Radiation-responsive self-assembling polypeptides, their hydrogels and applications

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to radiation-responsive self-assembling polypeptides, their hydrogels and applications. The radiation-responsive self-assembling polypeptide derivative hydrogel of the present invention has the advantages of simple synthesis method, good biocompatibility, and easy loading of drugs. The hydrogel can trigger pulsed drug release through radiation response, precisely regulate the release rate of drugs in a controllable time and space, and enhance the synergistic effect of radiotherapy and drug therapy. Due to its high biosafety, it can avoid long-term negative effects on the human body and has a very broad application prospect. Therefore, the radiation-responsive polypeptide hydrogel of the present invention can be widely used in the treatment of radiation-related diseases such as combined tumor radiotherapy, postoperative tissue repair and regeneration, nuclear drug delivery and nuclear medicine image-guided therapy, providing a more accurate and effective synergistic treatment plan.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a ray-responsive self-assembling polypeptide, a hydrogel thereof and applications. Background Art

[0002] With the continuous advancement of cancer treatment methods, radiotherapy (RT) has become a key approach for treating malignant tumors. Fractionated radiotherapy is widely used in clinical practice to improve therapeutic efficacy and minimize damage to normal tissues. During fractionated radiotherapy, tumor cells are progressively killed by varying radiation doses. However, the efficacy of radiotherapy often relies on the support of drug delivery systems, particularly for the delivery of adjuvant therapeutic agents such as chemotherapeutics and immunotherapies. Traditional drug delivery systems, such as oral administration and intravenous injection, suffer from unstable drug delivery processes, short drug retention times in lesions, and inaccurate peak drug concentrations. These systems cannot be precisely synchronized with the radiotherapy cycle, resulting in poor combined efficacy of radiotherapy and drugs. To address this critical issue, radiation-responsive drug delivery systems have been increasingly studied in recent years. These systems can utilize radiation to regulate drug release rates. Ideally, their design should exhibit excellent biocompatibility, responsiveness, and controllability, enabling precise drug release according to varying radiotherapy cycles and dose requirements.

[0003] In combined treatments such as radiotherapy, chemotherapy, and immunotherapy, achieving ideal therapeutic effects relies on the synergistic effect of radiation and drugs during the treatment cycle. However, existing radiation-responsive drug delivery systems have not yet taken into account the temporal and spatial synergy between drug release and radiation action, as well as the precise regulation of drug dosage. First, spatially, most radiation-responsive delivery systems cannot guarantee that the drug reaches an effective dose at the target site during irradiation. Second, existing radiation-responsive delivery systems mostly release drugs in bursts or continuously after irradiation, which cannot meet the time requirements for timely drug replenishment and maximization of drug utilization during multi-fraction radiotherapy.

[0004] Self-assembling peptides have recently become a key research area in drug delivery systems due to their excellent biocompatibility, biodegradability, and structural controllability. Furthermore, through modular design, self-assembling peptides can respond to external stimuli such as pH, temperature, radiation, and ion concentration, allowing for flexible control of drug release. Hydrogels based on self-assembling peptides hold great promise for precise drug delivery and controlled release. Summary of the Invention

[0005] The purpose of the present invention is to provide a radiation-responsive self-assembling polypeptide.

[0006] Another object of the present invention is to provide a method for preparing the above polypeptide.

[0007] Another object of the present invention is to provide a hydrogel containing the above polypeptide.

[0008] Another object of the present invention is to provide a radiation-responsive drug delivery system containing the above-mentioned hydrogel. Based on the above-mentioned hydrogel, precise pulsed disassembly and drug release under multiple radiation stimulations are achieved, which has high biosafety and good degradability and is suitable for drug delivery in tumor treatment and radiotherapy-assisted treatment.

[0009] The present invention provides the following compounds:

[0010]

[0011] R1 is a sulfur-containing amino acid end cap.

[0012] Preferably, R1 is selected from (2-naphthylthio)acetic acid, phenylthioacetic acid, benzothiopheneacetic acid, thiopheneacetic acid, 4-methylthiophenylacetic acid, 3-(benzylthio)propionic acid, 3-(4-methylthiobenzoyl)propionic acid, and 3-(tritylthio)propionic acid.

[0013] n is an integer greater than 1, preferably, n is 1, 2, 3, 4, 5, 6, 7, 8 or 9.

[0014] R2 is a structural sequence that can assemble with phenylalanine.

[0015] Preferably, R2 is phosphorylated tyrosine, glycine, or alanine.

[0016] Preferably, based on the above-mentioned general formula compound, the present invention provides a radiation-responsive self-assembling polypeptide, the structure of which is selected from formula (II) or formula (III):

[0017]

[0018] Formula (II): Nap-S-GFF P YGG;

[0019]

[0020] Formula (III): Ben-S-GFF P YGG.

[0021] In the present invention, the synthesis of polypeptides of Formula (I), Formula (II), or Formula (III) is performed using the classic Fmoc solid-phase synthesis method for amino acids. During the reaction, HBTU is used as an activator for the amino acid carboxyl group, DIEA is used as a catalyst, and piperidine is used to remove the Fmoc protecting group, thereby exposing the amino group, which then undergoes a cross-linking condensation reaction with the carboxyl group of the next amino acid activated with HBTU to form a peptide bond. Once the peptide chain is complete, it is cleaved from the dichlororesin using 1% trifluoroacetic acid (TFA).

