Sulfatase-driven polypeptide condensate with chemotherapy sensitization effect and preparation method of sulfatase-driven polypeptide condensate

Abstract

The invention discloses a sulfatase-driven polypeptide condensate with a chemotherapy sensitization effect and a preparation method thereof, and belongs to the field of biological medicines. According to the principle that sulfatase can catalyze hydrolysis of sulfate bonds, main polypeptide YSO4F responded by sulfatase enzyme is designed and synthesized; and the enzyme response aggregate m-YSO4F-LSG is blended with a protein G3BP2 ligand FGDF-YSO4F to prepare the enzyme response aggregate m-YSO4F-LSG with a sorafenib chemosensitization effect. Tumor cells can generate stress particles with a chemotherapy drug resistance effect under the stimulation of a chemotherapy drug sorafenib, the treatment effect of the sorafenib is weakened, and the protein G3BP2 is one of core proteins of the stress particles. After the m-YSO4F-LSG enters tumor cells, sulfate groups are hydrolyzed under the catalytic action of sulfatase overexpressed in tumor cell lysosomes, so that phase separation liquid drops d-YF-LSG with high affinity to protein G3BP2 are formed in the cells in situ, the phase separation liquid drops d-YF-LSG act with the protein G3BP2 and are fused with stress particles, chemotherapy drug resistance generated by the stress particles is inhibited, and the chemotherapy effect is improved. The treatment effect is improved.

Description

Technical Field

[0001] The invention relates to a sulfatase-driven polypeptide aggregate with chemosensitization effect, belonging to the field of biomedicine. Background Art

[0002] Liquid-liquid phase separation is a natural phenomenon whereby synthetic and natural polymers form coacervates by thermodynamically concentrating macromolecules from a homogeneous solution driven by long- and short-range interactions. Phase separation of proteins and nucleic acids in eukaryotic cells leads to the formation of biomolecular condensates that are compartmentalized into membraneless organelles within the cell. Over the past decade, membraneless organelles have been shown to play important roles in regulating myriad biological functions ranging from signal transduction, gene transcription / translation to stress responses. Dysfunction of membraneless organelles due to excessive or insufficient phase separation may affect a variety of fundamental physiological processes in cells and further contribute to many diseases, including cancer, neurodegenerative diseases, and infectious diseases. Therefore, the important role of membraneless organelles in manipulating cell fate makes them target structures for therapeutic intervention. Although small molecule drugs have been developed to bind to the constituent proteins of membraneless organelles or modulate the signaling pathways of protein expression, methods to directly bind to abnormal membraneless organelles and modulate their biological activities remain understudied.

[0003] Conventional membraneless organelles in living cells mainly include P bodies, germ granules and stress granules, as well as nucleoli, nuclear speckles and Cajal bodies located in the nucleus. Among these membraneless organelles, stress granules are condensed by translation-stalled mRNA, RNA-binding proteins and various translation initiation factors after being stimulated by stress. The rapid assembly of stress granules protects proteins and mRNA from degradation, while the decomposition of stress relief helps release the protected components for reuse. The whole process helps cell survival and is particularly important for cancer cells. Because cancer cells are often under various stressors, including hypoxia, oxidative stress, endoplasmic reticulum stress, etc. Chemotherapeutic drugs such as sorafenib can also induce the formation of stress granules in cancer cells, leading to chemotherapy resistance in cancer, which means that stress granule targeted cancer treatment has potential. Due to the fusion-fission properties of droplets, the in situ generation of liquid-like droplets in cancer cells may allow therapeutic drugs to fuse with stress granules, which can provide a strategy to directly inhibit the function of stress granules and solve the problem of drug resistance during cancer chemotherapy. Summary of the invention

[0004] The purpose of the present invention is to provide a sulfatase-driven polypeptide aggregate with a chemosensitizing effect based on the problem that the current stress granules lack a targeted treatment method, which causes cancer cells to produce chemotherapeutic drugs and limits the effect of tumor treatment. At the same time, a method for preparing the enzyme-responsive aggregate is provided.

[0005] The technical solution of the present invention is:

[0006] The first aspect of the present invention aims to provide a sulfatase-driven polypeptide aggregate having a chemosensitizing effect, wherein the polypeptide enzyme response aggregate is mY SO4 FL SG , through peptide Y SO4 F and FGDF-Y SO4 F is prepared by blending, and the structural formula of each component is as follows:

[0007]

[0008] Among them, polypeptide Y SO4 F and FGDF-Y SO4 The volume ratio of F is 95:5.

[0009] The second aspect of the present invention is to provide the above-mentioned polypeptide enzyme response condensate mY SO4 FL SG The preparation method comprises the following steps:

[0010] Step 1: Peptide Y SO4 F and FGDF-Y SO4 Synthesis of F;

[0011] (1) Peptide Y SO4 F is synthesized by liquid phase reaction. First, 1 equivalent of aminoethyl sulfide and 3 equivalents of DIPEA are added to a DMF solution containing 2 equivalents of Boc-Phe-OH, 2 equivalents of HBTU and 2 equivalents of HOBT relative to aminoethyl sulfide, and the reaction mixture is poured into 200 ml of water to produce Boc-FsF-Boc precipitate, which is collected by filtration and washed with water. After vacuum drying, the crude product Boc-FsF-Boc is dissolved in a TFA solution and stirred for 3 hours to remove the amino protecting group. After rotary evaporation to remove DCM and TFA, icy ether is added and frozen for 30 minutes. The precipitate is collected by centrifugation and washed with icy ether to obtain a crude product FsF, which is directly used for the next step of synthesis without purification; then 2 equivalents of Fmoc-Tyr(SO4)-OH, 2 equivalents of HOBT, 2 equivalents of HATU, 1 equivalent of FsF and 3 equivalents of DIPEA are added to 5 ml of 4% ethanol. DMF solution, stirred at room temperature for 5 hours, the reaction mixture was poured into 200 ml of water, the precipitate was collected by filtration and washed with water, and the product Fmoc-Y SO4 F was dehydrated in vacuo and dissolved in piperidine solution. The amino protecting group was removed by stirring for 1 h. After the solvent was removed by rotary evaporation, icy ether was added and the mixture was frozen and precipitated for 30 min. The precipitate was collected by centrifugation and washed with cold ether to obtain the crude product Y. SO4 F, purified by HPLC and verified by UPLC-MS;

[0012] In this step, the DCM / TFA ratio in the TFA solution is 1:1; the piperidine / DCM ratio in the piperidine solution is 1:2;

