TIM-3 scFv bionic nanoparticles loaded with A-D-A organic sound-sensitive agent as well as preparation method and application of TIM-3 scFv bionic nanoparticles loaded with A-D-A organic sound-sensitive agent

Abstract

The invention discloses TIM-3 scFv bionic nanoparticles loaded with an A-D-A organic sound-sensitive agent and a preparation method and application of the TIM-3 scFv bionic nanoparticles loaded with the A-D-A organic sound-sensitive agent. The preparation method comprises the steps that a tetrahydrofuran solution of CH-H and a tetrahydrofuran solution of DSPE-PEG are added into deionized water, ultrasonic treatment is carried out, then rotary evaporation is carried out, concentration and drying are carried out, and the A-D-A organic sound-sensitive agent nanoparticles are obtained; the method comprises the following steps: obtaining a cell membrane of a macrophage with stable overexpression of an EGFP-TIM-3 scFv gene, then mixing the cell membrane with A-D-A organic sound-sensitive agent nanoparticles, and extruding through an extruder; the organic sound-sensitive agent can efficiently generate ROS and can directly kill tumor cells and induce ICD, meanwhile, TIM-3 scFv blocks TIM-3 signals on cDC1 cells, the antigen presentation function of the cDC1 cells is recovered, positive feedback circulation of sonodynamic killing, antigen release, cross presentation and T cell activation is formed, and the aim of treating colorectal cancer is achieved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a TIM-3 scFv biomimetic nanoparticle loaded with ADA organic sound-sensing agent, its preparation method, and its application. Background Technology

[0002] Colorectal cancer is one of the most common malignant tumors worldwide, with high incidence and mortality rates, and has become the third most common cancer globally. Current clinical treatments, such as surgery, radiotherapy, and chemotherapy, while improving patient survival rates, still face challenges including limited efficacy, recurrence and metastasis, and significant side effects. The rapid development of tumor immunotherapy, particularly the clinical application of immune checkpoint inhibitors (ICIs) such as PD-1 / PD-L1 antibodies, has brought new hope to colorectal cancer treatment; however, their objective response rate remains below 20%, a treatment bottleneck that profoundly reflects the complexity and heterogeneity of tumor immune escape mechanisms.

[0003] Sonodynamic therapy (SDT) uses ultrasound to activate a sonosensitive agent to generate reactive oxygen species (ROS), such as hydroxyl radicals (·OH) and superoxide anions (·O2). - These reactive oxygen species (ROS) can damage the DNA, proteins, and cell membranes of tumor cells, ultimately leading to cell death. Simultaneously, SDT induces immunogenic cell death (ICD), prompting tumor cells to release tumor-associated antigens (TAAs) and damage-associated molecular patterns (DAMPs), including calreticulin (CRT), high mobility group box 1 (HMGB1), and adenosine triphosphate (ATP), thereby activating dendritic cells (DCs) and enhancing anti-tumor immune responses. However, traditional inorganic somatosensitizers, such as titanium dioxide (TiO2) and cadmium sulfide (CdS), while capable of generating ROS, suffer from poor biocompatibility and long-term safety concerns.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a biomimetic nanoparticle of TIM-3 scFv loaded with ADA organic acoustic sensitizer, its preparation method, and its application. The organic acoustic sensitizer in the biomimetic nanoparticles of this invention can efficiently generate ROS, directly kill tumor cells and induce ICD, prompting tumor cells to release tumor-associated antigens and damage-associated molecular patterns, activating the innate immune response. Simultaneously, TIM-3 scFv blocks TIM-3 signaling on cDC1 cells, restoring their antigen-presenting function and promoting CD8+. + The proliferation and activation of T cells form a positive feedback loop of "sonic killing - antigen release - cross-presentation - T cell activation", thereby achieving the therapeutic goal of colorectal cancer.

