Polypeptide, tumor microenvironment response polypeptide drug delivery system containing polypeptide and application

Abstract

The invention discloses a polypeptide, a tumor microenvironment response polypeptide drug delivery system containing the polypeptide and application of the tumor microenvironment response polypeptide drug delivery system. The polypeptide has a structure as shown in a formula HC-Y-X-Y '; wherein HC is a hydrophobic pi conjugated group; x is an LARKS fragment, and the amino acid sequence of X is ATGGRG; y and Y'are flexible connecting peptide fragments, and amino acid sequences of the flexible connecting peptide fragments are RG and DY. HC-Y-X-Y 'can be converted into more stable liquid crystals by loading small molecule drugs, and after HC-Y-X-Y' is delivered to tumor cells, drugs are released in a responsive manner through a tumor microenvironment, so that the tumor cells are killed.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. Specifically, this invention relates to a polypeptide, a polypeptide-containing tumor microenvironment-responsive polypeptide drug delivery system, and its applications. Background Technology

[0002] Low-complexity domains (LCDs) of natural disordered proteins (IDPs) can drive various phase transitions. Among them, LARKS (low-complexity, amyloid-like reversible kinks) are short peptide fragments with simple sequences, rich in aromatic amino acids, and possessing dynamic kinetic structures, capable of driving reversible fibrosis. Proteins containing LARKS (such as keratin, hnRNPA1, and FUS) can undergo reversible transitions between monomers, droplets, and gels with temperature changes, showing potential as phase transition driving units. However, the design of phase transition biomaterials encoded by LARKS remains largely unexplored.

[0003] Stimulus-responsive peptide assemblies hold significant promise for targeted drug delivery within the tumor microenvironment (TME). The tumor microenvironment typically exhibits weak acidity and high glutathione (GSH) expression, characteristics that enable drug delivery systems to sense tumor-specific signals and release drugs through programmable phase transitions. In recent years, peptide assemblies have demonstrated potential in the intracellular transport of proteins and nucleic acids due to their ability to evade lysosomal phagocytosis, good cell permeability, and long retention time, showing promise in the treatment of malignant tumors. However, their liquid nature and inherent metastable state often lead to insufficient drug encapsulation and hinder the controlled release of small molecule drugs, limiting their further clinical application.

[0004] Therefore, developing a phase-change regulated drug delivery system for cancer treatment is very promising. Summary of the Invention

[0005] This invention provides a polypeptide, a polypeptide-containing tumor microenvironment-responsive polypeptide drug delivery system, and its applications. The aggregated drug delivery system of this invention can effectively encapsulate small molecule drugs, particularly Erastin, an inducer of ferroptosis.

[0006] The technical solution provided by this invention is as follows: In a first aspect, the present invention provides a polypeptide having the structure shown in the formula HC-YX-Y'; wherein HC is a hydrophobic π-conjugated group; X is a LARKS fragment with the amino acid sequence ATGGRG (SEQ ID NO.2); and Y and Y' are flexible linker peptides with the amino acid sequences RG and DY, respectively.

[0007] The hydrophobic π-conjugated group is a group with aggregation-induced emission properties.

[0008] Preferably, the group having aggregation-induced emission properties is a tetraphenylethylene (TPE) group.

[0009] The HC-YX-Y' is a compound formed by attaching a tetraphenylethylene group to the N-terminus of the amino acid sequence shown in SEQ ID NO.1.

[0010] The polypeptide can self-assemble into liquid condensates under physiological conditions, and the phase of these condensates can change in response to external stimuli. The polypeptide can undergo reversible liquid-liquid phase separation to form dynamic biomolecular condensates with multiple environmental signal responses.

[0011] The external stimuli include at least one of temperature, pH value and reducing environment; preferably, the polypeptide at a concentration of 1-2 mg / mL forms a microgel at 25-34°C and a liquid aggregate at 35-45°C.

