL.

[0130] 10. Circular dichroism spectroscopy (CD):

[0131] At 25 °C, the CD spectra of the samples were obtained using a Chirascan-Plus circular dichroism spectrometer (Applied Photophysics). The samples were resuspended in PBS at a concentration of 0.2 mg / ml. A quartz cuvette with a 1 cm optical path length was used to measure the spectra from 260 nm to 190 nm at a resolution of 0.1 nm.

[0132] Results:

[0133] We selected ferritin (HFn) as the drug carrier because it has been widely proven to have good biocompatibility, tumor targeting ability mediated by the CD71 receptor, and the ability to load therapeutic drugs in its lumen. We hypothesized that linking NERiP to HFn and loading Pt(IV) in the same carrier could achieve dual-targeted delivery and synergistically enhance the anti-tumor effect on cisplatin-resistant cells ( Figure 3 A).

[0134] Figure 3 In, A. Schematic diagram of the synthesis of HFn-NERiP-Pt(IV) nanodrugs. B. Synthetic route of NERiP-SS-Mal. C. Transmission electron microscope (TEM) images of HFn, HFn-Pt(IV), HFn-NERiP, and HFn-NERiP-Pt(IV) nanodrugs. Scale bar = 50 nm. D. Zeta potential measurements of HFn, HFn-Pt(IV), HFn-NERiP, and HFn-NERiP-Pt(IV) nanodrugs. E. Ultraviolet absorption spectra of HFn, HFn-Pt(IV), HFn-NERiP, and HFn-NERiP-Pt(IV) nanodrugs. F. Size exclusion chromatography (SEC) analysis of HFn, HFn-Pt(IV), HFn-NERiP, and HFn-NERiP-Pt(IV) nanodrugs. G. Circular dichroism spectroscopy analysis of HFn, HFn-Pt(IV), HFn-NERiP, and HFn-NERiP-Pt(IV) nanodrugs.

[0135] To promote the conjugation of NERiP with ferritin, we modified NERiP with a tumor microenvironment-responsive maleimide (Mal) group to synthesize Mal-SS-NERiP. Specifically, the hydroxyl group of the VHL ligand in NERiP was used as a suitable linker point. By introducing a linker containing a GSH-sensitive disulfide bond, compound S9 was obtained to ensure the efficient release of NERiP. Subsequently, compound S9 was reacted with OH-PEG 4 -Mal under the action of DMAP and EDCI, and finally the target compound Mal-SS-NERiP (compound S10) was obtained. The specific synthetic route is as Figure 3 shown in Scheme B. Mal-SS-NERiP was conjugated to the surface of ferritin through a maleimide–thiol reaction. Meanwhile, Pt(IV) was encapsulated into the ferritin cavity through a thermally controlled drug channel to obtain HFn-NERiP-Pt(IV)( Figure 3 Scheme A).

[0136] Transmission electron microscopy measurements showed that HFn, HFn-NERiP, HFn-Pt(IV), and HFn-NERiP-Pt(IV) all maintained a uniform spherical structure ( Figure 3 Scheme C). Their zeta potentials were similar to those of unmodified ferritin, indicating that their surface topologies remained unchanged ( Figure 3 Scheme D). In addition, ultraviolet spectroscopy showed a characteristic absorption peak at 340 nm, verifying the successful conjugation of NERiP ( Figure 3 Scheme E). Size exclusion chromatography confirmed the absence of impurities after modification, and circular dichroism (CD) showed that the secondary structure of ferritin in these nanodrugs was stable ( Figure 3 Schemes F–G).

[0137] Example 4

[0138] Responsive drug release of the HFn-NERiP-Pt(IV) delivery system.

[0139] Experimental method:

[0140] 1. Study on the reductive release of NERiP from NERiP-SS-Mal

[0141] 50 μM of NERiP-SS-Mal was incubated with 10 mM of DTT (methanol:water = 1:9, v / v) at 37 °C for a certain time, and then the concentration of the released NERiP was determined by HPLC (mobile phase: A: water containing 0.1% TFA, C: methanol; flow rate: 1 mL / min; column temperature: 30 °C; ultraviolet detection wavelength: 340 nm; elution gradient: 0 - 10 minutes, from 90% A to 10% A; 20 - 25 minutes, from 10% A to 90% A).

