Polypeptide self-assembled nanoparticles as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of genetic engineering and biological medicine, and particularly discloses polypeptide self-assembled nanoparticles as well as a preparation method and application thereof. The polypeptide self-assembled nano-particle provided by the invention is mainly constructed by amphiphilic compound molecules based on polypeptide, and comprises two functional polypeptides: a targeting peptide (CR) based on tumor tissue high expression CXCR4, used for realizing precise targeting on tumor; a CD24 blocking peptide (CB) containing an MMP-2 responsive restriction enzyme cutting site releases LAG-CSBP polypeptide under the action of MMP-2 in a tumor microenvironment, so that the interaction of CD24 / Siglec-10 is blocked. Under the hydrophobic action force, a hydrophobic SLC7A11 inhibitor erastin is encapsulated by the two, and the nano-particles CBR and E are formed through self-assembly. According to the system, the tumor radiotherapy tolerance can be improved through multiple synergistic effects, and the treatment accuracy and safety are improved.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and biomedicine technology, and specifically relates to a polypeptide self-assembling nanoparticle and a preparation method thereof, as well as the application of the nanoparticle in preparing a drug for reversing the radiotherapy resistance and immune escape of tumor stem cells. Background Art

[0002] Radiation therapy (RT) is one of the three major treatments for malignant tumors, but its clinical efficacy is often limited by radioresistance. Studies have shown that cancer stem cells (CSCs) with the potential for self-renewal and multidirectional differentiation are the key biological basis for mediating radioresistance. CSCs can significantly reduce radiosensitivity by enhancing DNA damage repair capacity, upregulating the expression of the antioxidant stress response-related protein SLC7A11, and activating multiple survival signaling pathways. In addition to endogenous factors of the cell, CSCs can also inhibit macrophage-mediated phagocytosis through binding to the immunosuppressive receptor sialic acid-binding immunoglobulin-like lectin-10 (Siglec-10) through the highly expressed CD24 on the surface, promoting immune escape and limiting the cascade amplification of the anti-tumor effect of radiotherapy. This also makes CSCs one of the most challenging targets in current cancer treatment.

[0003] To improve the efficacy of anti-CSCs immunotherapy, Chinese patent application CN117959419A (Shenzhen Bay Laboratory) discloses a genetically engineered cell membrane nanoparticle that induces anti-tumor stem cell immunity, including nanoparticles with the function of alleviating tumor hypoxia (such as hMnO2 nanoparticles, catalase nanoparticles, MOF nanoparticles, graphyne nanoparticles, manganese iron oxide nanoparticles, etc.) and cell membrane vesicles coated thereon (specifically, cell membrane vesicles derived from cancer stem cells that are genetically engineered to overexpress SIRPα variants). These nanoparticles have both hypoxia alleviation and CD47-SIRPα immune checkpoint blockade functions, which can trigger powerful anti-tumor immunity, thereby eliminating CSCs and inhibiting tumor growth. In addition, Chinese patent application CN118593721A (Shanghai Institute of Ceramics, Chinese Academy of Sciences) also discloses a hydroxyethyl starch-stabilized CuET nanoparticle, which is formed by coating CuET nanocrystals with hydroxyethyl starch. The killing ability of CuET on tumor cells and cancer stem cells is enhanced by increasing the water solubility of CuET. However, none of the above solutions can effectively improve the tolerability of tumor radiotherapy. Summary of the Invention

[0004] The main technical problem solved by the present invention is to provide a polypeptide self-assembling nanoparticle that has the functions of targeting both tumors and CD24 / Siglec-10 immune checkpoints. The nanoparticle is mainly constructed by a polypeptide-based amphiphilic composite molecule and contains two functional polypeptides: a targeting peptide (CR) based on the high expression of CXCR4 in tumor tissue, which is used to achieve precise targeting of tumors; and a CD24 blocking peptide (CB) containing an MMP-2 responsive enzyme cleavage site, which releases the LAG-CSBP peptide under the action of MMP-2 in the tumor microenvironment, thereby blocking the CD24 / Siglec-10 interaction.

[0005] Secondly, the present invention provides a method for preparing polypeptide self-assembled nanoparticles.

[0006] Finally, the present invention provides an application of polypeptide self-assembled nanoparticles in the preparation of anti-tumor drugs.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] Polypeptide self-assembling nanoparticles, the raw materials for preparing the nanoparticles include: a first composite molecule and a second composite molecule;

[0009] The first composite molecule comprises a CXCR4 targeting peptide;

[0010] The second composite molecule comprises a CD24 / Siglec-10 blocking peptide.

[0011] As a preferred embodiment of the present invention, the first composite molecule further comprises a hydrophobic chain, which is connected to the CXCR4 targeting peptide to form a first (amphiphilic) composite molecule.

[0012] Specifically, the hydrophobic chain is a C16 (hexadecyl) chain.

[0013] Specifically, the CXCR4 targeting peptide is: LGASWHRPDK (SEQ ID NO: 1).

[0014] Specifically, the first complex molecule is: C16-GGG-LGASWHRPDK.