[0013] (2) Synthetic peptide FGDF-Y SO4 F was prepared by reacting the peptide fragment FGDF (LTFGDFDEG) with the purified polypeptide Y SO4 F was synthesized by coupling in solution, while the peptide fragment was obtained by standard Fmoc solid phase synthesis. During the synthesis, 2-chlorotrimethylbenzyl chloride resin was swollen in anhydrous DCM for 30 minutes, and 4 times the equivalent of the first amino acid from the C-terminus to the N-terminus and 6 times the equivalent of DIEA dissolved in anhydrous DCM were added to it. After reacting for 3 hours, the first amino acid residue from the C-terminus to the N-terminus was connected, and methanol was added and shaken for 30 minutes. The remaining amino acid coupling reaction was carried out by adding 4 times the amino acid, 4 times the HBTU and 6 times the equivalent of DIEA dissolved in anhydrous DCM relative to the resin. DIEA was dissolved in DMF and shaken for 1.5 hours. A 25% piperidine DMF solution was added and shaken for 20 minutes to deprotect Fmoc. After all coupling reactions were completed, a mixture of 85% DCM, 10% TFA, 2.5% TIPS and 2.5% H2O was used for 3 hours. The peptide fragment was cut from the resin, and the excess solvent was removed by rotary evaporation and icy ether was added to obtain a precipitate. The last amino acid residue from the C-terminus to the N-terminus was protected with a Boc agent. During the solution coupling process, the crude peptide fragment and 1 equivalent of Y SO4 F. A mixture of 6 equivalents of DIEA and 4 equivalents of HBTU was dissolved in 10 ml of DMF. The reaction mixture was stirred at room temperature for 8 hours, then poured into 50 ml of water, and the precipitate was collected by filtration. After washing with excessive water, the precipitate was redissolved in a TFA solution and stirred for 3 hours to remove the protecting group. DCM and TFA were removed by rotary evaporation, and then glacial ether was added. The precipitate was frozen for 30 minutes, and the precipitate was collected by centrifugation and washed with glacial ether to obtain a crude product FGDF-YF or FGDF-Y SO4 F, purified by HPLC and verified by UPLC-MS;

[0014] In this step, the synthesized polypeptide FGDF-Y SO4 F is obtained by reconstructing the peptide fragment FGDF, the amino acid sequence of the peptide fragment FGDF is LTFGDFDEG, and the reconstructed polypeptide FGDF-Y SO4 The amino acid sequence of F is: LTFGDFDEG-Y SO4 FWf SO4

[0015] In this step, the TFA / DCM ratio in the TFA solution is 1:1;

[0016] Step 2: By adding peptide Y SO4F and FGDF-Y SO4 F blending preparation mY SO4 FL SG ;

[0017] First, the peptide FGDF-Y SO4 F and Y SO4 The lyophilized powder of F was dissolved into the same concentration of FGDF-Y SO4 F and Y SO4 F peptide master solution; mixed with peptide FGDF-Y at a volume ratio of 95:5 SO4 F and Y SO4 The mother liquor of F was used to prepare mY SO4 FL SG For the mother solution, after preparing the mixed solution, the pH value of the solution was adjusted to 7.4 with sodium hydroxide, and the total peptide concentration was quantified to be 1.4 mM.

[0018] The third aspect of the present invention aims to provide the use of the above-mentioned polypeptide enzyme response coacervate in sulfatase response.

[0019] The mY prepared by the present invention SO4 FL SG Based on the principle that sulfatase can catalyze the hydrolysis of sulfate ester bonds, the main peptide Y that responds to sulfatase was designed and synthesized. SO4 F, and protein G3BP2 ligand FGDF-Y SO4 F was mixed to prepare peptide enzyme responsive aggregates mY with sorafenib chemotherapy sensitization effect SO4 FL SG Under the stimulation of the chemotherapy drug sorafenib, tumor cells will produce stress granules with chemotherapy resistance, weakening the therapeutic effect of sorafenib, and the protein G3BP2 is one of the core proteins of stress granules. SO4 FL SG After entering tumor cells, the sulfate groups are hydrolyzed under the catalysis of sulfatase overexpressed in tumor cell lysosomes, thereby forming phase-separated droplets with high affinity for protein G3BP2 in situ in the cells. SG , by interacting with the protein G3BP2 and fusing with stress granules, it inhibits the chemotherapy resistance produced by stress granules and improves the therapeutic effect.

[0020] Sulfatase-driven polypeptide aggregate mY with chemotherapy sensitization effect obtained by the present invention SO4 FL SG Characterization, including optical microscopy and laser confocal microscopy, is described in detail in the specific implementation manner.

[0021] Advantages and beneficial effects of the present invention:

[0022] (1) The sulfatase-driven polypeptide aggregates with chemosensitizing effect formed by the present invention have the advantages of biocompatibility and low immunogenicity.

[0023] (2) All the reaction conditions of the present invention are very mild, the preparation method is simple, and the operation is easy.

[0024] (3) The sulfatase-driven polypeptide aggregates with chemosensitizing effect obtained by the present invention can inhibit the chemotherapeutic drug resistance produced by stress granules in cancer cells, thereby improving the therapeutic effect of sorafenib.

[0025] (4) The sulfatase-driven polypeptide aggregates with chemosensitizing effect prepared by the present invention have a selective chemosensitizing effect on tumor cells and will not produce additional toxic side effects on normal tissue cells, thereby establishing a reliable strategy for future efficient cancer targeted therapy.

[0026] The advantages and effects of the present invention will be described below through examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 .Sulfatase-driven polypeptide aggregate mY with chemotherapy sensitization effect of the present invention SO4 FL SG Chemical structures and characterization of each component molecule involved molecules YF, FGDF-YF, FITC-YF and FITC-Y SO4 The chemical structure of F.

[0028] Figure 2 OM images of phase separation peptide YF undergoing liquid-liquid phase separation at different concentrations, 1.3, 1.4, 1.5, 1.6 and 1.8 mM from left to right.

[0029] Figure 3 .(A and B) Sulfated polypeptide Y SO4 UPLC spectrum (A) and corresponding mass spectrum (B) of F before and after sulfatase (50 U / ml) treatment. (C) Sulfated peptide Y SO4 F (1.4 mM) OM images before and after sulfatase (50 U / ml) treatment for 24 h.

[0030] Figure 4 .(A and B) Sulfated polypeptide Y SO4 CLSM images of F (1.4 mM) after sulfatase (50 U / ml) treatment for 24 hours and its post-photobleaching recovery process (A) and droplet fusion process (B). (C) Sulfated peptide Y SO4 F and ligand peptide FGDF-Y SO4 F is mixed to make mY SO4 FL SGCLSM images after sulfatase (50 U / ml) treatment for 24 hours and the recovery process after photobleaching. 2% FITC-Y SO4 F was used as a fluorescent probe.

