Preparation method of polypeptide hydrogel and application thereof in tumor treatment

Abstract

The application discloses a preparation method of a polypeptide hydrogel and application of the polypeptide hydrogel in tumor treatment, and belongs to the technical field of biotechnology and new medicine technology.The polypeptide hydrogel is formed by co-assembly of a polypeptide gelation factor and Nin, and is an FGFR1-responsive polypeptide hydrogel (Gel Y / Nin).The polypeptide hydrogel (Gel Y / Nin) has good biocompatibility and biodegradability, and is suitable for local administration; the polypeptide hydrogel (Gel Y / Nin) is converted into Nap-Phe-Phe-Phe-Glu-Thr-Glu-Leu-Tyr(H2PO3)-OH (referred to as Nap-Yp) through an FGFR1-triggered phosphorylation reaction, so that the polypeptide hydrogel (Gel Y / Nin) is disintegrated and continuously releases Nin, thereby effectively inhibiting the growth, invasion and metastasis of tumor cells; in-vitro and in-vivo studies show that the polypeptide hydrogel (Gel Y / Nin) has a significant therapeutic effect on inhibition of survival and metastasis of lung cancer cells, and shows application potential in clinical tumor treatment.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and new medicine, and relates to a method for preparing polypeptide hydrogels and their application in tumor treatment. Background Technology

[0002] Lung cancer is one of the leading causes of cancer death worldwide, and its high invasiveness and metastasis are major reasons for the difficulty in treatment. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases, and its standard treatment includes surgery and neoadjuvant or adjuvant chemotherapy. However, despite surgery and platinum-based chemotherapy, the five-year survival rate for NSCLC patients has only improved by about 5%. For patients diagnosed at an advanced stage, conventional chemotherapy has limited efficacy and is accompanied by significant toxic side effects, resulting in a survival time of only 6 to 18 months and a five-year survival rate as low as 15%. To address these issues, various targeted therapy strategies have emerged, including small molecule inhibitors and monoclonal antibodies; in recent years, molecularly targeted therapy has become the main strategy for treating NSCLC, especially with the introduction of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib, which have significantly prolonged progression-free survival (PFS) for certain subgroups of NSCLC patients. However, squamous cell carcinoma of the lung (SQCC) typically does not produce oncogenic drivers such as EGFR and anaplastic lymphoma kinase (ALK), making NSCLC treatment a significant challenge. Later, fibroblast growth factor receptor 1 (FGFR1) was discovered as an important molecular target and is highly expressed in approximately 20% of SQCC patients. Overactivation of FGFR1 promotes epithelial-mesenchymal transition (EMT) and is closely associated with lung cancer grade and stage. As part of the tyrosine kinase receptor family, the FGFR1 gene is located on chromosome 8p. Upon binding to its ligand, FGFR1 dimers, autophosphorylates, and activates downstream signaling pathways such as the RAS-RAF-MAPK and PI3K-AKT-mTOR pathways, thereby regulating cell cycle progression, apoptosis, and autophagy. These signaling pathways play crucial roles in cell proliferation, migration, invasion, and survival, making FGFR1 a promising target for NSCLC treatment. Although second-generation FGFR inhibitors such as AZD4547, BGJ398, brevanib, dovitinib, lenvatinib, and nintedanib (Nin) have been introduced into clinical use, Nin has garnered more attention due to its multi-target inhibitory efficacy. Nin effectively overcomes drug tolerance issues in NSCLC by selectively inhibiting the ATP-binding pocket, simultaneously inhibiting vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and FGFR. However, the therapeutic effect of Nin is significantly limited by its poor water solubility, low bioavailability, and short serum half-life. Furthermore, high-dose oral administration inevitably increases the metabolic burden on the liver, impacting patients' quality of life. Summary of the Invention

[0003] To address the aforementioned problems, the present invention aims to propose a polypeptide hydrogel (i.e., a polypeptide hydrogel that triggers local sustained-release of drugs by FGFR1) to improve the therapeutic effect of FGFR1 inhibitors and reduce their systemic toxicity.

[0004] The technical solution of the present invention is as follows: A polypeptide hydrogel for tumor treatment, comprising polypeptide gelling factor (Nap-Y) and nintedanib (Nin for short), a small molecule inhibitor containing phosphorylated kinase fibroblast growth factor receptor 1 (FGFR1), wherein the polypeptide gelling factor and nintedanib co-assemble through non-covalent interactions to form a polypeptide hydrogel (Gel Y / Nin).