[0015] As a preferred embodiment of the present invention, the second composite molecule further comprises a DSPE-PEG chain, which is connected to the CD24 / Siglec-10 blocking peptide to form a second (amphiphilic) composite molecule.

[0016] Specifically, the DSPE-PEG chain is connected to the CD24 / Siglec-10 blocking peptide via an MMP-2 responsive enzyme cleavage site.

[0017] Specifically, the DSPE-PEG chain is DSPE-PEGn-NHS, where n=1000-3000.

[0018] Specifically, the MMP-2 responsive enzyme cleavage site is PLGLAG (SEQ ID NO: 2) or PGGGAG (SEQ ID NO: 5).

[0019] (Please confirm whether the description is appropriate. If it is inappropriate, please modify it directly)

[0020] Specifically, the CD24 / Siglec-10 blocking peptide (CSBP) is LDVFLYSE (SEQ ID NO: 3).

[0021] Specifically, the second complex molecule is: DSPE-PEG2000-GGG-PLGLAG-LDVFLYSE.

[0022] Specifically, the second complex molecule is: DSPE-PEG2000-GGG-PGGGAG-LDVFLYSE.

[0023] As a preferred embodiment of the present invention, the mass ratio of the first composite molecule to the second composite molecule is 1-2:1-2.

[0024] As a preferred embodiment of the present invention, the raw materials for preparing the nanoparticles further include a hydrophobic pharmacological component, such as an SLC7A11 inhibitor.

[0025] Specifically, the SLC7A11 inhibitors include but are not limited to erastin (a ferroptosis promoter that can target SLC7A11 to induce ferroptosis) and sulfasalazine.

[0026] As a preferred embodiment of the present invention, the mass ratio of the first composite molecule to the hydrophobic medicinal component is 1:0.1-0.6.

[0027] As a preferred embodiment of the present invention, the size of the nanoparticles is 50-200 nm.

[0028] The preparation method of polypeptide self-assembled nanoparticles comprises the following steps:

[0029] The first composite molecule, the second composite molecule and the hydrophobic medicinal component are mixed and self-assembled through hydrophilic-hydrophobic interaction to obtain nanoparticles.

[0030] As a preferred embodiment of the present invention, the first composite molecule is synthesized by Fmoc solid phase method, specifically: C16-GGG-LGASWHRPDK.

[0031] As a preferred embodiment of the present invention, the second composite molecule is prepared by reacting DSPE-PEGn-NHS (phospholipid polyethylene glycol active ester, n=1000-3000), M-CSBP (GGG-PLGLAG-LDVFLYSE, SEQ ID NO: 4) or NM-CSBP (GGG-PGGGAG-LDVFLYSE, SEQ ID NO: 6). Both M-CSBP and NM-CSBP can be synthesized by the Fmoc solid phase method.

[0032] Specifically, the second complex molecule is: DSPE-PEG2000-GGG-PLGLAG-LDVFLYSE.

[0033] Specifically, the second complex molecule is: DSPE-PEG2000-GGG-PGGGAG-LDVFLYSE.

[0034] Specifically, the mass ratio of the DSPE-PEGn-NHS to M-CSBP is 1:1-3.

[0035] As a preferred embodiment of the present invention, the method for preparing the nanoparticles comprises the following steps:

[0036] The first composite molecule, the second composite molecule and the SLC7A11 inhibitor are dissolved in DMSO, and then ultrasonically treated after adding water. During the ultrasonication process, the first composite molecule, the second composite molecule and the SLC7A11 inhibitor are self-assembled through hydrophilic-hydrophobic interaction forces, and nanoparticles are obtained after standing.

[0037] Specifically, the SLC7A11 inhibitor (such as erastin) is encapsulated in the hydrophobic core cavity of the nanoparticles due to its good hydrophobicity. After the ultrasonication, the solution is allowed to stand at room temperature for a period of time to further stabilize the formed nanoparticles.

[0038] Application of polypeptide self-assembled nanoparticles in the preparation of anti-tumor drugs.

[0039] As a preferred embodiment of the present invention, the application includes but is not limited to one or more of the following aspects:

[0040] (1) Application in the preparation of drugs for reversing the radiotherapy resistance and immune escape of tumor stem cells;

[0041] (2) Application in the preparation of drugs for enhancing the oxidative stress level of tumor tissues;

[0042] (3) Application in the preparation of drugs for promoting radiotherapy-mediated cell apoptosis;

[0043] (4) Application in the preparation of drugs for promoting the phagocytosis of CSCs by macrophages;

[0044] (5) Application in the preparation of drugs for inhibiting tumor growth (including reducing tumor weight and tumor volume).

[0045] As a preferred embodiment of the present invention, the anti-tumor drug further comprises other anti-tumor pharmaceutical components.