[0142] 2. In vitro drug release and stability study

[0143] For in vitro drug release, briefly, 2 mL of HFn-NERiP-Pt(IV) was transferred into a dialysis bag, which was then placed into a beaker containing 200 mL of PBS buffer with different pH values (7.4 and 5.0). It was heated and stirred at 37 °C. At different time points, 2 mL of the PBS buffer in the beaker was taken out, and the Pt concentration was detected by ICP / MS.

[0144] Experimental results:

[0145] The experimental results are as Figure 4 shown: A. Schematic diagram of GSH-responsive release of NERiP-SS-Mal. B. High-performance liquid chromatography (HPLC) chromatogram of the reduction and release of NERiP from NERiP-SS-Mal under the action of 5 mM DTT. C. Quantitative analysis of the NERiP release percentage. D. Platinum release curves in HFn-NERiP-Pt(IV) treated with PBS buffer (pH 7.4) or PB buffer (pH 5.0).

[0146] In the synthetic design, the free hydroxyl groups on the VHL ligand module in NERiP were initially occupied by the GSH-responsive linker, temporarily inhibiting the ability of the VHL ligand to recognize the E3 ligase. However, in the reduced tumor microenvironment, the linker was cleaved by intracellular GSH, further rearranged, and the active hydroxyl groups were restored, thus reactivating the ability of PROTAC to recruit the E3 ligase and promote the degradation of the target protein ( Figure 4 A). HPLC analysis showed that 97% of NERiP was released after incubation with DTT for two hours, confirming the high reduction sensitivity of this disulfide-bonded PROTAC ( Figure 4 B–C). In addition, ferritin provided pH-sensitive dissociation, promoting the release of platinum under acidic conditions. As Figure 4 shown in D, the platinum release reached 82% at pH 5.0, while the release was negligible at pH 7.4, indicating that the surface modification of NERiP did not affect the pH responsiveness of the carrier. Overall, these results verified the ability of HFn-NERiP-Pt(IV) to release NERiP and platinum drugs in the tumor microenvironment. By integrating the advantages of PROTAC-mediated protein degradation with ferritin-facilitated platinum delivery, this system provides a solid theoretical basis for targeted therapy against cisplatin-resistant tumors.

[0147] Example 5

[0148] Experiment on the use of the HFn-NERiP-Pt(IV) delivery system for in vitro targeted degradation of ERCC1 / XPF protein and increasing the accumulation of Pt in tumor cells.

[0149] Experimental methods:

[0150] 1. Cellular immunofluorescence

[0151] Seed EC9706 / CDDP cells in a bottom glass culture dish and incubate until they reach 40% confluence. Add different concentrations of HFn-NERiP and continue to incubate for 48 hours, or add FITC-labeled HFn-NERiP at different time points. Paraformaldehyde was used to fix the cells, followed by permeabilization with 0.1% Triton X-100, and then blocking with 5% BSA. Then, the cells were incubated with primary and secondary antibodies and stained with Phalloidin (red) and DAPI (blue). Images were captured using a laser confocal microscope.

[0152] 2. Detection of GSH level

[0153] Seed EC9706 drug-resistant cells in a 10 cm cell culture dish. After the cells adhered, add different compounds. Use a cell scraper to collect the cells. Half of the cells were used for protein extraction and determination of protein concentration, and the other half were used to detect the intracellular GSH level. Briefly, by separately detecting the total GSH and GSSG contents in the cells, the reduced GSH level was obtained by subtracting the two. Incubate 10 μL of the sample with 200 μL of the detection working solution (0.25 mM DTNB and 0.125 mg / mL NADPH in T-GSH detection buffer) at 25 °C for 5 min, measure the absorbance at 412 nm, and substitute it into the standard curve to determine the GSH concentration of the sample.

[0154] 3. Cellular uptake

[0155] Seed EC9706 drug-resistant cells in a 10 cm cell culture dish and grow them to 80% density. Then, add different compounds and continue to incubate for 6 hours, and then wash with PBS. Use 0.25% trypsin to digest and collect the cells and count them with a hemocytometer. After digestion with aqua regia, the Pt concentration was detected by ICP-MS.