[0005] Furthermore, the polypeptide gelling factor (Nap-Y) has the following amino acid sequence: 2-naphthaleneacetic acid-phenylalanine-phenylalanine-phenylalanine-glutamic acid-threonine-glutamic acid-leucine-tyrosine (Nap-Phe-Phe-Phe-Glu-Thr-Glu-Leu-Tyr-OH).

[0006] Furthermore, the preparation steps are as follows:

[0007] Step (1): Synthesize the impurity-containing peptide gelling factor (Nap-Y) by solid-phase peptide synthesis method;

[0008] Step (2): Purify the peptide gelling factor (Nap-Y) containing impurities from the previous step using a semi-preparative high performance liquid chromatography system to obtain a pure peptide gelling factor (Nap-Y) in white powder form;

[0009] Step (3): Formation of polypeptide hydrogel (Gel Y / Nin).

[0010] Further, in step (1), the specific synthesis process is as follows: First, 2-chlorotriphenylmethyl chloride resin (2g) is swollen in 20mL of N,N-dimethylformamide (DMF) for 20 minutes. Then, the first amino acid Fmoc-Tyr(OtBu)-OH (3.2mmol) is grafted onto the resin in a DMF solution containing N,N-diisopropylethylamine (DIPEA, 800μL) for 1 hour. After washing three times with DMF to remove ungrafted amino acids from the reaction solution, the resin is capped with a capping solution (DMF:methanol = 19:1) for 30 minutes.

[0011] Next, the Fmoc protecting group was removed with a 20% piperidine-DMF solution for 30 minutes, and then washed three times with DMF to remove residual piperidine from the reaction solution;

[0012] Next, HOBt / HBTU / DIPEA was used as a coupling agent to couple the next Fmoc-protected amino acid, Fmoc-Leu-OH (3.2 mmol), to the free amino group; peptide chain growth was achieved by repeating these coupling and deprotection steps.

[0013] Finally, after washing with DMF (5 times), isopropanol (5 times), and n-hexane (5 times), the synthesized peptide was cleaved from the resin twice with 95% trifluoroacetic acid-dichloromethane solution for 30 minutes each time. Since each wash could not completely remove unreacted compounds, the cleaved peptide gelling factor (Nap-Y) contained impurities. Because the cleaved peptide gelling factor (Nap-Y) contained some impurities, further purification was still required.

[0014] Furthermore, in step (2), the specific synthesis process is as follows: using water-acetonitrile with 0.1% TFA added as the mobile phase eluent (from 60:40 to 0:100) to purify the polypeptide solution containing impurities; after obtaining the purified polypeptide solution, using a rotary evaporator to remove the acetonitrile in the purified polypeptide solution, and drying it in a freeze dryer for 24 hours to remove the water in the purified polypeptide solution, finally obtaining a pure polypeptide gelling factor (Nap-Y) in white powder form.

[0015] Furthermore, in step (3), the specific synthesis process is as follows: the peptide gelling factor (Nap-Y) and the small molecule inhibitor nintedanib are mixed in a molar ratio of 1:125 in phosphate-buffered saline (PBS) with a pH of 7.4. Then, the solution is heated to 80°C and cooled to room temperature (around 25°C). During the heating and cooling process, the small molecule inhibitor nintedanib can co-assemble with the peptide gelling factor Nap-Y through non-covalent interactions, forming a dense nanofiber network structure on a microscopic level, and finally forming a peptide hydrogel (Gel Y / Nin) on a macroscopic level.

[0016] Furthermore, the peptide hydrogel (Gel Y / Nin) can specifically respond to FGFR1, which is specifically highly expressed in human lung squamous cell carcinoma A549 cells, and undergo degelatination. The specific mechanism is that FGFR1 can specifically recognize peptide gelling factors and phosphorylate them, thereby enhancing their water solubility, which leads to the degelatination of supramolecular hydrogels and ultimately achieves the slow release of nintedanib, thereby achieving enhanced treatment of human lung squamous cell carcinoma A549 cells.

[0017] Furthermore, the polypeptide hydrogel (Gel Y / Nin) triggers a phosphorylation reaction of polypeptide gelling factors in vivo or in vitro via FGFR1 phosphorylation kinase, converting Nap-Y into Nap-Phe-Phe-Phe-Glu-Thr-Glu-Leu-Tyr(H2PO3)-OH (abbreviated as Nap-Yp), thereby initiating the disintegration of the hydrogel and continuously releasing Nin; Nap-Yp is a hydrophilic molecule, causing the hydrogel to change from a gel state to a solution state, thus achieving the continuous release of Nin.

