Nanoparticle with surface modified with double polypeptides as well as preparation method and application of nanoparticle

Abstract

The invention provides a nanoparticle with the surface modified with double polypeptides as well as a preparation method and application of the nanoparticle, and belongs to the technical field of nanoparticles. The preparation method of the nanoparticle with the surface modified with the double polypeptides comprises the following steps: preparing the nanoparticle by taking an aggregation-induced emission material as a mother nucleus and a surfactant as a carrier, and connecting Pep47 and PepP polypeptides to the surface of the nanoparticle to obtain the nanoparticle with the surface modified with the double polypeptides. The nanoparticles with the surfaces modified with the double polypeptides have multifunctionality, can combine two modes of inducing cell immunogenicity death (ICD) and bispecific immune checkpoint blocking (ICB), enhance the anti-tumor immunity of a body and show a strong anti-tumor effect, and meanwhile, fluorescence signals of AIE molecules can deeply trace tumor boundaries.

Description

Technical Field

[0001] The present invention relates to the technical field of nanoparticles, and in particular to a nanoparticle with a surface modified with a double polypeptide, and a preparation method and application thereof. Background Art

[0002] Antibodies refer to proteins with protective effects produced by the body due to the stimulation of antigens. They have the functions of immune regulation, anti-infection protection, tumor treatment and disease diagnosis. However, the production cost of antibodies, especially monoclonal antibodies and bispecific antibodies, is high, and requires a complex cell culture process, which is time-consuming and expensive. Antibodies may also cause immune responses, causing patients to produce anti-drug antibodies, affecting the treatment effect. Due to the large molecular weight of antibodies, it is difficult for them to pass through the cell membrane and enter the cell, so their effect is limited when targeting intracellular targets. Antibodies may degrade in vivo and in vitro, affecting their effectiveness and storage life. Bispecific antibodies have a complex structure, are difficult to design and optimize, and have technical challenges in production and purification. It can be seen that the current application of antibodies in tumor treatment has the defects of high production cost, immunogenicity, difficulty in drug delivery, poor stability and high complexity.

[0003] Peptides are small molecule active substances with relatively simple synthesis process, low cost and easy to mass produce. Peptides generally have low immune activity and are not easy to cause immune response. They are easy to pass through cell membranes and effectively target intracellular targets. The stability of peptides can be significantly improved and the half-life of peptides in the body can be extended through modification. Peptide sequences can also be modified and optimized according to needs to increase their specific affinity. It can be seen that compared with antibodies, peptides have the advantages of low production cost, low immunogenicity, strong targeting ability, good stability and easy modification and optimization.

[0004] Therefore, modifying peptides onto nanoparticles to simulate bispecific antibodies in clinical practice may have better biocompatibility and safety compared to the direct use of bispecific antibodies, and can provide a new and effective method for the treatment of tumors and the diagnosis of diseases. Summary of the invention

[0005] The purpose of the present invention is to provide a nanoparticle with a surface modified with a double polypeptide and a preparation method and application thereof, so as to provide a new method for the treatment of tumors and the diagnosis of diseases.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing nanoparticles with dual polypeptides modified on the surface, wherein the nanoparticles are prepared by using an aggregation-induced luminescence material as a parent core and a surfactant as a carrier, and then Pep47 and PepP polypeptides are connected on the surface of the nanoparticles to obtain nanoparticles with dual polypeptides modified on the surface;

[0008] The amino acid sequence of the Pep47 polypeptide is shown in SEQ ID NO.1;

[0009] The amino acid sequence of the PepP polypeptide is shown in SEQ ID NO.2.

[0010] Preferably, the aggregation-induced emission material is any one of 2,4,6-tris{(diphenylamino)phenylvinyl}-methylpyridinium salt, tetraphenylethylene, and triphenylamine; and the surfactant is an amphiphilic surfactant.

[0011] Preferably, the amphiphilic surfactant is DSPE-PEG2000-Mal.

[0012] Preferably, the method for preparing nanoparticles comprises the following steps:

[0013] (1) dissolving an aggregation-induced emission material and a surfactant in an organic solvent, and adding the solvent to ultrapure water under ultrasonic conditions to obtain a nanoparticle solution containing the aggregation-induced emission material;

[0014] (2) The nanoparticle solution containing the aggregation-induced emission material obtained in step (1) is dialyzed and subjected to centrifugal ultrafiltration to separate the solid from the liquid, and the liquid portion is collected to obtain a purified nanoparticle solution.

[0015] Preferably, in step (1), the mass ratio of the aggregation-induced emission material to the surfactant is 0.8-1.2:2.5-3.5; the volume ratio of the total mass of the aggregation-induced emission material and the surfactant to the organic solvent is 3.5-4.5 mg:0.8-1.2 mL; the organic solvent is tetrahydrofuran or dimethyl sulfoxide; the power of the ultrasound is 12-20 W, the ultrasound is performed for 6-8 seconds and then stopped for 2-4 seconds, and the total ultrasound time is 2.5-3.5 min; the volume of the ultrapure water is 8-10 times the volume of the organic solvent;

[0016] The molecular weight cutoff during dialysis in step (2) is 3000-4000Da, and the dialysis time is 28-44h; the molecular weight cutoff during the centrifugal ultrafiltration is 80-120kDa, the rotation speed is 4000-6000rpm, the number of times is 3-7 times, and the time of each centrifugal ultrafiltration is 10-20min; the solid-liquid separation is filtration, and the pore size of the filter membrane used in the filtration is 0.45μm.

[0017] Preferably, when Pep47 and PepP polypeptides are connected to the surface of nanoparticles, a solution containing Pep47 and PepP polypeptides is mixed with a nanoparticle solution and reacted, and then dialyzed and centrifuged and ultrafiltered to obtain nanoparticles with dual polypeptides modified on the surface.

[0018] Preferably, the mass ratio of Pep47 and PepP in the solution containing Pep47 and PepP polypeptides is 4-6:1; the concentration of the solution containing Pep47 and PepP polypeptides is 3-5 mmol / L; the volume ratio of the solution containing Pep47 and PepP polypeptides and the nanoparticle solution when mixed is 0.8-1.2:0.8-1.2; the concentration of the nanoparticle solution is 1.5-2.5 mmol / L; the temperature during the reaction is 23-27°C, the rate is 150-250 rpm, and the time is 10-14 h; the molecular weight cutoff during dialysis is 3000-4000 Da, and the dialysis time is 20-28 h; the molecular weight cutoff during centrifugal ultrafiltration is 80-120 kDa, the rotation speed is 4000-6000 rpm, and the time is 10-20 min.