[0006] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: The first aspect of this invention provides a method for preparing TIM-3 scFv biomimetic nanoparticles loaded with ADA organic acoustic sensor, the preparation method comprising the following steps: (a) The recombinant vector expressing the EGFP-TIM-3 scFv gene was packaged with lentivirus, transfected, and screened to obtain macrophages that stably overexpressed the EGFP-TIM-3 scFv gene; (b) The tetrahydrofuran solution of CH-H and DSPE-PEG was added to deionized water and ultrasonically treated under water bath conditions. The organic solvent was then removed by rotary evaporation. The solution was then concentrated by membrane filtration, ultrafiltration, and drying to obtain ADA organic acoustic sensitizer nanoparticles. (c) Obtain the cell membrane of macrophages that stably overexpress the EGFP-TIM-3 scFv gene, mix the cell membrane with ADA organic sound-sensitizing agent nanoparticles and extrude them through an extruder to obtain TIM-3scFv biomimetic nanoparticles loaded with ADA organic sound-sensitizing agent.

[0007] Preferably, in step (a), the recombinant vector expressing the EGFP-TIM-3 scFv gene is constructed from the EGFP-TIM-3 scFv gene and a lentiviral expression vector.

[0008] Preferably, in step (b), the concentrations of CH-H and DSPE-PEG in the tetrahydrofuran solution are 0.3~0.8 mg / ml, respectively; and the volume ratio of the tetrahydrofuran solution of CH-H and DSPE-PEG to deionized water is 1:(8~10).

[0009] Preferably, in step (b), the water bath temperature is 35~40℃; the ultrasonic treatment lasts for 15~25 minutes, the ultrasonic power is 150~250W, and the frequency is 30~50Hz.

[0010] Preferably, in step (b), the membrane used for membrane filtration concentration has a pore size of 0.45 μm; and the ultrafiltration membrane used for ultrafiltration concentration has a molecular weight cutoff of 90~110 KD.

[0011] Preferably, in step (c), the mass ratio of cell membrane proteins to ADA organic acoustic sensor nanoparticles is 1:(0.8~1.2).

[0012] A second aspect of the present invention provides TIM-3scFv biomimetic nanoparticles loaded with ADA organic acoustic sensor prepared by the above preparation method.

[0013] The third aspect of this invention provides the application of TIM-3scFv biomimetic nanoparticles loaded with ADA organic acoustic sensitizer prepared by the above-mentioned preparation method in the preparation of drugs for treating colorectal cancer.

[0014] The fourth aspect of the present invention provides a medicament for treating colorectal cancer, the medicament comprising TIM-3 scFv biomimetic nanoparticles loaded with ADA organic sonosensitive agent prepared by the above preparation method and pharmaceutically acceptable excipients.

[0015] Preferably, the dosage form of the drug for treating colorectal cancer includes injections, powders, granules, capsules, or tablets.

