Phase separation polypeptide as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to phase separation polypeptide and a preparation method and application thereof. The structure of the phase separation polypeptide is shown as a formula (I). According to the polypeptide compound provided by the invention, liquid-liquid phase separation can be carried out to form condensed liquid drops, and DNA damage response proteins are enriched and separated in cells. The polypeptide compound disclosed by the invention is simple in synthesis method and good in biocompatibility, and liquid-liquid phase separation can be carried out; dNA damage response protein can be captured in cells, DNA repair is interfered, and the tumor radiotherapy effect is enhanced. Therefore, the polypeptide compound provided by the invention can be used for tumor radiotherapy, chemotherapy and other treatment methods.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to phase-separated polypeptides, their preparation methods, and applications. Background Technology

[0002] Liquid-liquid phase separation is a fundamental mechanism in the dynamic formation of intracellular droplet-like biomolecule condensates. Proteins containing intrinsically disordered regions (IDRs) assemble in spatially confined environments, inducing local molecular concentration increases exceeding a critical threshold, thereby triggering thermodynamically driven liquid-liquid phase separation and further promoting the formation of biomolecule condensates—membrane-free organelles (MLOs). Over the past few decades, research has revealed that MLOs regulate various key cellular processes in spatiotemporal dimensions, such as transcriptional regulation, RNA metabolism, and cellular adaptation to environmental stresses. With a deeper understanding of the molecular mechanisms driving biomolecule condensate formation in recent years, it has become possible to develop artificial condensates with controllable physicochemical properties. These condensates, with their ability to efficiently enrich and separate biomolecules, can play a role in regulating biochemical reactions, gene expression, and cellular pathways.

[0003] The intrinsically disordered regions in phase-separating proteins play a crucial role in driving phase separation and the formation of biomolecular condensates. These intrinsically disordered regions are characterized by highly repetitive sequences, indeterminate conformations, high hydrophilicity, and significant charge density. Studies have shown that phase-separating short peptides (typically less than 15 amino acids) can be rationally designed and synthesized based on the inherent amino acid sequences within these intrinsically disordered regions. Besides their structural simplicity and ease of synthesis, these short peptides can exhibit controllable functional properties by regulating their amino acid composition and sequence, thus becoming an important tool for elucidating the relationship between peptide sequences and phase-separated condensates. Simultaneously, peptide condensates also exhibit characteristics similar to other condensates in enriching and separating biomolecules, demonstrating the potential to regulate cellular function.

[0004] Liquid-liquid phase separation precisely controls various key cellular processes in the spatiotemporal dimensions by regulating the formation of dynamic membrane-free condensates composed of biomacromolecules. DNA damage response is a core mechanism in eukaryotic cells for recognizing, transmitting signals, and repairing DNA damage, and is crucial for maintaining genome stability. Recent studies have found that the activation of the DNA damage response is finely regulated by liquid-liquid phase separation of related proteins within specific temporal and spatial ranges. Therefore, enriching and separating key proteins of the DNA damage response using artificially constructed peptide condensates, thus distancing them from DNA damage sites and interfering with the DNA damage response mechanism, is a potential strategy to improve the radiosensitivity of tumors.

[0005] Currently, peptide condensates primarily rely on typical sequence motifs found in naturally occurring intrinsically disordered proteins (IDPs). However, the limited variety of peptides restricts their potential in terms of sequence diversity and functional complexity. Therefore, developing more diverse peptide-based condensates not only contributes to a deeper understanding of phase separation behavior and its molecular mechanisms but also opens up possibilities for more complex functional applications.

[0006] In tumor cells, the ability to respond to DNA damage is often enhanced as an adaptive response to genomic instability, which is directly related to the sensitivity of tumors to radiotherapy and chemotherapy. Although small molecule inhibitors targeting the DNA damage response have been developed, their efficacy is often limited by single-target action and the development of adaptive resistance. Summary of the Invention

[0007] The purpose of this invention is to provide a phase-separated polypeptide.

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned polypeptide compound.

[0009] One object of the present invention is to provide the application of the above-mentioned polypeptide compound.