[0019] The invention provides a surface-modified dual-polypeptide nanoparticle prepared according to the method.

[0020] The present invention also provides a reagent comprising nanoparticles with the surface-modified dual polypeptides.

[0021] The present invention further provides the use of the nanoparticles or the reagent in preparing products for killing tumor cells.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The present invention packages the sonogenic AIE material into nanoparticles, and modifies the dual polypeptides targeting immune checkpoints CD47 and PD-L1 on the surface thereof, thereby simulating the bispecific antibodies in clinical practice. The surface-modified dual polypeptide nanoparticles prepared by the present invention are multifunctional, and can combine the two methods of inducing cell immunogenic death (ICD) and bispecific immune checkpoint blockade (ICB), thereby enhancing the body's anti-tumor immunity and demonstrating a powerful anti-tumor effect. At the same time, the fluorescent signal of the AIE molecule can deeply trace the tumor boundary.

[0024] 2. The nanoparticles prepared by the present invention are efficiently and rapidly enriched at the tumor site in vivo, and the metabolic time after the action is short, which reduces the risk of long-term accumulation and has good biocompatibility and in vivo safety.

[0025] 3. The nanoparticles prepared by the present invention have a certain biosafety in the absence of light and ultrasound through in vitro CCK8 experiments, and can efficiently kill tumor cells under ultrasound conditions. Immunofluorescence, Western Blot and ATP detection results show that nanoparticles can efficiently produce ROS in cells under ultrasound conditions, inducing cell ICD (immunogenic death). In vivo experiments show that in a mouse bilateral tumor model, the sonodynamic therapy mediated by the nanoparticles prepared by the present invention can effectively play a tumor-suppressing role, stimulate immune response and prevent immune escape, showing a systemic anti-tumor immunotherapy effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0027] Figure 1 is the structural formula of 3TPA-E-Py in Example 1;

[0028] Figure 2 The detection results of intracellular ROS in Example 2;

[0029] Figure 3 This is the detection result of the calreticulin externalization level in Example 2, and the scale bar in the figure is 50 μm;

[0030] Figure 4 The detection result of ATP in the cell supernatant in Example 2;

[0031] Figure 5 The results of detecting HMGB1 and HSP70 proteins in the cell supernatant in Example 2;

[0032] Figure 6 The particle size distribution (left) and apparent morphology (right, scale bar is 200 μm) of the nanoparticles in Example 4;

[0033] Figure 7 The potential measurement result of the nanoparticles prepared in Example 3 in Example 4;

[0034] Figure 8 The promoting effect of different nanoparticles on BMDC maturation ability in Example 5;

[0035] Fig. 9 This is a flow cytometric quantitative graph of Raw264.7 engulfing CSFE-labeled SCC7 cells in Example 5;

[0036] Fig.10 The killing effect of different dual polypeptide ratios on tumor cells in Example 6;

[0037] Fig.11 The results of measuring cell viability after culturing SCC7 cells with nanoparticle solutions of different concentrations in Example 7;

[0038] Fig.12 The comparison results of blood routine and liver function indexes of mice in different treatment groups in Example 8;

[0039] Fig.13 The results of H&E staining of liver, spleen and kidney sections of mice in different treatment groups in Example 8;

[0040] Fig.14 This is a typical fluorescent image of the tumor site of the same mouse after the tail vein injection of nanoparticles in Example 9;

[0041] Fig.15 is the relationship curve between fluorescence intensity and time after injection in Example 9;

[0042] Fig.16 The tumor growth curves of mice in different treatment groups after tail vein injection of nanoparticles in Example 9;

[0043] Fig.17 The volume of the primary tumor of the mouse in Example 9 is about 50 mm 3 The growth curve of the tumor

[0044] Fig.18 These are the results of related immune index determination obtained by flow cytometry analysis in Example 9. DETAILED DESCRIPTION

[0045] The present invention provides a method for preparing nanoparticles with dual polypeptides modified on the surface, wherein the nanoparticles are prepared by using an aggregation-induced luminescence material as a parent core and a surfactant as a carrier, and then Pep47 and PepP polypeptides are connected on the surface of the nanoparticles to obtain nanoparticles with dual polypeptides modified on the surface;

[0046] The amino acid sequence of the Pep47 polypeptide is: D C D F D FG D A D W D SATWSNYW D R D H;

[0047] The amino acid sequence of the PepP polypeptide is: D C D F D FG D ND Y D S D K D P D T D D D R D Q D Y D H D F.

[0048] In the present invention, the aggregation-induced emission material is any one of 2,4,6-tris{(diphenylamino)phenylvinyl}-methylpyridinium salt, tetraphenylethylene, and triphenylamine, preferably 2,4,6-tris{(diphenylamino)phenylvinyl}-methylpyridinium salt; and the surfactant is an amphiphilic surfactant.

[0049] In the present invention, the amphiphilic surfactant is DSPE-PEG2000-Mal.

[0050] In the present invention, the method for preparing nanoparticles comprises the following steps:

[0051] (1) dissolving an aggregation-induced emission material and a surfactant in an organic solvent, and adding the solvent to ultrapure water under ultrasonic conditions to obtain a nanoparticle solution containing the aggregation-induced emission material;

[0052] (2) The nanoparticle solution containing the aggregation-induced emission material obtained in step (1) is dialyzed and subjected to centrifugal ultrafiltration to separate the solid from the liquid, and the liquid portion is collected to obtain a purified nanoparticle solution.

[0053] In the present invention, the mass ratio of the aggregation-induced emission material and the surfactant in step (1) is 0.8-1.2:2.5-3.5, preferably 0.9-1.1:2.8-3.2, and more preferably 1:3; the volume ratio of the total mass of the aggregation-induced emission material and the surfactant to the organic solvent is 3.5-4.5 mg:0.8-1.2 mL, preferably 3.8-4.2 mg:0.9-1.1 mL, and more preferably 4 mg:1 mL; the organic solvent is tetrahydrofuran or dimethyl sulfoxide, preferably tetrahydrofuran; the power of the ultrasound is 12-20 W, preferably 14-18 W, more preferably 15-17 W, and more preferably 16 W, and the ultrasound is stopped for 2-4 seconds every 6-8 seconds, and preferably for 3 seconds every 7 seconds, and the total ultrasound time is 2.5-3.5 min, preferably 2.8-3.2 min, and more preferably 3 min; the volume of the ultrapure water is 8-10 times the volume of the organic solvent, and preferably 9 times;

[0054] The molecular weight cutoff during dialysis in step (2) is 3000-4000Da, preferably 3200-3800Da, more preferably 3400-3600Da, and more preferably 3500Da; the dialysis time is 28-44h, preferably 30-42h, more preferably 32-40h, and more preferably 36h; the molecular weight cutoff during the centrifugal ultrafiltration is 80-120kDa, preferably 90-110kDa, and more preferably 100kDa; the rotation speed is 4000-6000rpm, preferably 4500-5500rpm, and more preferably 5000rpm; the number of times is 3-7 times, preferably 4-6 times, and more preferably 5 times; the time of each centrifugal ultrafiltration is 10-20min, preferably 12-18min, more preferably 14-16min, and more preferably 15min; the solid-liquid separation is filtration, and the pore size of the filter membrane used in the filtration is 0.45μm.