[0046] Beneficial effects of the present invention:

[0047] The polypeptide self-assembling nanoparticles provided by the present invention are mainly constructed from amphiphilic polypeptide-based composite molecules and contain two functional polypeptides: a targeting peptide (CR) based on the high expression of CXCR4 in tumor tissue, which is used to achieve precise tumor targeting; and a CD24 blocking peptide (CB) containing an MMP-2 responsive enzyme cleavage site. Under the action of MMP-2 in the tumor microenvironment, it releases the LAG-CSBP peptide, thereby blocking the CD24 / Siglec-10 interaction. Under hydrophobic interaction, the two encapsulate the hydrophobic SLC7A11 inhibitor erastin and self-assemble to form nanoparticles CBR@E. This system improves tumor radiotherapy tolerance through synergistic multiple effects: (1) inhibiting SLC7A11-mediated cystine / glutamate reverse transport, blocking the glutathione synthesis pathway to enhance radiotherapy sensitivity; (2) targeting and blocking the CD24 / Siglec-10 immune checkpoint pathway, reshaping the anti-tumor immune microenvironment.

[0048] The multifunctional peptide-based self-assembling nanoparticles CBR@E provided by the present invention simultaneously have the functions of tumor targeting, inhibiting SLC7A11, and targeting the CD24 / Siglec-10 immune checkpoint. The CXCR4 targeting peptide in the outer layer can accurately deliver chemotherapy drugs to the tumor site, improving treatment efficiency. At the same time, to achieve precise drug release at the tumor site, an MMP-2-specific enzyme cleavage sequence is introduced into the nanoparticles. This sequence can respond to the highly expressed MMP-2 enzyme in the tumor microenvironment and trigger the targeted release of the drug, thereby minimizing nonspecific damage to normal tissues, significantly improving the accuracy and safety of treatment, and achieving efficient targeted treatment effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Schematic diagram of the synthesis scheme of polypeptide self-assembled nanoparticles CBR@E in the examples.

[0050] Figure 2 Characterization of polypeptide self-assembled nanoparticles CBR@E in the experimental example.

[0051] Figure 3 is the ability of CBR@E to induce cellular oxidative stress in the experimental example.

[0052] Figure 4 In the experimental example, CBR@E effectively induced apoptosis of CT26 cells.

[0053] Figure 5 In the experimental example, CBR@E promotes the phagocytosis of tumor cells by macrophages.

[0054] Figure 6 The in vivo distribution of CBR@E in the experimental example.

[0055] Figure 7 In the experimental example, CBR@E effectively inhibited the progression of CT26 tumors.

[0056] To more clearly illustrate the technical solutions of the embodiments of the present invention, the above briefly introduces the drawings obtained in the experimental examples. It should be understood that the above drawings only illustrate certain experimental examples of the present invention and should not be construed as limiting the scope of protection of the claims. Those skilled in the art can also derive other relevant drawings based on these drawings without inventive effort. DETAILED DESCRIPTION

[0057] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments and experimental examples. However, it should be understood by those skilled in the art that the embodiments are only used to illustrate the technical solution of the present invention and should not be regarded as limiting the scope of protection of the present invention. Based on the following embodiments, all other embodiments obtained by those of ordinary skill in the art without making creative work, such as modifications, deformations, or simple replacements, should fall within the scope of protection of the present invention.

[0058] Unless otherwise specified, the experimental methods used in the following examples and experimental examples are all conventional methods; the raw materials (including biological materials), reagents, culture media, instruments, etc. used are all commonly used in the art and commercially available to the public unless otherwise specified; the terms and abbreviations involved have the conventional meanings in the art, such as PBS buffer is phosphate buffered saline.

[0059] Example

[0060] This embodiment provides a polypeptide self-assembled nanoparticle, the preparation raw materials include: a first composite molecule, a second composite molecule and a hydrophobic pharmacological component;

[0061] The first complex molecule is: C16-GGG-LGASWHRPDK;

[0062] The second complex molecule is: DSPE-PEG2000-GGG-PLGLAG-LDVFLYSE;

[0063] The hydrophobic pharmacological component is the SLC7A11 inhibitor erastin;

[0064] The mass ratio of the first composite molecule, the second composite molecule and the hydrophobic medicinal component is 1:1:0.4.

[0065] This embodiment also provides a method for preparing polypeptide self-assembled nanoparticles (synthesis scheme schematic diagram as shown in Figure 1 ), comprising the following steps:

[0066] (1) Preparation of polypeptide (including the first composite molecule CR peptide):

[0067] The CR (C16-GGG-LGASWHRPDK) peptide and the M-CSBP (GGG-PLGLAG-LDVFLYSE) peptide were synthesized by the Fmoc solid-phase method. The peptides were separated and purified by a reverse-phase high-performance liquid chromatography system. The purified peptides were identified by mass spectrometry to determine whether the sequences of the synthesized peptides were correct.

[0068] (2) Preparation of the second composite molecule (CB peptide):

[0069] Accurately weigh appropriate amounts of M-CSBP peptide and DSPE-PEG2000-NHS (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., commercial parameters: D163641-100 mg), the ratio of M-CSBP peptide to DSPE-PEG2000-NHS is 2:1 (mass ratio), and DMF is added to fully dissolve them. The pH of the peptide solution is adjusted to weak alkaline (about pH 8.0-8.5) with triethylamine, and the pH-adjusted peptide solution is slowly added dropwise to the DSPE solution. Maintain uniform stirring during the addition process to avoid excessive local concentration. Stir and react at room temperature in the dark for 8 hours; after the reaction is complete, the product is placed in a dialysis bag with a molecular weight cutoff of 3500 Da for dialyzation to remove free peptide segments. The dialyzed sample is freeze-dried and stored.

