Diagnosis and treatment integrated immunoregulation material, preparation method and application thereof

Abstract

This invention relates to the field of biomedical technology, and more particularly to a therapeutic immunomodulatory material, its preparation method, and its application. This therapeutic immunomodulatory material uses nanoparticles P as its core, with the surface of the P nanoparticles coated with NV. CD47 / CRT NV CD47 / CRT The surface is coated with tannins and Mn 2+ Therapeutic immunomodulatory materials integrate activatable PA molecular probes with bispecifically clamped engineered nanovesicles, enabling potent immune activation and / or real-time monitoring. The nanovesicles are coated with a pH-responsive tannic acid-manganese shell. This protective coating remains intact under physiological conditions but rapidly disintegrates in an acidic TME, exposing functional ligands for targeted tumor binding. The therapeutic immunomodulatory material provided by this invention not only effectively eliminates tumors but also possesses durable immune memory.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to diagnostic and therapeutic immunomodulatory materials, their preparation methods, and applications. Background Technology

[0002] Cancer immunotherapy remains limited in its effectiveness against solid tumors due to the complex immunosuppressive network of the tumor microenvironment (TME). Innate immune cells, particularly dendritic cells (DCs) and macrophages, play crucial roles. Once recruited to the tumor site, they are often conditioned by the TME into dysfunctional or tumor-supporting phenotypes. DCs are essential for initiating cytotoxic T-lymphocyte (CTL) responses, but due to insufficient activation stimulation, they are often immature or tolerant, weakening adaptive anti-tumor immunity at its source. Tumor-associated macrophages (TAMs), as a significant component of the tumor-infiltrating immune cell population, typically exhibit an M2-like phenotype, further promoting tumor immune escape and progression. Therefore, impaired function and abnormal phenotypes of DCs and macrophages are major obstacles to the success of immunotherapy.

[0003] Dendritic cells (DCs) and macrophages can cross-present antigens or directly eliminate tumors by phagocytosing tumor cells. However, tumor cells have evolved complex immune escape mechanisms, with hijacking phagocytic checkpoints being a key pathway. CD47, a transmembrane protein widely expressed on the surface of tumor cells, can bind to signal regulatory protein α (SIRPα) on the surface of macrophages and DCs, transmitting anti-phagocytic signals to inhibit phagocytosis. Currently, the strategy of blocking the CD47-SIRPα axis with monoclonal antibodies has been used to break this immune escape, but because CD47 is also widely expressed in normal healthy cells, this treatment has limitations such as poor selectivity and high off-target toxicity. In addition, blocking CD47 alone often fails to trigger effective phagocytosis, because the phagocytic process not only requires the removal of anti-phagocytic signals but also depends on the participation of pro-phagocytic signals. Calreticulin (CRT), as a key pro-phagocytic signal, is exposed on the surface of immunogenically dead tumor cells and initiates phagocytosis by binding to receptors such as CD91 on phagocytes. However, the expression levels of CRT and other pro-phagocytic ligands are usually low in solid tumors. Therefore, achieving CD47 blockade and pro-phagocytic signal activation simultaneously in a spatiotemporal synergistic manner may be a more effective strategy to fully unleash the potential of DC and macrophage immunotherapy.

[0004] Besides the limitations of treatment strategies, the lack of tools for non-invasive, real-time tracking of key immune events also severely restricts the evaluation and optimization of immunotherapy. Conventional monitoring indicators (such as changes in tumor volume and immunohistochemical analysis) are either lagging or invasive, failing to capture the real-time dynamics of immune cells within the tumor microenvironment. Although some studies have explored non-invasive visualization of tumor-associated tumors (TAMs) by targeting macrophage receptors with PET or MRI tracers, increasing evidence suggests that the polarization state of TAMs is more prognostic and predictive than their quantity. Therefore, there is an urgent need for imaging strategies that can track phenotypic changes in immune cells within tumors. In recent years, photoacoustic (PA) imaging, as a hybrid imaging technique, has become an ideal non-invasive tool for capturing the immune dynamics of solid tumors due to its superior spatiotemporal contrast and penetration depth compared to conventional fluorescence imaging. However, most existing PA probes are normally luminous, and their signal generation is unaffected by biological background, resulting in low diagnostic specificity. Activable PA probes that can respond to immune-specific signals and switch between "on" and "off" modes can provide more accurate real-time information on immune activity and have broad application prospects. Currently, designing an integrated strategy that can both enhance anti-tumor immunity and visualize in vivo immune dynamics in real time, thereby accurately guiding treatment plans, remains a technical problem that urgently needs to be solved. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in related technologies. Therefore, the first objective of this invention is to provide a method for preparing a diagnostic and therapeutic immunomodulatory material; the second objective is to provide a diagnostic and therapeutic immunomodulatory material; and the third objective is to provide applications of the diagnostic and therapeutic immunomodulatory material.

[0006] To achieve the first objective, the technical solution adopted by this invention is as follows: The preparation method of therapeutic immunomodulatory materials includes: With probe Using the probe as a raw material, nanoparticles P containing the probe were prepared; Construct a cell line that stably co-expresses CD47 and CRT; CRT stands for calreticulin; Nanovesicles (NVs) were prepared using cell lines that stably co-express CD47 and CRT. CD47 / CRT ; Utilizing the nanovesicles NV CD47 / CRT Encapsulating the nanoparticle P yields nanoparticle PN. CD47 / CRT ; Using the coordination system of tannic acid and manganese ions, the nanoparticle PN CD47 / CRT Encapsulation yields a therapeutic immunomodulatory material.

[0007] The probe is a novel molecular probe based on a diaminophenyl core, which reacts with NO to generate a triazole structure. The probe consists of alkoxytriphenylamine as the donor (D) and benzothiadiazole as the acceptor (A). The highly conjugated trithiophene acts as a π-bridge unit, which not only promotes a conjugated planar molecular structure but also facilitates efficient intramolecular charge transfer (ICT). Upon exposure to NO, NO treatment triggers a pronounced bright near-infrared absorption centered at approximately 700 nm, resulting from the enhanced ICT effect. This newly emerging strong near-infrared absorption makes the probe suitable as a material for NO-responsive PA sensors.

[0008] The nanovesicles (NVs) can block the CD47-SIRPα anti-phagocytic axis and present CRT-mediated pro-phagocytic signals. Nanovesicles co-presenting anti-CD47 nanobodies and CRT, while simultaneously encapsulating the NO-responsive PA probe, were successfully constructed, enabling simultaneous immune regulation and real-time immune monitoring. To protect the surface-expressed ligands and specifically enhance immune regulation at tumor sites, PN... CD47 / CRT Encapsulated within a pH-responsive tannic acid-manganese (TA-Mn) shell, this shell rapidly forms on the vesicle surface via an in-situ coordination reaction between tannic acid and manganese ions, creating a biocompatible protective coating. This design aims to shield expressed anti-CD47 nanobodies and CRT molecules during systemic circulation, minimizing off-target interactions and potential immune-related adverse reactions. Importantly, the TA-Mn shell remains stable under physiological conditions but disintegrates in an acidic TME, ensuring on-demand exposure of functional ligands for local immune modulation. Furthermore, the Mn released during shell degradation… 2+ Ions can serve as powerful immune stimulation cues, activating the cGAS-STING axis and amplifying the anti-tumor immune response.

[0009] Furthermore, the synthetic route of the probe is as follows: .