[0156] Experimental results:

[0157] The experimental results are as follows Figure 5Shown: A. Confocal microscopy images showing the co - localization of nuclei (blue), cytoskeleton (red), and FITC - labeled HFn - NERiP (green) in EC9706 / CDDP cells at different time points. Scale bar = 20 μm. B - C. Confocal microscopy images of EC9706 / CDDP cells after 48 - hour treatment with different concentrations of HFn - NERiP, showing the co - localization of nuclei (blue), cytoskeleton (red), and ERCC1 (green) or XPF (green). Scale bar = 20 μm. D. Quantitative analysis of intracellular fluorescence intensities of ERCC1 and XPF. E. Western blot analysis of the changes in ERCC1 and XPF protein levels in EC9706 / CDDP cells after 48 - hour treatment with different concentrations of HFn - NERiP. F. Changes in intracellular GSH levels in EC9706 / CDDP cells after treatment with different compounds. G. ICP - MS analysis of platinum content in EC9706 / CDDP cells after 6 - hour treatment with CDDP, HFn - Pt(IV), and HFn - NERiP - Pt(IV).

[0158] Before evaluating the anti - tumor activity of HFn - NERiP - Pt(IV), it is necessary to first study its intracellular behavior. As a ligand for the CD71 receptor, HFn is endocytosed after binding to tumor cells, making it an ideal carrier for targeted delivery of anti - tumor drugs. To evaluate the cellular uptake of HFn modified with NERiP, we labeled HFn - NERiP with FITC. Fluorescence microscopy results showed that within 8 hours, the intracellular distribution of HFn - NERiP in EC9706 / CDDP cells gradually increased ( Figure 5 A).

[0159] To explore the protein degradation effect of HFn - NERiP in EC9706 / CDDP cells, we used confocal microscopy to observe the changes in intracellular fluorescence signals of ERCC1 and XPF. As the concentration of HFn - NERiP increased, the fluorescence intensities of these proteins gradually decreased ( Figure 5 B and C). Fluorescence quantitative analysis indicated that HFn - NERiP effectively reduced the intracellular levels of ERCC1 and XPF proteins ( Figure 5 D). Western blot analysis further revealed a dose - dependent decrease in ERCC1 and XPF protein levels, with DC 50 values of 6.19 μM and 6.3 μM, respectively ( Figure 5 E).

[0160] Since Pt(IV) prodrugs are pharmacologically inert and need to be reduced intracellularly to exert cytotoxic effects, we measured the changes in GSH levels in EC9706 / CDDP cells. AsFigure 4 As shown in Figure F, after treatment with HFn-NERiP-Pt(IV), the intracellular GSH level was significantly lower than that of the control group. This GSH depletion demonstrated the conversion of Pt(IV) to active Pt(II). In addition, the intracellular platinum content analyzed by ICP-MS showed that the platinum level accumulated in the cells treated with HFn-NERiP-Pt(IV) was significantly higher than that of the free CDDP group ( Figure 5 G). These results indicated that HFn-NERiP could effectively trigger the activation of Pt(IV) while enhancing platinum uptake, thereby improving the overall efficacy of platinum-based chemotherapeutic drugs in overcoming drug resistance.

[0161] Example 6

[0162] This example provides an experiment on the HFn-NERiP-Pt(IV) delivery system for overcoming platinum drug resistance.

[0163] Experimental method:

[0164] Cell viability detection

[0165] The changes in cell viability under the influence of CDDP, HFn-CDDP, and HFn-Pt(IV) nanodrugs were detected by CCK8. EC109, EC9706, TE1 parental cells, and EC109 / CDDP, EC9706 / CDDP, TE1 / CDDP drug-resistant cells were seeded in 96-well plates at a cell density of 4000 cells / well and cultured for 24 h. After the cells adhered and grew, the old medium was discarded, and 200 μL of fresh complete medium containing the drug was added. The final concentrations of the drugs were 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.0625 μM, a total of 8 concentrations, and the cells were cultured for another 48 h. The drug-containing medium was discarded, 100 μL of serum-free medium containing CCK8 was added to each well, and the reaction was carried out for about 2 h. The OD value was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The percentage was calculated by comparing with the OD value of the blank control group to obtain the corresponding cell survival rate, and then the half-maximal inhibitory concentration (IC 50 ) was calculated using SPSS software.

[0166] Transcriptome sequencing and data analysis

[0167] EC9706 drug-resistant cells were seeded in 10-cm cell culture dishes. After the cells adhered, CDDP and HFn-NERiP-Pt(IV) were added respectively. After incubation for 48 hours, the cells were collected using a cell scraper. Total RNA was isolated from the cells using TRIzol reagent (Solarbio, R1100), and then quickly frozen in liquid nitrogen. RNA sequencing analysis was completed by Biomarker Technologies (Beijing).