[0022] The present invention provides the use of the radiation-responsive self-assembling polypeptide in preparing drugs for treating tumors or auxiliary radiotherapy preparations.

[0023] In the present invention, cancer treatment drugs mainly include radiotherapy drugs, which generally refer to drugs used during radiotherapy to enhance the radiotherapy effect, reduce radiotherapy side effects or treat radiotherapy-related symptoms, mainly including: radiosensitizers, radiation protectants, symptomatic treatment drugs, etc.

[0024] Radiosensitizers include metronidazole and its derivatives, such as metronidazole and penidazole, and nitroimidazoles, such as misonidazole and etanercept. 5-Fluorouracil is not only a chemotherapy drug but also a radiosensitizer. Cisplatin, a commonly used chemotherapy drug, also has radiosensitizing effects.

[0025] Radiation protectants include amifostine, reduced glutathione, etc.

[0026] Symptomatic treatment drugs include ondansetron, granisetron, metoclopramide, etc.

[0027] The present invention provides a radiation-responsive polypeptide hydrogel, which contains at least one of the polypeptides of formula (II) or formula (III), that is, the hydrogel contains the polypeptide of formula (II), or the hydrogel contains the polypeptide of formula (III), or the hydrogel contains the polypeptides of formula (II) and formula (III) at the same time.

[0028] Furthermore, in the hydrogel, the mass ratio of the polypeptide of formula (II) to the polypeptide of formula (III) is 1:1-10. Preferably, the mass ratio of the polypeptide of formula (II) to the polypeptide of formula (III) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or any value within the above range, which are not listed here one by one.

[0029] According to a specific embodiment of the present invention, a method for preparing a radiation-responsive polypeptide hydrogel comprises the following steps:

[0030] (1) mixing the polypeptide of formula (II) and the polypeptide of formula (III) to prepare a polypeptide solution;

[0031] (2) adding alkaline phosphatase to the polypeptide solution to induce polypeptide self-assembly to form the radiation-responsive polypeptide hydrogel.

[0032] In step (1), the polypeptide of formula (II) and the polypeptide of formula (III) are dissolved in a buffer solution, wherein the buffer solution can be selected from Tris-HCl buffer solution, phosphate buffer solution (PBS), citric acid-disodium hydrogen phosphate buffer solution, glycine-sodium hydroxide buffer solution, borax-boric acid buffer solution, etc. After obtaining the polypeptide solution, the pH of the polypeptide solution is adjusted to 7.8-8.2.

[0033] The mass ratio of the polypeptide of formula (II) to the polypeptide of formula (III) is 1:1-10. Preferably, the mass ratio of the polypeptide of formula (II) to the polypeptide of formula (III) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or any value within the above range, which is not detailed here.

[0034] Preferably, the concentration of the polypeptide solution is 0.5-5 mg / mL, more preferably, the concentration of the polypeptide solution is 1-3 mg / mL, or the concentration of the polypeptide solution is 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 mg / mL, or any value within the above range, which are not listed here one by one.

[0035] In step (2), alkaline phosphatase (ALP) is added to the polypeptide solution to remove phosphate groups from the polypeptide molecules, triggering the self-assembly of the polypeptide molecules to form a hydrogel material. The amount of alkaline phosphatase added is 0.5-20 U / mL. Preferably, the amount of alkaline phosphatase (ALP) added is 0.5-5 U / mL.

[0036] According to a specific embodiment of the present invention, the radiation-responsive pulsed drug delivery system comprises the above-mentioned hydrogel and lipid nanoparticles, or the drug delivery system is made of the above-mentioned polypeptide and lipid nanoparticles.

[0037] A method for preparing a radiation-responsive pulsed drug delivery system comprises the following steps:

[0038] (1) mixing the polypeptide of formula (II) and the polypeptide of formula (III) to prepare a polypeptide solution;

[0039] (2) Add lipid nanoparticle solution to the polypeptide mixed solution, then add ALP, and let it stand.

[0040] Preferably, the volume ratio of the polypeptide solution to the lipid nanoparticle solution is 1-10:1. More preferably, the ratio range of the polypeptide solution to the lipid nanoparticle solution is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, or any value within the above range, which are not detailed here.

[0041] According to a specific embodiment of the present invention, the radiation-responsive pulsed drug delivery system further comprises a radiotherapy agent. For example, radiotherapy drugs applicable to the drug delivery system include cisplatin, 5-fluorouracil, paclitaxel, PD-L1, and the like.

[0042] Beneficial effects of the present invention:

[0043] The self-assembling polypeptide provided by the present invention contains a thiol blocking group at the N-terminus, which can realize the transformation from a gel to a solution in response to radiation and reactive oxygen species. The structure does not contain boron and iron elements, has good biocompatibility, and releases drugs through disassembly in response to radiation and ROS, thereby increasing the synergistic effect, and can be used to treat diseases related to radiation or oxidative damage.

[0044] The present invention provides self-assembling polypeptide derivatives with varying radiation sensitivities. By adjusting their ratios, these peptide derivatives can continuously respond to repeated radiation exposures and precisely release drugs in a pulsed manner. After forming into hydrogels, the peptide derivatives can self-assemble into micro-nanostructures, and during multiple radiotherapy sessions, radiation stimulation can trigger pulsed drug release.