[0012] The polypeptide is responsive to the tumor microenvironment and can undergo aggregate disintegration under conditions of pH 5.5-6.5 and the presence of 1-10 mM glutathione.

[0013] In a second aspect, the present invention provides a tumor microenvironment-responsive peptide drug delivery system comprising the peptides described above.

[0014] The system is responsive to the tumor microenvironment and can cause aggregate disintegration under conditions of pH 5.5-6.5 and containing 1-10 mM glutathione.

[0015] The system also includes a hydrophobic drug encapsulated in an aggregate formed by the polypeptide.

[0016] Preferably, the hydrophobic drug is a drug that can induce ferroptosis.

[0017] More preferably, the drug is Erastin.

[0018] The molar ratio of the polypeptide to the drug is (2:1) to (8:1); preferably (8:1).

[0019] The loading of the drug can induce the transformation of the liquid aggregates of the peptide into a liquid crystal state, manifested as the appearance of birefringent signals and a slower fluorescence recovery rate.

[0020] Thirdly, the present invention provides the application of the above-mentioned polypeptide drug delivery system, namely, for the preparation of drugs that induce ferroptosis in tumor cells.

[0021] The tumor cells are A375 melanoma cells.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention utilizes targeted screening of amino acid sequences and alters key amino acids in the LARKS fragment. For example, replacing ATGGRG with ATGHRG resulted in the peptide remaining in a monophase state at 37°C; replacing ATGGRG with ATGGCG resulted in its insolubility in PBS at 37°C; while TPE-RGATGGRGDY could stably dissolve and form liquid aggregates under the same conditions. This indicates that the types of amino acids in the LARKS fragment directly affect the solubility and phase transition behavior of the peptide, confirming the core regulatory role of the LARKS fragment in peptide function. This design overcomes the technical bottlenecks of poor targeting and insufficient stability in traditional peptide delivery systems, realizing a tumor microenvironment-responsive liquid crystal delivery system based on LARKS-encoded peptides, providing a novel strategy for precision tumor treatment.

[0023] 2. This invention promotes the transformation of aggregates into more stable liquid crystals during drug loading. After delivery to tumor cells, the drugs are released in response to the tumor microenvironment, inducing ferroptosis in tumor cells. At the same time, this delivery system has excellent biocompatibility, which can reduce toxic side effects on normal cells. It has strong targeting, can accurately accumulate in tumor sites, and improve drug utilization efficiency. It has a high drug encapsulation rate, which can effectively load hydrophobic small molecule drugs. Moreover, the liquid crystal structure has good stability, which can avoid premature leakage of drugs during in vivo transportation, significantly improving the safety and effectiveness of drug delivery. Attached Figure Description