[0168] RNA Extraction and RT-qPCR Analysis

[0169] EC9706 drug-resistant cells were seeded in 10-cm cell culture dishes. After the cells adhered to the wall, CDDP and HFn-NERiP-Pt(IV) were added respectively. After incubation for 48 hours, the cells were collected using a cell scraper. Total RNA in the cells or tissues was extracted using the TRIzol kit, and 1 μg of RNA was reverse transcribed into cDNA using the HiScript II Q RT SuperMix reverse transcription kit. The reverse transcription reaction system was set as follows: 1 μg of RNA, 1 μL of reverse transcriptase, 1 μL of oligo(dT)15, 1 μL of dNTP mixture, and an appropriate amount of reverse transcription buffer was added to make the volume up to 20 μL. The temperature control conditions were: 25°C for 5 min, 50°C for 30 min, 85°C for 5 min, and stored at 4°C. Subsequently, qPCR was performed. Each reaction system (20 μL) contained: 10 μL of 2×SYBR Green Master Mix, 0.5 μL of forward primer (10 μM), 0.5 μL of reverse primer (10 μM), 2 μL of cDNA template, and the remaining volume was filled with RNase-free water. The relative mRNA expression level was calculated by the comparative cycle threshold method (ΔΔCT method). All experiments were repeated three times.

[0170] Experimental Results:

[0171] The experimental results are as Figure 6 shown: A - B. Viability curves of EC9706 (A) and EC9706 / CDDP (B) cells after treatment with HFn, CDDP, HFn-NERiP, HFn-Pt(IV), and HFn-NERiP-Pt(IV) for 48 hours at equivalent platinum concentrations. C. Western blot analysis of the changes in the protein levels of XPF, ERCC1, γ-H2AX, and Cleaved Caspase-3 in EC9706 / CDDP cells after treatment with different compounds, and band gray scale analysis was performed. D - E. Volcano plots showing the differential gene expression profiles of EC9706 / CDDP cells after treatment with CDDP (D) and HFn-NERiP-Pt(IV) (E), identifying significantly upregulated and downregulated genes. F - G. KEGG enrichment analysis of the pathways of upregulated and downregulated genes in EC9706 / CDDP cells after treatment with HFn-NERiP-Pt(IV). H. Heat map analysis of the expression changes of genes related to DNA replication and repair in EC9706 / CDDP cells after treatment with CDDP and HFn-NERiP-Pt(IV). I. mRNA expression of EC9706 / CDDP cells after treatment with CDDP, NERiP, and HFn-NERiP-Pt(IV) for 48 hours.

[0172] We further evaluated the in vitro anti-tumor effect of HFn-NERiP-Pt(IV). Figure 6 Figures 6A and 6B show the cytotoxicity curves of CDDP, HFn-Pt(IV), and HFn-NERiP-Pt(IV) against EC9706 and EC9706 / CDDP cells at different platinum concentrations. The results showed that HFn-NERiP-Pt(IV) exhibited significant growth inhibitory effects in both cell lines, which were superior to HFn-Pt(IV). The IC 50 in EC9706 / CDDP cells was 4.89 μM, which was close to the IC of CDDP in EC9706 cells, 50 indicating its potential to overcome CDDP resistance in esophageal squamous cell carcinoma.

[0173] Western blot analysis showed that HFn-NERiP-Pt(IV) based on HFn-NERiP effectively degraded XPF and ERCC1. Compared with CDDP at the same platinum dose, HFn-NERiP-Pt(IV) significantly increased the levels of the DNA damage marker γ-H2AX and the apoptosis-related protein cleaved Caspase-3. This indicated that HFn-NERiP-Pt(IV) could target and degrade the ERCC1-XPF protein, inhibit the NER mechanism in drug-resistant tumor cells, resulting in more severe DNA damage and ultimately inducing apoptosis ( Figure 6 C).