[0031] Figure 5 (A) Flow cytometry analysis of A549 cells responding to peptide Y at different time points SO4 Cellular uptake of F and YF. (B) Flow cytometry study of the uptake of Y SO4 Quantitative fluorescence intensity of A549 cells incubated with F or YF. (C) SO4 CLSM images of cells incubated in the presence of F for 2, 4, 8, and 12 hours, and lysosomes were stained with Lyso-Red. (D) SO4 PCC values ​​between Lyso-Red stain and FITC signals in A549 cells treated with F or YF. (E) Y SO4 F CLSM of A549 cells after 12 h of treatment and the post-photobleaching recovery process of droplets formed by cells in situ.

[0032] Figure 6 .(A) Sulfated polypeptide Y SO4 F and ligand peptide FGDF-Y SO4 F is mixed to make mY SO4 FL SG After entering A549 cells, the cells undergo in situ liquid-liquid phase separation, and the stress granules are targeted to enhance the killing effect of sorafenib. (B and C) Ligand FGDF-YF (B) or droplet d-YF-L SG (C) MST curve of the binding affinity to protein G3BP2. (D) Different concentrations of Y SO4 F, dY SO4 FL SG or m-YF-L SG Cell viability of A549 cells after 24 h of treatment. (E) A549 cells were treated with sorafenib (Sor:1) alone for 12 h, or with Y SO4 F(2), FGDF-Y SO4 F(3),dY SO4 FL SG (4), or m-YF-L SG (5) After 12 hours of treatment, the viability of A549 cells was further incubated with sorafenib for 12 hours. The IC50 values ​​of each treatment group are shown in the figure. (F and G) The viability of A549 cells treated with m-YSO4F-LSG (F) or Y SO4 F(G) CLSM images of A549 cells treated for 12 hours and then incubated with sorafenib for 2 hours. G3BP2 protein was stained by immunofluorescence.

[0033] Figure 7 .(A and B) use mY SO4 FL SG (A) or Y SO4 F(B) CLSM images of A549 cells treated with sorafenib for 12 hours and stimulated with sorafenib for 2 hours, TIA-1 protein was stained by immunofluorescence. (C) NT-647 was used to label the protein G3BP2 and then to stain with d-YF-L droplets. SG CLSM images of the cells after 2 h of incubation. (D) Using mY SO4 FL SG CLSM images of A549 cells after treatment, G3BP2 protein was stained by immunofluorescence. (E and F) PBS (1), mY SO4 FL SG (2) A549 cells were treated with Sor (3) for 24 h or 12 h, and then with d-YF-L SG (4), FGDF-Y SO4 F(5) or mY SO4 FL SG (6) After 12 hours of treatment, the cells were co-incubated with sorafenib for another 12 hours, and the levels of p38, P-p38, Caspase-3, and Cleaved-Caspase-3 in the cells were detected by Western Blot (E) and semi-quantitatively analyzed (F). Statistical analysis was performed using one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001.

[0034] Figure 8 . Schematic diagram of the design, schematic diagram of the in situ phase separation of peptides into droplets in living cells, which target membraneless organelle stress granules for cancer chemotherapy in combination with sorafenib. Sulfated peptide Y SO4 F contains two sulfated tyrosine residues and is responsive to sulfatase. Stress granule ligand peptide FGDF-Y SO4 F and sulfated polypeptide Y SO4 F blending preparation SO4 FL SG , which undergoes sulfatase-induced liquid-liquid phase separation to form droplets d-YF-L SG Under sorafenib stimulation, the generated droplets fuse with stress granules driven by ligand-G3BP2 interaction, thereby inhibiting the chemoresistance function of stress granules on cells and promoting cell apoptosis through the Caspase-3 pathway. DETAILED DESCRIPTION

[0035] Embodiment 1:

[0036] 1. Preparation of a sulfatase-driven polypeptide aggregate with chemosensitizing effect, the main steps are as follows:

[0037] 1) Peptide Y SO4 F and FGDF-Y SO4 Synthesis of F;

[0038] (1) Peptide Y SO4 F is synthesized by liquid phase reaction. First, 1 equivalent of aminoethyl sulfide and 3 equivalents of DIPEA are added to a DMF solution containing 2 equivalents of Boc-Phe-OH, 2 equivalents of HBTU and 2 equivalents of HOBT relative to aminoethyl sulfide. The reaction mixture is poured into 200 ml of water to produce a Boc-FsF-Boc precipitate, which is collected by filtration and washed with water. After vacuum drying, the crude product Boc-FsF-Boc is dissolved in a TFA solution (DCM / TFA: 1:1) and stirred for 3 hours to remove the amino protecting group. After rotary evaporation to remove DCM and TFA, ice ether is added and frozen for 30 minutes. The precipitate is collected by centrifugation and washed with ice ether to obtain a crude product FsF, which is directly used for the next step of synthesis without purification. Then 2 equivalents of Fmoc-Tyr(SO4)-OH, 2 equivalents of HOBT, 2 equivalents of HATU, 1 equivalent of FsF and 3 equivalents of DIPEA are added to 5 ml of DMF solution. After stirring at room temperature for 5 hours, the reaction mixture was poured into 200 ml of water, and the precipitate was collected by filtration and washed with water. SO4 F was vacuum dried and dissolved in piperidine solution (piperidine / DCM: 1:2), stirred for 1 h to remove the amino protecting group, and the solvent was removed by rotary evaporation. Then, icy ether was added and frozen to precipitate for 30 min. The precipitate was collected by centrifugation and washed with cold ether to obtain the crude product Y. SO4 F, purified by HPLC and verified by UPLC-MS.