[0045] The radiation-responsive, self-assembling polypeptide derivative hydrogel of the present invention has the advantages of a simple synthesis method and good biocompatibility. The hydrogel can trigger pulsed drug release through radiation response, precisely regulating the drug release rate and enhancing the synergistic effect of radiotherapy and drug therapy. Due to its high biosafety, it can avoid long-term negative effects on the human body and has a very broad application prospect. Therefore, the radiation-responsive polypeptide hydrogel of the present invention can be widely used in tumor radiotherapy, treatment of radiation-related diseases, and treatment of oxidative damage diseases, providing a more precise and effective treatment plan. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0047] Figure 1 Display Nap-S-GFF P The structure of YGG was confirmed;

[0048] A is Nap-S-GFF P Mass spectrum of YGG;

[0049] B is Nap-S-GFF P Infrared spectrum of YGG.

[0050] Figure 2 Show Ben-S-GFF P The structure of YGG is confirmed; among them,

[0051] A is for Ben-S-GFF P Mass spectrum of YGG;

[0052] B is for Ben-S-GFF P Infrared spectrum of YGG.

[0053] Figure 3 Display Nap-S-GFF P YGG and Ben-S-GFF P The responsive high performance liquid chromatography (HPLC) characterization of YGG, wherein,

[0054] A is Nap-S-GFF P HPLC spectra of YGG after treatment with 0.25%, 0.5%, and 1% H2O2;

[0055] B is Nap-S-GFF P HPLC spectrum of YGG after 6Gy γ-ray treatment;

[0056] C is for Ben-S-GFF P HPLC spectra of YGG after treatment with 0.25%, 0.5%, and 1% H2O2;

[0057] D is Ben-S-GFF P HPLC spectrum of YGG after 6Gy γ-ray treatment.

[0058] Figure 4 Display NBS Gel Characterize the results before and after the ray response, where

[0059] A is NBS Gel the form before the rays;

[0060] B is NBS Gel The shape after the rays.

[0061] Figure 5 Display NBS Gel Viscosity and storage modulus / loss modulus maps before and after ray response, where A is NBS Gel Changes in viscosity before and after ray response;

[0062] B is NBS Gel Storage modulus / loss modulus plots before and after ray response.

[0063] Figure 6 Display NBS Gel Drug release results before and after different doses of γ-ray response, among which,

[0064] A is NBS Gel The drug release results of sustained release from 0 to 72 hours;

[0065] B is NBS Gel Drug release results after each of the three irradiations.

[0066] Figure 7 Display NBS Gel Transmission electron microscopy (TEM) images of drug release.

[0067] Figure 8 Display NBS Gel The results of cell transfection with mRNA-LNP vector before and after radiation response.

[0068] Figure 9 Display NBS Gel Confocal images of pulse release and cell transfection results as mRNA-LNP carriers.

[0069] Figure 10 NBS prepared with different ratios of NapS and BenS Gel Comparison of the pulse release effects of mRNA-LNP vectors. DETAILED DESCRIPTION

[0070] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without inventive effort are within the scope of protection of the present invention.

[0071] The present invention provides a method for preparing a radiation-responsive pulsed drug delivery system, comprising the following steps:

[0072] (1) mixing the polypeptide of formula (II) and the polypeptide of formula (III) to prepare a polypeptide solution;

[0073] (2) Add lipid nanoparticle solution to the polypeptide mixed solution, then add ALP solution and let it stand.

[0074] In step (1), the polypeptide of formula (II) and the polypeptide of formula (III) are dissolved in a buffer solution, wherein the buffer solution can be selected from Tris-HCl buffer solution, phosphate buffer solution (PBS), citric acid-disodium hydrogen phosphate buffer solution, glycine-sodium hydroxide buffer solution, borax-boric acid buffer solution, etc. After obtaining the polypeptide solution, the pH of the polypeptide solution is adjusted to 7.8-8.2.

[0075] The mass ratio of the polypeptide of formula (II) to the polypeptide of formula (III) is 1:1-10. Preferably, the mass ratio of the polypeptide of formula (II) to the polypeptide of formula (III) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or any value within the above range, which are not listed here one by one.

[0076] Preferably, the concentration of the polypeptide solution is 0.5-5 mg / ml, more preferably, the concentration of the polypeptide solution is 1-3 mg / ml, or the concentration of the polypeptide solution is 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 mg / ml, or any value within the above range, which are not listed here one by one.

[0077] Preferably, in step (2), the volume ratio of the polypeptide solution to the lipid nanoparticle solution is 1-10:1. More preferably, the ratio of the polypeptide solution to the lipid nanoparticle solution is in the range of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, or any value within the above ranges, which are not detailed here.

[0078] In step (2), 3-4 μL of 1 U / mL ALP solution was added.

[0079] In the present invention, the lipid nanoparticle solution is a liquid system formed by dispersing lipid nanoparticles as the main component in a solvent, and its components include lipid materials, solvents and other components.

[0080] Lipid materials: usually include phospholipids, such as distearoylphosphatidylcholine, which can form the basic structure of nanoparticles; cholesterol, which can regulate the fluidity and stability of lipid membranes; and some functional lipids, such as PEGylated lipids.