[0174] After observing that HFn-NERiP-Pt(IV) could effectively induce DNA damage and promote apoptosis of EC9706 / CDDP cells, we further investigated its potential transcriptomic changes using RNA-seq technology. The volcano plot showed that there were relatively small differences in gene expression in EC9706 / CDDP cells before and after CDDP treatment ( Figure 6 D). However, after treatment with HFn-NERiP-Pt(IV), significant changes in gene expression were observed, with a total of 2,040 genes upregulated and 2,176 genes downregulated ( Figure 6 E). Subsequent KEGG gene enrichment analysis further clarified that these differentially expressed genes were mainly involved in functional pathways related to CDDP resistance, such as the cell cycle and nucleotide excision repair, especially DNA replication ( Figure 6 F). Further data analysis showed that treatment with HFn-NERiP-Pt(IV) significantly downregulated genes related to DNA replication and repair downstream of ERCC1-XPF, including genes of the POLE, POLE2, POLD1, POLD2, PCNA, and RFC families ( Figure 5H). Subsequently, we detected the changes in the mRNA levels of these genes in EC9706 / CDDP cells after treatment with CDDP, NERiP, and HFn-NERiP-Pt(IV), and the results were consistent with the predictions of the KEGG gene enrichment analysis ( Figure 6 I).

[0175] In addition, Figure 6 G also demonstrated significant gene expression changes in the apoptosis, inflammatory response, and oxidative stress pathways. This indicates that HFn-NERiP-Pt(IV) may enhance its anti-cancer effect by activating the apoptosis signaling pathway. Overall, these findings suggest that HFn-NERiP-Pt(IV) effectively reduces DNA repair capacity, enhances platinum-induced DNA damage, thereby accelerating the apoptosis process, providing an effective strategy to overcome CDDP resistance.

[0176] Example 7

[0177] This example provides an experiment on the use of the HFn-NERiP-Pt(IV) delivery system to extend the in vivo plasma half-life of NERiP and platinum prodrugs.

[0178] Experimental method:

[0179] 1. Experimental animals

[0180] The BALB / c-nu mice used in the experiment were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China). All animal experiments were conducted in accordance with the experimental protocol established by the Animal Ethics Committee of Zhengzhou University.

[0181] 2. Plasma pharmacokinetic analysis

[0182] To obtain the pharmacokinetics of NERiP, HFn-NERiP, CDDP, HFn-Pt(IV), and HFn-NERiP-Pt(IV), healthy BALB / c mice were randomly grouped according to body weight and age, with three mice in each group. Different drugs (5 mg Pt / kg or 40 mg NERiP / kg) were intravenously injected into the mice. 20 μL of blood samples were repeatedly collected at 0.08, 1, 2, 4, 8, 12, 24, and 48 h by tail clipping. Analysis was performed using ICP / MS, and the blood half-life was calculated using GraphPad Prism 8.0.

[0183] 3. In vivo and ex vivo biodistribution imaging

[0184] The BALB / c-nu mice bearing EC9706 / CDDP cell xenograft tumors were divided into 3 groups, with 3 mice in each group. Once the tumor volume reached 200 mm 3, Intravenous injection of PBS, free-ICG, and ICG-HFn-NERiP, and the volume was calculated as L×W / 2, where L represents the maximum diameter of the tumor and W represents the minimum diameter of the tumor. At 0, 1, 2, 4, 6, 8, 12, and 24 h after injection, mice were observed using an IVIS in vivo imaging system (Lumina XR series, Perkin Elmer). At 24 h after intravenous injection, the mice were euthanized, and tumors and major organs (heart, liver, spleen, lung, and kidney) were collected for ex vivo imaging. 2 / 2 was calculated, where L represents the maximum diameter of the tumor and W represents the minimum diameter of the tumor. At 0, 1, 2, 4, 6, 8, 12, and 24 h after injection, mice were observed using an IVIS in vivo imaging system (Lumina XR series, Perkin Elmer). At 24 h after intravenous injection, the mice were euthanized, and tumors and major organs (heart, liver, spleen, lung, and kidney) were collected for ex vivo imaging.

[0185] 4. In vivo biodistribution of Pt

[0186] BALB / c-nu mice bearing EC9706 / CDDP xenograft tumors were divided into 3 groups, with 3 mice in each group. Once the tumor volume reached 200 mm 3 , CDDP, HFn-Pt(IV), and HFn-NERiP-Pt(IV) (2.5 mg Pt / kg) were intravenously injected. At 24 h after intravenous injection, the mice were euthanized, and tumor tissues were collected to measure the Pt concentration by ICP / MS.