[0018] Furthermore, the polypeptide hydrogel (Gel Y / Nin) is used for local administration to improve the bioavailability and targeting of Nin and reduce systemic toxicity.

[0019] The beneficial effects of this invention are: by triggering hydrogel disintegration and sustained release of Nin through FGFR1, this invention significantly inhibits the invasion, migration and survival of lung cancer cells, and shows enhanced therapeutic effects in in vivo studies; in addition, this hydrogel has good biocompatibility and biodegradability, making it suitable for clinical applications. Attached Figure Description

[0020] Figure 1 This is a flowchart illustrating the preparation process of the polypeptide hydrogel in this invention;

[0021] Figure 2 This is a synthetic route diagram of the polypeptide gelling factor (Nap-Y) in this invention;

[0022] Figure 3 This is a comparison chart of the frequency (strain: 1.0%) correlation between the dynamic energy storage modulus (G') and loss modulus (G”) of Gel Y in the embodiments of the present invention.

[0023] Figure 4 This is a comparison chart of the frequency (strain: 1.0%) correlation between the dynamic energy storage modulus (G') and loss modulus (G”) of Gel Y / Nin in the embodiments of the present invention.

[0024] Figure 5 This is an in vitro drug release curve of Nin in an embodiment of the present invention;

[0025] Figure 6 This is a statistical chart of cell viability after A549 cells were co-incubated with different groups in the embodiments of the present invention;

[0026] Figure 7 This is a diagram of dead / live staining (Calcein-AM / PI double staining) of A549 cells in an embodiment of the present invention;

[0027] Figure 8This is a graph showing the relative volume of the tumor over time after intratumoral injection of the drug into A549 tumor-bearing nude mice in an embodiment of the present invention.

[0028] Figure 9 This is a graph showing the blood routine analysis results of A549 tumor-bearing nude mice after different treatments in an embodiment of the present invention;

[0029] Figure 10 These are representative H&E staining images of major organs obtained from healthy mice treated with PBS and A549 tumor-bearing nude mice treated with Gel Y / Nin and sacrificed at the end of the experiment, as described in this embodiment of the invention. Detailed Implementation

[0030] The specific technical solution of the present invention will be further described in detail below with reference to specific examples.

[0031] Example 1: Synthesis and purification of the peptide gelling factor Nap-Y:

[0032] 1. Materials and Equipment:

[0033] Required reagents: Fmoc-protected amino acids, 2-chlorotriphenylmethyl chloride resin, HBTU, HOBt, N,N-dimethylformamide, dichloromethane, trifluoroacetic acid, isopropanol, n-hexane, etc.

[0034] Equipment: Solid-phase peptide synthesizer, high-performance liquid chromatography (HPLC), mass spectrometry (MS), refrigerated centrifuge, ultrapure water system, nuclear magnetic resonance spectrometer (600MHz), etc.

[0035] 2. Steps:

[0036] Solid-phase peptide synthesis: A peptide gelling agent with the sequence Nap-Phe-Phe-Phe-Glu-Thr-Glu-Leu-Tyr-OH (Nap-Y) was synthesized using a solid-phase peptide synthesizer; firstly, 2-chlorotriphenylmethyl chloride resin (2g) was swollen in 20mL of N,N-dimethylformamide (DMF) for 20 minutes.

[0037] The first amino acid, Fmoc-Tyr(OtBu)-OH (3.2 mmol), was then grafted onto resin containing N,N-diisopropylethylamine (DIPEA, 800 μL) in DMF solution for 1 hour. After washing three times with DMF to remove ungrafted amino acids from the reaction solution, the resin was capped with a capping solution (DMF:methanol = 19:1) for 30 minutes.

[0038] Next, the Fmoc protecting group was removed with a 20% piperidine-DMF solution for 30 minutes, followed by washing three times with DMF to remove residual piperidine from the reaction solution. Then, the next Fmoc-protected amino acid, Fmoc-Leu-OH (3.2 mmol), was coupled to the free amino group using HOBt / HBTU / DIPEA as a coupling agent. Peptide growth was achieved by repeating these coupling and deprotection steps.