[0010] Furthermore, The synthetic route is shown below: .

[0011] Furthermore, the method for preparing the nanoparticle P includes: adding an organic solution containing poloxamer and the probe to water and subjecting it to ultrasonic treatment, followed by collecting the nanoparticle P through a membrane filter.

[0012] Furthermore, a lentiviral packaging system was used to transfect CD47 and CRT-related plasmids into HEK293T cells to construct the cell line that stably co-expresses CD47 and CRT.

[0013] Furthermore, the lentivirus packaging system is a third-generation plasmid lentivirus system, and the transfection reagent required for transfection is Liposome 2000; The third-generation plasmid lentivirus system includes four plasmids: psPAX2, pMD2.G, pCDH-GFP-CD47, and pCDH-RFP-CRT.

[0014] Furthermore, the nanovesicles NV were prepared using a membrane extrusion method. CD47 / CRT .

[0015] To achieve the second objective, the technical solution adopted by this invention is as follows: Therapeutic immunomodulatory material is prepared using any of the above-described methods for preparing therapeutic immunomodulatory materials, with the nanoparticle P as the core and the nanovesicles NV coated on the surface of the nanoparticle P. CD47 / CRT The nanovesicles NV CD47 / CRT The outer shell covering the surface; The shell layer is composed of tannic acid and Mn. 2+ Complexes formed through coordination.

[0016] To achieve the third objective, the technical solution adopted by this invention is as follows: The application of a therapeutic immunomodulatory material, wherein at least one of the following products is prepared using the therapeutic immunomodulatory material: Integrated tumor diagnosis and treatment preparations, anti-tumor immunomodulators, anti-tumor immunotherapeutic agents, in vivo tumor immune dynamics real-time tracking and reporting reagents, immune adjuvants or immune enhancement carriers.

[0017] Furthermore, the immunomodulatory material has at least one of the following functions: It promotes the body's anti-tumor immune response, enhances the phagocytic activity of dendritic cells, strengthens the phagocytic capacity of macrophages in the tumor microenvironment, and increases the intensity of tumor imaging signals.

[0018] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: The present invention provides a diagnostic and therapeutic immunomodulatory material and its preparation method. The diagnostic and therapeutic immunomodulatory material has nanoparticles P as the core, and the surface of the nanoparticles P is coated with nanovesicles NV. CD47 / CRT Nanovesicles NV CD47 / CRT The surface is covered with a shell, which is composed of tannic acid and Mn. 2+ Coordination-formed complexes. This therapeutic immunomodulatory material integrates activatable PA molecular probes with bispecifically clamped engineered nanovesicles, thereby enabling potent immune activation and / or real-time monitoring.

[0019] Specifically, the genetically engineered membrane nanovesicles NV provided by this invention CD47 / CRT This study showcases an anti-CD47 nanobody and CRT signaling. This bifunctional nanovesicle targets tumor cells by blocking CD47 and recruits phagocytes via CRT-mediated phagocytic signaling, thus bridging tumor cells and immune cells. This synergistic regulation not only promotes dendritic cell maturation but also facilitates the conversion of cells to CD8+. + T-cell antigen cross-presentation, and driving TAMs to shift to the pro-inflammatory M1 phenotype, co-engineer a potent anti-tumor immune response.

[0020] To avoid premature immune recognition and clearance during systemic circulation, the nanovesicles are coated with a pH-responsive tannic acid-manganese (TA-Mn) shell. This protective coating remains intact under physiological conditions but rapidly disintegrates in the acidic TME, thereby exposing functional ligands for targeted tumor binding. Simultaneously, the degradation of the shell releases Mn... 2+ Ions, as potent immunostimulants, activate the cyclic GMP-AMP synthase-interferon gene stimulating factor (cGAS-STING) pathway, inducing a strong production of pro-inflammatory cytokines. This is achieved through CD47 blockade, CRT-mediated phagocytosis, and Mn... 2+ The synergistic effect between the activation of the STING pathway induces a robust and coordinated adaptive immune response.

[0021] The diagnostic and therapeutic immunomodulatory material provided by this invention not only effectively clears tumors in multiple mouse models, but also induces durable immune memory against tumor re-challenge and metastasis.

[0022] Overall, the diagnostic and therapeutic immunomodulatory material provided by this invention couples potent immune regulation with dynamic immune monitoring, offering a new paradigm for precision-guided personalized cancer immunotherapy.

[0023] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0024] Figure 1 T provided in Embodiment 2 of the present invention CD47 / CRT A schematic diagram of the cell preparation process.

[0025] Figure 2 The native HEK-293T cells and T cells provided in Example 2 of this invention CD47 / CRTRepresentative flow cytometry and quantitative analysis results of GFP and RFP reporter positivity rates in cells; Figure A is a two-parameter scatter plot of flow cytometry, Figure B is a statistical bar chart of double positivity rates, Figure C is a graph of Western blotting verification results, and Figure D is a single-parameter flow cytometry histogram of CRT expression.

[0026] Figure 3 Embodiment 2 of the present invention provides native HEK-293T cells and T cells respectively. CD47 / CRT Nanovesicles prepared from cells were co-incubated with 4T1 tumor cells, and then the images were obtained by immunofluorescence staining (IF) after staining with CD47-PE antibody.

[0027] Figure 4 The TTB probe and PN nanoparticles provided in Example 1 of this invention CD47 / CRT NO response characteristics; where A is a schematic diagram of the reaction process between TTB and NO, B is the absorption spectrum of TTB solution before and after NO treatment, and C is the PN... CD47 / CRT Absorption spectra after exposure to different concentrations of NO, Figure D shows the absorption spectra of PN. CD47 / CRT The absorption intensity at 720 nm after treatment with different substances, E figure represents PN CD47 / CRT The PA spectra in solution before and after NO treatment, with F showing the NO-treated PA spectrum. CD47 / CRT The curve showing the change in PA signal amplitude with NP concentration.

[0028] Figure 5 The PN provided in Example 2 of this invention CD47 / CRT A study on the pH-responsive TA-Mn shell coating and tumor microenvironment response characteristics; where Figure A represents PN CD47 / CRT Representative DLS and TEM results for @S NPs, Figure B shows the PN. CD47 / CRT HAADF-STEM images and corresponding element mappings of @S NPs, with Figure C representing PN. CD47 / CRT @S NPs during 12h treatment of Mn under different pH conditions 2 + In vitro cumulative release curve of ions.

[0029] Figure 6 The PN provided in Example 3 of this invention CD47 / CRT@S NPs promote dendritic cell activation and anti-tumor immune initiation; Figure A shows the BMDC isolation and maturation assessment after co-culturing with B16-F10 tumor cells treated with different nanoparticles; Figure B shows the expression of STING signaling protein in BMDCs after different treatments by Western blot determination; Figure C shows the quantitative analysis of IFN-β levels in the supernatant of BMDC-tumor co-culture after different treatments; Figure D shows PN CD47 / CRT Figure 1 shows the enhanced ability of BMDCs to recognize and engulf tumor cells. Figure 2 shows the representative flow cytometry results and quantification of BMDCs (PE-CD11c staining) engulfing B16-F10 tumor cells (CFSE staining) after different treatments. Figure 3 shows the representative flow cytometry results and quantification of mature DCs after different treatments.