[0039] (2) Synthetic peptide FGDF-Y SO4 F was prepared by reacting the peptide fragment FGDF (LTFGDFDEG) with the purified polypeptide Y SO4F was synthesized by coupling in solution, while the peptide fragment was obtained by standard Fmoc solid phase synthesis. During the synthesis, 2-chlorotrimethylbenzyl chloride resin was swollen in anhydrous DCM for 30 minutes, and 4 times the equivalent of the first amino acid and 6 times the equivalent of DIEA were added to it in anhydrous DCM relative to the resin. After the first amino acid residue was connected after 3 hours of reaction, methanol was added and continued to shake for 30 minutes. The remaining amino acid coupling reaction was carried out by dissolving 4 times the amino acid, 4 times the HBTU and 6 times the DIEA relative to the resin in DMF and shaking for 1.5 hours. Fmoc was deprotected by adding 25% piperidine DMF solution and shaking for 20 minutes. After all coupling reactions were completed, the peptide fragment was cut off from the resin using a mixture of 85% DCM, 10% TFA, 2.5% TIPS and 2.5% H2O for 3 hours. Excess solvent was removed by rotary evaporation and precipitate was obtained by adding ice ether. The last amino acid residue was protected using Boc agent. In the solution coupling process, the crude peptide fragment, 1 equivalent of Y SO4 F, a mixture of 6 equivalents of DIEA and 4 equivalents of HBTU was dissolved in 10 ml of DMF. The reaction mixture was stirred at room temperature for 8 hours and then poured into 50 ml of water. The precipitate was collected by filtration, washed with excessive water, and then redissolved in a TFA solution (TFA / DCM: 1 / 1) and stirred for 3 hours to remove the protecting group. DCM and TFA were removed by rotary evaporation, and then ice ether was added. The precipitate was frozen for 30 minutes, and the precipitate was collected by centrifugation and washed with ice ether to obtain a crude product FGDF-YF or FGDF-Y SO4 F, purified by HPLC and verified by UPLC-MS. The obtained peptide structure is shown below

[0040]

[0041] 2) By SO4 F and FGDF-Y SO4 F blending preparation mY SO4 FL SG ;

[0042] First, the peptide FGDF-Y SO4 F and Y SO4 The lyophilized powder of F was dissolved into the same concentration of FGDF-Y SO4 F and Y SO4 F peptide master solution; mixed with peptide FGDF-Y at a volume ratio of 95:5 SO4 F and Y SO4 The mother liquor of F was used to prepare mY SO4 FL SG After the mixed solution was prepared, the pH of the solution was adjusted to 7.4 with sodium hydroxide, and the total peptide concentration was quantified to be 1.4 mM.

[0043] The structural formulas of YF, FITC-YF and FGDF-YF involved in the present invention are as shown in the attached drawings. Figure 1 shown.

[0044] 2. Sulfatase-driven peptide aggregates mY with chemosensitizing effect SO4 FL SG Characterization, including optical microscopy, laser confocal microscopy, etc. The specific methods are as follows

[0045] 1) Optical Microscopy (OM): OM study samples were prepared directly using the above stock solution. Images were taken using an OLYMPUS BX53 microscope with a 10x or 40x objective magnification. Specifically, 10 μL of solution was dropped onto a glass slide and first observed with a 10x objective to roughly determine the focal range, then switched to a 40x objective and images were taken using WT-1000GM software.

[0046] 2) Confocal laser scanning microscopy (CLSM) images of sulfatase-induced mY SO4 FL SG Fluorescence recovery after photobleaching (FRAP) of the hydrolyzed droplets: mY SO4 FL SG The solution was added to a NaOAc buffer (0.5M, pH=5) solution containing a sulfatase solution (Helix pomatia, EC3.1.6.1, 50U / mL, 34mM NaCl) and incubated at 37°C for 24 hours. The total concentration of the polypeptide was 1.4mM. During imaging, 10μL of the solution was added to a glass slide and then placed under a microscope detector. Images were captured at an excitation wavelength of 488nm. A circular ROI was selected in the middle of the droplet, and four pulse bleaching (100μs per pulse) was performed at 100% intensity under a 488nm excitation wavelength laser, and the fluorescence recovery of the droplet was imaged at intervals of 15 seconds. Images were recorded using a 100x oil immersion objective under a NikonA1+ confocal laser scanning microscope.

[0047] 3) Cell culture method: Human A549 and HUVEC were cultured in DMEM medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin and 100 mg / mL streptomycin at 37°C in an environment of 5% CO2. All cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences.

[0048] 4) Characterization of in vitro cytotoxicity: The cytotoxicity of treatment to A549 cells was evaluated using a standard MTT assay. Cells were seeded in 96-well plates at a density of 5000 cells per well in 100 μL DMEM culture medium. After a 24-hour pre-incubation period, different concentrations of peptide drugs were added to the cells for 24 hours. For the treatment group containing both sorafenib and peptides, the cells were first treated with a total concentration of 1.4 mM peptide for 12 hours, and then different concentrations of sorafenib were added for another 12 hours of co-incubation. After the incubation was completed, fresh MTT solution (10 μL, 5 mg / mL) was added and the cells were cultured for another 4 hours. After that, the supernatant was removed and 100 μL DMSO was added to each well to dissolve the purple formazan crystals. Cell viability was determined by measuring the absorbance at a wavelength of 495 nm using a microplate reader (TECAN Infinite M Nano).

[0049] 5) CLSM microscopy to determine endosome / lysosome co-localization

[0050] In DMEM medium, the density was 1×10 5 A549 cells were seeded in confocal glass bottom plates. After 24 h of pre-incubation, peptides containing the corresponding FITC-labeled peptides (total concentration of 1.4 mM) were added to the culture dishes and incubated for 2, 4, 8, and 12 h, respectively. The cells were washed three times with PBS and further incubated with 50 nM Lyso-Red lysosomal stain for 30 min at 37 °C. After washing three times with PBS, the cells were imaged using a 100x oil immersion objective under a Nikon A1+ confocal laser scanning microscope.

[0051] 6) Determination of cellular peptide uptake using flow cytometry

[0052] Quantitative analysis of YF, Y SO4 Cellular uptake of F. A549 cells were seeded in 6-well plates at a density of 1×10 5 After overnight incubation, A549 cells were used with YF, Y SO4 F, d-YF-L SG mY SO4 FL SG , peptides containing the corresponding FITC-labeled peptides were added to the cells at a total concentration of 1.4 mM and incubated for 2, 4, 8, and 12 hours. The cells were then washed twice with PBS, collected, and analyzed by flow cytometry (BD LSR Fortessa).

[0053] 7) CLSM microscopy to measure the recovery of cell droplets after photobleaching: In DMEM medium, a cell droplet with a density of 1×10 5A549 cells were seeded in confocal glass bottom plates. After pre-incubation for 24 h, peptide Y containing the corresponding FITC-labeled peptide (total concentration of 1.4 mM) was added. SO4 F was added to the cells and incubated for 12 h. Photobleaching recovery was performed following the same protocol as for solution experiments, and images were recorded using a Nikon A1+ confocal laser scanning microscope with a 100x oil immersion objective.