[0039] Finally, after washing with DMF (5 times), isopropanol (5 times), and n-hexane (5 times), the synthesized peptide was cleaved from the resin twice with 95% trifluoroacetic acid-dichloromethane solution for 30 minutes each time. Because each wash could not completely remove unreacted compounds, the cleaved peptide solution contained some impurities (peptide gelling factor (Nap-Y) containing impurities), requiring further purification (e.g., ...). Figure 2 (as shown);

[0040] Purification: The peptide gelling factor (Nap-Y) containing impurities from the previous step was purified by semi-preparative high-performance liquid chromatography (HPLC). Specifically, water-acetonitrile containing 0.1% TFA was used as the mobile phase eluent (from 60:40 to 0:100) to purify the peptide gelling factor (Nap-Y) containing impurities. After obtaining the purified peptide gelling factor (Nap-Y), the acetonitrile in the purified peptide gelling factor (Nap-Y) was removed by rotary evaporation, and the solution was dried in a lyophilizer for 24 hours to remove water from the purified peptide solution, finally obtaining a pure peptide gelling factor (Nap-Y) in white powder form.

[0041] Structural Confirmation: The correctness of Nap-Y synthesis was confirmed by mass spectrometry, and the molecular structure of Nap-Y was determined by nuclear magnetic resonance (NMR) spectroscopy. Specifically, the molecular weight of Nap-Y is 1260.4 m / z, and the result of hydrogen addition, 1261.4 m / z, is consistent with the observed result, proving the correctness of the synthesis. Secondly, the specific observation results of its proton NMR spectrum are as follows: 1H NMR(500MHz,DMSO)δ(ppm):8.31–8.17(m,2H),8.10–8.04(m,1H),8.03–7.99(m, 1H),7.98–7.94(m,1H),7.94–7.90(m,2H),7.89–7.85(m,2H),7.79(dd,J=8.2,5 .2Hz,1H),7.75(dd,J=8.4,2.4Hz,1H),7.59(d,J=4.1Hz,1H),7.52–7.45(m,2H) ,7.30–7.11(m,17H),7.04–6.98(m,2H),6.68(t,J=5.1Hz,2H),4.67–4.51(m,1H) The values ​​are 4.47–4.41 (m, 2H), 4.37–4.27 (m, 4H), 4.04–3.98 (m, 1H), 3.60–3.48 (m, 3H), 3.10–2.99 (m, 2H), 2.93–2.69 (m, 5H), 2.35–2.22 (m, 4H), 2.02–1.91 (m, 2H), 1.87–1.74 (m, 2H), 1.60 (dt, J = 13.2, 6.7 Hz, 1H), 1.45 (dt, J = 14.7, 4.5 Hz, 2H), 1.07 (t, J = 5.6 Hz, 3H), and 0.86 (dt, J = 24.6, 5.1 Hz, 6H), consistent with the predicted results, proving the purity and correctness of the synthesized Nap-Y molecular structure.

[0042] Example 2: Measurement of the critical aggregation concentration (CAC) of peptide gelling factor Nap-Y

[0043] 1. Materials and Equipment:

[0044] Required reagents: peptide gelling factor Nap-Y, PBS buffer (pH 7.4);

[0045] Equipment: Ultraviolet-Vis Spectrophotometer

[0046] 2. Steps:

[0047] Measurement of critical aggregation concentration (CAC): Nap-Y solutions with concentrations ranging from 2 mM to 1 μM were prepared in PBS (10 mM, pH 7.4); the transmittance of different solutions at wavelengths from 500 nm to 700 nm was then measured using a UV-Vis spectrophotometer; the CAC of Nap-Y was calculated using the transmittance at 650 nm.

[0048] Example 3: Co-assembly of nintedanib and peptide gelling factor Nap-Y

[0049] 1. Materials and Equipment:

[0050] Required reagents: peptide gelling factor Nap-Y, nintedanib, PBS buffer (pH 7.4), etc.

[0051] Equipment: Metal bath heater;

[0052] 2. Steps:

[0053] Preparation of hydrogel: Nap-Y was dissolved in PBS buffer at a concentration of 1.0 wt%; after heating to 80°C, nintedanib was added at a molar ratio of 1:125, and then cooled to room temperature. The formation of a transparent and stable supramolecular hydrogel (Gel Y / Nin) could be observed with the naked eye.