[0055] In the present invention, when Pep47 and PepP polypeptides are connected to the surface of nanoparticles, a solution containing Pep47 and PepP polypeptides is mixed with a nanoparticle solution and reacted, and then dialyzed and centrifuged and ultrafiltered to obtain nanoparticles with double polypeptides modified on the surface.

[0056] In the present invention, the mass ratio of Pep47 to PepP in the solution containing Pep47 and PepP polypeptides is 4-6:1, preferably 5:1; the concentration of the solution containing Pep47 and PepP polypeptides is 3-5mmol / L, preferably 4mmol / L; the volume ratio of the solution containing Pep47 and PepP polypeptides and the nanoparticle solution when mixed is 0.8-1.2:0.8-1.2, preferably 0.9-1.1:0.9-1.1, and more preferably 1:1; the concentration of the nanoparticle solution is 1.5-2.5mmol / L, preferably 2mmol / L; the temperature during the reaction is 23-27°C, preferably 24-26°C, and more preferably 25°C, and the speed is 150-250rpm, preferably 170-230rpm, and more preferably 190-21 0rpm, more preferably 200rpm, time is 10-14h, preferably 11-13h, more preferably 12h; the molecular weight cutoff during dialysis is 3000-4000Da, preferably 3200-3800Da, more preferably 3400-3600Da, more preferably 3500Da, and the dialysis time is 20-28h, preferably 22-26h, more preferably 24h; the molecular weight cutoff during centrifugal ultrafiltration is 80-120kDa, preferably 90-110kDa, more preferably 100kDa, the rotation speed is 4000-6000rpm, preferably 4500-5500rpm, more preferably 5000rpm, the time is 10-20min, preferably 12-18min, more preferably 14-16min, more preferably 15min.

[0057] The invention provides a surface-modified dual-polypeptide nanoparticle prepared according to the method.

[0058] The present invention also provides a reagent comprising nanoparticles with the surface-modified dual polypeptides.

[0059] The present invention further provides the use of the nanoparticles or the reagent in preparing products for killing tumor cells.

[0060] The technical solutions provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention. The SCC7 cells used in the following examples were purchased from Hangzhou Junke Biotechnology Co., Ltd.

[0061] Example 1

[0062] A method for preparing nanoparticles, comprising the following steps:

[0063] (1) 1.0 mg of 2,4,6-tris{(diphenylamino)phenylvinyl}-methylpyridinium salt (3TPA-E-Py, abbreviated as 3TEP, with the structural formula Figure 1 As shown, the sonogenic AIE material) and 3.0 mg of phospholipid polyethylene glycol maleimide (DSPE-PEG2000-Mal) were dissolved in 1 mL of tetrahydrofuran (THF) to obtain a mixed solution;

[0064] (2) the mixed solution was added dropwise into 9 mL of ultrapure water under the ultrasonic action of a micro-tip probe sonicator, the ultrasonic power was 16 W, the ultrasonic was continued for 7 seconds and then stopped for 3 seconds, and the total time was 3 minutes, to prepare a clear and transparent solution containing AIE nanoparticles;

[0065] (3) The solution containing AIE nanoparticles was placed in a dialysis bag with a molecular weight cutoff of 3500Da and dialyzed for 36h to further replace the organic solvent in the nanoparticle solution. The retentate was then placed in an ultrafiltration centrifuge tube with a molecular weight cutoff of 100kDa and centrifuged and washed repeatedly at 5000rpm for 5 times, each time for 15min, to remove the residual organic solvent and excess molecules.

[0066] (4) The retentate in the ultrafiltration centrifuge tube was purified by filtration using a 0.45 μm filter membrane, and the volume was fixed to 2 mmol / L with ddH2O to obtain a purified nanoparticle solution, which was recorded as NPs and placed in a 4°C refrigerator for future use.

[0067] Example 2

[0068] The purpose of this example is to detect the effect of the NPs prepared in Example 1 on the immunogenic death of tumor cells.

[0069] 1. Immunofluorescence detection of intracellular ROS

[0070] In order to further explore the ability of NPs to generate reactive oxygen species (ROS) in SCC7 cells under ultrasound, DCFH-DA was used as an indicator for ROS detection. DCFH-DA can be converted into 2',7'-dichlorofluorescein (DCF) that emits green fluorescence under the oxidation of ROS, thereby realizing the visualization of ROS levels.

[0071] SCC7 cells were cultured at 1×10 5The cells were cultured at a density of 100 μM NPs / mL in a confocal dish. Three treatment groups were set up. After the cells adhered to the wall, they were washed three times with sterile PBS. The first group was added with serum-free culture medium, the second group was added with serum-free culture medium containing 20 μM NPs, and the third group was added with serum-free culture medium containing 20 μM HMME. The three treatment groups were placed in an incubator (5% CO2, 37°C) and incubated for 4 hours, then washed three times with sterile PBS. After that, serum-free culture medium containing 20 μM DCFH-DA was added to each group of cells for staining for 30 minutes, and then washed three times with sterile PBS. The three groups of cells after the above treatment were ultrasonically treated for 3 minutes (1 MHz, 50% duty cycle, 0.5 W / cm 2 ), the cells were imaged using a confocal laser scanning microscope (CLSM) to detect the fluorescence of DCF (excitation wavelength: 488 nm, emission wavelength: 530±20 nm). The above process was carried out under light-protected conditions.

[0072] Test results such as Figure 2 As shown, it can be seen that cells treated with ultrasound with only DCFH-DA did not show obvious green fluorescence signals, indicating that in the absence of external drug effects, the level of ROS in cells was low when cells were treated with ultrasound alone; cells treated with NPs showed significantly enhanced green fluorescence signals, which far exceeded the fluorescence emitted by cells treated with the same concentration of traditional sonosensitizer HMME. This comparison result intuitively proves that NPs can efficiently generate ROS in SCC7 cells under ultrasound excitation, and its effect is significantly better than the existing sonosensitizer HMME on the market.