[0010] According to a specific embodiment of the present invention, the phase-separated polypeptide has the following structure:

[0011]

[0012] Where n is selected from integers.

[0013] Preferably, n is selected from an integer between 0 and 20; more preferably, n is selected from 0, 1, 2, 3, 4 or 5.

[0014] According to a specific embodiment of the present invention, a method for preparing phase-separated peptides is employed using an amino acid Fmoc solid-phase synthesis method. During the reaction, HBTU is used as an activator for the carboxyl groups of the amino acids, DIEA is used as a catalyst, and piperidine is used to remove the Fmoc protecting groups, thereby exposing the amino groups to undergo cross-linking and condensation reactions with the carboxyl groups of the next amino acid activated by HBTU, forming peptide bonds. Once the peptide chain is fully formed, it is cleaved from the dichloropolymer resin using 1% trifluoroacetic acid (TFA).

[0015] This invention also provides the use of phase-separated peptides in any of the following:

[0016] (1) Application in the preparation of DNA damage sensitizers;

[0017] (2) Application in the preparation of radiosensitizing drugs for tumor radiotherapy.

[0018] DNA damage can occur due to factors such as UV radiation, IR radiation, X-rays, reactive oxygen species, depurination, depyrimidine, single-strand breaks, double-strand breaks, cytosine deamination, O6-methylguanine, base alkylation, DNA cross-linking, replication errors, or free radicals. The phase-separated polypeptide of this invention can assemble into aggregated droplets and enrich intracellularly to separate DNA damage response proteins, thereby interfering with DNA repair and sensitizing tumor radiotherapy.

[0019] Cancer treatments using chemotherapy or radiation therapy can target and disrupt the function of tumor cell DNA by inducing adducts or DNA double-strand or single-strand breaks. Cancer cells can overcome these therapies by developing resistance mechanisms, which can be induced or inherent to the cancer cells.

[0020] Preferably, the phase-separated polypeptide sensitizes γ-ray-induced DNA damage in tumor cells. After γ-ray irradiation, the aggregates formed by the phase-separated polypeptide can significantly reduce the survival rate of tumor cells, thereby enhancing the killing effect of radiation on tumor cells.

[0021] Cancer is a group of related diseases characterized by uncontrolled cell proliferation and the potential to metastasize throughout the body. These cancers include lung cancer, stomach cancer, breast cancer, liver cancer, pancreatic cancer, colorectal cancer, skin cancer, esophageal cancer, nasopharyngeal carcinoma, ovarian cancer, bile duct cancer, bladder cancer, malignant melanoma, or kidney cancer.

[0022] Although different cancers can occur in almost any body tissue, the basic processes that lead to cancer can be similar in all forms of disease. Cancer begins when cells break free from the normal restrictions on cell division and begin to grow and divide abnormally. Gene mutations in cells can prevent cells from controlling their division or initiating apoptosis, leading to uncontrolled cell growth and division.

[0023] According to a specific embodiment of the present invention, the tumor radiosensitizing drug comprises the aforementioned phase-separated polypeptide.

[0024] The present invention also provides a polypeptide aggregate solution, wherein the polypeptide aggregate solution is obtained by dissolving the above-mentioned phase-separated polypeptide in a buffer solution.

[0025] Preferably, the concentration of the phase-separated polypeptide is 10-50 mM.

[0026] Preferably, the pH of the polypeptide aggregate solution is 6.5-7.5, and more preferably, the pH of the polypeptide aggregate solution is 7.0.

[0027] Preferably, the buffer solution is selected from phosphate buffer, Tris buffer, or MES buffer.

[0028] The preparation method of polypeptide aggregate solution includes the following steps:

[0029] (1) At room temperature, the phase-separated peptides were dissolved in Tris buffer to prepare a solution;

[0030] (2) Adjust the pH of the solution obtained in step (1) by gradually increasing the pH value until the solution becomes turbid, thus obtaining the aggregate solution (WY). PS Preferably, the pH value is approximately 7 at this time.