[0030] Figure 7 The PN provided in Example 4 of this invention CD47 / CRT The effects of @S NPs on BMDM's phagocytosis of tumor cells and M1 polarization effect are shown in Figure A, which is a schematic diagram of BMDM extraction and its phagocytic activity against tumor cells. Figure B shows the representative flow cytometry results and quantification of 4T1 cells (CFSE staining) phagocytosed by BMDM (PE-CD11b staining) after different nanoparticle treatments. Figure C shows the representative flow cytometry results and quantification of BMDM polarization after different nanoparticle treatments. Figure D shows the representative flow cytometry results and quantification of NO production in BMDM after different nanoparticle treatments.

[0031] Figure 8 The PN provided in Example 5 of this invention CD47 / CRT The in vivo distribution and tumor-targeting properties of @S NPs; Figure A shows the intravenous injection of PN and PN. CD47 / CRT or PN CD47 / CRT Representative images of 4T1 tumor-bearing mice at different time points after @S. Image B shows representative images of the main organs and tumors isolated 24 hours after intravenous injection of different nanoparticles. Image C shows the quantitative results of tumor fluorescence intensity at different time points corresponding to Image A. Image D shows the quantitative results of fluorescence intensity of isolated organs corresponding to Image B.

[0032] Figure 9 PN provided for test example 6 of the present invention CD47 / CRT @S NPs in vivo in situ imaging of macrophage polarization; Figure A is a schematic diagram of the in vivo PA imaging experiment, and Figure B shows bilateral tumor-bearing mice under different pretreatments and PN. CD47 / CRTRepresentative PA images after @SNPs injection (scale bar: 1mm), C shows the corresponding tumor PA signal intensity, D shows the quantitative status of M1 macrophages in the tumor after different treatments, and E shows the intratumoral nitric oxide level measured by the Griess method under different treatments.

[0033] Figure 10 PN provided for test example 7 of the present invention CD47 / CRT @S NPs' potential for dynamic self-monitoring of macrophage activation; Figure A shows the effect of intravenous injection of PN. CD47 / CRT Representative PA images of tumor sites at different time intervals after @S (scale bar: 1mm). Image B shows the corresponding PA intensity, Image C shows the quantitative analysis results of PA intensity in different groups of tumors 24h after injection, and Image D shows the quantitative analysis results of macrophage content.

[0034] Figure 11 The PN provided for test example 8 of the present invention CD47 / CRT @S NPs report the assessment results of the ability of different treatments to induce tumor immune activation; among them, Figure A is a representative PA image of the tumor after different treatments (scale bar: 1 mm), Figure B is a representative flow cytometry result of M1 macrophages in the tumors of mice in different treatment groups, Figure C is the corresponding PA signal intensity, Figure D is the quantitative data, and Figure E is the correlation between the content of M1 macrophages in the tumor and the PA signal intensity by linear regression analysis.

[0035] Figure 12 The PN provided for test example 9 of the present invention CD47 / CRT @S The investigation of in vivo anti-tumor efficacy; where A is a schematic diagram of the experimental protocol for evaluating the therapeutic effect of different nanoparticles in 4T1 tumor-bearing mice, B is the tumor growth curve (n=5 mice in each group), C is the individual tumor volume of 4T1 tumor-bearing mice after different treatments, D is the quantitative data of tumor weight of different groups on day 25 (n=5), E is the representative image of tumor sections stained with TUNEL and the corresponding quantitative analysis results of different groups (n=3), F is the survival curve of different groups of 4T1 tumor-bearing mice (n=8), and G is the change in body weight of treated mice during the study (n=5).

[0036] Figure 13 The PN provided in Example 10 of this invention CD47 / CRT @S's regulatory effect on the tumor immune microenvironment (TME); Figure A shows the flow cytometry analysis results of representative mature DC populations in lymph nodes of different groups of mice; Figure B shows the flow cytometry analysis results of M2 macrophages in different groups of tumors; Figure C shows the flow cytometry analysis results of M1 macrophages in tumors; Figure D shows the flow cytometry analysis results of cytotoxic T cell populations in tumors; Figure E shows the flow cytometry analysis results of IFN-γ CD8+ in tumors. +The results of flow cytometry analysis of the T cell population are shown in Figure F, which shows the results of flow cytometry analysis of the Treg cell population in the tumor. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention, but cannot be used to limit the scope of this invention.

[0038] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available, unless otherwise specified, and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0039] Example 1 I. Synthesis of 4-bromo-N,N-bis(4-(octyloxy)phenyl)aniline The synthetic route is shown below: ; 1. Synthesis of 1-iodo-4-(octyloxy)benzene The process is as follows: Under argon protection, 4-iodophenol (90.9 mmol), 1-bromooctane (100 mmol), and K₂CO₃ (272.7 mmol) were thoroughly mixed, and anhydrous DMF (100 mL) was added. The reaction system was then heated to approximately 120 °C and stirred for 24 h. After the reaction was completed, the mixture was cooled to room temperature (approximately 25 °C), and deionized water was added. The mixture was extracted three times with CH₂Cl₂. The organic phases were combined, dried over MgSO₄, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography using CH₂Cl₂ / n-hexane (v / v = 1:10) as the eluent, yielding a colorless solid 1-iodo-4-(octyloxy)benzene (reaction yield 83%). The characterization data are shown below: 1 ¹H NMR (400 MHz, chloroform-d): δ = 7.42 (s, 2H), 6.56 (d, J = 8.9 Hz, 2H), 3.79 (t, J = 6.6 Hz, 2H), 1.67 (d, J = 12.5 Hz, 2H), 1.28 (dd, J = 55.3, 7.1 Hz, 10H), 0.86–0.77 (m, 3H); 13C NMR (101MHz, chloroform-d): δ=159.11, 138.22, 117.02, 82.54, 68.18, 31.96, 29.50, 29.39, 29.31, 26.16, 22.82, 14.29.

[0040] 2. The synthesis of 4-bromo-N,N-bis(4-(octyloxy)phenyl)aniline is as follows: Under argon protection, 1-iodo-4-(octoxy)benzene (85.2 mmol) and 4-bromoaniline (28.4 mmol) were dissolved in toluene (100 mL). Then, potassium hydroxide (568 mmol), cuprous iodide (28.4 mmol), and o-phenanthroline (28.4 mmol) were added sequentially to the system. After the additions were complete, the reaction system was heated to approximately 110 °C and stirred at this temperature for 24 h. The mixture was then allowed to cool naturally to room temperature. Deionized water was added to quench the reaction, and dichloromethane was used for extraction. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. This crude product was purified by silica gel column chromatography using dichloromethane / n-hexane (v / v = 1:6) as the eluent, ultimately yielding the colorless oily target compound 4-bromo-N,N-bis(4-(octoxy)phenyl)aniline (reaction yield 36%). The characterization data are shown below. 1 H NMR (400MHz, chloroform-d): δ=7.20 (s, 2H), 7.00 (d, J=8.5Hz, 4H), 6.79 (t, J=9.0Hz, 6H), 3.91 (t, J=6.5Hz , 4H), 1.76 (p, J=6.7Hz, 4H), 1.45 (t, J=7.6Hz, 4H), 1.36–1.25 (m, 16H), 0.89 (t, J=6.5Hz, 6H); 13 C NMR (101MHz, chloroform-d): δ=155.75, 148.09, 140.45, 140.43, 131.81, 126.65, 121.93, 115.43, 68.31, 31.94, 29.50, 29.47, 29.37, 26.22, 26.20, 22.80, 22.78, 14.25, 14.23.