[0054] Example 4: Observation of the micro-nanostructure of Gel Y / Nin

[0055] 1. Materials and Equipment:

[0056] Required reagents: Gel Y / Nin, PBS, etc.;

[0057] Equipment: Transmission electron microscope (TEM);

[0058] 2. Steps:

[0059] TEM sample preparation: Gel Y / Nin was diluted 10 times and dropped onto a carbon-coated copper grid and air-dried overnight; then it was observed and TEM images were obtained using a JEM-2100 transmission electron microscope (JEOL, Japan).

[0060] Example 5: Secondary structure measurement of Gel Y / Nin

[0061] 1. Materials and Equipment:

[0062] Required reagents: Gel Y / Nin, Na2CO3-NaH2PO4 buffer (pH=7.4), etc.

[0063] Equipment: Circular dichroism (CD) spectrometer;

[0064] 2. Steps:

[0065] CD measurement: First, a 1 wt% polypeptide hydrogel (GelY / Nin) was prepared using Na2CO3-NaH2PO4 buffer (pH=7.4). Then, the GelY / Nin was diluted tenfold using the same buffer. Data was recorded with a bandwidth of 0.1 nm in the wavelength range of 180-260 nm without thermocouple temperature control.

[0066] Example 6: Measurement of the rheological properties of Gel Y / Nin

[0067] 1. Materials and Equipment:

[0068] Required reagents: Gel Y / Nin, PBS, etc.;

[0069] Equipment: Rheometer;

[0070] 2. Steps:

[0071] Rheological testing: Rheological tests were performed using a Haake RheoStress 6000 instrument (Thermo Scientific); in short, 1 wt% Gel Y / Nin was first prepared, and then it was uniformly spread on the rheometer stage for rheological testing (e.g., Figure 3 , Figure 4 (As shown).

[0072] Example 7: FGFR1-triggered hydrogel degellation

[0073] 1. Materials and Equipment:

[0074] Required biological materials: A549 cells, T25 cell culture flasks, etc.;

[0075] Required reagents: A549 cell lysis buffer, DMEM medium, fetal bovine serum (FBS), Gel Y / Nin, PBS;

[0076] Equipment: High-performance liquid chromatography (HPLC), cell culture incubator, TEM, etc.;

[0077] 2. Steps:

[0078] Cell culture: A549 human cancer cells were preserved in DMEM; the cells were cultured in a humidified incubator at 37°C and 5% CO2, supplemented with 10% fetal bovine serum (Gibco) and 100 U / mL penicillin / streptomycin;

[0079] A549 cell lysis: First, A549 cells cultured in T25 cell culture flasks were lysed using RIPA lysis buffer. The cells were pipetted several times to ensure full contact between the lysis buffer and the cells. After full lysis, the tissue homogenate was transferred to a pre-cooled centrifuge tube and centrifuged at 10,000 rpm for 5 min at 4°C to obtain A549 cell lysis buffer.

[0080] Gel Y / Nin degelatination: Gel Y / Nin was incubated with A549 cell lysate overnight at 37°C; the degelatination of the hydrogel was observed macroscopically; then, the changes in the nanostructure of Gel Y / Nin were observed by TEM, and the product Nap-Yp, which is phosphorylated by the highly expressed FGFR1 in A549 cell lysate, was analyzed by HPLC at different time points.

[0081] Example 8: Measurement of the sustained-release rate of nintedanib under the action of FGFR1

[0082] 1. Materials and Equipment:

[0083] Required biological materials: A549 cells, 6-well cell culture plates;

[0084] Required reagents: DMEM medium, fetal bovine serum (FBS), Gel Y / Nin, PBS;

[0085] Equipment: Cell culture incubator, high-performance liquid chromatography, etc.;

[0086] 2. Steps:

[0087] Release assay: 10 μL of Gel Y / Nin was co-incubated with 8 × 10^5 A549 cells in 1000 μL of culture medium. Samples were taken at different time points (0.5, 1, 3, 6, 12, 24, 48, and 72 hours), and the release amount of Nin was quantitatively analyzed by HPLC (e.g., ...). Figure 5 (As shown).

[0088] Example 9: CCK-8 assay for A549 cell viability

[0089] 1. Materials and Equipment:

[0090] Required biological materials: A549 cells, 6-well cell culture plates;

[0091] Required reagents: CCK-8, DMEM medium, fetal bovine serum (FBS), Gel Y / Nin, PBS;

[0092] Equipment: Cell culture incubator, ELISA reader, fluorescence microscope, etc.