[0016] Compared with the prior art, the beneficial effects of the present invention include at least the following: This invention establishes a polyclonal cell line overexpressing TIM-3 single-chain fragment variable (scFv) on RAW264.7 macrophages, obtains the cell membrane of the cell line, and constructs TIM-3scFv biomimetic nanoparticles (HNPs@M) loaded with ADA organic acoustic sensitizer by co-extrusion of the cell membrane and HNPs. The core mechanism is as follows: (1) Sonodynamically mediated tumor killing and immunogenic death (ICD) induction: HNPs efficiently generate ROS under ultrasound activation, directly kill tumor cells and induce ICD, prompting tumor cells to release tumor-associated antigens and damage-associated molecular patterns, and activate innate immune response. The acoustic sensitizer encapsulated in the macrophage membrane improves targeting and intratumoral accumulation. (2) TIM-3 scFv blocks TIM-3 signaling on cDC1 cells, restores its antigen presentation function, and promotes CD8. +The proliferation and activation of T cells form a positive feedback loop of "sonodynamic killing - antigen release - cross-presentation - T cell activation". TIM-3 scFv can also directly act on tumor-infiltrating T cells, blocking TIM-3-mediated exhaustion signals and enhancing T cell function. This invention develops a highly efficient and safe colorectal cancer treatment platform, providing an innovative solution for the treatment of colorectal cancer and offering a reference for the treatment of other solid tumors. It has significant scientific value and clinical translational potential. Attached Figure Description To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0017] Figure 1 This is a map of the recombinant vector expressing the EGFP-TIM-3 scFv gene in an embodiment of the present invention; Figure 2 These are Western blot images of RAW-T and RAW in the experimental examples of this invention; Figure 3 This is a Western blot image of RAW-T and RAW cell membranes in an experimental example of the present invention; Figure 4 This is a confocal microscopy image of macrophages stably overexpressing the EGFP-TIM-3 scFv gene in the experimental examples of this invention; Figure 5 These are TEM images of HNPs, M, and HNPs@M in the experimental examples of this invention; Figure 6 The results of the Zeta potential measurement of HNPs and HNPs@M in the experimental examples of this invention; Figure 7 This is a particle size distribution diagram of HNPs and HNPs@M in the experimental examples of this invention; Figure 8 This illustrates the formation of ·OH from HNPs at different concentrations and power levels in the experimental examples of this invention. Figure 9 In the experimental examples of this invention, HNPs were used at different concentrations and power levels. 2- Generation status; Figure 10 In the experimental examples of this invention, DMPO is used as ·OH and ·O. 2- The ESR spectrum of HNPs after ultrasonic treatment of the cleaning agent; Figure 11The effects of different concentrations of HNPs and different treatment groups on the activity of CT26, MFC, MOC1, AKR, and NCM460 cells in the experimental examples of this invention; Figure 12 This is a bar chart showing the effect of different concentrations of HNPs on the viability of CT26, MFC, MOC1, AKR, and NCM460 cells in the experimental examples of this invention. Figure 13 These are fluorescence images of intracellular ROS generated by CLSM in CT6 cells of different treatment groups in the experimental examples of this invention; Figure 14 These are representative CLSM images of DNA damage in γ-H2AX-stained CT26 cells after different treatments in the experimental examples of this invention. Figure 15 These are representative CLSM images of CT26 cells from different treatment groups exposed to CRT in the experimental examples of this invention; Figure 16 The results of FCM on apoptosis of CT26 cells after different treatments in the experimental examples of this invention; Figure 17 The results of FCM analysis on the effects of different treatments on DC maturation in the experimental examples of this invention; Figure 18 These are tumor images of colorectal cancer in mice after different treatment groups in the experimental examples of this invention. Detailed Implementation

[0018] The embodiments of the technical solution of the present invention will be described in detail below with reference to the examples. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore only examples, and should not be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0019] This invention provides a method for preparing TIM-3 scFv biomimetic nanoparticles loaded with ADA organic sound-sensing agent, the preparation method comprising the following steps: (a) The recombinant vector expressing the EGFP-TIM-3 scFv gene was packaged with lentivirus, transfected, and screened to obtain macrophages that stably overexpressed the EGFP-TIM-3 scFv gene; (b) The tetrahydrofuran solution of CH-H and DSPE-PEG was added to deionized water and ultrasonically treated under water bath conditions. The organic solvent was then removed by rotary evaporation. The solution was then concentrated by membrane filtration, ultrafiltration, and drying to obtain ADA organic acoustic sensitizer nanoparticles. (c) Obtain the cell membrane of macrophages that stably overexpress the EGFP-TIM-3 scFv gene, mix the cell membrane with ADA organic sound-sensitizing agent nanoparticles and extrude them through an extruder to obtain TIM-3scFv biomimetic nanoparticles loaded with ADA organic sound-sensitizing agent.