[0070] (3) Full vector construction

[0071] 0.5 mg of CB peptide, 0.5 mg of CR peptide and 0.2 mg of erastin were weighed and placed in a 2 mL EP tube. All components were fully dissolved with 50 μL of DMSO, and deionized water was added to make up the total volume to 1 mL. The EP tube containing the solution was placed in an ultrasonicator and sonicated at 40 kHz for 10 min at 25°C. During the ultrasonic process, CB, CR and erastin self-assembled into nanoparticles through hydrophilic-hydrophobic interactions. Erastin was encapsulated in the hydrophobic core cavity of the nanoparticles due to its good hydrophobicity. After the ultrasonication, the solution was allowed to stand at room temperature for 20 min to further stabilize the formed nanoparticles.

[0072] This embodiment also provides a use of polypeptide self-assembled nanoparticles in the preparation of a drug for reversing the radiotherapy resistance and immune escape of tumor stem cells.

[0073] In other embodiments of the present invention, the mass ratio of the first composite molecule, the second composite molecule, and the hydrophobic pharmaceutical component can be adjusted arbitrarily within a given range without substantially affecting the physicochemical properties, biological functions, and therapeutic efficacy of the nanoparticles. For example, the second composite molecule is: DSPE-PEG2000-GGG-PGGGAG-LDVFLYSE.

[0074] In other embodiments of the present invention, the raw materials and preparation parameters of the nanoparticles can be adjusted arbitrarily within a given range without substantially affecting the physicochemical properties, biological functions, and therapeutic effects of the nanoparticles. The synthesized polypeptide sequences are shown in Table 1.

[0075] Table 1 Synthesized peptide sequences

[0076]

[0077] Comparative Example

[0078] The preparation method of CBR is as follows: 0.5 mg of CB peptide and 0.5 mg of CR peptide were weighed and placed in a 2 mL EP tube, all components were fully dissolved with 50 μL of DMSO, and deionized water was added to make the total volume 1 mL; the EP tube containing the solution was placed in an ultrasonic instrument and sonicated at 40 kHz at 25°C for 10 min.

[0079] The preparation method of CB@E is as follows: 0.5 mg of CB peptide and 0.2 mg of erastin are weighed and placed in a 2 mL EP tube, all components are fully dissolved with 50 μL of DMSO, and deionized water is added to make the total volume 1 mL; the EP tube containing the solution is placed in an ultrasonicator and sonicated at 40 kHz at 25°C for 10 minutes.

[0080] The preparation method of CSR@E is as follows: 0.5 mg of CS peptide (CSBP: LDVFLYSE), 0.5 mg of CR peptide and 0.2 mg of erastin are weighed and placed in a 2 mL EP tube, all components are fully dissolved with 50 μL of DMSO, and deionized water is added to make the total volume 1 mL; the EP tube containing the solution is placed in an ultrasonicator and sonicated at 40 kHz at 25°C for 10 min.

[0081] The preparation method of CB@DIR is as follows: 0.5 mg of CB peptide and 200 μL of DIR are weighed and placed in a 2 mL EP tube, all components are fully dissolved with 50 μL of DMSO, and deionized water is added to make the total volume 1 mL; the EP tube containing the solution is placed in an ultrasonicator and sonicated at 40 kHz for 10 minutes at 25°C.

[0082] The preparation method of CBR@DIR is as follows: 0.5 mg of CB peptide, 0.5 mg of CR peptide and 200 μL of DIR are weighed and placed in a 2 mL EP tube, all components are fully dissolved with 50 μL of DMSO, and deionized water is added to make the total volume 1 mL; the EP tube containing the solution is placed in an ultrasonicator and sonicated at 40 kHz at 25°C for 10 min.

[0083] Experimental example

[0084] 1. Experimental Methods

[0085] 1. Zeta potential and particle size distribution: After the nanoparticles prepared in the example were ultrasonically treated, 1 mL of sample was placed in a potential cup and a particle size cup respectively to measure the particle size and potential. The results are as follows: Figure 2 shown.

[0086] TEM morphology characterization: Take a copper mesh with intact grid and place it in a bacterial dish covered with sealing film. Gently add 10μL of sample to the surface of the copper mesh and let it stand for 2 minutes to allow the sample to fully adsorb on the surface of the support film. Repeat 3 times. Then use qualitative filter paper to touch the edge of the copper mesh and carefully absorb the excess liquid. Add uranyl acetate solution to the surface of the copper mesh for negative staining. Let it stand for 1 minute to allow the dye to fully contact the sample. After drying, place the copper mesh in a fume hood to dry. Use TEM for observation and take pictures of representative fields of view. The results are as follows Figure 2 shown.