[0024] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 The phase changes of the LARKS-encoded polypeptide of the present invention at 27°C and 37°C; Figure 1 Image a shows fluorescence bleaching recovery images at 27℃ and 37℃. Figure 1 b is Figure 1 Quantitative fluorescence recovery curve of fluorescence image in a; Figure 1 c represents the quantitative growth curves of ThT fibers at 27℃ and 37℃; Figure 1 In the image, d represents scanning electron microscope images taken at 27℃ and 37℃. Figure 1 In the image, e represents the polarization signal and fusion status over time at 27℃ and 37℃. Figure 2 Tumor microenvironment responsiveness of the LARKS-encoded peptide of the present invention; Figure 2 Image a shows fluorescence and bright-field images under normal physiological conditions (pH 7.4) and tumor microenvironment conditions (pH 5.5 + GSH); Figure 2Figure b shows the phase diagrams corresponding to different GSH concentrations and different peptide concentrations at pH 7.4. Figure 2 In the diagram, c represents the phase diagrams corresponding to different GSH concentrations and different peptide concentrations at pH 5.5. Figure 3 The present invention relates to LARKS-encoded peptide loading and release of drugs, and drug loading promoting the conversion of aggregates into liquid crystals; Figure 3 In the figure, 'a' represents the encapsulation efficiency of the peptide and Erastin drug at different molar ratios. Figure 3 Image b shows polarized light images of the peptide before and after drug loading; Figure 3 In the middle c, the fluorescence bleaching recovery curves are shown before and after the peptide is loaded with the drug. Figure 3 In the middle, d represents the fluorescence bleaching recovery images of the peptide before and after drug loading; Figure 4 The liquid crystal that the LARKS-encoded peptide of the present invention transforms into after loading a drug is more stable; Figure 4 Image a shows the phase changes of unloaded and loaded drug-containing peptides after heating from 37°C to 42°C. Figure 4 In Figure b, the anti-dilution ability curves of the unloaded drug peptide and the drug-loaded peptide are shown. Figure 5 The LARKS-encoded peptide drug delivery system of the present invention induces ferroptosis in A375 tumor cells; Figure 5 In the figure, 'a' represents a bar chart showing the cell survival rate after co-incubating tumor cells with different concentrations of peptides for 24 or 48 hours. Figure 5 In the middle b, there are bar charts showing the cell viability of the control group (no treatment), the drug-only group (Erastin), and the group treated with the LARKS-encoded peptide drug delivery system (Erastin@LEPCs). Figure 5 In the middle, c represents the bar chart of lipid peroxidation rate under different treatment groups; Figure 5 In the middle, d represents the fluorescence images of lipid peroxidation (BODIPY-C11) and the fluorescence images of mitochondrial damage (JC-1) under different treatment groups. Figure 5 In the figure, e represents the expression of GPX4 protein under different treatment groups; Figure 6 The phase transition behavior characterization diagrams are shown for the peptide prepared in Example 1 and the peptides prepared in Comparative Examples 1-3; wherein, Figure 6 In Figure a, the phase diagram of TPE-RGATGGRGDY in Example 1 is shown under different peptide concentrations (0.001-50 mg / mL) and different temperatures (17-77 °C). Figure 6In Figure b, the phase diagrams of RGATGGRGDY without TPE modification in Comparative Example 1 are shown under different peptide concentrations (0.001-50 mg / mL) and different temperatures (17-77℃). The two insets above and below correspond to the bright field microscopy images of 1 mg / mL peptide and 10 mg / mL peptide at 37℃, respectively (magnification: ). Figure 6 Comparison diagram of the phase states of Example 1, Comparative Examples 2 and 3; Figure 7 The mass spectrum of the peptide TPE-RGATGGRGDY prepared in Example 1; Figure 8 The mass spectrum of RGATGGRGDY without TPE modification is shown in Comparative Example 1. Figure 9 The peptide TPE-RGATGG is from Comparative Example 2. C GDY mass spectrum; Figure 10 The peptide TPE-RGATGG is the peptide of Comparative Example 3. H GDY mass spectrum. Detailed Implementation

[0025] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0026] Example 1: Peptide Synthesis Synthesis of the target peptide TPE-RGATGGRGDY (SEQ ID NO.1): The tetraphenylethylene (TPE) luminescent group is an aggregation-induced luminescent molecule. During phase separation, the peptide is enriched in the condensed phase, and the fluorescence is greatly enhanced during condensation. ATGGRG (SEQ ID NO.2) is a LARKS fragment that can promote the phase transition of the peptide.

[0027] The solid-state synthesis method was used for synthesis, and the specific steps are as follows: 1. Resin pretreatment: Weigh 0.25 mmol Rink resin (833 mg, degree of substitution 0.3 mmol / g) and add it to a solid-phase synthesis tube as a solid substrate. Swell the resin with a mixture of N,N-dimethylformamide (DMF) / dichloromethane (DCM) (1:1) for half an hour, remove the solvent by vacuum filtration, and wash the resin three times with DMF and filter dry.