[0081] Solvent: Generally water or various buffer solutions, which provide a dispersion medium for lipid nanoparticles.

[0082] Other ingredients: These include active substances such as the encapsulated drug and nucleic acid, as well as additives for adjusting the properties of the solution, such as salts and sugars. For example, the examples provide lipid nanoparticles encapsulating fluorescently labeled mRNA. The mRNA used above can also be replaced with other therapeutic drugs, such as radiosensitizers, radioprotectants, and symptomatic treatment drugs.

[0083] Example 1 Nap-S-GFF P Synthesis and purification of YGG

[0084] 1.1 Preparation process

[0085] Weigh 1g of dichlororesin into a solid-phase synthesis tube. Add approximately 10mL of dichloromethane (DCM) and allow the tube to swell on a shaker for 5 minutes. Use an ear bulb to squeeze out the solvent. Weigh 1mmol of the first amino acid, Fmoc-Gly-OH, into a 20mL vial. Add approximately 10mL of DCM, followed by 2mmol (400μL) of DIEA. Pipette thoroughly with a plastic dropper to completely dissolve the mixture, then add it to the solid-phase synthesis tube and allow it to react at room temperature for 2 hours. After the reaction, squeeze out the reaction solution, wash twice with DCM and three times with N,N-dimethylformamide (DMF). Add approximately 10mL of freshly prepared methanol solution (DCM:CH3OH:DIEA = 17:2:1) to block any unreacted active chlorine atoms on the dichlororesin. Allow to react at room temperature for 30 minutes. The reaction mixture was extruded and washed five times with DMF. Approximately 8 mL of 20% piperidine was added and allowed to react at room temperature for 30 minutes to remove the Fmoc protecting group on the amino group of the first amino acid, exposing it. The piperidine reaction mixture was extruded and washed five times with DMF to remove any residual piperidine. Then, 2 mmol of the second amino acid, Fmoc-Gly-OH, and 2 mmol of HBTU were weighed into a vial. Approximately 10 mL of DMF and 4 mmol (800 μL) of DIEA were added. After thorough dissolution, the mixture was added to a solid-phase synthesis tube and allowed to react at room temperature for 2 hours. (HBTU was used as a coupling agent and DIEA as a catalyst in all subsequent reactions.)

[0086] Repeat the above steps of "washing-deprotection-washing-adding amino acid", and add amino acids (Fmoc-Tyr(H2PO3)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, (2-naphthylthio)acetic acid) in sequence to react.

[0087] After the reaction of the last blocked (2-naphthylthio)acetic acid at the amino terminus of the peptide chain was completed, the reaction solution was squeezed out and washed with DMF 5 times to clean the unreacted amino acid raw material. Then, it was washed 5 times with DCM to clean the DMF on the resin. Then, freshly prepared 95% TFA solution (TFA:TIS:ddH2O=95%:2.5%:2.5%) was added to cut the peptide chain from the resin, and the resin was repeatedly washed with 95% TFA (10 min / time, for a total of 6 times).

[0088] Collect the reaction solution in an eggplant-shaped flask and use a vacuum rotary evaporator to remove TFA to obtain a viscous liquid. Add an appropriate amount of icy ether to the eggplant-shaped flask for precipitation. After standing at room temperature for a period of time, carefully pour off the anhydrous ether supernatant and vacuum-dry the remaining solid-liquid mixture. The resulting solid is the crude product and is stored in a refrigerator at -20°C.

[0089] 1.2 Purification process

[0090] The crude product was dissolved in methanol and filtered using an organic phase filter membrane (0.22 μm). Samples were collected by HPLC and the target peak was identified by high-resolution mass spectrometry.

[0091] HPLC mobile phase configuration: Aqueous phase A: 95% ultrapure water + 5% methanol + 0.5% trifluoroacetic acid; Organic phase B: 99.5% methanol + 0.5% trifluoroacetic acid. The injection concentration was 100 mg / mL, and 100 μL was injected each time.

[0092] The target peak solution was collected and freeze-dried by vacuum rotary evaporator to obtain pure Nap-S-GFF. P YGG, its structural formula is as follows:

[0093]

[0094] 1.3 Structure confirmation

[0095] The target peak solution collected by HPLC was detected by ultra-high pressure liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS). Figure 1 As shown in A, the molecular ion peak (942.27) of the polypeptide can be found in the mass spectrometry spectrum.

[0096] Fourier transform infrared spectroscopy (FI-TR) characterization of peptides

[0097] Control group Nap-S-GFFP YGG polypeptide solution (2 mg of pure polypeptide was dissolved in 1 mL of PBS to obtain polypeptide solution), the experimental group was Nap-S-GFF oxidized with 1% H2O2 P YGG polypeptide solution. The control group and experimental group were placed in a freeze vacuum dryer to freeze-dry the obtained white solid, which was then crushed and the infrared spectrum of the sample was recorded using the KBr tablet method. The infrared spectrum of the sample was measured using a Nicolet 6700 Fourier transform infrared spectrophotometer at 4000-500 cm -1 The infrared spectrum of the range is as follows Figure 1 As shown in B.

[0098] After adding 1% H2O2 to the experimental group, the Nap-S hydrogen peroxide treatment group had a -1 There is a characteristic absorption peak at , indicating that the sulfur in the polypeptide is oxidized to sulfoxide or sulfone.