[0187] Experimental results:

[0188] The experimental results are as Figure 7 shown: A. In vivo pharmacokinetic curves within 48 h after intravenous injection of NERiP or HFn-NERiP. B. In vivo pharmacokinetic curves within 48 h after intravenous injection of CDDP, HFn-Pt(IV), or HFn-NERiP-Pt(IV). C. In vivo fluorescence imaging of mice after intravenous injection of ICG, ICG-HFn, or ICG-HFn-NERiP at different time points (n = 3 mice). D-E. Fluorescence imaging (D) and quantitative analysis of fluorescence intensity (E) showed the distribution of ICG, ICG-HFn, or ICG-HFn-NERiP in tumors and major organs after 24 h (n = 3). F. Platinum concentration in tumor tissues 24 h after intravenous injection of CDDP, Pt(IV), HFn-Pt(IV), or HFn-NERiP-Pt(IV).

[0189] After confirming that HFn-NERiP-Pt(IV) degrades DNA repair proteins in vitro and enhances platinum-induced cytotoxicity, we next evaluated its pharmacokinetic properties, tumor targeting, and biosafety in vivo.

[0190] Analysis of blood samples by HPLC showed that the small molecule NERiP was rapidly cleared (half-life: 0.2 h), while HFn-NERiP remained in the blood circulation for a longer time (half-life: 4.2 h), indicating that HFn could significantly prolong the systemic exposure of NERiP. Figure 7 A). Similarly, Pt in HFn-NERiP-Pt(IV) exhibited a half-life of 4.9 h, much longer than that of free CDDP, further demonstrating that ferritin could improve the bioavailability of NERiP and platinum drugs. Figure 7 B).

[0191] To investigate the biodistribution of HFn-NERiP, we used ICG-labeled HFn and HFn-NERiP. Twenty-four hours after injection, these compounds gradually accumulated at the tumor site. In contrast, the signal of free ICG disappeared within 12 h. Figure 7 C). In vitro near-infrared imaging further confirmed the tumor-specific localization of HFn-NERiP after 24 h. Figure 7 D). Quantitative fluorescence measurements showed that the intensities of ICG-labeled HFn and HFn-NERiP in tumors were similar and significantly higher than that of free ICG, indicating that NERiP did not affect the targeting ability of ferritin. Figure 6 E).

[0192] Next, we evaluated the platinum accumulation in tumor tissues. As shown in Figure 6 F, the platinum concentrations in the tumors of mice injected with HFn-Pt(IV) and HFn-NERiP-Pt(IV) were 4.8-fold and 4.9-fold higher than those in the CDDP group, respectively. These results highlighted the superior platinum delivery efficiency of HFn-NERiP-Pt(IV). Figure 6 F).

[0193] Overall, these findings indicated that HFn-NERiP-Pt(IV) prolonged the blood circulation time of NERiP, enhanced the tumor accumulation of the drug, and supported its clinical application prospects in the treatment of drug-resistant tumors.

[0194] Example 8

[0195] This example provides an experiment on the in vivo safety and antitumor activity of the HFn-NERiP-Pt(IV) delivery system.

[0196] Experimental method:

[0197] EC9706 / CDDP cells (2×10 6 cells) were injected into the right back of BALB / c-nu female mice to establish a CDDP-resistant cell xenograft tumor model. When the tumor volume reached 100 mm 3, mice were divided into 7 groups (n = 6): PBS group, HFn group, CDDP group (2 mg Pt / kg), NERiP group, NERiP+CDDP group (administered in combination with 2 mg Pt / kg), HFn-Pt(IV) group (2 mg Pt / kg), and HFn-NERiP-Pt(IV) group (2 mg Pt / kg). They were injected intravenously once a week for three weeks. The tumor volume and weight of the mice were recorded every two days. After 21 days of treatment, all mice were euthanized and the tumor tissues were collected for analysis. The main organs were collected for H&E staining, and the tumors were collected for immunohistochemistry and TUNEL staining.

[0198] Experimental results:

[0199] The experimental results are as Figure 8 shown: A. Schematic diagram of the process of establishing a CDDP-resistant ESCC CDX model. B. Curve of tumor volume change during treatment. C-I. Curves of tumor volume change in each group during treatment. J. Representative photos of tumors after resection on day 21 (n = 6 mice). K. Tumor weights of mice in each group after the end of treatment. Data are expressed as mean ± standard deviation (SD) (n = 6 mice). L. Immunohistochemical staining of main organs and TUNEL staining of tumor tissues in different groups. Scale bar = 100 μm. M. Body weight changes of mice in each group during treatment. Data are expressed as mean ± standard deviation (n = 6 mice).