[0028] 2. Coupling of the first amino acid: Add a DMF solution of the first Fmoc-protected amino acid (i.e., Fmoc-Tyr(tBu)-OH, 4 equivalents, 1.0 mmol). Then add the condensing agent HBTU / HOBt (or HATU, 4 equivalents) and the activating base DIPEA (8 equivalents). React at room temperature under nitrogen bubbling or mechanical stirring for 60 minutes to ensure that the carboxyl group of the first amino acid is covalently linked to the amino group of the resin. After the reaction is complete, filter to remove the reaction solution and wash the resin thoroughly with DMF 3-4 times to remove excess reagents and byproducts.

[0029] 3. Removal of amino protecting groups Fmoc: The resin with amino acid coupling in step 2 is soaked in 20% (v / v) piperidine / DMF solution for 15 minutes, then washed five times with DMF and filtered dry; the removal of Fmoc protecting groups is tested by ninhydrin.

[0030] 4. Gradual growth of the peptide chain: Following the target sequence RGATGGRGDY from the C-terminus to the N-terminus, the following operations are performed cyclically, adding Fmoc-protected amino acids one by one (Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH). Specifically as follows: Weigh out 4 times the excess of amino acids, the amount of which is 0.25 × 4 × the molecular weight of amino acids; then weigh out 364.16 mg of benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), add the above substances to a solid-phase synthesis tube, add 10 mL of DMF and 500 μL of N,N-diisopropylethylamine (DIEA), and react on a shaker for at least 2 hours; test the amino acid linkage by ninhydrin.

[0031] The ninhydrin test is used to check the completion of the coupling reaction and the removal of the Fmoc group.

[0032] Add 20 to a 1.5 mL centrifuge tube 30 resin beads; add 5 wt.% ninhydrin solution (dissolved in ethanol), 80% phenol solution (dissolved in ethanol), and anhydrous pyridine as three detection reagents to a centrifuge tube in a volume ratio of 2:1:1; heat the centrifuge tube at 110℃ for 2 minutes. The complete reaction of the amine terminal groups can be seen from the colorimetric analysis of the product. a) If the product is blue, it indicates the presence of unreacted primary amine (positive); b) If the product is brown or red, it indicates the presence of secondary amine (positive); c) If the product is pale yellow, it indicates the absence of terminal amine (negative).

[0033] If a positive sample is obtained after the coupling reaction, a second coupling reaction with the same amino acid is required. If the ninhydrin test is negative, the new amino acid has been successfully coupled, and peptide synthesis should continue to remove the Fmoc group for the next coupling reaction. This step is repeated until the peptide sequence RGATGGRGDY is obtained.

[0034] 5. Tetraphenylcarboxylic acid (TPE) Coupling of COOH: TPE Treating COOH as an amino acid, weigh out 4 times the excess amount, which is 0.25 × 4 × TPE. COOH molecular weight; connect using the same method as steps 2 and 3.

[0035] 6. Cutting, precipitation and purification: TPE After the COOH reaction was complete, the solvent was removed by vacuum filtration. The solid was washed 3 times with DMF and 5 times with DCM, then dried and transferred to a clean vial. A peptide cleavage solution (trifluoroacetic acid:triisopropylsilane:H₂O, volume ratio: 92.5:5:2.5) was prepared and added to the vial. The mixture was stirred for 3 hours, vacuum filtered, and the filtrate was collected. The trifluoroacetic acid was removed by air, and 30 mL of ice-cold diethyl ether was added to precipitate the solid. The solid was collected by centrifugation. The solid was then washed twice with ice-cold diethyl ether and collected by centrifugation. The solid was then analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC). The target peptide was further purified by HPLC. The molecular weight of the product was confirmed by mass spectrometry (MS). MALDI-MS detection showed m / z 1367.51 [M+H]¹⁺, indicating a positively charged peak representing a peptide bound to one proton (see [M+H]¹⁺). Figure 7 ).