[0020] This invention does not impose strict limitations on lentivirus packaging, transfection, and screening; those skilled in the art can employ conventional lentivirus packaging, transfection, and screening methods. In one embodiment, lentivirus packaging includes the following steps: After mixing solutions A and B, the mixture was added to HEK293T cells. Viral supernatants were collected and purified after 48 h and 72 h, respectively. Solution A was prepared by adding pSPAX2, pMD2.G, and the recombinant vector expressing the EGFP-TIM-3 scFv gene in a mass ratio of 1:3:4 to 300 μL of serum-free DMEM. The total amount of pSPAX2, pMD2.G, and the recombinant vector expressing the EGFP-TIM-3 scFv gene was 11-12 μg. Solution B was prepared by adding 6 μl of PEI to 300 μL of serum-free DMEM.

[0021] In one embodiment, the MOI during transfection is 12-18, and the transfected cells are RAW 264.7 cells.

[0022] In one embodiment, screening involves changing the medium 48 hours after transfection, at which point the cells can be cultured in complete medium containing 3.5 μg / mL puromycin to screen for positive cells expressing TIM-3 scFv. After the selection pressure from puromycin, macrophages stably overexpressing the EGFP-TIM-3 scFv gene are obtained. To maintain long-term stable expression of the TIM-3 scFv gene, macrophages stably overexpressing the EGFP-TIM-3 scFv gene are cultured in complete medium containing 2.5 μg / mL puromycin.

[0023] In one embodiment, in step (a), the recombinant vector expressing the EGFP-TIM-3 scFv gene is constructed from the EGFP-TIM-3 scFv gene and a lentiviral expression vector.

[0024] The method for constructing the recombinant vector expressing the EGFP-TIM-3 scFv gene is not strictly limited in this invention. Those skilled in the art can construct it using conventional methods. In one embodiment, the map of the recombinant vector expressing the EGFP-TIM-3 scFv gene is as follows: Figure 1 As shown.

[0025] In one embodiment, in step (b), the concentrations of CH-H and DSPE-PEG in the tetrahydrofuran solution of CH-H and DSPE-PEG can be 0.3~0.8 mg / ml, respectively; the volume ratio of the tetrahydrofuran solution of CH-H and DSPE-PEG to deionized water can be 1: (8~10).

[0026] The structural formula of CH-H in this invention is as follows: .

[0027] In this invention, CH-H is an existing product; see the existing literature High-Performance ElectronAcceptors Containing Transition Metals (Web of Science).

[0028] In this invention, the molecular weight of DSPE-PEG is 2000~5000.

[0029] In one embodiment, in step (b), the water bath temperature is 35~40℃; the ultrasonic treatment lasts for 15~25 minutes, the ultrasonic power is 150~250W, and the frequency is 30~50Hz.

[0030] In one embodiment, in step (b), the membrane filtration concentration uses a membrane with a pore size of 0.45 μm; the ultrafiltration concentration uses an ultrafiltration membrane with a molecular weight cutoff of 90~110 KD.

[0031] In one embodiment, in step (c), the mass ratio of cell membrane proteins to ADA organic acoustic sensor nanoparticles is 1:(0.8~1.2).

[0032] Another embodiment of the present invention provides TIM-3 scFv biomimetic nanoparticles loaded with ADA organic acoustic sensor prepared by the above preparation method.

[0033] Another embodiment of the present invention provides the application of TIM-3 scFv biomimetic nanoparticles loaded with ADA organic acoustic sensitizer prepared by the above preparation method in the preparation of drugs for treating colorectal cancer.

[0034] Another embodiment of the present invention provides a drug for treating colorectal cancer, the drug comprising TIM-3 scFv biomimetic nanoparticles loaded with ADA organic sonosensitive agent prepared by the above preparation method and pharmaceutically acceptable excipients.

[0035] In one embodiment, the dosage form of the drug for treating colorectal cancer includes injections, powders, granules, capsules, or tablets.