[0099] 1.4 Investigation of radiation-responsive drug release as mRNA-LNP carriers

[0100] 5 mg of pure polypeptide was dissolved in 1 mL of ddH2O to obtain a polypeptide solution. After adding Cy5.5-labeled liposome particle solution containing 1 μg of mRNA to the polypeptide solution (LNP components and ratios are DOTAP: CHO: DSPC: DSPE-PEG2k = 50%: 38.5%: 10%: 1.5%. It was synthesized by thin film hydration method, that is, the various components in the above ratio were weighed and dissolved in chloroform. After forming a uniform thin film by rotary evaporation, DEPC water was added, and the probe was ultrasonicated and filtered through a 220 μm filter membrane.), Na2CO3 was added to adjust the solution pH to 8.0, and 3 μL of ALP solution was added. After standing and waiting for gelation, it was named NapS. Gel @mLNP, and add 1mL PBS solution on top of it.

[0101] The cells were irradiated with 2 Gy or 6 Gy of gamma rays at 0, 24, and 48 h. The fluorescence intensity of the PBS solution on the gel was measured at 22 time points (0, 2, 4, 6, 8, 12, 16, 24, 26, 28, 30, 32, 36, 40, 48, 50, 52, 54, 56, 60, 64, and 72 h) under the conditions of excitation wavelength of 680 nm and emission wavelength of 710 nm to plot the drug release curve.

[0102] like Figure 10 As shown in the figure, compared with the control group, the groups receiving 2Gy and 6Gy of γ-ray irradiation could significantly increase the drug release ratio.

[0103] Example 2Ben-S-GFF P Synthesis and purification of YGG

[0104] 2.1Ben-S-GFF P Preparation of YGG

[0105] Weigh 1g of dichlororesin into a solid-phase synthesis tube. Add approximately 10mL of dichloromethane (DCM) and allow the tube to swell on a shaker for 5 minutes. Use an ear bulb to squeeze out the solvent. Weigh 1mmol of the first amino acid, Fmoc-Gly-OH, into a 20mL vial. Add approximately 10mL of DCM, followed by 2mmol (400μL) of DIEA. Pipette thoroughly with a plastic dropper to completely dissolve the mixture, then add it to the solid-phase synthesis tube and allow it to react at room temperature for 2 hours. After the reaction, squeeze out the reaction solution, wash twice with DCM and three times with N,N-dimethylformamide (DMF). Add approximately 10mL of freshly prepared methanol solution (DCM:CH3OH:DIEA = 17:2:1) to block any unreacted active chlorine atoms on the dichlororesin. Allow to react at room temperature for 30 minutes. The reaction mixture was extruded and washed five times with DMF. Approximately 8 mL of 20% piperidine was added and allowed to react at room temperature for 30 minutes to remove the Fmoc protecting group on the amino group of the first amino acid, exposing it. The piperidine reaction mixture was extruded and washed five times with DMF to remove any residual piperidine. Then, 2 mmol of the second amino acid, Fmoc-Gly-OH, and 2 mmol of HBTU were weighed into a vial. Approximately 10 mL of DMF and 4 mmol (800 μL) of DIEA were added. After thorough dissolution, the mixture was added to a solid-phase synthesis tube and allowed to react at room temperature for 2 hours. HBTU was used as a coupling agent and DIEA as a catalyst in all subsequent reactions.

[0106] Repeat the "wash-deprotection-wash-add amino acid" steps above, adding amino acids (Fmoc-Tyr(H2PO3)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, phenylthioacetic acid) in sequence to react. After the reaction of the last phenylthioacetic acid blocking the amino terminus of the peptide chain is complete, squeeze out the reaction solution and wash with DMF five times to remove unreacted amino acid starting material. Then wash with DCM five times to remove DMF from the resin. Then, add freshly prepared 95% TFA solution (TFA:TIS:ddH2O = 95%:2.5%:2.5%) to cleave the peptide chain from the resin. Repeat the 95% TFA washes (10 min / time, for a total of 6 times). Collect the reaction solution in an eggplant-shaped flask and use a vacuum rotary evaporator to remove TFA to obtain a viscous liquid. Add an appropriate amount of icy ether to the eggplant-shaped flask for precipitation. After standing at room temperature for a period of time, carefully pour off the anhydrous ether supernatant and vacuum-dry the remaining solid-liquid mixture. The resulting solid is the crude product and is stored in a refrigerator at -20°C.

[0107] The crude product was dissolved in methanol and filtered using an organic phase filter membrane (0.22 μm). Samples were collected by HPLC and the target peak was identified by high-resolution mass spectrometry.

[0108] HPLC mobile phase configuration: Aqueous phase A: 95% ultrapure water + 5% methanol + 0.5% trifluoroacetic acid; Organic phase B: 99.5% methanol + 0.5% trifluoroacetic acid. The injection concentration was 100 mg / mL, and 100 μL was injected each time.

[0109] The target peak solution was collected and lyophilized to obtain the pure peptide Ben-S-GFF. P The synthesis and purification of YGG, its structural formula is as follows:

[0110]

[0111] 2.2 Structure confirmation

[0112] The target peak solution collected by HPLC was detected by ultra-high pressure liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS). Figure 2 As shown in A, the molecular ion peak of the polypeptide can be found in the mass spectrometry spectrum.