[0093] 2. Steps:

[0094] Cell culture: A549 cells were cultured in DMEM medium containing 10% FBS;

[0095] Cell viability assay: A549 cells were seeded in 96-well plates (5000 cells / well); culture medium (Ctrl group), Gel Y (800 μM Nap-Y), Nin (6.4 μM), or Gel Y / Nin (800 μM Nap-Y + 6.4 μM Nin) were added to the plates respectively; cell proliferation was detected on days 1, 2, and 3 using the CCK-8 assay according to the kit instructions; in short, 100 μL LDMEM containing 10 μL CCK-8 was used to replace the culture medium in each well; after incubation at 37°C for 1 hour, the absorbance (OD) value at 450 nm was measured using a microplate reader (SpectraMax iD3, Molecular Devices); each experiment was performed at least three times (e.g., Figure 6 (As shown).

[0096] Example 10: Apoptosis detection in A549 cells

[0097] 1. Materials and Equipment:

[0098] Required biological materials: A549 cells, 6-well cell culture plates;

[0099] Required reagents: Calcein-AM / PI double staining kit, DMEM medium, fetal bovine serum (FBS), Gel Y / Nin, PBS;

[0100] Equipment: Cell culture incubator, fluorescence microscope, flow cytometer, etc.

[0101] 2. Steps:

[0102] To further investigate the cell death mechanisms induced by various treatments, apoptosis / necrosis assays were performed. In detail, A549 cells after different treatments were collected, washed three times with PBS, treated with an annexin V-FITC / propidium iodide (PI) apoptosis detection kit (KeyGen Biotech, China), and finally detected by flow cytometry.

[0103] Example 11: A549 cell dead / live staining

[0104] 1. Materials and Equipment:

[0105] Required biological materials: A549 cells, 6-well cell culture plates;

[0106] Required reagents: Cell dead / live staining kit, DMEM medium, fetal bovine serum (FBS), Gel Y / Nin, PBS;

[0107] Equipment: Cell culture incubator, fluorescence microscope, etc.

[0108] 2. Steps:

[0109] Cell viability was visually determined using a live / dead viability / cytotoxicity kit; specifically, A549 cells were seeded at a density of 50,000 cells per well in 24-well plates and incubated for 24 hours; then the cells were subjected to different treatments; finally, the cells were co-stained with 1 μM calcein acetoxymethyl ester (calcein AM, Ex / Em: 495 / 515 nm) and 2 μM propidium iodide (PI, Ex / Em: 535 / 617 nm) at room temperature for 30 minutes, washed three times with PBS, and then imaged using a fluorescence microscope (e.g., ...). Figure 7 (As shown).

[0110] Example 12: Detection of A549 cell migration and invasion in vitro

[0111] 1. Materials and Equipment:

[0112] Required biological materials: A549 cells, 6-well cell culture plates;

[0113] Required reagents: DMEM medium, fetal bovine serum (FBS), Gel Y / Nin, PBS, etc.

[0114] Equipment: Cell culture incubator, fluorescence microscope, etc.

[0115] 2. Steps:

[0116] Cell scratch assay: A549 cells were seeded in 6-well plates (1×10⁵ cells / well) and incubated for 24 hours with DMEM, GelY (800 μM Nap-Y), Nin (6.4 μM), or GelY / Nin (800 μM Nap-Y + 6.4 μM Nin); a scratch with a 200 μL yellow pipette tip was placed in the center of each well; the wells were then gently washed with PBS to remove cell debris; incomplete culture medium was then added to each well, and photographs were taken at 0 and 24 hours.

[0117] Transwell cell migration and invasion assays: For the transwell migration assay, 2 × 10⁵ cells (dispersed in FBS-free 1640 medium) were seeded in the upper chamber of a transwell (24-well insert; pore size, 8 μm); the lower chamber was filled with 550 μL of 1640 medium supplemented with 20% FBS as a chemical inducer; after incubation with different groups for 24 hours, the migrated cells in the lower chamber were fixed with 70% (v / v) ethanol for 30 min, stained with 0.1% (w / v) crystal violet for 30 min, and then observed under a microscope; next, the crystal violet was eluted with 33% acetic acid, and the absorbance was measured at 570 nm using a microplate reader; for the cell invasion assay, the protocol was similar to that for the cell migration assay, except that the upper chamber was coated with 50 μL of L Atrigel (BD Biosciences).