[0041] II. Synthesis of the probe compound 4,7-bis(6-(4-(bis(4-(octyloxy)phenyl)amino)phenyl)dithieno[3,2-b:2',3'-d]thien-2-yl)benzo[c][1,2,5]thiadiazole-5,6-diamine, denoted as TTB, with the following molecular structure: ; The synthetic route is shown below: 1. Synthesis of 4-(dithieno[3,2-b:2',3'-d]thiophen-2-yl)-N,N-bis(4-(octoxy)phenyl)aniline The synthesis process is as follows: Under argon protection, dithiopheno[3,2-b:2',3'-d]thiophene (2.04 mmol) was dissolved in anhydrous THF (20 mL). The reaction system was then cooled to approximately -78 °C and maintained at this temperature for 30 min. Next, a solution of n-butyllithium (2.24 mmol) in n-hexane (1.6 mol / L, 1.4 mL) was slowly added dropwise. After stirring for 2 h, tributyltin chloride (2.44 mmol) was added. The reaction system was then allowed to rise naturally to room temperature (approximately 25 °C), stirred overnight, and the reaction was quenched with water. The mixture was then extracted with dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain the crude product. Under an argon atmosphere, the crude product, 4-bromo-N,N-bis(4-(octoxy)phenyl)aniline (2.04 mmol), and Pd(PPh3)4 (0.04 mmol) were added to anhydrous THF (20 mL). The mixture was heated under reflux and stirred for 24 h. After the reaction was completed, the mixture was cooled to room temperature and water was added. The mixture was then extracted three times with dichloromethane. The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography using dichloromethane / n-hexane (v / v = 1:5) as the eluent to obtain a yellow solid 4-(dithienro[3,2-b:2',3'-d]thiophene-2-yl)-N,N-bis(4-(octoxy)phenyl)aniline (yield 46%). The characterization data are shown below: 1 H NMR (400MHz, chloroform-d): δ=7.33 (d, J=6.6Hz, 2H), 7.29–7.22 (m, 2H), 7.18 (d, J=5.5Hz, 1H), 6.99 (d, J=8.8Hz, 4H), 6.84 (d, J=9.4 Hz, 2H), 6.75 (d, J=8.8Hz, 4H), 3.91–3.82 (m, 4H), 1.71 (d, J=13.3Hz, 4H), 1.35 (s, 4H), 1.22 (s, 16H), 0.82 (d, J=4.1Hz, 6H); 13C NMR (101 MHz, chloroform-d): δ = 154.71, 147.66, 144.43, 141.22, 139.49, 139.24, 130.22, 129.87, 127.87, 125.80, 125.54, 125.30, 125.18, 124.40, 119.69, 119.13, 114.31, 67.25, 30.80, 28.36, 28.23, 25.07, 21.66, 13.09.

[0042] 2. Synthesis of 4,4'-((5,6-dinitrobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(dithienro[3,2-b:2',3'-d]thiophene-6,2-diyl))bis( N,N -Bis(4-(octyloxy)phenyl)aniline), its structural formula is shown below: ; Its synthesis process is as follows: Under argon protection, 2.04 mmol of 4-(dithienro[3,2-b:2',3'-d]thiophene-2-yl)-N,N-bis(4-(octoxy)phenyl)aniline was dissolved in 20 mL of anhydrous THF. The solution was cooled to -78 °C and kept at that temperature for 30 min. Then, 1.4 mL of 1.6 mol / L n-butyllithium hexane solution was slowly added dropwise. After stirring for 2 h, 2.44 mmol of tributyltin chloride was added. The reaction system was then allowed to rise naturally to room temperature (approximately 25 °C), stirred overnight, and quenched with water. The mixture was then extracted with dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated to obtain the crude tin-based product. .

[0043] Under argon protection, the above-mentioned crude tin-modified product (2.04 mmol), 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole (1.02 mmol), and Pd(PPh3)4 (0.04 mmol) were dissolved in anhydrous THF (40 mL). The mixture was heated under reflux with stirring for 24 h, then naturally cooled to room temperature (approximately 25 °C), water was added, and the mixture was extracted three times with dichloromethane. The combined organic phases were then subjected to anhydrous... Magnesium sulfate was dried and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using dichloromethane / n-hexane (volume ratio 1:5) as eluent to obtain a green solid 4,4'-(5,6-dinitrobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(dithienro[3,2-b:2',3'-d]thiophene-6,2-diyl))bis(N,N-bis(4-(octyloxy)phenyl)aniline). Its characterization data are shown below: 1H NMR (400MHz, chloroform-d): δ=7.36 (d, J=8.6Hz, 4H), 7.19 (s, 4H), 7.01 (d, J=8.9Hz, 8H), 6.86 (d, J=8.8Hz, 4H), 6.77 (d, J=8.9Hz, 8H), 3.87 (t, J=6.5Hz, 8H), 1.74–1.68 (m, 8H), 1.41–1.37 (m, 8H), 1.18 (s, 32H), 0.82 (d, J=6.2Hz, 12H); 13 C NMR (101 MHz, chloroform-d): δ = 166.73, 154.88, 139.07, 134.17, 131.45, 129.85, 127.78, 125.97, 125.41, 118.92, 114.35, 67.25, 30.82, 28.68, 28.37, 28.24, 27.91, 25.08, 22.74, 13.09.

[0044] 3. Synthesize TTB.

[0045] Under argon protection, 0.037 mmol of 4,4'-(5,6-dinitrobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(dithienro[3,2-b:2',3'-d]thiophene-6,2-diyl))bis(N,N-bis(4-(octoxy)phenyl)aniline) (and 1.11 mmol of iron powder were added to glacial acetic acid (20 mL). After stirring, the mixture was heated to reflux and reacted at this temperature for 15 min. Deionized water was then added to the reaction system, followed by extraction with dichloromethane. The organic phase was dried over anhydrous magnesium sulfate and concentrated. The crude product was purified by silica gel column chromatography using pure dichloromethane as the eluent to obtain red solid TTB (yield 52%). The characterization data are shown below: 1 H NMR (400MHz, chloroform-d): δ=7.74 (d, J=16.5Hz, 4H), 7.63 (d, J=18.6Hz, 4H), 7.29 (d, J=14.3Hz, 8H), 7.15 (d, J=14.6Hz, 4 H), 7.05 (d, J=14.4Hz, 8H), 4.75 (s, 4H), 4.21–4.08 (m, 8H), 1.99 (s, 8H), 1.67 (s, 8H), 1.50 (s, 32H), 1.09 (s, 12H); 13C NMR (101 MHz, chloroform-d): δ = 155.77, 150.62, 148.79, 146.08, 142.32, 140.27, 140.25, 139.62, 135.11, 132.51, 130.93, 126.86, 126.37, 122.25, 120.20, 115.37, 107.39, 68.31, 31.88, 29.77, 29.73, 29.42, 29.33, 26.16, 22.72, 14.19.

[0046] Example 2 The process for preparing a diagnostic and therapeutic immunomodulatory material is as follows: I. Preparation of nanoparticles P (denoted as PNPs).