[0031] In step (1), the Tris buffer contains 0.2M NaCl and pH=3; the concentration of the phase-separated polypeptide in the resulting solution is 30mM.

[0032] The beneficial effects of this invention are:

[0033] The polypeptide compound provided by this invention can be assembled to form aggregate droplets and enriched within cells to separate DNA damage response proteins.

[0034] The polypeptide compounds of this invention are simple to synthesize, biocompatible, and biodegradable; they can capture DNA damage response proteins within cells, interfere with DNA repair, and sensitize tumor radiotherapy. Therefore, the polypeptide compounds of this invention can be used in treatment methods such as tumor radiotherapy and chemotherapy. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 The analysis results for Pep-1 are as follows,

[0037] (A) Mass spectrum of Pep-1 (molecular weight 1381.70); (B) Optical microscope image of RGDVYGGRKDVY in Tris buffer (the image in the upper right corner is a picture of the solution appearance).

[0038] Figure 2 These are mass spectra of Pep-2, Pep-3, Pep-4, Pep-5, and Pep-6, among which...

[0039] A is the mass spectrum of Pep-2 (RGDVYGGWRKDVY);

[0040] B is the mass spectrum of Pep-3 (RGDVYWGGWRKDVY);

[0041] C is the mass spectrum of Pep-4 (RGDVYWGWRKDVY);

[0042] D is the mass spectrum of Pep-5 (WGDVYGGRKDVY);

[0043] E is the mass spectrum of Pep-6 (Ben-GDVYGGRKDVY).

[0044] Figure 3 This shows optical micrographs of Pep-2, Pep-3, Pep-4, Pep-5, and Pep-6 in Tris buffer (the image in the upper right corner shows the appearance of the solution).

[0045] Figure 4 These are the mass spectra of Pep-7, Pep-8, and Pep-9; among them,

[0046] A is the mass spectrum of Pep-7 (WGDVYGRGRKDVY);

[0047] B is the mass spectrum of Pep-8 (WGRGDVYGGRKDVY);

[0048] C is the mass spectrum of Pep-9 (WGRGRGDVYGGRKDVY).

[0049] Figure 5 This shows optical micrographs of Pep-7, Pep-8, and Pep-9 in Tris buffer (the image in the upper right corner shows the solution appearance).

[0050] Figure 6 (A) LLC cells against WY PS Laser confocal scanning microscopy images of condensates; (B) LLC cells against WY PS Representative flow cytometry plots and quantitative analysis results of condensate uptake. Data are expressed as mean ± standard deviation (n=3), and p-values ​​were determined using Tukey's HSD post-hoc test in a one-way ANOVA.

[0051] Figure 7 Display WY PS The effect of condensates on capturing the DNA damage response protein PARP1;

[0052] (A) Representative CLSM image of mCherry-PARP1 (purple) in LLC cells (scale bar = 20 μm);

[0053] (B)mCherry-PARP1 (purple) and WY PS @FITC (green) cellular localization in LLC cells (scale bar = 10 μm);

[0054] (C) Using ImageJ software to analyze WY PS Fluorescence colocalization analysis was performed on the aggregates (green), mCherry-PARP1 (purple), and cell nuclei (blue).

[0055] Figure 8 Display WY PS The effect of condensates interfering with DNA repair;

[0056] (A) LLC cells were treated with Tris buffer (0 Gy) and WY PS (0Gy), Tris buffer (2Gy), Pep-8 (2Gy) and WY PS Representative images of γ-H2AX immunofluorescence staining after (2Gy) treatment;

[0057] (B) Representative images of single-cell gel electrophoresis of LLC cells after treatment with Tris buffer (0 Gy), WYPS (0 Gy), Tris buffer (2 Gy), Pep-8 (2 Gy) and WYPS (2 Gy);

[0058] (C) Quantitative analysis results of single-cell gel electrophoresis experiment;

[0059] (D) LLC cells were treated with Tris buffer, Pep-8, and WY. PS Clonal images on day 8 after treatment with different doses of gamma rays, 4 hours later;