[0087] 2. MMP-2 enzyme cleavage assay: Weigh an appropriate amount of peptide, add TCNB buffer to dissolve to 200 μM, and mix with activated rhMMP-2 (0.2 μg / mL) in equal volumes to make the final concentration of peptide 100 μM and rhMMP-2 0.1 μg / mL. Place the reaction tube in a 37°C constant temperature incubator for 3 hours; centrifuge the mixture after enzyme cleavage at 12000 rpm and 4°C for 15 minutes, and take 100 μL of the supernatant to detect the effect of enzyme cleavage release by mass spectrometry. The results are as follows: Figure 2 shown.

[0088] 3. ROS content detection:

[0089] a) Plating of CT26 tumor spheres: CT26 tumor spheres induced to form spheres for 14 days were collected and digested with 0.5% trypsin at 37°C for 5 min. Digestion was terminated with DMEM medium containing 10% FBS, washed twice with PBS 7.2, and resuspended in dry culture medium and the cell density was adjusted to 2×10 4 cell / mL, cells were seeded in ultra-low adsorption 6-well plates (2 mL system per well);

[0090] b) Drug addition: CT26 cells and CT26 tumor spheres were collected and 2×10 5 The cells were plated at a density of 1000 cells / well in a 24-well plate. After 12 hours of cell stabilization, three replicate wells were set up in each group. After irradiation with 6 Gy for 48 hours, the cells were treated with different drugs for 12 hours. The experimental groups were as follows: PBS, RT, RT+E, RT+CB@E, and RT+CBR@E.

[0091] c) Dilute the ROS probe DCFH-DA to 10 μM using serum-free medium, add 50 μL of the diluted ROS probe to the 1.5 mL EP tube, and then incubate the 1.5 mL EP tube in a 37°C incubator for 20 minutes, inverting and mixing every 3-5 minutes to ensure full contact between the probe and the cells;

[0092] d) Wash the cells three times with serum-free culture medium to remove the extracellular residual DCFH-DA probe, take a portion of the cells and drop them onto a glass slide, select appropriate excitation and emission wavelengths, observe the fluorescence intensity using a fluorescence microscope, and transfer the remaining cells into a flow cytometry tube for flow cytometry detection. Figure 3 shown.

[0093] 4. CBR@E promotes cell apoptosis:

[0094] a) Digest CT26 cells in the logarithmic growth phase and adjust the cell number to 5×10 5 The cells were plated at a density of 1000 cells / well in a 6-well plate, and the plate was placed in a 37°C, 5% CO2 cell culture incubator for culture until the cells adhered to the wall.

[0095] b) After the cells were fully adhered, three replicates were set up in each group. The cells were irradiated with 6 Gy for 48 hours and then treated with different drugs for 12 hours. The experimental groups were as follows: PBS, RT, RT+E, RT+CB@E, and RT+CBR@E;

[0096] c) Collect the supernatant and cells in a 15 mL EP tube and centrifuge at 2000 rpm. Resuspend the cells in pre-chilled PBS 7.2 and transfer to a 1.5 mL EP tube for washing. Handle gently to avoid mechanical damage to the cells that could affect the accuracy of subsequent results.

[0097] d) Resuspend the cells in 200 μL of 1× Binding Buffer, add 5 μL of Annexin V (purchased from Hangzhou Lianke Biotechnology Co., Ltd.), gently vortex to mix, and incubate on ice in the dark for 15 min.

[0098] e) Add 10 μL of 7-AAD and incubate on ice in the dark for 5 minutes, transfer to the flow cytometry loading tube, and load the sample at a low speed for data analysis. Figure 4 shown.

[0099] 5. CSCs and BMDM co-culture experiment:

[0100] a) Induction of BMDM: The thighs and calves of mice were removed, and the bone marrow was blown out with a 1 mL syringe to disperse the bone marrow cells. The cell pellet was collected by centrifugation at 2000 rpm at 4°C for 5 min, and the cells were resuspended in 5 mL of 1× red blood cell lysis buffer and lysed at room temperature for 8 min. The cells were washed twice with PBS 7.2, the supernatant was removed, and the cells were resuspended in DMEM. The cytokine mGM-CSF (20 ng / mL) was added and cultured in an incubator for 7 days.

[0101] b) CSCs cultured for 13 days were irradiated with 6 Gy and then cultured in an incubator for 24 hours;

[0102] c) Macrophages induced to differentiate for 7 days were treated with serum-free medium for 2 hours, and then the cells were harvested, enriched by centrifugation, and the cells were counted and stored on ice until use;

[0103] d) CFSE staining: CSCs were digested into a single-cell suspension, centrifuged, and resuspended in 1 μM CFSE. The cells were incubated on ice in the dark for 10 min.

[0104] e) Stop staining: add pre-chilled serum to a final concentration of 30%, incubate on ice in the dark for 10 min, stop staining, and wash cells twice with DMEM;

[0105] f) Drug addition: The cells were treated with different drugs and the experimental groups were as follows: PBS, RT, RT+CB, RT+CBR@E, RT+CBR@E+rhMMP-2, RT+CSR@E, RT+CSR@E+rhMMP-2. After mixing, the cells were incubated on ice for 30 min.