[0047] Pluronic F-127 (5 mg) and probe TTB (1 mg) were dissolved in THF (1 mL) and stirred thoroughly until completely dissolved to obtain a homogeneous THF solution. Subsequently, the THF solution was slowly added dropwise to deionized water (10 mL) while continuously sonicating for 3 min during the addition process. After sonication, the mixed solution was placed in a fume hood and allowed to evaporate naturally at room temperature to remove THF, resulting in a coarse dispersion of PNPs. Finally, PNPs were collected by centrifugation using a membrane filter with a molecular weight cutoff of 10 kDa.

[0048] II. Construction of HEK293T cell line co-expressing CD47 and CRT.

[0049] like Figure 1 As shown, in order to construct a stable HEK293T cell line co-expressing CD47 and CRT, a third-generation plasmid lentivirus system was used to co-transfect plasmids encoding anti-CD47 nanoantibodies and CRT into HEK293T cells.

[0050] The specific steps are as follows: (1) Cell transfection: Using Lipofectamine 2000 reagent (Invitrogen), plasmids psPAX2, pMD2.G, pCDH-GFP-CD47 and pCDH-RFP-CRT were mixed in a ratio of 5:7.5:5:5 and transfected into HEK293T cells after thorough mixing.

[0051] (2) Virus harvest: After transfection, cell culture supernatant, i.e. virus supernatant, was collected 48h and 72h after transduction and stored at low temperature for later use.

[0052] (3) Cell screening and expansion: HEK293T cells transfected with puromycin and G418 reagent (Invitrogen, Carlsbad, CA, USA) were screened over a two-week period. Finally, a stable HEK293T cell line co-expressing CD47 and CRT was obtained and named T. CD47 / CRT .

[0053] Both were fused with different fluorescent reporter genes (GFP for anti-CD47 and mCherry for CRT) and membrane targeting sequences, and were co-transfected with lentiviral packaging plasmids.

[0054] After screening, a stable dual-expression cell line (TCD47 / CRT) was established; subsequently, the expression efficiency and membrane localization of the anti-CD47 nanobody and CRT were verified. Fluorescence reporter analysis confirmed that both plasmids were efficiently transfected in engineered cells. Figure 2 As shown in Figures A and B.

[0055] Western blot analysis confirmed the engineered T by detecting their respective fusion tags. CD47 / CRT Both anti-CD47 nanobody and CRT were strongly expressed in cells, while these signals were not detected in parental native 293T cells. Figure 2 As shown in Figure C.

[0056] Notably, both the anti-CD47 nanobody and CRT were highly enriched in cell membrane nanovesicles, indicating their effective presentation on the cell surface. Furthermore, flow cytometry using fluorescently labeled anti-CRT antibodies showed that almost all engineered T cells... CD47 / CRT All surfaces exhibit CRT (99.5%). Figure 2 As shown in Figure D.

[0057] III. Preparation of Nanovesicles (NVs) CD47 / CRT ).

[0058] Take T cells cultured to the logarithmic phase CD47 / CRT Cells were ruptured through continuous hypotonic treatment, and the resulting cell suspension was then extruded through a polycarbonate membrane. After extrusion, centrifugation was performed to remove cell debris and unbound impurities, yielding nanovesicles (NVs). The specific process is as follows: (1) T CD47 / CRT Cells were suspended in a hypotonic lysis buffer consisting of D-mannitol (225 mM), Tris-HCl (pH 7.5) (30 mM), EGTA (0.2 mM), and sucrose (75 mM), with a mixture of phosphatase and protease inhibitors added to prevent protein degradation.

[0059] (2) Cell disruption: Using a Kinematica Polytron PT10 / 35 probe homogenizer, the cell suspension was mechanically disrupted 15 times at 70% power in the same hypotonic lysis buffer to obtain cell homogenate.

[0060] (3) Centrifugation purification: The obtained cell homogenate was centrifuged at 10,000g for 25 minutes to precipitate cell nuclei and cell debris, and the supernatant was collected; then the supernatant was further centrifuged at 150,000g for 35 minutes to separate the cell membrane portion, the supernatant was discarded, and the precipitate was retained.

[0061] (4) Resuspension and preservation: The cell membrane precipitate was resuspended in an EDTA aqueous solution (0.2 mM) to obtain cell membrane nanovesicles, denoted as NV. CD47 / CRT The total protein concentration of cell membrane samples was determined using the BCA protein assay kit. After the assay, the membrane samples were frozen and stored at -80°C for later use.

[0062] Transmission electron microscopy (TEM) and dynamic light scattering (DLS) together indicate that NV CD47 / CRT The nanovesicles are spherical with an average diameter of nearly 200 nm. Competitive binding experiments verified that the CD47 nanobodies displayed on the nanovesicles can effectively bind to CD47 on tumor cells, confirming their surface expression and functional activity. Figure 3 As shown. Where N native (or abbreviated as N) refers to cell membrane nanovesicles prepared using native HEK-293T cells as raw materials, prepared by the same method as NV. CD47 / CRT NV CD47 To utilize HEK-293T cells that express only CD47 (the construction process of these cells is basically the same as that of T cells, except that CRT protein is not introduced), CD47 / CRT Nanovesicles prepared using cell-specific methods.

[0063] IV. Preparation of Nanoparticles (PN) CD47 / CRT ).

[0064] The aforementioned prepared nanoparticles P NPs and nanovesicles T CD47 / CRT After thorough vortex mixing (using a CRT protein to NPs mass ratio of 1:4), the mixture was extruded sequentially through aqueous filters with gradually decreasing pore sizes (800 nm, 400 nm, and 200 nm) to ensure complete incorporation and uniform dispersion. After extrusion, the resulting suspension was centrifuged at 15000 g for 20 min at 4°C. This centrifugation was repeated three times to ensure complete purification, yielding a nanoparticle solution (protein concentration of 1 μg / μL), denoted as PN. CD47 / CRT NPs (or abbreviated as PN)CD47 / CRT ).

[0065] PN CD47 NPs (or abbreviated as PN) CD47 Besides nanovesicles, NV CD47 The rest of the process is the same as PN. CD47 / CRT The preparation process of NPs.

[0066] PN NPs (or abbreviated as PN) are used in addition to nanovesicles with N native The rest of the process is the same as PN. CD47 / CRT The preparation process of NPs.

[0067] V. Preparation of therapeutic immunomodulatory materials.

[0068] The PN prepared above CD47 / CRT Add 200 μL of NPs to 10 μL of tannic acid (TA) aqueous solution (20 mg / mL), vortex for 60 s to ensure thorough mixing; then add 50 μL of MnSO4·H2O aqueous solution (10 mg / mL), and continue vortexing for 60 s to ensure complete reaction; finally, add 1 mL of PBS buffer, and centrifuge the mixture using a membrane filter with a molecular weight cutoff of 10 kDa. Repeat this washing process three times to remove unreacted impurities and excess reagents. The resulting solution is the therapeutic immunomodulatory material, denoted as PN. CD47 / CRT @S NPs (or abbreviated as PN) CD47 / CRT @S), store it at 4℃ for later use.

[0069] PN CD47 @S NPs (or abbreviated as PN) CD47 @S) In addition to PN CD47 / CRT NPs replaced with PN CD47 NPs, the rest of the process is the same as PN CD47 / CRT Preparation process of @S NPs.

[0070] PN@S NPs (or abbreviated as PN@S) are NPs that, in addition to PN CD47 / CRT Replace NPs with PN NPs, and the rest of the process is the same as PN. CD47 / CRT Preparation process of @S NPs.