[0060] (E)LLC cells were treated with Tris buffer, Pep-8, and WY. PS Quantitative analysis results of clones on day 8 after receiving different doses of gamma radiation 4 hours after treatment. Data are expressed as mean ± standard deviation (n=5), and p-values ​​were determined using Tukey's HSD post-hoc test in a one-way ANOVA. Detailed Implementation

[0061] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0062] This invention establishes a liquid-liquid phase-separated peptide library based on low-complexity, repetitive amino acid sequences. The main driving forces for phase separation include non-covalent interactions such as hydrogen bonding, electrostatic interactions, π-π interactions, cation-π interactions, and van der Waals forces. First, arginine (Arg, R) and tyrosine (Tyr, Y) are selected to provide cation-π and π-π interactions. Second, lysine (Lys, K) and aspartic acid (Asp, D), two amino acids with opposite charges, are introduced to provide electrostatic interactions. Valine (Val, V) is also introduced to provide hydrophobic interactions. Using the "sticker-spacer" principle, two similar repetitive sequences, "RGDVY" and "RKDVY," are constructed as "stickers," and two glycine (GG) sequences are inserted between them as "spacers," ultimately yielding the peptide sequence "RGDVYGGRKDVY" (named Pep-1). To further enhance the phase separation driving force of the peptide, the sequence of Pep-1 was continuously optimized, ultimately yielding the peptide compound of this invention, with the following structural formula:

[0063] Where n is selected from 0, 1, 2, 3, 4, or 5.

[0064] The mouse lung cancer cells LLC used in the embodiments of this invention were purchased from Cyagen (Guangzhou) Biotechnology Co., Ltd.

[0065] All reagents used in the embodiments of this invention were purchased.

[0066] Example 1: Preparation of Pep-1

[0067] Pep-1 was prepared using the classic Fmoc solid-phase synthesis method for amino acids.

[0068] Peptide Pep-1 was prepared using solid-phase peptide synthesis (SPPS). The specific steps are as follows:

[0069] (1) 1 mmol of Fmoc-protected tyrosine was mixed with 1 g of 2-chlorotriphenylmethyl resin (2 mmol) in dichloromethane (DCM) and diisopropylethylamine (DIEA) and reacted for 2 h.

[0070] (2) The resin was sealed using methanol (2 mL), DIEA (1 mL), and DCM (17 mL);

[0071] (3) Remove the Fmoc protecting group using a solution (15 mL) of N,N-dimethylformamide (DMF) containing 20% ​​piperidine;

[0072] (4) Subsequently, Fmoc-protected valine, Fmoc-protected aspartic acid, Fmoc-protected lysine, Fmoc-protected arginine, Fmoc-protected glycine, Fmoc-protected glycine, Fmoc-protected tyrosine, Fmoc-protected valine, Fmoc-protected aspartic acid, Fmoc-protected glycine, Fmoc-protected arginine, as well as O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU) and DIEA were added in sequence to allow them to undergo a coupling reaction with the amino groups of the above amino acids for 2 hours.

[0073] (5) Treat again with a DMF solution containing 20% ​​piperidine to remove the Fmoc protecting group, 30 min.

[0074] (6) After the reaction is complete, wash the resin 5 times with DCM and pyrolyze the resin for 30 min with a mixed solution of 95% trifluoroacetic acid (TFA): triisopropylsilane (TIS): water (19:0.5:0.5, v / v);

[0075] (7) The crude product was concentrated under vacuum and then purified by high performance liquid chromatography (HPLC) to obtain the target polypeptide.

[0076]

[0077] The amino acid sequence of Pep-1 is RGDVYGGRKDVY, and its mass spectrometry characterization is shown below. Figure 1 A.

[0078] At room temperature, Pep-1 was dissolved in Tris buffer, and the pH was gradually adjusted to neutral. The mixture was then observed using an inverted optical microscope.

[0079] The results show ( Figure 1 B) Pep-1 was still in solution, and no obvious droplet formation was observed under an optical microscope, indicating that the phase separation driving force of the polypeptide was significantly insufficient.