[0036] Example 2: Phase analysis of LARKS-encoded peptides Based on the TPE-RGATGGRGDY polypeptide successfully synthesized in Example 1, this example aims to investigate its phase behavior at different temperatures and to clarify whether the condensates formed by its liquid-liquid phase separation will undergo a liquid-solid phase transition.

[0037] The peptide prepared in Example 1 was dissolved in pH 7.4 phosphate-buffered saline (PBS) to a concentration of 1 mg / mL, heated to 80°C until completely dissolved, and then cooled to 37°C and 27°C for observation. Figure 1It can be seen that at 37℃, the peptides are aggregates because the fluorescence recovers quickly after fluorescent bleaching, the amount of internal fibers generated is small and slow, there is no polarized light signal, and the droplets can fuse rapidly, which is consistent with the characteristics of aggregates. However, at 27℃, the fluorescence does not recover after bleaching, a large number of fibers are generated, there is a clear polarized light signal, and there is almost no fusion, indicating that it is a microgel.

[0038] Figure 1 The phase changes of the LARKS-encoded peptide of the present invention at 27°C and 37°C are shown; Figure 1 Image a shows fluorescence bleaching recovery images at 27℃ and 37℃. Figure 1 b is Figure 1 Quantitative fluorescence recovery curve of fluorescence image in a; Figure 1 c represents the quantitative growth curves of ThT fibers at 27℃ and 37℃; Figure 1 In the image, d represents scanning electron microscope images taken at 27℃ and 37℃. Figure 1 In the image, e represents the polarization signal and fusion status over time at 27℃ and 37℃.

[0039] Example 3 Tumor microenvironment responsiveness of LARKS-encoded peptides This embodiment aims to investigate the responsiveness of the liquid condensate to a simulated tumor microenvironment (acidic pH and high reducing power) at 37°C. The specific steps are as follows: 1. Solution preparation: Normal physiological environment simulation solution: phosphate buffer solution with a pH of 7.4; Tumor microenvironment simulation solution: pH 5.5 phosphate buffer (containing 5 mM glutathione (GSH)) simulates the tumor microenvironment.

[0040] A 1 mg / mL peptide solution (the peptide was prepared in Example 1) was prepared using a normal physiological environment simulation solution and a tumor microenvironment simulation solution, and observed under a fluorescence microscope.

[0041] Figure 2 The tumor microenvironment responsiveness of the LARKS-encoded peptide of the present invention is demonstrated. Figure 2 Image a shows fluorescence and bright-field images under normal physiological conditions (pH 7.4) and tumor microenvironment conditions (pH 5.5 + GSH). Figure 2 In Figure b, the phase diagrams are shown for different GSH concentrations and different peptide concentrations at pH 7.4. Figure 2 In the diagram, c represents the phase diagrams corresponding to different GSH concentrations and different peptide concentrations at pH 5.5.

[0042] The results showed that the aggregates disintegrated in the tumor microenvironment simulation solution, transforming into a single-phase solution. Figure 2(a) Phase diagrams at different GSH concentrations (1-30 mM) and different peptide concentrations (0.001-50 mg / mL) at pH 7.4 and pH 5.5 revealed that peptides of the same concentration were more prone to disintegration under weakly acidic conditions, indicating that the tumor microenvironment responsiveness of peptide aggregates is a synergistic effect of GSH and weak acidity.

[0043] Example 4 Drug loading and release of LARKS-encoded peptides The peptide prepared in Example 1 was prepared into a 1 mg / mL solution using pH 7.4 phosphate-buffered saline (PBS), incubated at 37°C, and then Erastin was added in different molar ratios (the molar ratios of peptide to Erastin were 2:1, 4:1, and 8:1, respectively). The mixture was incubated for 15 minutes, centrifuged for 10 minutes, and the Erastin content in the supernatant was measured. The encapsulation efficiency of the drug at different molar ratios was calculated.