[0071] Test Example 1 TTB probe and nanoparticle PN CD47 / CRT NO response characteristics, such as Figure 4 As shown.

[0072] We designed and synthesized a novel NO-responsive molecular probe TTB based on a diaminobenzene core, which can specifically react with NO to generate a triazole structure.

[0073] like Figure 4 As shown in Figure A, this probe employs a donor-π-bridge-acceptor (D-π-A) conjugated structure: alkoxytriphenylamine serves as the electron donor (D), benzothiadiazole as the electron acceptor (A), and a highly conjugated trithiophene unit is introduced as a π-bridge. The trithiophene unit not only promotes the formation of a planar conjugated structure but also effectively mediates intramolecular charge transfer (ICT) processes. When exposed to NO, the probe undergoes a structural transition from a "strong D-weak A" configuration to a "strong D-strong A" configuration, leading to significant changes in its photophysical properties.

[0074] After reacting with NO, the color of the probe solution changed significantly from yellow to green, as shown below. Figure 4 As shown in Figure B, a significantly enhanced near-infrared absorption band appears at approximately 700 nm, a phenomenon attributed to the enhanced ICT effect following the structural transition. This activatable near-infrared absorption characteristic gives the probe the potential to function as a photoacoustic (PA) sensor for NO-related detection.

[0075] To verify the PN nanoparticles CD47 / CRT The NO response performance of the instrument was investigated, and its response behavior to different NO concentrations was examined. Figure 4 As shown in Figure C, this result indicates that the near-infrared absorption intensity of the nanoprobe is significantly positively correlated with the sodium nitrite concentration.

[0076] Meanwhile, selective experimental results showed that, compared to other bio-related substances, nanoparticles PN... CD47 / CRT It exhibits excellent response specificity to NO, such as Figure 4 As shown in Figure D.

[0077] Based on the enhanced near-infrared absorption characteristics after NO activation, this invention further evaluates the PN nanoparticles. CD47 / CRT PA performance, such as Figure 4 In the E diagram, before NO treatment, PN CD47 / CRT The system exhibited only a weak PA signal in the 680–950 nm spectral range; however, after NO activation, a significantly enhanced PA signal appeared in this range, confirming the NO-responsive PA activation capability of the nanoplatform.

[0078] This invention further quantitatively analyzed the correlation between PA signal intensity at 720 nm and NO concentration. The results showed that PA intensity increased linearly with increasing NO concentration, as shown in the figure below. Figure 4 As shown in Figure F.

[0079] In summary, the above results confirm the successful construction of cell membrane-encapsulated nanovesicles that simultaneously co-present anti-CD47 nanobodies and CRT, and encapsulate NO-responsive PA probes. CD47 / CRTIt can achieve simultaneous immune regulation and real-time immune monitoring.

[0080] Test Example 2 like Figure 5 As shown, PN CD47 / CRT Study on pH-responsive TA-Mn shell coating and tumor microenvironment response characteristics.

[0081] To protect the ligands expressed on the vesicle surface and specifically enhance the immunomodulatory effect at the tumor site, this invention uses PN CD47 / CRT Encapsulated within a pH-responsive tannic acid-manganese (TA-Mn) shell. This shell rapidly forms on the vesicle surface via an in-situ coordination reaction between tannic acid and manganese ions, constructing a biocompatible protective coating. Its design aims to shield surface-expressed anti-CD47 nanoantibodies and CRT protein molecules during systemic circulation, minimizing off-target interactions and potential immune-related adverse reactions. The TA-Mn shell remains stable under physiological conditions but disintegrates in the acidic tumor microenvironment (TME), enabling on-demand exposure of functional ligands and ensuring precise local immune regulation. Furthermore, the Mn released during shell degradation… 2+ Ions can serve as potent immunostimulatory signals, activating the cGAS-STING pathway and amplifying anti-tumor immune responses. TA-Mn-coated nanovesicles (PNVs) CD47 / CRT The average hydrated diameter of @S NPs was 152 nm, slightly larger than that of uncoated PNs. CD47 / CRT ,like Figure 5 As shown in Figure A; PN CD47 / CRT HAADF-STEM images of @S NPs and their corresponding element mappings, such as Figure 5 As shown in Figure B; To evaluate PN CD47 / CRT The pH-responsive degradation behavior of @S NPs, this invention will... CD47 / CRT @S NPs were incubated under physiological conditions (pH 7.4) and weakly acidic conditions (pH 6.0), respectively. The results showed that acidic conditions accelerated shell disintegration, and Mn was reduced within 12 hours. 2+ The release rate is approximately 58%, such as Figure 5 As shown in Figure C, this facilitates the exposure of surface ligands.

[0082] Test Example 3 PN CD47 / CRT @S NPs promote dendritic cell activation and anti-tumor immune initiation, such as Figure 6 As shown.

[0083] This invention explores PN CD47 / CRTCan @SNPs enhance anti-tumor immune responses by inducing dendritic cell (DC) activation and maturation? After DC maturation, it can upregulate co-stimulatory molecules and enhance antigen presentation capacity, initiating antigen-specific T-cell immune responses. Therefore, effectively promoting DC maturation is a key step in stimulating T-cell-mediated anti-tumor immunity. This study hypothesizes: CD47 immune blockade, CRT-mediated phagocytosis, and Mn... 2+ The STING pathway activated by the drive works synergistically to promote DC maturation.

[0084] like Figure 6 Figure A shows the isolation of bone marrow-derived dendritic cells (BMDCs) from the tibia and femur of mice.

[0085] First, evaluate PN at the cellular level. CD47 / CRT The activation effect of @S NPs on the STING signaling pathway. After co-culturing BMDCs with B16-F10 cells, different nanoparticle interventions were administered. Western blot was used to assess the expression levels of STING pathway-related signaling proteins. Results are as follows: Figure 6 As shown in Figure B, compared with the PBS control group, the nanoparticles containing the TA-Mn shell (PN@S, PN...) showed... CD47 @S and PN CD47 / CRT @S) can significantly upregulate the expression of phosphorylated STING (p-STING), phosphorylated TBK1 (p-TBK1), and phosphorylated IRF3 (p-IRF3) in BMDC. These results indicate that Mn can be released 2+ The TA-Mn nanoshell can effectively activate the cGAS-STING signaling pathway. Among them, PN... CD47 / CRT The upregulation of p-STING, p-TBK1, and p-IRF3 was most significant in the @S group.

[0086] ELISA test results further indicate that PN CD47 / CRT@S The highest IFN-β secretion levels were observed in the treated groups, namely the PBS group, the single PN group, the PN@S group, and the PN group. CD47 / CRT Group and PN CD47 @Group S's 7.63x, 7.34x, 2.56x, 4.12x, and 1.47x, such as Figure 6 As shown in Figure C. The above data fully confirms that PN CD47 / CRT @S can significantly activate the cGAS-STING signaling pathway within BMDC.