[0080] Therefore, the amino acid composition of Pep-1 was analyzed. Glycine accounted for 25.00%, anionic amino acids accounted for 16.67%, cationic amino acids accounted for 25.00%, aliphatic amino acids accounted for 16.67%, and aromatic amino acids accounted for 16.67%. It was found that the proportion of cationic amino acids (Arg, Lys) was significantly higher than that of aromatic amino acids (Y), resulting in insufficient cationic-π and π-π interactions in the driving force.

[0081] Example 2: Preparation of peptides Pep-2, Pep-3, Pep-4, Pep-5, and Pep-6

[0082] To enhance the cation-π and π-π interactions of the peptide, tryptophan with two aromatic rings and abundant π electrons was selected. One or two tryptophans (Trp, W) were introduced into the sequence of Pep-1 and placed in the middle position of the peptide to synthesize Pep-2 and Pep-3.

[0083] Pep-4 was synthesized by reducing the number of glycine residues in the "spacer" of the Pep-3 peptide to one.

[0084] In addition to adding tryptophan, this embodiment also uses tryptophan or phenylpropionic acid to replace the N-terminal Arg to synthesize Pep-5 and Pep-6.

[0085] The synthesis methods of peptides Pep-2, Pep-3, Pep-4, Pep-5, and Pep-6 are the same as in Example 1, and the sequences of the obtained peptides are shown in the table below.

[0086] compound amino acid sequence Pep-2 <![CDATA[RGDVYGG W RKDVY]]> Pep-3 <![CDATA[RGDVY W GG W RKDVY]]> Pep-4 <![CDATA[RGDVY W G W RKDVY]]> Pep-5 <![CDATA[ W GDVYGG RKDVY]]> Pep-6 <![CDATA[ Ben -GDVYWGWRKDVY]]>

[0087] Note: Compared with Pep-1, the underlined letters in the sequences of peptides Pep-2, Pep-3, and Pep-4 indicate newly introduced amino acids, while the underlined letters in the sequences of Pep-5 and Pep-6 indicate newly substituted amino acids.

[0088]

[0089] Mass spectra of Pep-2 (molecular weight 1567.78), Pep-3 (molecular weight 1753.86), Pep-4 (molecular weight 1696.84), Pep-5 (molecular weight 1412.67), and Pep-6 (molecular weight 1385.65) are shown below. Figure 2 .

[0090] Pep-2, Pep-3, Pep-4, Pep-5, and Pep-6 were dissolved in Tris buffer, and the pH was gradually adjusted to neutral. The results were observed using an inverted optical microscope. Figure 3 As shown, Pep-2, Pep-3, and Pep-4 formed numerous precipitates in solution, with no obvious droplet formation. Due to the N-terminus being blocked, Pep-6 formed a blocky gel in solution. Pep-5 did not form aggregates at room temperature, but in a Tris buffer solution at 4°C, due to weakened molecular thermal motion, Pep-5 formed biomimetic aggregates.

[0091] Example 3: Preparation of peptides Pep-7, Pep-8, and Pep-9

[0092] Arginine and glycine were introduced at the N-terminus or in the middle of peptide Pep-5, and peptides Pep-7, Pep-8 and Pep-9 were synthesized according to the method in Example 1.

[0093] compound amino acid sequence Pep-7 <![CDATA[WGDVYG R GRKDVY]]> Pep-8 <![CDATA[W GR GDVYGGRKDVY]]> Pep-9 <![CDATA[W GRGR GDVYGGRKDVY]]>

[0094] Note: Compared to Pep-5, the underlined letter indicates a newly introduced amino acid.

[0095]

[0096] Mass spectra of Pep-7 (molecular weight 1567.78), Pep-8 (molecular weight 1624.80), and Pep-9 (molecular weight 1793.92) are shown below. Figure 4 .

[0097] Pep-7, Pep-8, and Pep-9 were dissolved separately in Tris buffer, and the pH was gradually adjusted to neutral. The results were observed using an inverted optical microscope. Figure 5 As shown, Pep-7 formed numerous precipitates in solution with no obvious droplet formation. Pep-8 and Pep-9 could form biomimetic aggregates in Tris buffer solution at room temperature.