[0118] Example 13: Establishment of an animal model

[0119] 1. Materials and Equipment:

[0120] Required biological materials: A549 cells, Balb / c nude mice, etc.

[0121] Required reagents: DMEM medium, fetal bovine serum (FBS), PBS buffer (pH 7.4), etc.

[0122] Equipment: Animal experimental equipment, digital calipers, tissue slicer, microscope, etc.

[0123] 2. Steps:

[0124] Animal model establishment: 4-week-old female BALB / c nude mice (16±2g); all experimental procedures complied with the "Regulations on the Administration of Laboratory Animal Affairs in China"; to establish a tumor-bearing nude mouse model, 8×10 6 A549 cells (suspended in 100 μL PBS) were subcutaneously injected into the right back of each mouse;

[0125] Example 14: Evaluation of the therapeutic effect of Gel Y / Nin in vivo

[0126] 1. Materials and Equipment:

[0127] Required biological material: A549 tumor-bearing nude mice;

[0128] Required reagents: Gel Y / Nin, PBS, etc.;

[0129] Equipment: Animal experimental equipment, syringes, etc.

[0130] 2. Steps:

[0131] Drug administration experiment: After the tumor volume reached a certain size, nude mice were randomly divided into four groups and received local injections of PBS, Gel Y, Nin and Gel Y / Nin, respectively, once every three days for a total of four times;

[0132] Efficacy evaluation: Tumor volume and mouse body weight were measured periodically; after the experiment, the tumors were removed and their volume was analyzed to evaluate the tumor inhibition effect (e.g., Figure 8 (As shown).

[0133] Example 15: Biocompatibility and safety assessment of hydrogels

[0134] 1. Materials and Equipment:

[0135] Required reagents: mouse serum, H&E staining reagent, etc.;

[0136] Equipment: Biochemical analyzer, microscope, etc.

[0137] 2. Steps:

[0138] Tissue staining: On day 12 post-injection, A549 tumor-bearing mice subjected to the different treatments described above were sacrificed. Their tumors and major organs (heart, liver, spleen, lung, and kidney) were then excised and fixed with 4% paraformaldehyde solution. After embedding in paraffin S8 and sectioning, the obtained samples were stained with hematoxylin and eosin (H&E) according to standard protocols. Furthermore, tumor sections were analyzed using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL). Specifically, tumors from 4T1 tumor-bearing mice after different treatments were excised, and then… Fixed with 4% paraformaldehyde solution; the obtained tissue sections were embedded in paraffin and sectioned, then incubated three times in xylene (10 min), and dehydrated three times in pure ethanol (5 min), followed by dehydration in a gradient of 95%, 90%, 80%, and 70% ethanol; finally, the samples were washed with distilled water; obvious liquid in the target tissue sections was eliminated; the samples were covered with proteinase K working solution and then incubated at 37°C for 25 min; then, the samples were washed three times with PBS; excess liquid was eliminated; osmotic working solution (0.1% Triton) was added. Cover the tissue sections with X-100 and incubate at room temperature for 20 minutes; then wash the tissue sections three times with PBS; after drying the sections, cover the tissue sections with buffer and incubate at room temperature for 10 minutes; depending on the tissue size, add an appropriate amount of buffer from the TDT enzyme, dUTP, and tunel kit (ratio 1:5:50) to cover the tissue sections; place the target sections in a flat, humidified chamber and incubate at 37°C for 2 hours; wash the tissue sections three times with PBS and then stain with DAPI solution at room temperature for 10 minutes; after washing three times with PBS, block the tissue sections with anti-fading fixation medium; then obtain fluorescence images using CLSM.

[0139] Hemolysis assay: Blood samples (1 mL) were collected from healthy BALB / c mice; red blood cells (RBCs) were obtained from the blood samples by centrifugation (600 g, 5 min) and resuspended in PBS (50 mL) to obtain an RBC suspension; next, gel Y (800 μM Nap-Y), Nin (6.4 μM), or gel Y / Nin (800 μM Nap-Y + 6.4 μM Nin) were mixed with the RBC suspension (0.5 mL); after 2 hours, the supernatant was collected by centrifugation (600 g, 10 min); RBCs in 1% Triton X-100 and PBS were used as positive and negative controls, respectively.