[0113] Fourier transform infrared spectroscopy (FI-TR) characterization of peptides

[0114] The control group is Ben-S-GFF P YGG peptide solution (2 mg of pure peptide was dissolved in 1 mL of PBS to obtain peptide solution), the experimental group was Ben-S-GFF oxidized with 1% H2O2 P YGG polypeptide solution. The control group and the experimental group were placed in a freeze vacuum dryer to freeze-dry the obtained white solid, which was then crushed and the infrared spectrum of the sample was recorded using the KBr tablet method. The infrared spectrum of the sample was measured using a Nicolet 6700 Fourier transform infrared spectrophotometer at 4000-500 cm -1 Range of infrared spectrum, the results are as follows Figure 2 As shown in B. After adding 1% H2O2 to the experimental group, the Ben-S hydrogen peroxide treatment group showed a decrease in the ion density at 1040 cm -1 There is a characteristic absorption peak at , indicating that the sulfur in the polypeptide is oxidized to sulfoxide or sulfone.

[0115] 2.3 Investigation of radiation-responsive drug release as mRNA-LNP carriers

[0116] 5 mg of pure polypeptide was dissolved in 1 mL of ddH2O to obtain a polypeptide solution. After adding Cy5.5-labeled liposome particle solution containing 1 μg of mRNA to the polypeptide solution (LNP components and ratios are DOTAP: CHO: DSPC: DSPE-PEG2k = 50%: 38.5%: 10%: 1.5%. It was synthesized by thin film hydration method, that is, the various components in the above ratio were weighed and dissolved in chloroform, and a uniform film was formed by rotary evaporation. DEPC water was added, and the probe was ultrasonicated and filtered through a 220 μm filter membrane.), Na2CO3 was added to adjust the solution pH to 8.0, and 3 μL of ALP solution was added. After standing and waiting for gelation, it was named BenS. Gel @mLNP, and add 1mL PBS solution on top of it.

[0117] The cells were irradiated with 2 Gy or 6 Gy of gamma rays at 0, 24, and 48 h. The fluorescence intensity of the PBS solution on the gel was measured at 22 time points (0, 2, 4, 6, 8, 12, 16, 24, 26, 28, 30, 32, 36, 40, 48, 50, 52, 54, 56, 60, 64, and 72 h) under the conditions of excitation wavelength of 680 nm and emission wavelength of 710 nm to plot the drug release curve.

[0118] like Figure 10 As shown in the figure, compared with the control group, the groups receiving 2Gy and 6Gy of γ-ray irradiation could significantly increase the drug release ratio.

[0119] Example 3 Nap-S-GFF P YGG and Ben-S-GFF P Responsive HPLC Characterization of YGG

[0120] 2 mg of each of the purified polypeptides prepared in Examples 1 and 2 were dissolved in 1 mL of PBS to obtain two polypeptide solutions. Na2CO3 was added to adjust the pH of the solution to 8.0, and 3 μL of ALP solution was added. The solution was allowed to stand for gelation.

[0121] Take 100 μL of the two groups of peptide solutions respectively, add 900 μL of methanol solution to the control group, and add 900 μL of methanol solution and 30% H2O2 solution of different concentrations to the experimental groups, so that the final H2O2 concentrations are 0.25%, 0.5%, and 1%, respectively, and then let it stand for 10 minutes.

[0122] Dissolve 2 mg of pure peptide in 1 mL of PBS. Add Na2CO3 to adjust the pH to 8.0, then add 3 μL of ALP solution. Allow to stand until gelation occurs. Then, irradiate with 6 Gy of γ-rays and allow to stand for 10 minutes.

[0123] The samples of both the control group and the experimental group were filtered through an organic phase filter membrane (0.22 μm) and characterized by HPLC.

[0124] HPLC mobile phase: Aqueous phase A: 95% ultrapure water + 5% methanol + 0.5% trifluoroacetic acid; Organic phase B: 99.5% methanol + 0.5% trifluoroacetic acid. The injection concentration was 100 mg / mL, and 100 μL was injected each time.

[0125] like Figure 3 As shown in (A, C), after adding different concentrations of H2O2, the sulfur in the peptide is oxidized into sulfoxide or sulfone, the hydrophilicity increases, and the chromatographic peak shifts forward to varying degrees. Figure 3 (B, D) Ben-S-GFF after receiving 6Gy of γ-ray irradiation P The YGG chromatographic peak shifted significantly, while the Nap-S-GFF P YGG is not obvious, proving that Ben-S-GFF P YGG has better responsiveness under 6Gy γ-ray conditions.

[0126] Example 4 NBS Gel Preparation and radiation responsiveness verification

[0127] Take 1mg Nap-S-GFF P Pure YGG and 3mg Ben-S-GFF P YGG was dissolved in 2 mL of PBS to obtain a peptide solution. After adding Na2CO3 to adjust the solution pH to 8.0, 3 μL of ALP solution was added and allowed to stand for gel formation. The solution was named NBS. Gel .

[0128] like Figure 4 (A) shows NBS Gel Can form stable hydrogels.

[0129] NBS Gel The hydrogels were irradiated with 6Gy of gamma rays at 0, 24, and 48 hours, and the changes in appearance were observed. Figure 4 As shown in (B), the hydrogel softened and loosened after two irradiations, and the gel network collapsed after three irradiations, indicating that radiation can cause polypeptide disassembly. Gel Ray responsiveness.