[0098] Example 4 Cellular uptake of aggregates

[0099] WY PS The preparation method of FITC solution includes the following steps:

[0100] (1) Dissolve the polypeptide in 100 μL of 20 mM Tris buffer (containing 0.2 M NaCl, pH = 3) at room temperature (25 °C) to prepare a solution with a concentration of 30 mM.

[0101] (2) Add 1M NaOH solution dropwise to the polypeptide solution. As the pH value gradually increases, the solution becomes turbid, thus obtaining the aggregate solution (WY). PS At this point, the pH value is approximately 7.

[0102] (3) Prepare a 10 mM FITC DMSO stock solution, then dilute the FITC DMSO stock solution 50 times with PBS to prepare the FITC working solution. Finally, add 2 μL of FITC working solution to 100 μL of aggregate solution to obtain the FITC-encapsulated aggregate solution (WY). PS @FITC).

[0103] 30mM WY PS @FITC solution (prepared from Pep-8 peptide) was co-incubated with LLC cells for 45 min. Results of laser confocal scanning microscopy are shown below. Figure 6As shown in A, WY PS @FITC condensates can rapidly enter cells and distribute in the cytoplasm and nucleus within a short period of time.

[0104] Flow cytometry quantification results as follows Figure 6 As shown in Figure B, the positive cell rate after incubation with Tris buffer was 0.28%, the positive cell rate after incubation with FITC was 11.4%, and the positive cell rate after incubation with WY was... PS The positive cell rate after FITC incubation was 62.8%, indicating that approximately 60% of the cells contained WY. PS @FITC condensate indicates WY PS The condensate undergoes efficient cellular uptake.

[0105] Example 5: Enrichment of the DNA damage response protein PARP1 in aggregate cells

[0106] An LLC cell line overexpressing the Parp1 gene was constructed using a lentiviral vector, and mCherry was fused to observe the intracellular localization of the PARP1 protein.

[0107] 1. The day before transfection, 4×10 6 -6×10 6 One LLC cell was seeded into a 10cm cell culture dish and cultured to a density of 70%-80% at the time of packaging.

[0108] 2. Transfer the cultured target cells to a 24-well plate.

[0109] 3. Prepare a puromycin stock solution (1 mg / mL) and then prepare concentrations of 0.5, 1, 2, 3, 4, and 5 μg / mL, and add them to 24-well cells. The concentration at which all cells die after 48 hours is the puromycin selection concentration for target cells.

[0110] 4. Plate the cells as needed, aiming for a cell density of approximately 50% by the second day, and incubate overnight at 37°C.

[0111] 5. Before infection, remove the lentivirus from the -80°C freezer and thaw it on ice. Dilute it to the required concentration with PBS or serum-free culture medium and mix thoroughly by pipetting.

[0112] 6. Change the medium for the cells before infection, then add the diluted virus solution evenly to each well, shake gently, and incubate overnight at 37°C.

[0113] 7. 24 hours after infection, cells were isolated using trypsin and re-seeded at a low density. 3 hours after inoculation, puromycin was added to the culture medium to screen for positive cells. The culture medium was replaced with fresh medium the next day, and then the cells were passaged every other day to maintain the puromycin concentration.

[0114] 8. Select colonies formed from single-cell expansion for further cell proliferation, and continue maintenance selection culture with puromycin. Continue expansion culture. Select stable cell lines with good identification results and freeze them for preservation.

[0115] 9. Stain the nuclei of LLC cells with Hoechst 33342. Then stain with 30 mM WY PS @FITC solution (prepared from Pep-8 peptide) was co-incubated with LLC cells for 60 min, and the capture of PARP1 protein by droplets was observed using a laser confocal microscope.