[0073] 2. Immunofluorescence detection of ecto-CRT eversion level

[0074] SCC7 cells were cultured at 1×10 5 The cells were cultured at a density of 100 μg / mL in a confocal dish. Three treatment groups were set up. When the cells attached to the wall, the cell culture medium in the second group was replaced with a serum-free medium containing 20 μM NPs, and the cell culture medium in the third group was replaced with a serum-free medium containing 20 μM HMME and then incubated for 4 h. After washing with PBS, the cells were replaced with new serum-free medium. The cells in the three treatment groups were treated with ultrasound for 3 minutes (1 MHz, 50% duty cycle, 0.5 W / cm 2 After 12 hours of continuous culture in the incubator, the cells were washed three times with sterile PBS, fixed on ice for 20 minutes with 4% paraformaldehyde, washed three times with PBS, and anti-calreticulin antibody (ab2907, diluted 1:200 in 1×PBS) was added to the cells and kept overnight at 4°C. The cells were washed three times with PBS and fixed on ice for 20 minutes with 4% paraformaldehyde. The conjugated secondary antibody (1×PBS diluted 1:200) was continued to be incubated at room temperature for 2 hours, washed three times with PBS, stained with 10μg / mL DAPI for 30 minutes, washed twice, and immersed in PBS for fluorescence imaging under a laser scanning confocal microscope. The excitation wavelength of the fluorescence signal of Ecto-CRT is 633nm, and the emission wavelength is 640-670nm. The excitation wavelength of the fluorescence signal of DAPI is 405nm, and the emission wavelength range is 420-500nm. The above process was carried out under light-proof conditions.

[0075] Fluorescence imaging results Figure 3 As shown, no significant ecto-CRT production was observed in samples treated with ultrasound alone, indicating that these treatments themselves had little effect on promoting the expression of ecto-CRT on the cell surface; however, when the NPs-treated cells were subsequently treated with ultrasound, significantly increased ecto-CRT expression was observed compared to that treated with HMME, indicating that NPs-mediated SDT can efficiently induce the immunogenic death of SCC7 cells.

[0076] 3. Detection of ATP in cell supernatant

[0077] To comprehensively evaluate the ability of NPs-mediated sonodynamic therapy in inducing immunogenic cell death (ICD) of tumor cells, an ATP detection kit was used to quantify the ATP content in the supernatant of SCC7 cells under different treatment conditions.

[0078] SCC7 cells were cultured at 1×10 5 The cells were cultured at a density of 100 μL / well in a 12-well plate. Three treatment groups were set up. When the cells adhered to the wall, the cell culture medium in the second group was replaced with a serum-free medium containing 20 μM NPs, and the cell culture medium in the third group was replaced with a serum-free medium containing 20 μM HMME and continued to incubate for 4 h. After washing with PBS, each well was replaced with 500 μL of new serum-free medium. The cells in the three treatment groups were treated with ultrasound for 3 minutes (1 MHz, 50% duty cycle, 0.5 W / cm 2 ) and then continued to be cultured in the incubator for 12 hours, the cell supernatant was collected and centrifuged at 12000 rpm at 4°C for 10 minutes, and the ATP content in the supernatant was detected using a commercially available ATP bioluminescence detection kit and according to the operating instructions. The above process was carried out under light-proof conditions.

[0079] Test results such as Figure 4 As shown, it can be seen that compared with ultrasound alone or NPs alone, the group treated with NPs combined with ultrasound promoted SCC7 cells to secrete more ATP into the extracellular environment, showing a more significant effect than HMME combined with ultrasound treatment.

[0080] 4. Detection of HMGB1 and HSP70 proteins in cell supernatant

[0081] Western Blot analysis was used to detect the levels of high mobility group protein B1 (HMGB1) and heat shock protein 70 (HSP70) in the cell supernatant after treatment.

[0082] Add protease and phosphatase inhibitors to the cell supernatant collected above, then centrifuge at 12000rpm at 4℃ for 20 minutes, discard the precipitate, add loading buffer to the supernatant, heat in a 100℃ metal bath for 10 minutes, and store the samples in a -80℃ refrigerator. Load an equal volume (10μL) of the sample on a 10% sodium dodecyl sulfate separation polyacrylamide gel (SDS-PAGE), perform 80V constant voltage electrophoresis, wait for the protein to swim to the separation gel, adjust the voltage to 120V until the protein swims to the bottom of the gel, and then transfer the protein sample to a polyvinylidene fluoride (PVDF) membrane in an ice bath under 0.3A cross-flow conditions, and the transfer time is 25min. Use a fast blocking solution to block the PVDF membrane for 30 minutes, and wash 5 times (5 minutes / time) with 1×TBST (containing 0.1% Tween-20). The washed samples were divided into two groups, and anti-HMGB1 antibody (ab79823, diluted 1:1000 in 1×TBST) and anti-HSP70 antibody (ab181606, diluted 1:1000 in 1×TBST) were added respectively. The samples were placed in a 4°C refrigerator and shaken overnight. After rewarming for 1 hour the next day, they were washed 5 times with 1×TBST (5 minutes / time), incubated with HRP-conjugated secondary antibody at room temperature with shaking for 90 minutes, and washed 5 times with 1×TBST (5 minutes / time). Finally, the strips were evenly immersed in supersensitive exposure solution and placed in a chemical exposure instrument for exposure detection.

[0083] Test results such as Figure 5 As shown in the figure, the HMGB1 and HSP70 protein levels of SCC7 cells treated with NPs and ultrasound were significantly higher than those in the HMME-mediated sonodynamic therapy group. It can be seen that NPs-mediated sonodynamic therapy can not only effectively promote the release of a large amount of HMGB1 and HSP70 from SCC7 cells to the extracellular space, but also its promoting effect is significantly better than that of the traditional sonosensitizer HMME.

[0084] As can be seen from the above, 3TEP, as a new type of sonogenic ICD inducer, can not only efficiently produce reactive oxygen species under the triggering of ultrasound, thereby effectively killing tumor cells, but also induce immunogenic death of tumor cells by promoting the release of immune stimulatory factors such as ATP, HMGB1 and HSP70. This series of mechanisms of action provides a strong experimental basis for NPs to activate anti-tumor immune response in vivo, demonstrating its great application potential in the field of tumor treatment.