[0036] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0037] Example 1 This embodiment describes a method for preparing TIM-3 scFv biomimetic nanoparticles loaded with ADA organic sound-sensing agent. The preparation method includes the following steps: (a) pSPAX2, pMD2.G and a recombinant vector expressing the EGFP-TIM-3 scFv gene in a mass ratio of 1:3:4 (as shown in the figure) Figure 1 Solution A was prepared by adding 6 μl of PEI to 300 μL of serum-free DMEM, and solution B was prepared by adding 6 μl of PEI to 300 μL of serum-free DMEM. Solutions A and B were mixed and added to HEK293T cells. Viral supernatants were collected and purified after 48 h and 72 h, respectively. RAW 264.7 cells were transfected at an MOI of 15. The medium was changed 48 hours after transfection, and cells were then cultured in complete medium containing 3.5 μg / mL puromycin. Positive cells expressing TIM-3 scFv were screened. After the selection pressure of puromycin, macrophages stably overexpressing the EGFP-TIM-3 scFv gene were obtained. (b) Dissolve 0.5 mg CH-H and 0.5 mg DSPE-PEG (molecular weight 5000, manufacturer: Yusi Pharmaceutical) in 0.5 ml tetrahydrofuran, then mix them and add 9 ml deionized water. Sonicate in a water bath (37°C) for 20 min (ultrasonic power 200 W, frequency 40 Hz). Remove the organic solvent by rotary evaporation, then filter and concentrate using a 0.45 μm membrane, followed by ultrafiltration and drying to obtain ADA organic acoustic sensitizer nanoparticles (denoted as HNPs). (c) Macrophages stably overexpressing the EGFP-TIM-3 scFv gene were flash-frozen in liquid nitrogen for 3 min and then thawed in a water bath at 37 °C for 3 min. This process was repeated 5 times. After that, the cells were centrifuged at low speed (1000 g × 5 min) to remove cell debris and the supernatant was collected. The cells were then centrifuged at 4 °C and 3000 g for 5 min to remove mitochondria and other organelles and the supernatant was collected. The cells were then centrifuged at high speed (18000 g × 60 min) to enrich the cell membrane components. The cell membrane was then mixed with ADA organic acoustic sensitizer nanoparticles (the mass ratio of cell membrane protein to ADA organic acoustic sensitizer nanoparticles was 1:1) and extruded through an extruder to obtain TIM-3 scFv biomimetic nanoparticles loaded with ADA organic acoustic sensitizer (denoted as HNPs@M).

[0038] Comparative Example 1 This comparative example demonstrates a method for preparing cell membrane vesicles, which includes the following steps: Macrophages stably overexpressing the EGFP-TIM-3 scFv gene were flash-frozen in liquid nitrogen for 3 min and then thawed in a water bath at 37°C for 3 min. This process was repeated 5 times. Cell debris was removed by low-speed centrifugation (1000 g × 5 min), and the supernatant was collected. The cells were then centrifuged at 4°C and 3000 g for 5 min to remove mitochondria and other organelles. The supernatant was collected again and then centrifuged at high speed (18000 g × 60 min) to enrich cell membrane components. The cell membrane was then extruded through an extruder (i.e., without the addition of ADA organic sonication agent nanoparticles) to obtain cell membrane vesicles (denoted as M).

[0039] Experimental Example This experimental example demonstrates the functional study of TIM-3 scFv biomimetic nanoparticles loaded with ADA organic sound-sensing agent: 1. The expression and localization of TIM-3 scFv protein in RAW264.7 cells were verified by Western blot and confocal microscopy (CLSM); Western blot analysis was performed on macrophages stably overexpressing the EGFP-TIM-3 scFv gene (denoted as RAW-T) and macrophages (denoted as RAW). The results are as follows: Figure 2 As shown; Depend on Figure 2 It can be seen that TIM-3 scFv protein is significantly expressed in RAW264.7 cells.