[0106] g) adding equal proportions of macrophages to the above mixture, mixing well, plating the cells in a 24-well plate pre-placed with a sterilized cover slip, and incubating in an incubator overnight;

[0107] h) Discard the supernatant from the plate, wash twice with PBS 7.2, add anti-mouse CD11b APC antibody, incubate on ice in the dark for 30 minutes, wash away excess antibody with PBS 7.2, add 1 mL of 4% paraformaldehyde fixative to the side of the plate, fix at room temperature for 20 minutes, wash twice with PBS 7.2, add 1 mL of DAPI staining solution (working concentration 1 μg / mL), incubate in the dark for 10 minutes, and wash twice with PBS 7.2 after staining;

[0108] i) Add 10 μL of PBS 7.2 to the center of the slide, pick up the cover glass in the well plate with tweezers and place it upside down on the slide, and observe and photograph using a laser confocal microscope. Figure 5 shown.

[0109] 6. Study on the distribution of CBR@E in vivo:

[0110] a) Subcutaneous tumor-bearing mice: BALB / c female mice aged 6-8 weeks were selected for tumor-bearing, with a tumor load of 2×10 5 Use a pipette to gently pipette the cell suspension evenly, draw 200 μL of CT26 cell suspension with a 1 mL syringe, and carefully inoculate the cell suspension into the subcutaneous area on the right side of the mouse's back;

[0111] b) After successfully establishing the CT26 subcutaneous tumor model, monitor and record the growth of the mouse tumor. When the tumor volume grows to 150-200mm 3 Mice were randomly divided into three groups: free DIR group, CB@DIR group and CBR@DIR group. The drug concentration was quantitatively measured by DIR (0.5 mg / kg). The mice were anesthetized with gas at 1h, 4h, 8h, 12h, 24h and 48h after the injection of nanocarriers. After the mice were fully anesthetized, the distribution of nanocarriers in vivo was observed and recorded using a living imaging device. After 48h, the heart, liver, spleen, lungs, kidneys, brain and tumors of the mice were removed using surgical instruments for imaging. The results are shown in Figure 2. Figure 6 shown.

[0112] 7. Tumor-bearing mouse treatment model:

[0113] a) Establish a CT26 subcutaneous tumor-bearing mouse model. On the 8th day of tumor bearing, wait until the tumor grows to 40-80 mm. 3 At the same time, the mice were treated by tail vein injection and divided into the following groups: PBS group, RT group, RT+E group, RT+CSBP group, RT+CB group, RT+CB@E group and RT+CBR@E group;

[0114] b) Mice with dorsal tumors were treated with local radiotherapy at a dose of 6 Gy. 48 hours later, 200 μL of drug was drawn up using a 1 mL syringe and injected into the tail vein. The PBS group was injected with the same volume of PBS 7.2 as a control. The drug was administered once every 3 days for a total of 5 times.

[0115] c) During the dosing period, the mice were weighed and recorded every two days to facilitate the subsequent plotting of a weight change curve to observe the effect of the drug on the mice's weight. At the same time, a vernier caliper was used to measure the length (a) and width (b) of the dorsal tumor of each mouse. The formula: tumor volume = 0.5 × a × b 2 The tumor volume was calculated and the tumor growth curve was drawn. Figure 7 shown.

[0116] 2. Experimental Results

[0117] 1. Zeta potential, particle size distribution and morphology characterization

[0118] The physicochemical properties of the MMP-2 responsive peptide self-assembled nanocarriers CBR@E prepared in the example were characterized. Transmission electron microscopy (TEM) results showed that CBR and CBR@E had good dispersibility and exhibited a uniform spherical structure. Dynamic light scattering (DLS) measurements showed that the average hydrated particle sizes of CBR and CBR@E were 101.08±1.56nm and 112.06±1.29nm, respectively. Figure 2 A, B), after CBR was loaded with erastin, the zeta potential increased from 10.64±1.23mV to 32.63±1.86mV ( Figure 2 C), the above results all indicate the successful construction of CBR@E.

[0119] 2. MMP-2 enzyme responsiveness

[0120] To further evaluate whether the formation of nanoparticles affects the enzymatic response properties of the peptide, CBR@E was co-incubated with MMP-2 enzyme for 3 h. The results of LC-MS / MS showed that a new elution peak appeared on the liquid chromatography peak after CBR@E was treated with enzymes. Further analysis showed that its molecular weight was consistent with that of LAG-CSBP ( Figure 2The above results indicate that CBR@E can respond to MMP-2 enzyme and release the product LAG-CSBP.

[0121] 3. CBR@E induces cells to produce ROS

[0122] To verify the radiosensitization effect of CBR@E nanoparticles, the DCFH-DA fluorescent probe was used to detect the level of intracellular ROS. Fluorescence microscopy imaging results showed that the fluorescence intensity of intracellular ROS in the CBR@E combined with radiotherapy group was significantly enhanced compared with the radiotherapy group alone ( Figure 3 A). Flow cytometry was used to accurately quantify the intracellular ROS level. The results showed that the average fluorescence intensity of intracellular ROS in the free drug combined with radiotherapy group increased by 2.66 times compared with the radiotherapy group alone, indicating that erastin can effectively enhance the intracellular ROS level; the CBR@E combined with radiotherapy group increased by 4.23 times compared with the radiotherapy group alone and by 1.59 times compared with the free drug group. It is worth noting that compared with the CB@E group with unmodified targeting ligand, the CBR@E group improved the cellular uptake efficiency due to the modification of CXCR4 targeting peptide, which led to a significant increase in the intracellular ROS level ( Figure 3 B). This result confirms the important role of targeting peptide modification in enhancing drug delivery efficiency. Compared with the radiotherapy group alone, CBR@E treatment significantly increased the level of ROS in CSCs, reaching 2.4 times that of the radiotherapy group alone ( Figure 3 C).