[0085] Example 3

[0086] A method for preparing nanoparticles with double polypeptides modified on the surface comprises the following steps:

[0087] (1) Pep47 and PepP polypeptides were mixed in a mass ratio of 5:1 and dissolved in ddH2O containing 10% DMSO to a concentration of 4 mmol / L, heated to boiling, and then cooled to room temperature (about 25°C) to obtain a solution containing Pep47 and PepP polypeptides;

[0088] Among them, the amino acid sequence of Pep47 polypeptide is: D C D F D FG D A D W D SATWSNYW D R D H (SEQ ID NO. 1, targeting CD47);

[0089] The amino acid sequence of the PepP polypeptide is: D C D F D FG D N D Y D S D K D P D T D D D R D Q D Y D H D F (SEQ ID NO. 2, targeting PD-L1);

[0090] (2) The solution containing Pep47 and PepP polypeptides was mixed with the purified nanoparticle solution prepared in Example 1 in a volume ratio of 1:1, reacted at 200 rpm at room temperature for 12 h, and then placed in a dialysis tape with a molecular weight cutoff of 3500 Da for 24 h to remove excess organic solvent; the retentate in the dialysis bag was placed in an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa, and centrifuged and washed at 5000 rpm for 15 min to remove unbound polypeptides, thereby obtaining surface-modified dual-polypeptide nanoparticles, recorded as BiNPs (5:1).

[0091] The inventors also adjusted the mixing mass ratio of Pep47 and PepP polypeptides in step (1) to 1:1, and the prepared surface-modified bi-polypeptide nanoparticles were denoted as BiNPs (1:1).

[0092] At the same time, nanoparticles with surface modified Pep47 polypeptide or PepP polypeptide are prepared, and the steps are as follows:

[0093] The Pep47 polypeptide was dissolved in ddH2O containing 10% DMSO to a concentration of 4 mmol / L, heated to boiling and then cooled to room temperature (about 25°C) to obtain a solution containing the Pep47 polypeptide; the solution containing the Pep47 polypeptide was mixed with the purified nanoparticle solution prepared in Example 1 in a volume ratio of 1:1, reacted at room temperature at 200 rpm for 12 hours, and then placed in a dialysis tape with a molecular weight cutoff of 3500Da and dialyzed for 24 hours to remove excess organic solvent; the retentate in the dialysis bag was placed in an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa, and centrifuged and washed at 5000 rpm for 15 minutes to remove unbound polypeptides, thereby obtaining nanoparticles with surface-modified Pep47 polypeptide, recorded as NPs (αCD47).

[0094] The PepP polypeptide was dissolved in ddH2O containing 10% DMSO to a concentration of 4 mmol / L, heated to boiling and then cooled to room temperature (about 25°C) to obtain a solution containing the PepP polypeptide; the PepP polypeptide-containing solution was mixed with the purified nanoparticle solution in a volume ratio of 1:1, reacted at 200 rpm at room temperature for 12 hours, and then placed in a dialysis belt with a molecular weight cutoff of 3500Da and dialyzed for 24 hours to remove excess organic solvent; the retentate in the dialysis bag was placed in an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa, centrifuged and washed at 5000 rpm for 15 minutes to remove unbound polypeptides, and nanoparticles with surface-modified PepP polypeptide were obtained, which were recorded as NPs (αPD-L1).

[0095] Example 4

[0096] The purpose of this example is to measure the physical properties of several nanoparticles prepared in Example 3.

[0097] The surface-modified bi-polypeptide nanoparticles BiNPs (5:1) prepared in Example 3 were observed under an electron microscope and the particle size distribution of the nanoparticles was measured. The results are as follows: Figure 6 As shown, the average particle size of the nanoparticles BiNPs (5:1) is 255nm (left), and the nanoparticles are stable and uniform in morphology under an electron microscope (right).

[0098] The Zera potential of the nanoparticles prepared in Example 3 was measured respectively. The results are shown in Figure 7 As shown. Figure 7 It can be seen that the potential of the nanoparticles is different when modified with different proportions of polypeptides, indicating that the proportion of polypeptides can be controlled.

[0099] Example 5

[0100] 1. Co-culture experiment of mouse bone marrow derived dendritic cells (BMDC) and tumor cells

[0101] (1) Extraction, separation and culture of mouse spleen immune cells: C3H mice (7-8 weeks) were killed by cervical dislocation, and quickly immersed in 75% ethanol for 5 minutes, then taken out and placed in a sterile culture dish in a clean bench; the mouse abdominal skin was picked up with tweezers, a small cut was made in the middle of the left ventral side of the mouse, and the skin was torn open to reveal the red long strip of spleen. The spleen was gently lifted with tweezers, and the connective tissue under the spleen was separated with ophthalmic scissors. The spleen was taken out and placed in a sterile culture dish containing 5 mL of RPMI 1640 culture medium; the spleen was gently chopped with ophthalmic scissors, placed on a 200-mesh cell sieve, and the spleen was gently ground with a 10 mL syringe needle. After squeezing, a small amount of RPMI was aspirated. The cell sieve was rinsed with 1640 medium, and the spleen cell suspension was rinsed into the culture dish; the spleen cell suspension in the culture dish was aspirated with a sterile pipette and placed in a 50 mL sterile centrifuge tube, centrifuged at 300 × g for 5 min at 4 °C, the supernatant was removed, 5 mL of red blood cell lysis buffer was added, and the red blood cell lysis buffer was gently blown and evenly mixed with a pipette, and the red blood cell lysis was performed at room temperature for 2 min (keep gently shaking), 15 mL of PBS was added and mixed, and the supernatant was discarded after centrifugation at 300 × g for 5 min at 4 °C, and the cells were washed twice with PBS, and the cells were collected and resuspended with RPMI 1640 complete medium (containing 10% FBS and 1% penicillin / streptomycin double antibody); the cell concentration was adjusted to 5.0 × 10 6 cells / mL, for future use.

[0102] (2) CFDA-SE labeling of SCC7 tumor cells: Take 5 μL of 10 mM CFDA-SE stock solution and add 5 mL PBS to prepare a 10 μM CFDA-SE solution (all subsequent steps must be strictly protected from light). Collect the trypsin-digested SCC7 cells, centrifuge at 800 rpm for 5 min, wash the cells three times with PBS, resuspend the cell pellet obtained by centrifugation with 1 mL PBS and then add 1 mL of 10 μM CFDA-SE solution, incubate at room temperature for 8 min and shake gently (after collecting SCC7 cells, serum components should be washed with PBS to remove them to avoid affecting the staining efficiency of CFDA-SE on cells later.) After the incubation, add 10 mL of RPMI 1640 complete medium and gently blow evenly to terminate CFDA-SE staining; centrifuge at 800 rpm for 5 min, remove the supernatant, and wash the cells twice with PBS; resuspend and count with RPMI 1640 complete medium; adjust the cell concentration to 1×10 5 cells / mL, for future use.