[0116] The results are as follows Figure 7 As shown in Figure A, the purple fluorescence of the mCherry-PARP1 fusion protein was evenly distributed in lentivirally transfected cells, confirming the successful establishment of the overexpression cell line. Next, WY... PS The aggregates were co-incubated with LLC cells overexpressing mCherry-PARP1. CLSM image ( Figure 7 B) showed abundant droplet-like purple fluorescence, with surface PARP1 protein enriched by aggregates. Fluorescence colocalization analysis ( Figure 7 C) This further confirms the strong co-localization of mCherry-PARP1 with the condensate. These findings collectively indicate that WY PS The aggregates can capture the DNA damage response protein PARP1 within living cells.

[0117] Example 6: Aggregate Interference with DNA Repair Enhances Tumor Radiotherapy

[0118] Will WY PS In combination with gamma rays, we will investigate whether condensates can enhance the effectiveness of radiotherapy in killing tumor cells by interfering with DNA repair.

[0119] The steps for the γ-H2AX immunofluorescence staining experiment are as follows:

[0120] (1) Mice's lung cancer LLC cells (2×10⁻⁶) 5 (Number of cells / well) were seeded in confocal culture dishes and incubated at 37°C for 24 h. Subsequently, the cells were treated with 500 μL Tris buffer, FITC solution, and WYG solution, respectively. PS @FITC solution, incubate for 4 hours;

[0121] (2) Discard the original culture medium and wash the cells twice with PBS buffer. Then, fix the cells with 4% paraformaldehyde at room temperature for 30 min, wash them three times with PBS, and then permeate them with ice-cold methanol for 15 min.

[0122] (3) Block non-specific binding sites with 1% BSA for 1 h at room temperature, and then incubate the cells with γ-H2AX primary antibody overnight at 4°C.

[0123] (4) The next day, wash the cells three times with PBS and incubate them with fluorescently labeled secondary antibody at room temperature for 1 hour.

[0124] (5) Wash the cells three times with PBS and stain the nuclei with DAPI for 5 min;

[0125] (6) Wash with PBS three times, observe and photograph the cells using a CLMS microscope.

[0126] The results are as follows Figure 8 As shown in Figure A, in the absence of gamma rays, Tris buffer and WY PS Cells in the group showed almost no γ-H2AX signal. When cells were exposed to 2 Gy of γ-rays, weak γ-H2AX signaling was observed in the Tris buffer and Pep-8 groups. When subjected to WY... PS Cells pretreated with condensates were exposed to 2 Gy of gamma rays, resulting in a significant increase in intracellular γ-H2AX signals. These results indicate that condensates can significantly enhance the degree of DNA damage following irradiation.

[0127] The single-cell gel electrophoresis experiment includes the following steps:

[0128] (1) The LLC cell line was divided into 3×10 4 Cells / wells were seeded in 6-well culture dishes and incubated at 37°C for 24 h. Afterward, they were treated with 500 μL Tris-HCl buffer, FITC-labeled, and WYG-labeled solutions, respectively. PS @FITC complex was subjected to a 4-hour gradient treatment;

[0129] (2) The culture was terminated immediately after irradiation with 2 Gy rays. The cell suspension was collected by trypsin digestion and the viable cell concentration was adjusted to 1 × 10⁻⁶. 6 / mL;

[0130] (3) Prepare high melting point agarose gel, and after gradient heating to complete melting state, take a predetermined amount and pour it into electrophoresis glass slide, and let it stand at room temperature until the colloid solidifies; simultaneously activate low melting point agarose reagent, and maintain a 37℃ constant temperature water bath system to ensure the stability of the colloidal liquid state;

[0131] (4) Mix the low melting point gel and cell suspension at a volume ratio of 7:3 (total volume 100 μL), take a small amount of the mixture and accurately drop it onto the surface of the solidified high melting point gel, transfer it to a culture dish containing lysis buffer, and complete the membrane permeation treatment for 2.5 h at 4°C in the dark.

[0132] (5) After rinsing the electrophoresis slide with pure water, place it horizontally in the electrophoresis tank, fill it with electrophoresis solution, unwind it for 20 minutes at a low temperature of 4℃, and then apply an electric field of 30V for 30 minutes of electrophoresis.