[0140] Serological analysis: Serum from mice treated with Gel Y / Nin was used for biochemical analysis to assess liver and kidney function and blood parameters (such as...). Figure 9 (as shown);

[0141] Histopathological analysis: Major organs (liver, kidney, heart, lung, spleen) were subjected to H&E staining to observe histopathological changes and assess the biocompatibility and safety of the hydrogel (e.g., Figure 10 (As shown).

Claims

1. A method for preparing a polypeptide hydrogel, characterized by, The polypeptide gelator and the small molecule inhibitor nintedanib containing a phosphorylating kinase fibroblast growth factor receptor 1 are co-assembled to form a supramolecular hydrogel through non-covalent interaction; The amino acid sequence of the polypeptide gelator is: 2-naphthaleneacetic acid-phenylalanine-phenylalanine-phenylalanine-glutamic acid-threonine-glutamic acid-leucine-tyrosine.

2. The method for preparing a polypeptide hydrogel according to claim 1, characterized in that, The preparation steps are as follows: Step (1): synthesizing the polypeptide gelator containing impurities through solid-phase peptide synthesis; Step (2): purifying the polypeptide gelator containing impurities through semi-preparative high-performance liquid chromatography, and finally obtaining the pure polypeptide gelator in the form of white powder; Step (3): mixing the polypeptide gelator and the prepared small molecule inhibitor nintedanib in phosphate buffered saline, heating and cooling, and finally forming a polypeptide hydrogel.

3. The method for preparing a polypeptide hydrogel according to claim 2, characterized in that, In step (1), the synthesis steps are as follows: First, swell the 2-chlorotrityl chloride resin in a DMF solution; Then, graft the first amino acid Fmoc-Tyr (OtBu)-OH to the resin in the DMF solution and react for 1 hour; after washing three times with the DMF solution, cap the resin with a capping solution for 30 minutes; Next, remove the Fmoc protecting group with a 20% piperidine-DMF solution for 30 minutes, then wash three times with the DMF solution, and then use HOBt / HBTU / DIPEA as a coupling reagent to couple the next Fmoc-protected amino acid Fmoc-Leu-OH to the free amino group; repeat the above coupling and deprotection steps to grow the peptide chain; Finally, wash five times in DMF solution, isopropyl alcohol, and n-hexane respectively, then use 95% trifluoroacetic acid-dichloromethane solution to cut the synthesized polypeptide from the resin, for a total of two times, 30 minutes each time, and the cut polypeptide is the polypeptide gelator containing some impurities.

4. The method for preparing a polypeptide hydrogel according to claim 2, characterized in that, In step (2), during the purification process: Use water-acetonitrile with 0.1% TFA added as the mobile phase eluent to purify the polypeptide gelator containing impurities; After obtaining the purified polypeptide gelator, use a rotary evaporator to remove the acetonitrile in the purified polypeptide gelator, and dry it in a freeze dryer for 24 hours to remove the water in the purified polypeptide gelator, finally obtaining the pure polypeptide gelator in the form of white powder.

5. The method for preparing a polypeptide hydrogel according to claim 2, characterized in that, In step (3), the molar ratio of the polypeptide gelator to the prepared small molecule inhibitor nintedanib is 1:125; The pH value of the phosphate buffered saline is 7.

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6. The method for preparing a polypeptide hydrogel according to claim 2, characterized in that, In step (3), mix the polypeptide gelator and the prepared small molecule inhibitor nintedanib in the phosphate buffered saline, heat to 80°C, and cool to room temperature; And during the heating and cooling process, the small molecule inhibitor nintedanib co-assembles with the polypeptide gelator through non-covalent interaction, forming a dense nanofiber network structure microscopically, and finally forming a polypeptide hydrogel macroscopically.

7. Use of a polypeptide hydrogel prepared by the method of any one of claims 1-6 in the preparation of a drug for treating human lung squamous carcinoma A549 cells. The specific mechanism is that the phosphorylation kinase fibroblast growth factor receptor 1 in the small molecule inhibitor nintedanib can specifically recognize the polypeptide glue factor and phosphorylate it, enhance its water solubility, thereby causing the ungluing of the polypeptide hydrogel, and finally realizing the release of the small molecule inhibitor nintedanib, thereby realizing the enhanced treatment of human lung squamous carcinoma A549 cells.

8. Use according to claim 7, characterized in that, The polypeptide hydrogel is used for local administration to improve the bioavailability and targeting of the small molecule inhibitor nintedanib and reduce systemic toxicity.

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