[0103] (3) Co-culture of spleen cells and CFDA-SE-labeled SCC7 tumor cells: 0.5 mL of 1×10 5cells / mL of CFDA-SE-labeled SCC7 cells and 0.5 mL of 5.0×10 6 cells / mL of spleen cells were shaken evenly and placed in a cell culture incubator for co-culture for 4 hours; after the culture was completed, different nanoparticles were given (among which, the Blank group was BMDC cells alone, and the Ctrl group was tumor cells and BMDC cells cultured alone), and the cells were placed in a cell culture incubator for incubation for 24 hours; after the incubation was completed, the culture medium in the wells was collected into a centrifuge tube, and the adherent SCC7 cells were digested with trypsin or scraped with a cell scraper, and then the cells were collected into a centrifuge tube; the cells were washed once with the buffer solution in the cell apoptosis PI staining kit (centrifugation at 800 rpm, 5 min), and the cells were resuspended with an appropriate amount of buffer solution; the cells were stained with the PI staining solution in the cell apoptosis PI staining kit, and the staining was carried out at room temperature in the dark for 10 min; according to flow cytometry analysis, the CFDA-SE+ / PI+ cell ratio was the proportion of tumor cells that died after the nanoparticles stimulated the spleen cells to exert anti-tumor immune effects.

[0104] The results of the test are as follows Figure 8 As shown. Figure 8 It can be seen that the BiNPs (5:1) group showed a stronger ability to promote BMDC maturation than other treatment groups.

[0105] 2. Co-culture experiment of RAW264.7 and tumor cells

[0106] SCC7 tumor cells were labeled with CFDA-SE fluorescent probe (same method as above) and 1×10 6 Cells were evenly plated in 24-well plates and divided into five groups (Ctrl group, NPs (αCD47) group, NPs (αPD-L1) group, BiNPs (1:1) group, and BiNPs (5:1) group). After they adhered to the wall, RPMI-1640 serum-free medium containing different nanoparticles was added to the other groups except the Ctrl group for 12 hours, and then washed with PBS. 1×10 6 After co-culture of RAW264.7 mouse macrophages for 2 h, the supernatant in the well plate was aspirated into a flow tube, washed twice with PBS, and the washing solution PBS was also collected into the flow tube, centrifuged at 1000 rpm for 5 minutes, and then resuspended with 300 μL PBS. Finally, the proportion of CFDA-SE+ cells was detected by flow cytometry to evaluate the phagocytic level of RAW264.7 on tumor cells.

[0107] Flow cytometric quantification of Raw264.7 phagocytosis of CSFE-labeled SCC7 cells Fig. 9As shown, the figure reflects the effect of different treatment groups on the phagocytic function of macrophages after the surface-modified polypeptide nanoparticles blocked the SCC7 cell immune checkpoint CD47 / PD-L1. It can be seen that the BiNPs (5:1) group exhibited better phagocytic function than other treatment groups.

[0108] Example 6

[0109] In order to further optimize the effect of dual-polypeptide modified nanoparticles in tumor immunotherapy, the inventors studied the effects of different dual-polypeptide ratios on immune response. The specific experimental method was the same as the co-culture experiment of dendritic cells (BMDC) extracted from mouse bone marrow and tumor cells in Example 5.

[0110] In preliminary experiments, nanoparticles with unmodified peptides (the added nanoparticles were not loaded with AIE molecules to avoid color crosstalk in subsequent flow cytometry experiments) were prepared as controls, as well as nanoparticles with different ratios of peptides modified on the surface (the ratios of Pep47:PepP were 1:0, 10:1, 5:1, 1:1, 1:5, 1:10, and 0:1, respectively). The results are as follows Fig.10 As shown in (a), when the peptide ratio is 5:1, the proportion of CFDA-SE+ / PI+ cells is significantly increased, which suggests that when Pep47 targeting CD47 accounts for a higher proportion in the dual peptide, it can more effectively activate the immune system and enhance the killing effect on tumor cells. Based on this finding, a series of dual peptide modified nanoparticles with different ratios were further prepared (ratios of 1:0, 15:1, 12.5:1, 10:1, 7.5:1, 5:1, 2.5:1, 1:1 and 0:1, respectively), and the co-culture experiment was repeated. The results are shown in Figure 2. Fig.10 As shown in (b), when the ratio of Pep47 to PepP is 5:1, the immune system is most activated and the tumor cell killing effect is most significant.

[0111] Therefore, we determined that 5:1 was the optimal ratio for double polypeptide modification of nanoparticle surface in subsequent experiments. This ratio not only optimized the activation of nanoparticles on the tumor immune microenvironment, but also provided important experimental basis for the next stage of experiments, demonstrating the potential of enhancing immune response by precisely regulating the ratio of double polypeptides.

[0112] Example 7

[0113] SCC7 cells were cultured in a 96-well plate at a density of 5000 cells per well. After the cells adhered to the wall, the culture medium was removed. The BiNPs (5:1) prepared in Example 3 were prepared into solutions with concentrations of 2.5, 5.0, 10.0, 20.0 and 40.0 μM using serum-free RPMI 1640 culture medium, and added to the 96-well plate containing SCC7 cells at a volume of 100 μL per well. The culture was continued for 4 h, and then the serum-free RPMI 1640 culture medium was replaced. Ultrasound (1 MHz, 50% duty cycle, 0.5 W / cm 2 ) for 3 minutes and then placed in an incubator for 24 hours; the control group was not subjected to ultrasonic treatment, and the solution after the culture medium was directly placed in an incubator for 24 hours. The culture was carried out in the dark. After the culture was completed, 10 μL of CCK8 solution with a final concentration of 0.5 mg / mL was added to each well. After standing in the incubator for 2 hours, the absorbance value of the solution in each well at 450 nm was measured using an enzyme marker.

[0114] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%,

[0115] Wherein, As: absorbance of experimental well (containing cells, culture medium, CCK-8 solution and drug solution); Ac: absorbance of control well (containing cells, culture medium, CCK-8 solution but not containing drug); Ab: absorbance of blank well (containing culture medium and CCK-8 solution but not containing cells or drug).

[0116] The results of cell viability determination after culturing SCC7 cells with different concentrations of nanoparticle solutions are shown in Fig.11 As shown, US refers to ultrasonic treatment. Fig.11 It can be seen that in the absence of ultrasound, the cytotoxicity of different concentrations of BiNPs (5:1) to SCC7 cells is almost negligible, which shows that BiNPs (5:1) itself has good biocompatibility and safety, and can effectively kill tumor cells under ultrasound conditions.

[0117] Example 8 Safety Testing

[0118] Healthy mice were used to evaluate whether BiNPs (5:1) had side effects in vivo. BiNPs (5:1) (200 μL, based on 1 mM 3TEP) were intravenously injected into healthy C3H mice on days 0 and 3. Blood tests and liver function index tests were performed on mice on day 10, and mice not treated with BiNPs (5:1) were used as controls (Untreated).