[0123] 4. CBR@E promotes cell apoptosis

[0124] Annexin V / 7-AAD double staining flow cytometry was used to quantitatively evaluate cell apoptosis in different treatment groups. The results showed that the cell apoptosis rate in the free erastin combined with radiotherapy group was significantly increased by 2.88 times compared with the radiotherapy alone group, confirming that erastin promotes radiotherapy-induced cell apoptosis by enhancing cellular oxidative stress. The CBR@E combined with radiotherapy group induced the highest cell apoptosis rate (27.15% ± 1.8%), which was significantly higher than that of the CB@E group (15.12% ± 0.3%), and was 1.79 times higher than that of the CB@E group ( Figure 4 A, B).

[0125] 5. CBR@E promotes macrophage phagocytosis of CSCs

[0126] To verify the regulatory effect of CBR@E on the ability of macrophages to phagocytose CSCs, an in vitro co-culture system was constructed. Primary macrophages isolated and purified from mouse bone marrow were used as effector cells, and tumor cells and CSCs labeled with CFSE fluorescent dye were used as target cells. The phagocytic activity of macrophages was verified by laser confocal microscopy and flow cytometry. Laser confocal imaging results showed that after CBR@E treatment, the phagocytic activity of CD11b-labeled macrophages (red fluorescence) towards CFSE-labeled CSCs (green fluorescence) was significantly enhanced, as manifested by the strongest green fluorescence signal ( Figure 5 A). Further flow cytometry results showed that after CBR@E treatment, the phagocytic rate of CSCs by macrophages increased to 2.1 times that of the control group ( Figure 5 These results indicate that the CBR@E nano-drug delivery system can effectively inhibit the CD24 / Siglec-10 interaction and relieve CD24-mediated immune escape, demonstrating its potential application value in enhancing anti-tumor immune responses.

[0127] 6. Distribution of CBR@E in vivo

[0128] In order to evaluate the in vivo tumor targeting of nanoparticles, a CT26 subcutaneous tumor-bearing mouse model was constructed, and the in vivo distribution of nanoparticles labeled with the near-infrared fluorescent dye DIR was studied. The experiment was divided into three groups: free DIR, CB@DIR, and CBR@DIR. After a single tail vein administration, the accumulation of nanoparticles in the tumor site was dynamically monitored at specific time points (2, 4, 8, 12, 24 and 48h) using an in vivo fluorescence imaging system (IVIS Spectrum). The results showed that in the targeted nanoparticle (CBR@DIR) group, obvious fluorescence signals could be detected at the tumor site 2h after administration, and the signal intensity gradually increased over time, reaching a peak at 12h ( Figure 6 A). 48 hours after administration, the heart, liver, spleen, lung, kidney, and tumor tissues of the mice were collected for in vitro fluorescence imaging analysis. The results showed that the fluorescence intensity of the CBR@DIR group in the tumor tissue was significantly higher than that of the CB@DIR group, about twice that of the latter ( Figure 6 B, C). These results demonstrate that the CBR@DIR nanocarrier system exhibits excellent tumor tissue-selective distribution. This nanoformulation effectively enhances drug accumulation at the lesion site, providing an excellent delivery platform for precision medicine.

[0129] 7. Inhibitory effect of CBR@E combined with radiotherapy on CT26 tumor growth

[0130] Based on the significant tumor cell growth inhibition effect and excellent tumor targeting performance of CBR@E in in vitro experiments, a CT26 subcutaneous tumor-bearing mouse model was further constructed to systematically evaluate its anti-tumor activity in vivo. On the 8th day after tumor inoculation, the tumor-bearing mice were randomly divided into 7 groups: PBS, RT, RT+E, RT+CSBP, RT+CB, RT+CB@E and RT+CBR@E. Except for the PBS control group, the mice in the other groups received 6Gy of local low-dose radiotherapy for the tumor. 48 hours after radiotherapy, the corresponding drug was injected into the tail vein for treatment. Thereafter, the drug was repeated every three days for a total of 5 treatment cycles ( Figure 7 A). During the entire treatment process, the weight changes and tumor growth of the mice were regularly monitored. The experimental results showed that there was no significant change in the weight of the mice in each group during the entire treatment cycle ( Figure 7 B). By analyzing tumor growth curves and in vitro tumor weight ( Figure 7 CE), the results showed that compared with the PBS control group, all treatment groups showed varying degrees of tumor growth inhibition. Among them, the radiotherapy group (RT) alone showed only a weak anti-tumor effect due to the low radiotherapy dose, suggesting the limitations of single low-dose radiotherapy in inhibiting tumor growth. In the combined treatment group, although both the free drug group and the free CSBP blocking peptide group showed a certain degree of tumor growth inhibition, their effects were relatively limited. In the CBR@E+RT group, the anti-tumor activity was most significant, as shown by a significant inhibition of tumor growth, and some tumors even regressed. Because CBR@E can effectively enhance the oxidative stress level of tumor cells, thereby synergizing radiotherapy-mediated cell apoptosis, the DCFH-DA fluorescent probe was used to detect the level of ROS generation in each group of tumor tissues, and TUNEL staining (green fluorescence) was used to evaluate the apoptosis in tumor tissues. The results showed that compared with the RT group and other control groups, the ROS fluorescence intensity of the CBR@E+RT group was significantly enhanced ( Figure 7 F, G). Cell apoptosis was further evaluated by TUNEL staining (green). The results showed that the cell apoptosis induced by the CBR@E+RT group was significantly higher than that of the other groups, as manifested by a significant increase in the green fluorescence signal in the tumor tissue ( Figure 7 These results demonstrate that CBR@E significantly promotes radiotherapy-induced apoptosis by enhancing oxidative stress in tumor tissue, resulting in a synergistic anti-tumor effect. This enhanced apoptosis is likely triggered by ROS-mediated oxidative damage, consistent with previous in vitro results.