[0054] 8) Microscale thermophoresis (MST) experiment: MST experiment was performed on Monolith NT.115 instrument. TM The protein G3BP2 was labeled using the RED Protein Labeling Kit. The labeled protein was diluted with PBS to an appropriate concentration to achieve a fluorescence signal that matches the typical detection requirements of IntegralNT. 16 peptide samples were prepared by gradient dilution of the stock solution (FGDF-YF concentration ranged from 30 nM to 200 μM). Specifically, for sample d-YF-L SG , keeping the concentration of peptide YF fixed at 1.4mM to maintain the condensed droplet structure, and changing the concentration of ligand FGDF-YF to maintain the same range as the MST control experiment using only the ligand. The diluted protein solution was then added to the 16 peptide samples at gradually changing concentrations and incubated at 25°C for another 30 minutes. The incubated mixtures were respectively loaded into Monolith TM standard capillaries and measured by the Monolith NT.115 system. The laser power and LED power were set to 20%, the final value of Fnorm for each sample was set to 100%, and the data for each group were normalized. The dissociation constant (Kd) value between the protein and the peptide ligand was calculated using MOAffinityAnalysis software.

[0055] 9) Detection of stress granule targeting after immunofluorescence staining using CLSM microscopy

[0056] In DMEM medium, the density was 1×10 5 A549 cells were seeded in confocal glass bottom plates. SO4 F's SO4 FL SG(total concentration of 1.4 mM) and incubated together for 12 hours. Subsequently, sorafenib (13 μM) was added to the cells and incubated for 2 hours. After washing the cells three times with PBS, the cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature. The cells were further washed three times with PBS and incubated with 0.2% Triton X-100 for 5 minutes at room temperature. After washing three times with PBS, the cells were incubated with blocking solution at room temperature for 1 hour and treated with G3BP2 or TIA-1 antibodies at 4°C overnight. After washing three times with PBS, the cells were incubated with secondary antibodies labeled with fluorescent dyes (561 nm) for 2 hours. After washing three times with PBS, the cells were imaged using a Nikon A1+ confocal laser scanning microscope.

[0057] 10) Analysis of intracellular protein expression levels after peptide treatment of A549 cells

[0058] 1×10 5 A549 cells were seeded in 6-well cell culture dishes at a density of 1.54 μg / mL and incubated for 24 hours before incubation with mY SO4 FL SG (total concentration is 1.4 mM), d-YF-L SG (total concentration is 1.4 mM), FGDF-Y SO4 F (70 μM) was used to treat the cells. After incubation with the peptide for 12 h, sorafenib (13 μM) was added to the cells and incubated for another 12 h. Afterwards, the cells were washed twice with PBS and treated with 2% SDS to estimate the protein. Before electrophoresis measurement, the resulting protein concentration was analyzed using a Bradford protein quantification detection kit (Sangon Biotechnology) and incubated with loading buffer at 95 °C for 10 min. Subsequently, the proteins in A549 cells treated with different samples were loaded on SDS-PAGE gels (Tris-Gly, 4–20%) in the same amount (n=3, 20 μg) and transferred to polyvinylidene fluoride (PVDF) membranes by electrophoresis. The membrane was washed with 5% skim milk in TBST (50×10-3M Tris-HCl, pH=7.5; 150×10 -3 The samples were blocked with 50% NaCl and 0.1% Tween 20 at 25°C for 1 hour, and then incubated overnight at 4°C in the presence of the corresponding specific primary antibody. The protein bands were incubated with horseradish peroxidase-labeled secondary antibodies at 25°C for 1 hour and then imaged on a Tanon-5200 chemiluminescent imaging system (Tanon Science and Technology).

[0059] 11) Y SO4 F and mY SO4 FL SG Sulfatase-responsive hydrolysis monitoring

[0060] When studying sulfatase-induced hydrolysis, peptide samples were diluted to a concentration of 100 μM and incubated for various times at 37° C. in the presence of sulfatase (Helixpomatia, EC 3.1.6.1, 50 U / mL, 34 mM NaCl) in NaOAc buffer (0.5 M, pH=5). UPLC-MS analysis was performed using a Waters UPLC-MS system, eluting with an aqueous acetonitrile medium containing 0.1% trifluoroacetic acid.

[0061] Embodiment 2:

[0062] Characterization of Peptide YF Liquid-Liquid Phase Separation Ability:

[0063] YF stock solutions were prepared with concentrations of 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM and 1.8 mM, respectively.

[0064] Test results: Figure 2 As shown, the optical microscopy results indicate that YF solutions with a concentration above 1.3 mM can undergo liquid-liquid phase separation, and the generation of droplet structures can be observed under an optical microscope. Finally, a lower concentration of 1.4 mM at which phase separation can occur was selected.

[0065] Embodiment 3:

[0066] Sulfatase-responsive peptide liquid-liquid phase separation precursor Y SO4 Characterization of the sulfatase responsiveness of F:

[0067] Y SO4 Study on the enzyme-dependent hydrolysis process of the sulfate group of F molecule: Y SO4 The F stock solution was diluted to a concentration of 100 μM and incubated in a 37°C constant temperature water bath for different time periods in the presence of sulfatase (Helixpomatia, EC 3.1.6.1, 50 U / mL, 34 mM NaCl) in NaOAc buffer (0.5 M, pH = 5), and the reaction was monitored by UPLC-MS.

[0068] Test results: Figure 3 (A) Figure 3 The UPLC trace in (B) shows that after the addition of sulfatase, peptide Y SO4 F was gradually converted to YF. Mass spectrometry studies showed that an intermediate with a single sulfate group was formed during the hydrolysis, indicating that the sulfate group was cleaved stepwise by sulfatase.

[0069] Y SO4 Study on the enzyme-responsive liquid-liquid phase separation process of F solution: Using 1.4mMY SO4The F solution was incubated in a 37°C constant temperature water bath for 24 h in the presence of sulfatase (Helix pomatia, EC 3.1.6.1, 50 U / mL, 34 mM NaCl). Sulfatase induces sulfate hydrolysis under acidic conditions, while liquid-liquid phase separation of YF occurs in neutral solutions. SO4 Enzyme-induced liquid-liquid phase separation of F was performed by first incubating the peptide with sulfatase in an acidic solution and then adjusting the solution to pH 7.4.

[0070] Measurement results: Optical microscope images show that Y SO4 F solution homogeneous phase Figure 3 (C), but after 24 h of treatment with sulfatase, clear droplets formed. These results demonstrate that polypeptide Y SO4 F has the ability to undergo liquid-liquid phase separation under the induction of sulfatase.