[0119] Test results such as Fig.12As shown, it can be seen that there is no significant difference in the blood routine and liver function indexes of mice in the BiNPs (5:1) group and the control group, indicating that the BiNPs (5:1) material prepared by the present invention has good safety.

[0120] At the same time, H&E staining was performed on the liver, spleen and kidney of mice in the BiNPs (5:1) group and the control group. The specific method is as follows:

[0121] (1) Dewaxing: The paraffin sections of renal tissue were placed in a 65°C oven for 30 min, followed by xylene for 10 min, xylene for 10 min, xylene for 10 min, anhydrous ethanol for 3 min, anhydrous ethanol for 3 min, 95% ethanol for 3 min, 85% ethanol for 3 min, 75% ethanol for 3 min, 50% ethanol for 3 min, and then placed in ddH2O for 5 min (interruption is allowed).

[0122] (2) Antigen repair: Dilute the Bio-Tech 50× antigen repair solution to 1×, immerse the slices completely in it, heat in a microwave oven until boiling, and keep boiling for 20 minutes (the liquid should cover the slices, and add liquid in time to prevent the slices from drying out), then leave at room temperature overnight.

[0123] (3) Staining: Wash the sections slowly with PBS (PBST) supplemented with Tween on a shaker for 3 times, 5 min each time; take out the sections and gently shake them, wipe the area around the tissue dry with the back of a paper, and quickly draw a circle with an immunohistochemistry pen 0.5 cm outside the tissue to completely surround the tissue, incubate with 3% hydrogen peroxide for 20 min, and wash three times with PBST shaker after the incubation, 5 min each time. After drying, add 40 ul of immunohistochemical blocking solution containing goat serum to each tissue, block at room temperature for 1 h, and then shake off to remove the blocking solution. Subsequently, add 40 μL of the primary antibody diluted according to the instructions to each tissue, and incubate at 4°C overnight (add water to the wet box to prevent the sections from drying out). On the second day, the wet box was taken out and rewarmed for 20 minutes. The primary antibody was recovered and washed on a PBST shaker for 3 times, 5 minutes each time. After drying, 40 μL of reaction enhancement solution was added to each tissue, and the tissue was allowed to stand at room temperature for 15 minutes. The wet box was washed three times with PBST, 5 minutes each time, and then dried. 40 μL of goat anti-rabbit / mouse IgG polymer secondary antibody was added to each tissue, and the tissue was incubated at room temperature for 1 hour (water was added to the wet box to prevent drying of the slides). The DAB reagent was prepared in the dark and added to the tissue under a microscope. After color development for about 30 seconds, the tissue was placed in clean water. After hematoxylin staining of the nucleus for about 3 minutes, the nucleus was differentiated with hydrochloric acid and alcohol (5 seconds), and the tissue was rinsed with tap water to return to blue (15-30 minutes).

[0124] (5) Dehydration and sealing: After staining, the paraffin sections were evenly immersed in 75% ethanol for 3 min, 85% ethanol for 3 min, 95% ethanol for 3 min, 100% ethanol for 3 min, 100% ethanol for 3 min, xylene for 3 min, xylene for 3 min, and xylene for 3 min. After drying in a biosafety cabinet, the sections were sealed with a mixture of neutral gum and xylene. After solidification, the sections were observed and photographed under a microscope for preservation.

[0125] The staining results are as follows Fig.13 As shown, it can be seen that the H&E staining results of the liver, spleen and kidney sections of the mice in the BiNPs (5:1) group and the control group did not show any damage or lesions, indicating that the BiNPs (5:1) material prepared by the present invention has good in vivo biosafety.

[0126] Example 9 Establishment of animal model and in vivo tumor inhibition experiment

[0127] 1. Construction of bilateral SCC7 head and neck squamous cell carcinoma subcutaneous tumor model in mice: 50 μL of 2×10 6 A suspension of 10 SCC7 cells was used as the primary tumor, and 50 μL containing 5 × 10 5 On the 10th day after the right tumor inoculation, the primary tumor volume reached 40mm. 3 The mice were randomly divided into groups for the next treatment experiment. After BiNPs (5:1) nanoparticles (1 mM, 200 μL) were injected into the tail vein, the primary tumor was treated with ultrasound (1 MHz, 1.5 W / cm 2 , 5min), and the contralateral side (simulated transfer side) was not ultrasounded.

[0128] Tumor volume calculation: The long and short diameters of the tumor were measured with a vernier caliper and determined using the following formula: volume = long diameter × short diameter × short diameter / 2.

[0129] Typical fluorescence images of the tumor site of the same mouse after tail vein injection of nanoparticles Fig.14 The relationship between fluorescence intensity and time after injection is shown in Fig.15 As shown, it can be seen that the nanoparticles have the ability to quickly accumulate in the tumor site, and the specific time point of ultrasound after intravenous injection was confirmed to be 12 hours.

[0130] 2. The mice bearing bilateral SCC7 tumors were transplanted into the uterus when the primary tumor volume was approximately 40 mm. 3The mice were randomly divided into four groups (5 in each group), namely: "control group", "NPs (αCD47) + US group", "NPs (αPD-L1) + US group" and "BiNPs (1:1) + US group". Except for the control group, the other three groups were injected with saline via the tail vein and then the tumor was locally treated with ultrasound (1MHz, 1.5W / cm 2 The tumor growth curves and tumor growth conditions of bilateral SCC7 tumor-bearing mice (n=5) in different treatment groups were counted. Fig.16 As shown, it can be seen that the nanoparticles prepared by the present invention not only have an anti-tumor effect on the primary tumor, but also induce a systemic immune response. The activated immune cells also have an anti-tumor effect on the contralateral side, and reflect the synergistic enhanced anti-tumor advantage of dual-polypeptide bispecific therapy; the sonodynamic therapy mediated by BiNPs (1:1) + US group showed a stronger bilateral tumor inhibition effect than the "Saline group", "NPs (αCD47) + US group" and "NPs (αPD-L1) + US group".

[0131] 3. The mice bearing bilateral SCC7 tumors were placed in the 40-well tubes at a primary tumor volume of approximately 50 mm. 3 The mice were randomly divided into three groups: control group, BiNPs (1:1) + US group, and BiNPs (5:1) + US group. The experimental results were obtained on the 21st day after the same experimental operation. Fig.17 As shown, we found that the "BiNPs (5:1) + US group" had a better tumor inhibition effect on bilateral tumors than the "BiNPs (1:1) + US group". Among the mice treated with the "BiNPs (1:1) + US group", 20% of the tumors were completely cured after ultrasound, which was consistent with the "BiNPs (5:1) + US group", and 60% of the distal tumors without ultrasound were completely cured, which was completely superior to the "BiNPs (1:1) + US group".