[0131] Although the technical solution of the present invention has been described in detail above using general descriptions, specific embodiments, and experimental examples, it should be noted that the embodiments and experimental examples are only intended to illustrate the technical solution and technical effects of the present invention and should not be construed as limiting the scope of protection of the present invention. Simple variations, modifications, or improvements based on the technical concept of the present invention are all within the scope of protection claimed by the present invention.

Claims

1. Polypeptide self-assembled nanoparticles, characterized by: The raw materials for preparing the nanoparticles include: a first composite molecule and a second composite molecule; The first composite molecule comprises a CXCR4 targeting peptide; The second composite molecule comprises a CD24 / Siglec-10 blocking peptide.

2. The polypeptide self-assembling nanoparticles according to claim 1, characterized in that: The first composite molecule further comprises a hydrophobic chain, which is connected to the CXCR4 targeting peptide to form the first composite molecule; The hydrophobic chain is a C16 chain; The CXCR4 targeting peptide is: LGASWHRPDK; And / or, the second composite molecule further comprises a DSPE-PEG chain, which is connected to the CD24 / Siglec-10 blocking peptide via an MMP-2 responsive enzyme cleavage site to form a second composite molecule; The DSPE-PEG chain is DSPE-PEGn-NHS, n=1000-3000; The MMP-2 responsive enzyme cleavage site is PLGLAG or PGGGAG; The CD24 / Siglec-10 blocking peptide is: LDVFLYSE.

3. The polypeptide self-assembling nanoparticles according to claim 1, characterized in that: The first complex molecule is: C16-GGG-LGASWHRPDK; And / or, the second complex molecule is: DSPE-PEG2000-GGG-PLGLAG-LDVFLYSE; And / or, the second complex molecule is: DSPE-PEG2000-GGG-PGGGAG-LDVFLYSE; The mass ratio of the first composite molecule to the second composite molecule is 1-2:1-2.

4. The polypeptide self-assembling nanoparticles according to claim 1, characterized in that: The raw materials for preparing the nanoparticles also include a hydrophobic pharmacological component; The mass ratio of the first composite molecule to the hydrophobic medicinal component is 1:0.1-0.

6.

5. The polypeptide self-assembling nanoparticles according to claim 4, characterized in that: The SLC7A11 inhibitors include but are not limited to erastin and sulfasalazine.

6. The polypeptide self-assembling nanoparticles according to claim 1, characterized in that: The size of the nanoparticles is 50-200 nm.

7. The method for preparing polypeptide self-assembled nanoparticles according to any one of claims 1 to 6, characterized in that: The following steps are involved: The first composite molecule, the second composite molecule and the hydrophobic medicinal component are mixed and self-assembled through hydrophilic-hydrophobic interaction to obtain nanoparticles.

8. The preparation method according to claim 7, characterized in that: The method for preparing the nanoparticles comprises the following steps: The first composite molecule, the second composite molecule and the SLC7A11 inhibitor are dissolved in DMSO, and then ultrasonically treated after adding water. During the ultrasonication process, the first composite molecule, the second composite molecule and the SLC7A11 inhibitor are self-assembled through hydrophilic-hydrophobic interaction forces, and nanoparticles are obtained after standing.

9. Use of the polypeptide self-assembled nanoparticles according to any one of claims 1 to 6 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that: The applications include but are not limited to one or more of the following aspects: (1) Application in the preparation of drugs for reversing the radiotherapy resistance and immune escape of tumor stem cells; (2) Application in the preparation of drugs for enhancing the oxidative stress level of tumor tissues; (3) Application in the preparation of drugs for promoting radiotherapy-mediated cell apoptosis; (4) Application in the preparation of drugs for promoting the phagocytosis of CSCs by macrophages; (5) Application in the preparation of drugs for inhibiting tumor growth; And / or, the anti-tumor drug further comprises other anti-tumor pharmaceutical components.

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