[0200] To evaluate the in vivo therapeutic potential of dual delivery of HFn-NERiP-Pt(IV) against cisplatin-resistant esophageal squamous cell carcinoma (ESCC), we established an EC9706 / CDDP tumor mouse model ( Figure 8 A). As Figure 8 shown in B to D, during the treatment period, the tumor volumes of mice in the PBS group and the HFn group increased rapidly, with average volumes reaching 863 mm 3 and 881 mm 3 . The tumor growth inhibition rate (TGI) of the CDDP group was only 29.8% ( Figure 8 E), indicating that this model has strong cisplatin resistance. Compared with the CDDP group, the NERiP+CDDP group did not show enhanced antitumor effects, which may be due to the poor pharmacokinetics of NERiP in vivo ( Figure 8 G). The HFn-Pt(IV) group showed significant antitumor effects, which may be attributed to the tumor-targeted accumulation of HFn ( Figure 8 H). Importantly, dual-targeted delivery of HFn-NERiP-Pt(IV) achieved the most effective inhibition of EC9706 / CDDP tumor growth ( Figure 8I and 8J), with a TGI of 75.4%. These results indicate that HFn-mediated delivery significantly improves the pharmacokinetics of NERiP, thereby enhancing the anti-tumor activity of platinum-based chemotherapy. Similarly, the HFn-NERiP-Pt(IV) group had the lowest average tumor weight (0.235 g), demonstrating its superior efficacy compared to all other treatments ( Figure 8 K).

[0201] To elucidate the mechanism of reversal of drug resistance, we performed immunohistochemical analysis on EC9706 / CDDP tumor sections, focusing on the detection of XPF, ERCC1, cleaved Caspase-3, and TUNEL staining. HFn-NERiP-Pt(IV) treatment significantly reduced the protein levels of XPF and ERCC1, accompanied by an increase in DNA damage and obvious apoptotic signals( Figure 8 L). Thus, by removing ERCC1-XPF, HFn-NERiP-Pt(IV) disrupted the NER pathway in vivo, effectively inducing the death of drug-resistant tumor cells.

[0202] We further evaluated the biosafety of HFn-NERiP-Pt(IV) by monitoring body weight changes and performing H&E staining. Although H&E staining of major organs did not show acute renal toxicity, body weight changes indicated that mice in the CDDP and NERiP+CDDP treatment groups had consistently lower body weights during weekly dosing, suggesting possible cumulative long-term toxic effects( Figure 8 M). In contrast, mice receiving HFn-NERiP-Pt(IV) maintained relatively stable body weights and showed no signs of major organ toxicity, demonstrating superior safety compared to free CDDP or NERiP+CDDP.

[0203] Finally, it should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article or apparatus comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or apparatus.

[0204] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0205] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A ferritin nanocomplex based on the synergistic effect of PROTAC and platinum drug, characterized in that: The nanocomplex includes a ferritin carrier, a platinum drug precursor, and a PROTAC molecule that targets and degrades ERCC1 protein and / or XPF protein; The platinum drug precursor is loaded in the inner cavity of the ferritin carrier; The PROTAC molecule is modified on the surface of the ferritin carrier; The PROTAC molecule is composed of an E3 ubiquitin ligase ligand and a ligand targeting ERCC1 protein and / or XPF protein connected by a linker.

2. The ferritin nanocomplex based on the synergistic effect of PROTAC and platinum drugs according to claim 1, characterized in that: In the PROTAC molecule, the ligand targeting the ERCC1 protein and / or the XPF protein includes the ligand NERi, and the structural formula of the ligand NERi is as follows:

3. A ferritin nanocomplex based on the synergistic effect of PROTAC and platinum drugs according to claim 1 or 2, characterized in that: The structural formula of the E3 ubiquitin ligase ligand is as follows: The PROTAC molecule includes at least one of C8-NERiP, C10-NERiP, PEG2-NERiP, PEG3-NERiP, PEG4-NERiP and PEG5-NERiP; The structural formulas of the C8-NERiP, the C10-NERiP, the PEG2-NERiP, the PEG3-NERiP, the PEG4-NERiP and the PEG5-NERiP are respectively as follows:

4. The ferritin nanocomplex based on the synergistic effect of PROTAC and platinum drugs according to claim 1, characterized in that: The platinum drug precursor includes platinum tetraiodide; The ferritin carrier includes the ferritin carrier HFn.