[0087] Subsequently, the present invention further examined PN CD47 / CRT The regulatory role of @S on the phagocytic uptake capacity of DC-mediated tumor cells, such as Figure 6As shown in Figure D. CFSE-labeled B16-F10 tumor cells were co-cultured with BMDCs for 24 h under different nanoparticle intervention conditions. DCs were then stained with PE-labeled anti-CD11c antibody, and phagocytosis was quantitatively analyzed by flow cytometry. The results are shown in Figure D. Figure 6 As shown in Figure E, it can be seen from the figure that compared with other groups, PN CD47 / CRT The proportion of CFSE-positive cells in the co-culture system of the @S treatment group was significantly increased, indicating that PN CD47 / CRT @S can enhance the phagocytic ability of BMDCs against tumor cells. PN CD47 / CRT@ The phagocytic index of group S was significantly higher than that of the nanovesicle group presenting only CD47 nanobodies. This result indicates that CD47 nanobodies and CRT can produce a synergistic effect, effectively promoting the phagocytosis and uptake of tumor cells by BMDCs. Based on this, this invention further analyzed the phenotype of BMDCs in each group using flow cytometry, and the results are as follows: Figure 6 As shown in Figure F.

[0088] Test Example 4 PN CD47 / CRT The influence of @S NPs on promoting phagocytosis of tumor cells by bone marrow-derived macrophages (BMDM) and the M1 polarization effect, such as Figure 7 As shown.

[0089] First, BMDM was isolated from mouse bone marrow, and then PN was systematically evaluated. CD47 / CRT The effect of @S NPs on the phagocytic capacity of tumor cells mediated by macrophages. In the experiment, tumor cells were labeled with CFSE and co-seeded with BMDM, followed by incubation with different nanoparticles, such as... Figure 7 As shown in Figure A.

[0090] Flow cytometry results, such as Figure 7 As shown in Figure B, compared to other groups, exposure to PN... CD47 / CRT In macrophages of 4T1 cells in group @S, the proportion of CFSE-positive signals was significantly increased, indicating that PN CD47 / CRT @S can effectively enhance the phagocytic activity of BMDM against tumor cells. Quantitative analysis results further confirm that PN CD47 The phagocytosis ratio of the @S group was 13.57, significantly higher than that of the PBS group (5.19), the PN group alone (5.21), and the PN@S group (6.87), suggesting that the presentation of CD47 nanobody can effectively promote macrophage-mediated tumor cell uptake. CD47 / CRT The phagocytosis ratio of the @S treatment group further increased to 21.80, compared to PN. CD47The 1.60-fold increase in the @S group indicates that CRT modification on the surface of nanovesicles can further enhance the ability of BMDM to phagocytose tumor cells. As a classic pro-phagocytic signaling molecule, CRT can promote the recognition and phagocytosis of tumor cells by macrophages through specific binding to scavenger receptors (such as LRP1, LOX-1, CD91, etc.) on the surface of macrophages. The results of this invention clearly demonstrate that the co-display of anti-CD47 nanobodies and CRT can significantly enhance the interaction between tumor cells and phagocytic cells.

[0091] Subsequently, this invention further investigated whether the synergistic effect of increased phagocytosis and STING pathway activation could regulate macrophage phenotypic polarization and functional state. Flow cytometry results showed that PN CD47 / CRT @S processing group CD86 + The proportions of BMDM (characteristic markers of M1 macrophages) were respectively in the PBS group, the PN group alone, the PN@S group, and the PN group. CD47 / CRT Group and PN CD47 @Group S's 3.27x, 3.26x, 2.35x, 1.49x, and 1.60x, such as Figure 7 As shown in Figure C.

[0092] Furthermore, detection and analysis using the NO-sensitive probe DAF-FM DA revealed that PN CD47 / CRT The NO release in BMDM treated with @S increased significantly, such as Figure 7 As shown in Figure D.

[0093] The above results not only confirm that: PN CD47 / CRT @S treatment can effectively reprogram BMDM to a pro-inflammatory M1 phenotype, and also suggests that it is feasible to use NO-responsive probes to monitor macrophage-mediated immune responses during immunotherapy.

[0094] Test Example 5 PN CD47 / CRT The in vivo distribution behavior and tumor-targeting enrichment ability of @SNPs, such as Figure 8 As shown.

[0095] First, different nanoparticles were labeled with DiO, and their pharmacokinetic and tissue distribution characteristics were compared and visualized using in vivo fluorescence imaging.

[0096] like Figure 8 As shown in Figure A, PN CD47 / CRT @S produced significant fluorescence enrichment signals at the tumor site, reaching a peak fluorescence level approximately 12 hours after intravenous injection. Quantitative analysis showed that at 12 hours, PN... CD47 / CRT The tumor fluorescence signal intensity in group S was approximately that of PN without a TA-Mn shell. CD47 / CRT1.67 times that of nanovesicles, such as Figure 8 As shown in Figure C, this result indicates that TA-Mn shell coating enhances tumor accumulation efficiency. Meanwhile, PN CD47 / CRT The tumor accumulation of @S was 2.59 times higher than that of blank PN nanovesicles without ligand expression. Upon encountering acidic TME, the pH-responsive TA-Mn coating disintegrated, exposing the CD47 nanobody and CRT protein, which could promote specific interactions with tumor and immune cells, thereby increasing tumor enrichment compared to the non-expression control; and a persistent tumor fluorescence signal could still be detected 24 hours after injection, indicating that its in vivo retention time was significantly prolonged.

[0097] To verify the in vivo results, major organs and tumor tissues of mice were dissected 24 hours after drug administration, and analyzed using an IVIS imaging system. The results are as follows: Figure 8 As shown in Figures B and D, the control group (non-expressing nanovesicles) exhibited weak fluorescence in tumor tissue, indicating that passive accumulation was insufficient for effective tumor delivery. CD47 / CRT While nanovesicles can enhance interactions with tumor cells and local immune cells, premature exposure during circulation may lead to off-target recognition, resulting in only a moderate increase in tumor accumulation compared to the non-expressing control. Although CD47 nanobodies and CRT modification can enhance interactions with phagocytes, PN and PN... CD47 / CRT Only slight differences were observed in liver and spleen accumulation between the two. Notably, the PN shielded by the TA-Mn shell... CD47 / CRT @S compared to PN alone and PN CD47 / CRT All groups showed significantly enhanced tumor signal, consistent with in vivo imaging results. These results indicate that a controlled shielding and pH-triggered on-demand exposure strategy can effectively promote tumor-targeted delivery.

[0098] Test Example 6 To evaluate PN CD47 / CRT @S in vivo in situ imaging of macrophage polarization, such as Figure 9 As shown.

[0099] Establish bilateral tumor models, such as Figure 9 As shown in Figure A, the tumor was pretreated with PBS or hydroxychloroquine HCQ, followed by intravenous injection of PN. CD47 / CRT PA imaging was performed at @S. To confirm NO-dependent PA activation, Carboxy-PTIO was subsequently injected into the HCQ-pretreated tumor, followed by intravenous administration of PN. CD47 / CRT @S NPs, and PA imaging was performed on two tumor sites, with the following results: Figure 9 As shown in Figures B and C, HCQ-pretreated tumors exhibited significantly enhanced PA signals, with an intensity 2.92 times higher than that of PBS-treated tumor sites.

[0100] Flow cytometry was used to quantitatively assess macrophage polarization in tumor tissues. Consistent with imaging results, the proportion of M1-polarized macrophages was higher in HCQ-pretreated tumor tissues than in the PBS group, confirming the presence of PN. CD47 / CRT @S NPs can report macrophage activation dynamics in vivo, such as Figure 9 As shown in Figure D.