[0132] 4. Flow cytometry analysis of related immune indicators

[0133] On the 10th day after ultrasound tumor in animal experiments, lymph nodes, tumors, and peripheral blood mononuclear cells from SCC7-bearing mice in different groups were collected. Lymph nodes and tumor tissues were ground into single cell suspensions, sieved through a 200-mesh nylon mesh, and then centrifuged at 1700 rpm for 5 minutes to collect cells. The cells were treated with red blood cell lysis buffer for 5 to 10 minutes to remove red blood cells, sieved through a 200-mesh nylon mesh again, and centrifuged at 1500 rpm for 5 minutes to collect cells. The cell concentration was adjusted to 1×10 with PBS. 6 Pieces / mL.

[0134] To investigate the proportion of mature DCs, lymph node cell suspensions from different treatment groups were incubated and stained with anti-CD11c-PE, anti-CD86-FITC, and anti-CD80-APC antibodies at room temperature in the dark.

[0135] To investigate the level of M1 / M2 infiltration in tumor cells, tumor single cell suspensions from different treatment groups were co-incubated with anti-F4 / 80-anti-CD11b-anti-CD86-FITC and anti-CD206-APC antibodies.

[0136] In order to detect the proportion of IFN-γ+CD8+T cells in the tumor, the tumor single cell suspension was mixed with perforation buffer, treated in the dark at room temperature for 30 minutes, and then incubated with anti-CD3-FITC, anti-CD8-APC and anti-IFN-γ-PE antibodies.

[0137] All the above antibody staining was performed at room temperature in the dark. After 17 minutes of staining, 2 mL of PBS was added to terminate the staining. The cells were centrifuged at 1700 rpm for 5 minutes, the supernatant was removed, and 500 μL of 1% paraformaldehyde was added to resuspend the cells, and the cells were loaded and analyzed on a flow cytometer.

[0138] The analysis results are as follows Fig.18 As shown in the figure, blue represents the control group (saline group), red represents the "BiNPs (1:1) + US group", and green represents the "BiNPs (5:1) + US group". It can be seen that both BiNPs (1:1) and BiNPs (5:1) induced CD8 + Increased T cells, IFNγ + The increase in T cells, maturation of DC cells, and increase in macrophages (increase in M1 / M2) indicate that the surface-modified dual-polypeptide nanoparticles prepared by the present invention can efficiently activate the body's immune system, and the ratio of the dual-polypeptides can be adjusted.

[0139] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing surface-modified dual-polypeptide nanoparticles, characterized in that: Nanoparticles are prepared by using an aggregation-induced luminescence material as a core and a surfactant as a carrier, and then Pep47 and PepP polypeptides are connected to the surface of the nanoparticles to obtain nanoparticles with double polypeptides modified on the surface; The amino acid sequence of the Pep47 polypeptide is shown in SEQ ID NO.1; The amino acid sequence of the PepP polypeptide is shown in SEQ ID NO.

2.

2. The method according to claim 1, characterized in that The aggregation-induced emission material is any one of 2,4,6-tri{(diphenylamino)phenylvinyl}-methylpyridinium salt, tetraphenylethylene, and triphenylamine; and the surfactant is an amphiphilic surfactant.

3. The method according to claim 2, characterized in that The amphiphilic surfactant is DSPE-PEG2000-Mal.

4. The method according to claim 3, characterized in that The method for preparing nanoparticles comprises the following steps: (1) dissolving an aggregation-induced emission material and a surfactant in an organic solvent, and adding the solvent to ultrapure water under ultrasonic conditions to obtain a nanoparticle solution containing the aggregation-induced emission material; (2) The nanoparticle solution containing the aggregation-induced emission material obtained in step (1) is dialyzed and subjected to centrifugal ultrafiltration to separate the solid from the liquid, and the liquid portion is collected to obtain a purified nanoparticle solution.

5. The method according to claim 4, characterized in that The mass ratio of the aggregation-induced emission material and the surfactant in step (1) is 0.8-1.2:2.5-3.5; the volume ratio of the total mass of the aggregation-induced emission material and the surfactant to the organic solvent is 3.5-4.5 mg:0.8-1.2 mL; the organic solvent is tetrahydrofuran or dimethyl sulfoxide; the power of the ultrasound is 12-20 W, the ultrasound is performed for 6-8 seconds and then stopped for 2-4 seconds, and the total ultrasound time is 2.5-3.5 minutes; the volume of the added ultrapure water is 8-10 times the volume of the organic solvent; The molecular weight cutoff during dialysis in step (2) is 3000-4000Da, and the dialysis time is 28-44h; the molecular weight cutoff during the centrifugal ultrafiltration is 80-120kDa, the rotation speed is 4000-6000rpm, the number of times is 3-7 times, and the time of each centrifugal ultrafiltration is 10-20min; the solid-liquid separation is filtration, and the pore size of the filter membrane used in the filtration is 0.45μm.

6. The method according to claim 4, characterized in that When Pep47 and PepP polypeptides are connected to the surface of nanoparticles, a solution containing Pep47 and PepP polypeptides is mixed with a nanoparticle solution and reacted, and then dialyzed and centrifuged and ultrafiltered to obtain nanoparticles with double polypeptides modified on the surface.

7. The method according to claim 6, characterized in that The mass ratio of Pep47 and PepP in the solution containing Pep47 and PepP polypeptides is 4-6:1; the concentration of the solution containing Pep47 and PepP polypeptides is 3-5 mmol / L; the volume ratio of the solution containing Pep47 and PepP polypeptides and the nanoparticle solution when mixed is 0.8-1.2:0.8-1.2; the concentration of the nanoparticle solution is 1.5-2.5 mmol / L; the temperature during the reaction is 23-27°C, the speed is 150-250 rpm, and the time is 10-14 h; the molecular weight cutoff during dialysis is 3000-4000 Da, and the dialysis time is 20-28 h; the molecular weight cutoff during centrifugal ultrafiltration is 80-120 kDa, the rotation speed is 4000-6000 rpm, and the time is 10-20 min.

8. A surface-modified dual-polypeptide nanoparticle prepared according to the method of any one of claims 1 to 7.

9. A reagent comprising nanoparticles with surface-modified dual polypeptides according to claim 8.

10. Use of the nanoparticles according to claim 8 or the reagent according to claim 9 in preparing a product for killing tumor cells.

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