[0071] Embodiment 4:

[0072] Sulfatase-driven peptide aggregates with chemosensitizing effects SO4 FL SG Characterization of Sulfatase-Responsive Liquid-Liquid Phase Separation:

[0073] Use 0.2% FITC-Y SO4 1.4mMY of F SO4 F and 1.4 mM mY SO4 FL SG The solution was incubated in a 37°C constant temperature water bath for 24 hours in the presence of sulfatase (Helix pomatia, EC 3.1.6.1, 50 U / mL, 34 mM NaCl). After the incubation, the solution was adjusted to pH 7.4 and characterized under a confocal microscope.

[0074] Measurement results: Fluorescence recovery and fusion after photobleaching are typical characteristics of condensed droplets. Figure 4 As shown in (A), Y SO4 After incubation in the presence of sulfatase and adjusting the solution pH to 7.4, CLSM images showed the formation of green spherical droplets. Irradiation of the central region of the droplets with high-intensity laser light resulted in complete bleaching of the fluorescence signal, which recovered within a few minutes. This phenomenon indicates the rapid exchange of peptides between the droplets and the surrounding medium, thus forming condensed droplets. In addition, CLSM images also show the fusion between different droplets, such as Figure 4 (B) Two small droplets were monitored to coalesce into a large droplet at 120 seconds. The above results directly prove that peptide Y SO4 FLLPS in response to enzymes after sulfatase-induced hydrolysis of sulfate groups on tyrosine residues. mY SO4 FLSG The same phenomenon was observed after incubation in the presence of sulfatase and adjusting the solution pH to 7.4.

[0075] Embodiment 5:

[0076] Sulfatase-responsive peptide liquid-liquid phase separation precursor Y SO4 F Study on in situ liquid-liquid phase separation in A549 cells:

[0077] 1. Cell uptake assay: A549 cells were seeded in 6-well plates at a density of 1×10 5 A549 cells were incubated overnight with a total concentration of 1.4 mM YF, SO4 F and FITC-labeled peptides containing the corresponding 2% molar ratio were added to the cells and incubated for 2, 4, 8 and 12 hours. The cells were then washed twice with PBS, collected and analyzed using a flow cytometer (BD LSRFortessa).

[0078] Test results: Figure 5 (A) As shown, FITC-Y SO4 F and FITC-YF as Y SO4 The probes of F and YF were used to measure the expression of peptide Y in A549 cells. SO4 Cellular uptake of F and YF. Flow cytometric analysis showed that A549 cells internalized these two peptides. Within the observed time range, the quantitative fluorescence signal intensity of the intracellular peptides showed that free YF was higher than YF. SO4 The cellular uptake of F is better Figure 5 (B).

[0079] 2. Lysosomal escape research:

[0080] In DMEM medium, the density was 1×10 5 A549 cells were seeded in confocal glass bottom plates. After 24 h of pre-incubation, Y SO4 F polypeptide was added to the culture dish and incubated for 2, 4, 8 and 12 hours. The cells were washed three times with PBS and further incubated with 50 nM Lyso-Red lysosomal stain for 30 minutes at 37°C. After washing three times with PBS, the cells were imaged with a 100x oil immersion objective lens under a NikonA1+ confocal laser scanning microscope.

[0081] Measurement results: Using Y SO4 The Pearson correlation coefficient (PCC) value between peptide FITC and Lyso-Red signals in F-treated A549 cells was estimated to be approximately 0.73 at the beginning and decreased to 0.23 after 12 h of incubation, as shown in Figure 2 . Figure 5 (C) and Figure 5 (D) These data indicate the colocalization of the peptide with lysosomes and its lysosomal escape, thus clarifying the role of peptide Y SO4 The lysosomal-mediated uptake pathway of F.

[0082] 3. Study on fluorescence recovery after photobleaching of droplets generated in situ in cells:

[0083] In DMEM medium, the density was 1×10 5 A549 cells were seeded in confocal glass bottom plates. After pre-incubation for 24 h, peptide Y containing the corresponding FITC-labeled peptide (total concentration of 1.4 mM) was added. SO4 F was added to the cells and incubated for 12 hours. Images were captured at 488 nm excitation wavelength. By selecting a circular ROI in the middle of the droplet, four pulses of bleaching (100 μs per pulse) were performed at 100% intensity under 488 nm excitation wavelength laser, and the fluorescence recovery of the droplet was imaged at 15 second intervals. Images were recorded using a 100x oil immersion objective under a Nikon A1+ confocal laser scanning microscope.

[0084] Test results: Figure 5 As shown in (E), the CLSM image of the treated A549 cells indeed showed spherical droplets, which was further confirmed by using a post-photobleaching recovery study: the green droplets in the A549 cells were exposed to high-intensity laser irradiation, causing the fluorescence signal to fade. After 120 seconds, the fluorescence signal in the droplets was observed to recover. This indicates that Y SO4 F In situ phase separation into droplets in A549 cells.

[0085] Embodiment 6:

[0086] FGDF-Y SO4 F polypeptide and droplet d-YF-L SG Binding affinity study on G3BP2 protein:

[0087] Using Monolith NT TM The protein G3BP2 was labeled using the RED Protein Labeling Kit. The labeled protein was diluted with PBS to an appropriate concentration to achieve a fluorescence signal that matches the typical detection requirements of IntegralNT. 16 peptide samples were prepared by gradient dilution of the stock solution (FGDF-YF concentration ranged from 30 nM to 200 μM). Specifically, for sample d-YF-L SG, the concentration of peptide YF was kept fixed at 1.4mM to maintain the condensed droplet structure, and the concentration of ligand FGDF-YF was changed to maintain the same range as the MST control experiment using only the ligand. The diluted protein solution was then added to the 16 peptide samples at gradually changing concentrations and incubated for another 30 minutes at 25°C. The incubated mixtures were loaded into Monolith TM standard capillaries and measured by the Monolith NT.115 system. The laser power and LED power were set to 20%, the final value of Fnorm for each sample was set to 100%, and the data for each group were normalized. The dissociation constant (Kd) value between the protein and the peptide ligand was calculated using MO AffinityAnalysis software.

[0088] Test results: Figure 6 (B) The results shown indicate a binding constant of 21.6 μM for FGDF-YF and demonstrate its potential targeting activity to stress granules. Considering the molecular recruitment capacity of condensed droplets, we sought to evaluate the activity of droplet d-YF-L SG The enhancement of ligand binding affinity. Incorporation of ligands into droplets d-YF-L SG The ligand-protein binding was enhanced by 3.9 times, and the binding constant reached 5.6 μM. Figure 6 (C) This demonstrates that droplets can recruit ligands and proteins and further promote their binding.