[0130] NBS Gel The hydrogels were irradiated with 6 Gy of γ-rays at 0, 24, and 48 hours, respectively, to serve as test samples. 100 μL of each hydrogel sample was placed on the sample stage and measured using a rotational rheometer, capturing images of its viscosity and storage / loss modulus.

[0131] like Figure 5 As shown in (A), with the increase of irradiation times, NBS Gel The viscosity of Figure 5 As shown in (B), with the increase of irradiation times, NBS Gel The storage modulus / loss modulus gradually decreased, indicating that radiation can cause polypeptide disassembly, NBS Gel Ray responsiveness.

[0132] Example 5 NBS Gel Pulse medication

[0133] 5.1 Release Investigation

[0134] 1 mg of pure Nap-S-GFFPYGG and 3 mg of Ben-S-GFFPYGG were dissolved in 2 mL of PBS to obtain a peptide solution. After adding a Cy5.5-labeled liposome particle solution containing 1 μg of mRNA to the peptide solution (LNP components and ratios are DOTAP: CHO: DSPC: DSPE-PEG2k = 50%: 38.5%: 10%: 1.5%. It was synthesized by the thin film hydration method, that is, the various components in the above ratio were weighed and dissolved in chloroform. After forming a uniform thin film by rotary evaporation, DEPC water was added, and the solution was filtered through a 220 μm filter membrane after probe sonication), Na2CO3 was added to adjust the solution pH to 8.0, and 3 μL of ALP solution was added. After standing and waiting for gelation, it was named NBS. Gel @mLNP, and add 1mL PBS solution on top of it.

[0135] The cells were irradiated with 2 Gy or 6 Gy of gamma rays at 0, 24, and 48 h. The fluorescence intensity of the PBS solution on the gel was measured at 22 time points (0, 2, 4, 6, 8, 12, 16, 24, 26, 28, 30, 32, 36, 40, 48, 50, 52, 54, 56, 60, 64, and 72 h) under the conditions of excitation wavelength of 680 nm and emission wavelength of 710 nm to plot the drug release curve.

[0136] like Figure 6 As shown in (A), compared with the control group, NBS Gel The release rate of the drug gradually increases, and when the irradiation condition is 6Gy, pulsed drug release can be achieved. Figure 6 As shown in (B), the drug release rates within 24 hours after three irradiations of 6 Gy were 36.43%, 20.96%, and 15.95%, respectively.

[0137] 5.2NBS Gel Transmission electron microscopy (TEM) detection of pulsed drug release

[0138] 10 μL of each sample from each of the above treatment groups, taken at 4, 28, and 52 hours, was dripped onto a TEM copper grid. After 3 minutes of stagnation, excess sample was removed using filter paper along the edge of the grid. 10 μL of uranyl acetate was then dripped onto the grid. The sample was negatively stained for 3 minutes, after which excess uranyl acetate was removed using a filter paper. After drying overnight, the sample morphology was observed and images were acquired using a TEM.

[0139] like Figure 7 As shown in the figure, with the increase of irradiation dose and number of times, the polypeptide fibers were obviously broken and the number of liposome particles decreased.

[0140] 5.3NBS Gel Investigation of the transfection efficiency of radiation-responsive cells as mRNA-LNP carriers

[0141] Take 1mg Nap-S-GFF P Pure YGG and 3mg Ben-S-GFF P YGG was dissolved in 2 mL of PBS to obtain a peptide solution. After adding a liposome particle (LNP) solution containing 1 μg of mRNA, the pH of the solution was adjusted to 8.0 by adding Na2CO3, and then 3 μL of LNP solution was added. The solution was allowed to stand for gelation and named NBS. Gel @mLNP.

[0142] NBS Gel @mLNPs were irradiated with 2 Gy and 6 Gy of γ-rays, respectively, and allowed to stand for 10 min.

[0143] 293T and DC2.4 cells with good growth were collected and resuspended in DMEM complete medium heated to 37°C in a water bath. The cells were plated into transwell 6-well plates at a density of 2×10 5 When the cells grow to 70-80% of the well plate, BenS Gel The mLNPs were placed in the transwell chamber and the original culture medium was replaced with serum-free Opti-MEM medium preheated to 37°C. Gel After 6 hours of co-culture with mLNP, the medium was replaced with complete DMEM. After 24 hours, the cells were harvested and the expression of EGFP protein in the cells was detected by flow cytometry.

[0144] like Figure 8As shown in the results, compared with the control group, the 2Gy and 6Gy γ-ray irradiation groups could increase the FITC-positive ratio of DC2.4 cells from 20% to 24.5% and 31.3%, respectively, and the FITC-positive ratio of 293T cells from 11.4% to 14.3% and 23.9%, respectively, indicating that radiation can cause polypeptide disassembly, thereby releasing the encapsulated drug, NBS Gel Ray responsiveness.

[0145] 5.4NBS Gel Investigation of pulsed release as an mRNA-LNP carrier

[0146] Take 1mg Nap-S-GFF P Pure YGG and 3mg Ben-S-GFF P YGG was dissolved in 2 mL of PBS to obtain a peptide solution. After adding a liposome particle (LNP) solution containing 1 μg of mRNA, the pH of the solution was adjusted to 8.0 by adding Na2CO3, and then 3 μL of ALP solution was added. The solution was allowed to stand for gelation and named NBS. Gel @mLNP, and add 1mL PBS solution on top of it.