[0044] Ultimately, the encapsulation efficiencies corresponding to the molar ratios of peptide to Erastin of 2:1, 4:1, and 8:1 were 76%, 80%, and 91%, respectively. Figure 3 (a) Given that the 8:1 molar ratio has the highest encapsulation efficiency, this condition was selected for all subsequent experiments.

[0045] When unloaded and drug-loaded peptides were observed under a polarizing microscope, liquid crystal signals were found to appear in the drug-loaded peptides. Figure 3 (b) Meanwhile, a fluorescence bleaching recovery experiment using confocal microscopy revealed that the fluorescence recovery of the drug-loaded peptide was significantly slower after laser bleaching. Figure 3 (cd). Therefore, drug loading promotes the transformation of condensates into liquid crystals.

[0046] When unloaded peptides and drug-loaded peptides were heated from 37°C to 42°C, the unloaded peptides changed directly from aggregates to a single-phase solution, while the drug-loaded peptides changed from liquid crystals to aggregates. Figure 4 (a) indicates that the thermal stability of the delivery system is improved after drug loading. Since peptides only exhibit strong fluorescence signals in the aggregated state, monitoring the fluorescence signal after gradient dilution of the peptide can reflect its anti-dilution ability before and after drug loading. Figure 4 As shown in Figure b, the unloaded peptide essentially transforms into a single phase after a dilution of approximately 64 times, while the drug-loaded peptide only becomes essentially single-phase after a dilution of approximately 512 times. These results indicate that the stability of peptides is enhanced after drug loading.

[0047] Example 5: LARKS-encoded peptide drug delivery system induces ferroptosis in tumor cells. The peptides prepared in Example 1 were prepared into peptide solutions of different concentrations (0-1 mg / mL) using pH 7.4 phosphate-buffered saline (PBS) and co-incubated with A375 tumor cells. When the peptide concentration was 0.1 mg / mL, the cell viability after 24 hours and 48 hours of incubation with tumor cells were 98% and 95%, respectively. Figure 5 (a) indicates that the peptide exhibits good biocompatibility at 0.1 mg / mL, and the concentration used in subsequent cell experiments was consistently 0.1 mg / mL. The efficacy of Erastin alone and Erastin delivered via a peptide carrier was then evaluated. The results showed that the peptide delivery system (Erastin@LEPCs) achieved a tumor cell killing rate of up to 86%, while the Erastin-only treatment group only achieved 55% (a). Figure 5 (b) Next, several indicators of ferroptosis were tested, and it was found that the Erastin@LEPCs treatment group had the highest levels of lipid peroxidation, mitochondrial damage, and GPX4 protein. Figure 5 The results (ce) indicate that the LARKS-encoded peptide drug delivery system effectively induced ferroptosis in tumor cells.

[0048] Comparative Example 1 The unmodified TPE polypeptide sequence, RGATGGRGDY, was obtained using the same solid-phase synthesis method as in Example 1. Mass spectrometry analysis (MALDI-MS) showed m / z 1230.74 [M], indicating the polypeptide was electroneutrally neutral (see [link]). Figure 8 ).

[0049] TPE-RGATGGRGDY and RGATGGRGDY peptides were prepared into peptide solutions of different concentrations (0.001-50 mg / mL) using pH 7.4 phosphate-buffered saline (PBS), and their phase states were then observed at different temperatures (17℃-77℃). Figure 6 a and Figure 6 As shown in Figure b, the 1 mg / mL RGATGGRGDY peptide is in a single-phase state at 37℃, while the 1 mg / mL TPE-RGATGGRGDY peptide is in a liquid condensate state at 37℃. This indicates that the modification of the TPE fluorophore reduces the phase transition concentration of the TPE-RGATGGRGDY peptide, enabling liquid-liquid phase separation to occur at a lower concentration.

[0050] Comparative Example 2 TPE-RGATGG was obtained by solid-state synthesis according to Example 1. C GDY (SEQ ID NO.3) peptide, which is about to contain the LARKS fragment (ATGG) RIn G), the R amino acid was replaced with a C amino acid. Mass spectrometry detection (MALDI-MS): m / z 1314.78 [M+H]¹⁺, indicating a peptide binding to one proton with a single positively charged peak (see...). Figure 9 ).