[0040] Cell membranes of macrophages stably overexpressing the EGFP-TIM-3 scFv gene (denoted as RAW-T) and macrophages (denoted as RAW) were extracted and extruded to obtain cell membrane vesicles. Western blot analysis was then performed, and the results are as follows: Figure 3 As shown; Depend on Figure 3 It can be seen that TIM-3 scFv protein is still significantly expressed on cell membrane vesicles.

[0041] The expression and localization of TIM-3 scFv protein in macrophages stably overexpressing the EGFP-TIM-3 scFv gene were verified using confocal microscopy (CLSM). The results are as follows: Figure 4 As shown; Depend on Figure 4 It can be seen that EFGP-labeled TIM-3 scFv and Dil-stained cell membranes have good co-localization, indicating that TIM-3 scFv is successfully expressed on the cell membrane surface.

[0042] 2. The morphology and structure of HNPs, M and HNPs@M were characterized by transmission electron microscopy (TEM), and the particle size distribution and stability of HNPs and HNPs@M were analyzed by dynamic light scattering (DLS), and the Zeta potential was measured. Transmission electron microscopy scanning results as follows Figure 5 As shown, by Figure 5 It can be seen that the cell membrane successfully encapsulates the spherical HNPs.

[0043] The zeta potentials of HNPs and HNPs@M are as follows: Figure 6 As shown, the particle size distribution is as follows Figure 7 As shown; by Figures 6-7 It can be seen that the Zeta potential of HNPs@M is approximately -10mV to -20mV, the particle size of HNPs@M is 100~400nm, and the average particle size is about 190nm.

[0044] 3. Research on the excellent acoustic dynamic effects of HNPs under ultrasound; The formation of ·OH was detected using 3,3',5,5'-tetramethylbenzidine (TMB). When HNPs were at 1.0 W / cm², the formation of ·OH was determined. 2 Under ultrasonic intensity, the absorption peak of TMB increased significantly after 5 minutes of irradiation (e.g. Figure 8 As shown in Figure B), at different concentrations, the TMB absorption peak also increases significantly with increasing concentration (e.g., ...). Figure 8 (As shown in A).

[0045] Simultaneously, using diphenylisobenzofuran (DPBF) as a probe, the effect of ultrasound on O2 was evaluated. 2- The ability to generate HNPs; when the HNP concentration increased from 0 μg / mL to 25 μg / mL, the absorption peak of DPBF decreased by 70% within 4 minutes (e.g., Figure 9 (As shown in Figure A). Even at the lower concentration of 6.25 μg / mL, the absorption peak decreased by 50%. The DPBF absorbance of the same concentration of HNPs at different power settings also decreased significantly with increasing power (e.g., ...). Figure 9 (As shown in Figure B); the results indicate that HNPs can efficiently generate ROS under ultrasonic excitation.

[0046] The type of reactive oxygen species generated was further identified using electron spin resonance (ESR) technology. 2,2,6,6-Tetramethylpiperidine (TEMP) was used as a spin trapping agent; the triplet signal characteristic of TEMP-1-oxyl was detected, indicating... 1 O 2 DMPO as ·OH and ·O 2- The spin trap of the free radical shows that ·OH (e.g.) Figure 10 (A) and (O)2- (like Figure 10 B) Free radical signal.

[0047] 4. To study the in vitro biocompatibility and antitumor therapeutic effects of HNPs; To ensure the safety of HNPs treatment, CT26, MFC, MOC1, AKR, and NCM460 cells were seeded in 96-well plates. After 24 hours, different concentrations of HNPs and different drugs were added for treatment. Cell viability was measured using the CCK8 assay to preliminarily assess its toxicity. Figure 11 China A and Figure 12 As shown, even at the highest concentration of 50 μg / mL, HNPs did not affect cell viability in the absence of laser irradiation, with survival rates exceeding 90% for each cell type. Significantly reduced cell viability was observed in the HNPs+US and HNPs@M+US groups under different drug treatments, while no significant changes were observed in the Control, US, HNPs, and HNPs@M groups (e.g., ...). Figure 11 As shown in Figure B), it demonstrates good biocompatibility and potential for biomedical applications.