[0147] The experimental groups were irradiated with 2 or 6 Gy of γ-rays at 0, 24, and 48 h, and PBS solutions were collected for six time periods: 0–12 h, 12–24 h, 24–36 h, 36–48 h, 48–60 h, and 60–72 h.

[0148] 293T and DC2.4 cells with good growth were collected and resuspended in DMEM complete medium heated to 37°C in a water bath. The cells were plated into confocal microplates and 6-well plates at a density of 2×10 5 Once the cells have grown to 70-80% of their size in the confocal dish or six-well plate, add the PBS solution to the confocal dish or six-well plate, and replace the original culture medium with serum-free Opti-MEM medium preheated to 37°C. After 6 hours of culture, replace the culture medium with complete DMEM. After 24 hours, observe fluorescence using a confocal microscope or harvest the cells and analyze EGFP protein expression using flow cytometry.

[0149] like Figure 9 As shown on the left, the confocal microscopy results showed that compared with the control group, the fluorescence quantity in the 0-12h, 24-36h, and 48-60h groups was the largest, while the fluorescence quantity in the 12-24h, 36-48h, and 60-72h groups was less. Figure 9As shown on the right, the flow cytometry results showed that the FITC positive ratio in the 0-12h, 24-36h, and 48-60h groups was the highest and significantly higher than that in the 12-24h, 36-48h, and 60-72h groups. The above results together indicate that the EGFP mRNA-LNP released within 12h after each irradiation is the most, and NBS Gel Pulse release of ray response can be achieved.

[0150] Control group 1 (NapS): 1 mg Nap-S-GFF P Pure YGG was dissolved in 2 mL of PBS to obtain a polypeptide solution.

[0151] Control group 2 (BenS): 3 mg Ben-S-GFF P YGG was dissolved in 2 mL of PBS to obtain a polypeptide solution.

[0152] Control group 3 (NapS:BenS=5:1): 5 mg Nap-S-GFF P Pure YGG and 1mg Ben-S-GFF P YGG was dissolved in 2 mL of PBS to obtain a polypeptide solution.

[0153] Control group 4 (NapS:BenS=3:1): 3 mg Nap-S-GFF P Pure YGG and 1mg Ben-S-GFF P YGG was dissolved in 2 mL of PBS to obtain a polypeptide solution.

[0154] Control group 5 (NapS:BenS=1:1): 1 mg Nap-S-GFF P Pure YGG and 1mg Ben-S-GFF P YGG was dissolved in 2 mL of PBS to obtain a polypeptide solution.

[0155] Control group 6 (NapS:BenS=1:5): 1 mg Nap-S-GFF P Pure YGG and 5mg Ben-S-GFF P YGG was dissolved in 2 mL of PBS to obtain a polypeptide solution.

[0156] Other experimental procedures are the same as those in 5.4.

[0157] The results are as follows Figure 10 As shown, it can be seen that when the ratio of NapS:BenS is 5:1, 3:1, and 1:3 and the irradiation condition is 6 Gy, pulse release can be achieved, and the pulse release effect is best when the ratio is 1:3.

[0158] 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 modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A radiation-responsive self-assembling polypeptide, characterized in that: The structure of the polypeptide is selected from any one of the following structures: Formula (II), Formula (III).

2. The method for preparing the radiation-responsive self-assembling polypeptide according to claim 1, wherein: The polypeptide is prepared by Fmoc solid phase synthesis.

3. Use of the radiation-responsive self-assembling polypeptide according to claim 1 in preparing a delivery system for treating tumor drugs or in preparing an adjuvant radiotherapy preparation.

4. A radiation-responsive polypeptide hydrogel, characterized in that: The hydrogel contains at least one of the polypeptides according to claim 1.

5. The radiation-responsive polypeptide hydrogel according to claim 4, characterized in that In the hydrogel, the mass ratio of the polypeptide of formula (II) to the polypeptide of formula (III) is 1:1-10.

6. The radiation-responsive polypeptide hydrogel according to claim 4, characterized in that The hydrogel is prepared by the following method: Mixing the polypeptide of formula (II) and the polypeptide of formula (III) to prepare a polypeptide solution; Alkaline phosphatase is added to the polypeptide solution to induce polypeptide self-assembly to form the radiation-responsive polypeptide hydrogel.

7. The method for preparing the radiation-responsive polypeptide hydrogel according to claim 4, characterized in that: The method comprises the following steps: Mixing the polypeptide of formula (II) and the polypeptide of formula (III) to prepare a polypeptide solution; Alkaline phosphatase is added to the polypeptide solution to induce polypeptide self-assembly to form the radiation-responsive polypeptide hydrogel.

8. A radiation-responsive pulsed drug delivery system, characterized in that: The drug delivery system comprises the hydrogel according to any one of claims 4 to 6 and lipid nanoparticles.

9. The radiation-responsive pulsed drug delivery system according to claim 8, characterized in that: The volume ratio of hydrogel and lipid nanoparticles was 10-1:

1.

10. The radiation-responsive pulsed drug delivery system according to claim 8, wherein: The drug delivery system further contains a radiotherapy adjuvant agent.

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