[0133] (6) After terminating electrophoresis, rinse with pure water and transfer to a culture dish containing neutralization solution, and maintain at 4°C for 20 min;

[0134] (7) After rinsing with PBS buffer, stain with EB dye for 5 min, and then perform microscopic imaging after terminating the reaction with ultrapure water.

[0135] (8) The level of DNA double-strand breaks was assessed by quantifying parameters such as comet tail length and tail moment using the CometScore professional image analysis system.

[0136] The results are as follows Figure 8 As shown in B and C, after WY PS Cells pretreated with condensates showed a significantly increased proportion of comet tail fragments after irradiation, indicating that condensates significantly increased the number of radiation-induced DNA fragments and exacerbated DNA damage.

[0137] The clone formation experiment was used to investigate WY PS The effect of condensate combined with radiotherapy on cell survival.

[0138] Cloning experiments include the following steps:

[0139] (1) LLC cells were seeded in a single-cell suspension of 500 cells / well in a 12-well plate and cultured for 24 hours;

[0140] (2) Remove the culture medium from the well plate and add 500 μL each of Tris buffer, FITC solution and WY buffer. PS @FITC solution, incubate for 3 hours, then aspirate the solutions from each group and add fresh culture medium;

[0141] (3) After treatment, the cells were subjected to 2, 4 and 6 Gy of γ-ray radiation, respectively.

[0142] (4) After irradiation treatment, continue culturing for 6 to 10 days. During this period, observe the formation of clonal clusters daily until multiple clonal clusters that are visible to the naked eye appear in the control group.

[0143] (5) Discard the cell culture medium, add 200 μL of crystal violet staining solution, and stain for 30 min at room temperature.

[0144] (6) Then discard the dye solution and gently rinse the cloning plate 2 to 3 times;

[0145] (7) After drying, use a gel imaging device to take pictures and count the cells. Calculate the radiosensitization ratio (SER) based on the single-target model. Determine the survival rate of proliferating cells by forming spherical clones.

[0146] The results are as follows Figure 8 Figures D and E show that the survival fractions of LLC cells under 2 Gy irradiation and 4 Gy irradiation were approximately 0.60 and 0.56, respectively. In contrast, 2 and 4 Gy irradiation combined with WY... PS Aggregates significantly reduced the survival fraction to approximately 0.42 and 0.18, respectively. These results indicate that WY PS It can significantly improve the killing effect of radiation on tumor cells by interfering with DNA repair in the cell nucleus.

[0147] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A phase-separated polypeptide, characterized in that, The structure of the phase-separated polypeptide is shown in (Ⅰ): Where n is selected from integers.

2. The phase-separated polypeptide according to claim 1, characterized in that, The value of n is selected from 0, 1, 2, 3, 4 or 5.

3. The method for preparing the phase-separated polypeptide according to any one of claims 1-2, characterized in that, The preparation method employs the amino acid Fmoc solid-phase synthesis method.

4. The use of the phase-separated polypeptide according to claim 1 or 2 in any of the following: (1) Application in the preparation of DNA damage sensitizers; (2) Application in the preparation of tumor radiotherapy and chemotherapy sensitizing drugs.

5. The application according to claim 4, characterized in that, The phase-separated polypeptide sensitizes γ-ray-induced DNA damage in tumor cells.

6. The application according to claim 4, characterized in that, The tumors mentioned are lung cancer, stomach cancer, breast cancer, liver cancer, pancreatic cancer, colorectal cancer, skin cancer, esophageal cancer, nasopharyngeal cancer, ovarian cancer, bile duct cancer, bladder cancer, malignant melanoma, or kidney cancer.

7. A tumor radiosensitizing drug, characterized in that, The drug comprises the phase-separated polypeptide according to any one of claims 1-2.

8. The tumor radiosensitizing drug according to claim 7, characterized in that, The tumors mentioned are lung cancer, stomach cancer, breast cancer, liver cancer, pancreatic cancer, colorectal cancer, skin cancer, esophageal cancer, nasopharyngeal cancer, ovarian cancer, bile duct cancer, bladder cancer, malignant melanoma, or kidney cancer.

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