[0048] 5. Further investigation was conducted on the oxidative damage and sonodynamic effects of different treatments on cells, including Control, US, HNPs, HNPs+US, HNPs@M, and HNPs@M+US. 2,7-Dichlorofluorescein (DCFH-DA) was used as a ROS probe to assess intracellular ROS levels in CT6 cells. Results are as follows: Figure 13 As shown, by Figure 13 It can be seen that abundant green fluorescence signals were observed in the cells of the HNPs+US and HNPs@M+US groups in the fluorescence images, while the green fluorescence was weak in the cells of the Control, US, HNPs and HNPs@M groups. The results indicate that HNPs exhibit excellent ability to induce intracellular ROS production under ultrasound.

[0049] 6. During sonodynamic processes, tumor cells undergo apoptosis, and changes in nuclear DNA damage are often indicators of apoptosis. To further explore the mechanism of cell damage induced by HNPs ultrasound treatment, γ-H2AX immunofluorescence staining was used to detect DNA damage in CT6 cells from different treatment groups: Control, US, HNPs, HNPs+US, HNPs@M, and HNPs@M+US. The results are as follows: Figure 14 As shown, by Figure 14 Significant DNA damage was observed in both the HNPs+US and HNPs@M+US groups.

[0050] 7. SDT can induce tumor ICD death or the release of large amounts of tumor antigens and damage-associated molecular patterns (DAMPs) from dying cells, mainly calreticulin (CRT) exposed on the cell membrane surface. CT26 cells (1×10⁻⁶) 5 After culturing for 24 h, the culture medium was changed to Control, US, HNPs, HNPs+US, HNPs@M, and HNPs@M+US for 12 h. After washing with PBS, the culture medium was changed, and the treatments were repeated for another 4 h. The culture was then stained with anti-CRT primary antibody, Alexa Fluor 488-labeled secondary antibody, and DAPI. The CLSM results were observed as follows: Figure 15 As shown, by Figure 15 It can be seen that after CRT staining, the cell membranes of the HNPs+US and HNPs@M+US groups showed abundant green fluorescence, while the green fluorescence intensity of the Control, US, HNPs and HNPs@M groups was weaker.

[0051] 8. The Annexin V-FITC apoptosis detection kit was used to detect cell apoptosis in different treatment groups: Control, US, HNPs, HNPs+US, HNPs@M, and HNPs@M+US. Results are as follows: Figure 16 As shown, by Figure 16 It was found that the proportions of early and late apoptotic cells increased in the HNPs+US and HNPs@M+US groups, with late apoptotic cells accounting for 40.3% and 40.8%, respectively, significantly higher than in other groups. This indicates that HNPs@M+US has a significant tumor-killing ability.

[0052] 9. To verify the ability of HNPs@M+US to stimulate DC maturation, bone marrow-derived cells were isolated from male Balb / c mice and incubated in RPMI 1640-based medium. Immature BMDCs were seeded at the bottom of 24-well plates, and CT26 cells were seeded at the top. After CT26 cell culture and incubation with different drugs for 24 h, the collected BMDCs were analyzed using FCM. The results are shown below. Figure 17 As shown, Figure 17 A represents the FCM display result. Figure 17 B in the chart is a bar chart, composed of... Figure 17 It can be seen that, compared with other groups, the HNPs@M+US group has a higher concentration of DC (CD11c). + CD80 + CD86 + The maturity of HNPs@M+US was significantly improved, reaching 52.0%, indicating that HNPs@M+US can significantly stimulate DC maturation.

[0053] 10. Next, we evaluated the ability of HNPs@M+US to inhibit tumor growth in an orthotopic colorectal cancer model using CT26-Luc cells.