[0051] TPE-RGATGG C GDY peptide and TPE-RGATGGRGDY peptide were each prepared into 1 mg / mL peptide solutions using pH 7.4 phosphate-buffered saline (PBS), and their phase states at 37°C were then observed. From... Figure 6 As can be seen from c, 1 mg / mL of TPE-RGATGG C The GDY peptide was insoluble in PBS at 37°C, while 1 mg / mL of the TPE-RGATGGRGDY peptide was in a liquid aggregate state at 37°C. This indicates that the LARKS fragment plays a crucial role in the phase transition of the TPE-RGATGGRGDY peptide.

[0052] Comparative Example 3 TPE-RGATGG was obtained using the same solid-state synthesis method as in Example 1. H GDY (SEQ ID NO.4) peptide, which is about to contain the LARKS fragment (ATGG) R In G), the R amino acid was replaced with the H amino acid. Mass spectrometry detection (MALDI-MS): m / z 1348.83 [M+H]¹⁺, indicating a peptide binding to one proton with a single positively charged peak (see...). Figure 10 ). TPE-RGATGG H GDY peptide and TPE-RGATGGRGDY peptide were each prepared into 1 mg / mL peptide solutions using pH 7.4 phosphate-buffered saline (PBS), and their phase states at 37°C were then observed. From... Figure 6 As can be seen from c, 1 mg / mL of TPE-RGATGG H The GDY peptide was in a single-phase state at 37℃, while the 1 mg / mL TPE-RGATGGRGDY peptide was in a liquid aggregate state at 37℃. This indicates that the hydrophilicity of the amino acids in the LARKS fragment affects the phase change of the TPE-RGATGGRGDY peptide, suggesting that the LARKS fragment plays a crucial role in the phase transition of the TPE-RGATGGRGDY peptide.

[0053] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A polypeptide, characterized in that, It has the structure shown in formula HC-YX-Y'; wherein, HC is a hydrophobic π-conjugated group; X is a LARKS fragment with the amino acid sequence ATGGRG; Y and Y' are flexible linker peptides with the amino acid sequences RG and DY, respectively.

2. The polypeptide according to claim 1, characterized in that, The hydrophobic π-conjugated group HC is a group with aggregation-induced emission properties; Preferably, the group having aggregation-induced emission properties is a tetraphenylethylene group.

3. The polypeptide according to claim 1, characterized in that, The polypeptide can self-assemble into a liquid condensate under physiological conditions, and the phase of the condensate can change in response to external stimuli.

4. The polypeptide according to claim 3, characterized in that, The external stimuli include at least one of temperature, pH value, and reducing environment.

5. The polypeptide according to claim 4, characterized in that, The polypeptide is responsive to the tumor microenvironment and can undergo aggregate disintegration under conditions of pH 5.5-6.5 and the presence of 1-10 mM glutathione.

6. A tumor microenvironment-responsive peptide drug delivery system comprising the peptide of any one of claims 1-5.

7. The polypeptide drug delivery system according to claim 6, characterized in that, The system also includes a hydrophobic drug encapsulated in an aggregate formed by the polypeptide; Preferably, the hydrophobic drug is a drug that can induce ferroptosis; More preferably, the drug is Erastin.

8. The polypeptide drug delivery system according to claim 6, characterized in that, The molar ratio of the polypeptide to the drug is 2:1 to 8:1, preferably 8:

1.

9. The polypeptide drug delivery system according to claim 6, characterized in that, The loading of the drug can induce the transformation of the liquid aggregates of the peptide into a liquid crystal state.

10. The application of a polypeptide drug delivery system as described in any one of claims 6-9, characterized in that, Used to prepare drugs that induce ferroptosis in tumor cells.

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