[0089] Embodiment 7:

[0090] Sulfatase-driven peptide aggregates with chemosensitizing effects SO4 FL SG Chemosensitization effect on human non-small cell lung cancer A549:

[0091] Cytotoxicity assay: The cytotoxicity of treatment to A549 cells was evaluated using a standard MTT assay. Cells were seeded in 96-well plates at a density of 5000 cells per well in 100 μL DMEM medium. After a 24-hour preincubation period, different concentrations of peptide drugs were added to the cells for 24 hours. For the treatment group containing both sorafenib and peptides, the cells were first treated with peptides at a total concentration of 1.4 mM for 12 hours, and then different concentrations of sorafenib were added for another 12 hours of co-incubation. After the incubation was completed, fresh MTT solution (10 μL, 5 mg / mL) was added and the cells were cultured for another 4 hours. After that, the supernatant was removed and 100 μL DMSO was added to each well to dissolve the purple formazan crystals. Cell viability was determined by measuring the absorbance at a wavelength of 495 nm using a microplate reader (TECAN Infinite M Nano).

[0092] Test results: Figure 6 (D) as shown, with Y SO4 F, mY SO4 FL SG or d-YF-L SG Treatment of A549 cells did not significantly reduce cell viability, indicating that these peptides had low cytotoxicity to cancer cells. At the same time, sorafenib showed moderate cytotoxicity to A549, with a median lethal dose (IC50) value of approximately 13 μM. Figure 6 (E). However, using mY SO4 FL SG or d-YF-L SG Pretreatment of A549 cells followed by incubation in the presence of sorafenib resulted in extensive cell death. In particular, mY SO4 FL SG The combined treatment with sorafenib reduced the IC50 value of sorafenib to 3 μM. These data suggest that in situ droplet formation has a cytotoxic sensitization effect.

[0093] Embodiment 8:

[0094] Sulfatase-driven peptide aggregates with chemosensitizing effects SO4 FL SG Research on the mechanism of chemotherapy sensitization effect:

[0095] 1. Targeted study of stress granules under CLSM microscope: In DMEM culture medium, a density of 1×10 5 A549 cells were seeded in confocal glass bottom plates. The cells were incubated with 1.4 mM FITC-Y SO4 F's SO4 FL SG or Y SO4 F was incubated together for 12 hours. Subsequently, sorafenib (13 μM) was added to the cells and incubated for 2 hours. After washing the cells three times with PBS, the cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature. The cells were further washed three times with PBS and incubated with 0.2% Triton X-100 for 5 minutes at room temperature. After washing three times with PBS, the cells were incubated with blocking solution at room temperature for 1 hour and treated with G3BP2 or TIA-1 antibodies at 4°C overnight. After washing three times with PBS, the cells were incubated with secondary antibodies labeled with fluorescent dyes (561 nm) for 2 hours. After washing three times with PBS, the cells were imaged using a NikonA1+ confocal laser scanning microscope.

[0096] Measurement results: In mY SO4 -L SG A549 cells were incubated in the presence of and then stimulated with sorafenib, resulting in obvious formation of condensed droplets in A549 cells, such as Figure 6(F). Fluorescence line scanning analysis also showed that the staining signals of peptide FITC and G3BP2-IF overlapped significantly. SO4 F After incubation, condensed droplets were also formed in A549 cells, showing no overlapping FITC and IF staining signals, e.g. Figure 6 (G) At the same time, the droplets also have a targeting effect on TIA-1, another classic protein that makes up stress granules. Figure 7 (A) and (B).

[0097] 2. d-YF-L SG Study on G3BP2 protein recruitment in solution:

[0098] Using Monolith NT TM The protein G3BP2 was labeled with the RED protein labeling kit. The protein was then mixed with d-YF-L containing 2% FITC-YF at a total concentration of 1.4 mM. SG After 2 h of co-incubation, the cells were dropped onto a glass slide and imaged using a 60x oil immersion objective lens under a Nikon A1+ confocal laser scanning microscope.

[0099] Measurement results: CLSM images of mixed proteins and droplets showed a high degree of overlap between the green FITC and red NT-647 fluorescence signals, as shown in Figure 2. Figure 7 (C), showing that droplets recruit G3BP2.

[0100] 3. Western blot (WB) detection: 1×10 5 A549 cells were seeded in 6-well cell culture dishes at a density of 1.54 μg / mL and incubated for 24 hours before incubation with mY SO4 FL SG (total concentration is 1.4 mM), d-YF-L SG (total concentration is 1.4 mM), FGDF-Y SO4 F (70 μM) was used to treat the cells. After incubation with the peptide for 12 h, sorafenib (13 μM) was added to the cells and incubated for another 12 h. Afterwards, the cells were washed twice with PBS and treated with 2% SDS to estimate the protein concentration. Before electrophoresis measurement, the resulting protein concentration was analyzed using a Bradford protein quantification assay kit (Sangon Biotechnology) and incubated with loading buffer at 95 °C for 10 min. Subsequently, the proteins in A549 cells treated with different samples were loaded on SDS-PAGE gels (Tris-Gly, 4–20%) in the same amount (n=3, 20 μg) and transferred to polyvinylidene fluoride (PVDF) membranes by electrophoresis. The membrane was washed with 5% skim milk in TBST (50×10-3MTris-HCl, pH=7.5; 150×10 -3The samples were blocked with 50% NaCl and 0.1% Tween 20 at 25°C for 1 hour, and then incubated overnight at 4°C in the presence of the corresponding specific primary antibody. The protein bands were incubated with horseradish peroxidase-labeled secondary antibodies at 25°C for 1 hour and then imaged on a Tanon-5200 chemiluminescent imaging system (Tanon Science and Technology).

[0101] Test results: Figure 7 (E) and (F) show that WB experiments showed that mY SO4 FL SG Treatment of A549 cells significantly increased the levels of P-p38 and Cleaved-Caspase-3, while decreasing the level of native p38 ( Figure 5 F). These results confirm that in situ droplet formation enhances the caspase-dependent apoptotic pathway of sorafenib. Mechanistic studies have shown that mY SO4 FL SG The targeting properties of in situ formed droplets and their bioactivity can enhance the cytotoxicity of the drug Sorafenib by inhibiting stress granule function, thereby improving cell survival.