[0054] This study has been approved by the Ethics Committee of Tianjin University and uses Balb / c mice to study the therapeutic efficacy of colorectal cancer in mice.

[0055] In an orthotopic colorectal cancer model, CT26 cells carrying luciferase (2×10⁻⁶) 6 Cells (per mouse) were implanted into the cecum of female BALB / c mice (8 weeks old, 20-22 g). Mice were randomly divided into 6 groups of 5 each. Each group received one of 6 different treatments to treat colorectal cancer. After treatment, the mice were euthanized, and their intestines were dissected and photographed. Tumor images of colorectal cancer in mice after different treatment groups are shown below. Figure 18 As shown, by Figure 18 It can be seen that the colorectal tumors in the HNPs@M+US group were significantly smaller than those in all other groups, further emphasizing the effectiveness of ultrasound-stimulated HNPs@M+US in in vivo treatment of colorectal cancer.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for preparing TIM-3 scFv biomimetic nanoparticles loaded with ADA organic sound-sensing agent, characterized in that, The preparation method includes the following steps: (a) The recombinant vector expressing the EGFP-TIM-3 scFv gene was packaged with lentivirus, transfected, and screened to obtain macrophages that stably overexpressed the EGFP-TIM-3 scFv gene; (b) The tetrahydrofuran solution of CH-H and DSPE-PEG was added to deionized water and ultrasonically treated under water bath conditions. The organic solvent was then removed by rotary evaporation. The solution was then concentrated by membrane filtration, ultrafiltration, and drying to obtain ADA organic acoustic sensitizer nanoparticles. (c) Obtain the cell membrane of macrophages that stably overexpress the EGFP-TIM-3 scFv gene, mix the cell membrane with ADA organic acoustic sensitizer nanoparticles and extrude them through an extruder to obtain TIM-3 scFv biomimetic nanoparticles loaded with ADA organic acoustic sensitizer.

2. The production method according to claim 1, characterized by, In step (a), the recombinant vector expressing the EGFP-TIM-3scFv gene is constructed from the EGFP-TIM-3 scFv gene and a lentiviral expression vector.

3. The production method according to claim 1, characterized by, In step (b), the concentrations of CH-H and DSPE-PEG in the tetrahydrofuran solution are 0.3~0.8 mg / ml, respectively; the volume ratio of the tetrahydrofuran solution of CH-H and DSPE-PEG to deionized water is 1:(8~10).

4. The method of claim 1, wherein, In step (b), the water bath temperature is 35~40℃; the ultrasonic treatment lasts for 15~25 minutes, the ultrasonic power is 150~250W, and the frequency is 30~50Hz.

5. The preparation method according to claim 1, characterized in that, In step (b), the membrane used for membrane filtration concentration has a pore size of 0.45 μm; the ultrafiltration membrane used for ultrafiltration concentration has a molecular weight cutoff of 90~110 KD.

6. The method of claim 1, wherein, In step (c), the mass ratio of cell membrane proteins to ADA organic acoustic sensor nanoparticles is 1:(0.8~1.2).

7. TIM-3 scFv biomimetic nanoparticles loaded with ADA organic acoustic sensor prepared by the preparation method according to any one of claims 1 to 6.

8. The use of TIM-3 scFv biomimetic nanoparticles loaded with ADA organic acoustic sensitizer prepared by any one of claims 1 to 6 in the preparation of drugs for treating colorectal cancer.

9. A medicament for treating colorectal cancer, characterized by comprising the compound of claim 1. The drug for treating colorectal cancer comprises TIM-3 scFv biomimetic nanoparticles loaded with ADA organic sonosensitive agent prepared by the preparation method of any one of claims 1 to 6, and pharmaceutically acceptable excipients.

10. The medicament for treating colorectal cancer according to claim 9, wherein The dosage forms of the drugs used to treat colorectal cancer include injections, powders, granules, capsules, or tablets.

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