5. The ferritin nanocomplex based on the synergistic effect of PROTAC and platinum drug according to claim 1, characterized in that: The PROTAC molecule is connected to the COOH-SS-Mal group, and the maleimide in the COOH-SS-Mal group can react with the sulfhydryl group on the surface of the ferritin carrier to achieve the connection between the PROTAC molecule and the ferritin carrier; The connection between the PROTAC molecule and the COOH-SS-Mal group is achieved by esterification reaction between the carboxyl group in the COOH-SS-Mal group and the hydroxyl group on the E3 ubiquitin ligase ligand. The structural formula of the COOH-SS-Mal group is as follows: The platinum drug precursor is loaded into the inner cavity of the ferritin carrier through a temperature-controlled drug channel.

6. The ferritin nanocomplex based on the synergistic effect of PROTAC and platinum drug according to claim 1, characterized in that: In the nanocomplex, the molar ratio of the ferritin carrier, the platinum drug precursor and the PROTAC molecule is 1:18:

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7. Use of a ferritin nanocomplex based on the synergistic effect of PROTAC and platinum drugs as described in any one of claims 1 to 6 in the preparation of anti-tumor drugs.

8. An anti-tumor drug, characterized in that: The active ingredient of the drug includes a ferritin nanocomplex based on the synergistic effect of PROTAC and platinum drug according to any one of claims 1-6.

9. A method for preparing a ferritin nanocomplex based on the synergistic effect of PROTAC and platinum drugs according to any one of claims 1 to 6, characterized in that: The preparation method comprises: S1. Dissolve 2-Carboxybenzaldehyde and 9-fluorenone hydrazone in an organic solvent, react for a certain period of time, perform solid-liquid separation, wash and dry the obtained solid, and obtain the ligand NERi; S2. 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecan-16-oic acid, HATU and DIPEA are dissolved in an organic solvent, and then (S,R,S)-AHPC-Me is added to react to obtain a mixed solution; S3. The mixed solution was diluted, washed, dried, filtered, concentrated under reduced pressure and purified by silica gel column chromatography to obtain AHPC-PEG4-BOC; S4. The AHPC-PEG4-BOC is dissolved in an organic solvent and then reacted with DMAP and 4-nitrophenyl carbonochloridate. The resulting reaction solution is diluted, washed, dried, filtered, concentrated under reduced pressure and purified by silica gel column chromatography to obtain compound S5; S5. 2,2′-Disulfanediylbis(ethan-1-ol) and DMAP are co-dispersed in an organic solvent, and then the compound S5 is added to react. The resulting reaction solution is diluted, washed, dried, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain compound S6; S6. The compound S6 is dissolved in an organic solvent, and then CF3COOH is added to react. After the reaction, the solvent is removed and purified by silica gel column chromatography to obtain compound S7; S7. The ligand NERi, HATU and DIPEA are dissolved in an organic solvent, and then the compound S7 is added to react. The resulting reaction solution is diluted, washed, dried, filtered, concentrated under reduced pressure and purified by silica gel column chromatography to obtain compound S8; S8. The compound S8, succinic anhydride and DMAP are dissolved in an organic solvent, reacted for a certain period of time, and then the solvent is removed and purified by silica gel column chromatography to obtain a compound S9; S9. The compound S9, EDC, Mal-PEG4-OH and DMAP are dissolved in an organic solvent, reacted for a certain period of time, and then the solvent is removed and purified by silica gel column chromatography to obtain compound S10, namely PEG4-NERiP-SS-Mal; S10. The PEG4-NERiP-SS-Mal and the ferritin carrier HFn are co-dispersed in a buffer solution, and after a certain reaction time, purified to obtain HFn-NERiP; S11. Using platinum drug precursor solution and HFn-NERiP solution as raw materials, the HFn-NERiP-Pt(IV) nanocomposite was prepared by heating to open the drug channel; The structural formulas of the ligand NERi, the AHPC-PEG4-BOC, and the compounds S5 to S10 are as follows:

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