[0102] Based on the above embodiments, the present invention provides a sulfatase-driven polypeptide aggregate with chemosensitization effect and a preparation method thereof, and the prepared enzyme-responsive aggregate mY SO4 FL SG Based on the principle that sulfatase can catalyze the hydrolysis of sulfate ester bonds, the main peptide Y that responds to sulfatase was designed and synthesized. SO4 F, and protein G3BP2 ligand FGDF-Y SO4 F was mixed to prepare enzyme-responsive condensates mY SO4 FL SG .like Figure 8 As shown, sulfated polypeptide Y SO4 F contains two sulfated tyrosines and is responsive to sulfatase. Stress granule ligand peptide FGDF-Y SO4 F and sulfated polypeptide Y SO4 F blending mY SO4 FL SG , which undergoes sulfatase-induced liquid-liquid phase separation to form droplets d-YF-L SG Under sorafenib stimulation, the generated droplets fuse with stress granules driven by ligand-G3BP2 interaction, thereby inhibiting the chemoresistance function of stress granules on cells and promoting cell apoptosis through the Caspase-3 pathway.

[0103] It should be further explained that the above implementation modes are only used to understand the technical solution of the present invention, and are not used to limit the protection scope of the present invention. Any obvious adjustments and modifications made to the technical solution of the present invention that belong to the technical concept of the present invention should also fall within the protection scope of the present invention.

Claims

1. A sulfatase-driven polypeptide aggregate with chemosensitizing effect, wherein the polypeptide aggregate is mY SO4 FL SG , through peptide Y SO4 F and FGDF-Y SO4 F is prepared by blending, and the structural formula of each component is as follows: Among them, polypeptide Y SO4 F and FGDF-Y SO4 The volume ratio of F is 95:

5.

2. The method for preparing the sulfatase-driven polypeptide aggregate with chemosensitizing effect according to claim 1, characterized in that: Here are the steps: Using peptide FGDF-Y SO4 F and Y SO4 The lyophilized powder of F was dissolved into the same concentration of FGDF-Y SO4 F and Y SO4 F peptide master solution; mixed with peptide FGDF-Y at a volume ratio of 95:5 SO4 F and Y SO4 The enzyme-responsive aggregate mY was prepared from the mother solution of F SO4 FL SG After preparing the mixed solution, the pH value of the solution was adjusted to 7.4 with sodium hydroxide, and the total peptide concentration was quantified to be 1.4 mM; wherein polypeptide Y SO4 F and FGDF-Y SO4 F has the structure shown in claim 1.

3. The method for preparing the sulfatase-driven polypeptide aggregate with chemosensitizing effect according to claim 2, characterized in that: Peptide Y SO4 F is synthesized by a liquid phase reaction. First, 1 equivalent of aminoethyl sulfide and 3 equivalents of DIPEA are added to a DMF solution containing 2 equivalents of Boc-Phe-OH, 2 equivalents of HBTU and 2 equivalents of HOBT relative to aminoethyl sulfide. The reaction mixture is poured into 200 ml of water to produce a Boc-FsF-Boc precipitate. The precipitate is collected by filtration and washed with water. After vacuum drying, the crude product Boc-FsF-Boc is dissolved in a TFA solution and stirred for 3 hours to remove the amino protecting group. After rotary evaporation to remove DCM and TFA, glacial ether is added and frozen for 30 minutes. The precipitate is collected by centrifugation and washed with glacial ether to obtain a crude product FsF, which is directly used in the next step of synthesis without purification. Then 2 equivalents of Fmoc-Tyr(SO4)-OH, 2 equivalents of HOBT, 2 equivalents of HATU, 1 equivalent of FsF and 3 equivalents of DIPEA are added to 5 ml of DMF solution, stirred at room temperature for 5 hours, the reaction mixture was poured into 200 ml of water, the precipitate was collected by filtration and washed with water, and the product Fmoc-Y SO4 F was dehydrated in vacuo and dissolved in piperidine solution. The amino protecting group was removed by stirring for 1 h. After the solvent was removed by rotary evaporation, icy ether was added and the mixture was frozen and precipitated for 30 min. The precipitate was collected by centrifugation and washed with cold ether to obtain the crude product Y. SO4 F, purified by HPLC and verified by UPLC-MS.

4. The method for preparing the sulfatase-driven polypeptide aggregate with chemosensitizing effect according to claim 3, characterized in that: The DCM / TFA ratio in the TFA solution was 1:1; the piperidine / DCM ratio in the piperidine solution was 1:

2.

5. The method for preparing the sulfatase-driven polypeptide aggregate with chemosensitizing effect according to claim 2, characterized in that: Peptide FGDF-Y SO4 F was prepared by reacting the peptide fragment FGDF (LTFGDFDEG) with the purified polypeptide Y SO4 F was synthesized by coupling in solution, wherein the peptide fragment was obtained by standard Fmoc solid phase synthesis. During the synthesis, 2-chlorotrimethylbenzene chloride resin was swollen in anhydrous DCM for 30 minutes, and 4 times the equivalent of the first amino acid and 6 times the equivalent of DIEA were added to it in anhydrous DCM. After the reaction for 3 hours, the first amino acid residue was connected, and methanol was added and shaken for 30 minutes. The remaining amino acid coupling reaction was carried out by dissolving 4 times the amino acid, 4 times the HBTU and 6 times the DIEA in DMF and shaking for 1.5 hours. A 25% piperidine DMF solution was added and shaken for 20 minutes to deprotect Fmoc. After all coupling reactions were completed, a mixture of 85% DCM, 10% TFA, 2.5% TIPS and 2.5% H2O was used for reaction for 3 hours, and the peptide fragment was cut from the resin. The excess solvent was removed by rotary evaporation and precipitate was obtained by adding ice ether. The last amino acid residue was protected with Boc agent. During the solution coupling process, the crude peptide fragment and 1 times the equivalent of Y SO4 F. A mixture of 6 equivalents of DIEA and 4 equivalents of HBTU was dissolved in 10 ml of DMF. The reaction mixture was stirred at room temperature for 8 hours, then poured into 50 ml of water, and the precipitate was collected by filtration. After washing with excessive water, the precipitate was redissolved in a TFA solution and stirred for 3 hours to remove the protecting group. DCM and TFA were removed by rotary evaporation, and then glacial ether was added. The precipitate was frozen for 30 minutes, and the precipitate was collected by centrifugation and washed with glacial ether to obtain a crude product FGDF-YF or FGDF-Y SO4 F, purified by HPLC and verified by UPLC-MS.

6. The method for preparing the sulfatase-driven polypeptide aggregate with chemosensitizing effect according to claim 5, characterized in that: The TFA / DCM ratio in the TFA solution is 1:

1.

7. Use of the sulfatase-driven polypeptide aggregate with chemosensitizing effect according to claim 1 in sulfatase response.

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