[0101] To further verify that the observed PA signal enhancement originates from NO generated during M1 polarization, this invention administers the selective NO scavenger Carboxy-PTIO to HCQ-pretreated tumor sites. Notably, the PA signal was significantly reduced in the Carboxy-PTIO treatment group, such as... Figure 9 As shown in Figures B and C, quantitative analysis of tissue NO levels revealed a strong correlation between PA signal intensity and local NO concentration in each group, as illustrated in Figures B and C. Figure 9 As shown in Figure E, the PA response of the nanoprobe is closely related to local NO levels. These results suggest that activated PA imaging strategies have potential applications in deep tumors.

[0102] Test Example 7 PN CD47 / CRT @S studies its potential for dynamic self-monitoring of macrophage activation, such as Figure 10 As shown.

[0103] Specifically, tumor-bearing mice were given PBS or PN via intravenous route. CD47 / CRT @S, the result is as follows Figure 10 As shown in Figures A and B, PN CD47 / CRT The PA signal within the tumor increased over time in the @S treatment group, reaching its maximum at 24 h. In contrast, the PBS group showed persistently weak PA intensity throughout the observation window.

[0104] To further confirm the macrophage-dependent nature of PA signaling activation, this invention established an additional macrophage depletion cohort (named the MD group) by selectively depleting phagocytes with clophosphate liposomes. Notably, PA signaling was significantly reduced in the MD group, confirming that the observed PA signaling enhancement was dependent on macrophage activation. Furthermore, flow cytometry was used to analyze the proportion of M1 macrophages in tumor tissues from different treatment groups, such as... Figure 10 As shown in Figures C and D, the obtained trend is consistent with the PA imaging results.

[0105] Test Example 8 This invention further evaluates PN CD47 / CRT @S NPs report the ability of different treatments to induce tumor immune activation, such as Figure 11 As shown.

[0106] In this experiment, tumor-bearing mice subcutaneously inoculated with 4T1 tumors were randomly divided into 6 groups and treated with PBS, N alone, N@S, NCD47 / CRT, NCD47@S, or NCD47 / CRT@S via intravenous injection, respectively. After two consecutive rounds of treatment, all mice were intravenously injected with PNCD47 / CRT@S for photoacoustic (PA) imaging to monitor the M1 polarization state of macrophages in the tumor microenvironment (TME) under different treatment regimens in real time.

[0107] like Figure 11 As shown in Figures A and C, these figures reveal that the PA imaging intensity in the N group and the N@S group was almost significantly different from that in the PBS control group, indicating that simple N treatment and the simple combination of N and S had no significant inducing effect on macrophage M1 polarization. CD47 / CRT and N CD47 Following @S treatment, enhanced PA signals were observed at tumor sites, with signal intensities increasing 4.30-fold and 3.98-fold compared to the PBS control group, respectively. This suggests that both treatments can effectively induce macrophage M1 polarization and enhance tumor immune activation. It is noteworthy that N... CD47 / CRT The @S treatment group exhibited the highest PA signal intensity, amplified 7.15 times compared to the PBS control group, and significantly higher than all other treatment groups. This significant signal enhancement indicates that the activation level of macrophages and the M1 polarization level in the tumor microenvironment of this treatment group reached the highest levels.

[0108] To verify the reliability of the above PA imaging results, this invention further employs flow cytometry for quantitative analysis to accurately depict the polarization characteristics of macrophages in tumor tissue after different treatment regimens. The results are as follows: Figure 11 As shown in Figures B and D, we can see from these two figures that: N CD47 / CRT CD45 in tumor tissue of the S treatment group + CD11b + F4 / 80 + CD86 + The proportion of macrophages (i.e. M1 macrophages) was the highest, increasing by 3.42 times, 3.17 times, 2.74 times, 1.45 times, and 1.62 times respectively compared to the PBS control group, N alone treatment group, N@S treatment group, NCD47 / CRT treatment group, and NCD47@S treatment group, which is highly consistent with the PA imaging results.

[0109] Linear regression analysis further confirmed a very strong positive correlation between PA signal intensity and M1 macrophage infiltration density (R0). 2 =0.93), such as Figure 11 As shown in the E-plot, this result strongly supports PN. CD47 / CRT@S can not only act as an inducer of M1 macrophage activation, but also as a reporter molecule for real-time monitoring of dynamic changes in tumor immunity.

[0110] Test Example 9 PN CD47 / CRT An investigation into the in vivo antitumor efficacy of @S, such as Figure 12 As shown.

[0111] like Figure 12 As shown in Figure A, on day 0, 1×10 6 4T1 cells were subcutaneously injected into the right flank of female BALB / c mice to establish a 4T1 tumor-bearing mouse model. On day 7, the mice were randomly divided into six groups and given different treatments, as shown below: PBS group, PN group, PN@S group, PN CD47 / CRT Group, PN CD47 @S group or PN CD47 / CRT @Group S, tumor volume was measured every two days to dynamically monitor tumor growth.

[0112] like Figure 12 As shown in Figures B and C, compared with the rapid tumor progression in the PBS group, the PN and PN@S groups showed only negligible tumor suppression effects, while the PN group... CD47 @S group showed moderate tumor suppression effect; notably, PN CD47 / CRT Group S showed the most significant tumor-suppressing effect, significantly superior to other groups. By day 25, PN CD47 / CRT The mean tumor volume of the @S group was approximately the same as that of the PBS group, PN group, PN@S group, and PN group. CD47 / CRT Group and PN CD47 The results of 1 / 7.28, 1 / 6.70, 1 / 6.61, 1 / 3.22, and 1 / 3.98 in the @S group further confirm its excellent tumor suppression effect.

[0113] To further verify the effects of different treatments on tumor growth, tumors were dissected from mice in each group after the experiment and their weight was measured. The results were consistent with the trend observed in tumor volume monitoring, such as... Figure 12 As shown in Figure D, this confirms the differences in antitumor effects among different treatment groups.

[0114] To further investigate the antitumor effect, this invention employs terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) staining to detect tumor tissue damage and tumor cell proliferation, such as... Figure 12 As shown in Figure E, from which we can see that: PN CD47 / CRT @S treatment can significantly induce significant tumor tissue damage and strongly inhibit the proliferation of tumor cells.

[0115] Survival outcome as Figure 12 As shown in Figure F, the mice in the PNCD47 / CRT@S treatment group had significantly better survival than the other groups. More than 50% of the mice in this group survived for more than 80 days, while the survival rates of mice treated with PBS, PN alone, PN@S, and PN... CD47 / CRT and PN CD47 @S group mice all died within 30, 31, 34, 59, and 50 days, fully demonstrating PN CD47 / CRT @S significantly prolongs the survival time of tumor-bearing mice.

[0116] Furthermore, throughout the treatment process, all treatment groups of mice showed a slight trend of weight gain, suggesting that the administered nanoparticles had good biocompatibility. Figure 12 As shown in the G diagram.

[0117] Test Example 10 To further elucidate PN CD47 / CRT The potential antitumor mechanism of @S was further explored in this invention, specifically its regulatory role in the tumor immune microenvironment (TME). After each treatment, mouse tumor tissues and tumor-draining lymph nodes were collected, and the activation status of immune cells in vivo was assessed using flow cytometry.

[0118] like Figure 13 As shown in Figure A, PN CD47 / CRT Mature dendritic cells (DCs, phenotype CD45) in the @S treatment group + CD11c + CD80 + CD86 + The highest proportion was observed in the PBS control group (10.9%), PN group (11.2%), PN@S group (15.4%), and PN... CD47 / CRT Group (27.4%) and PN CD47 @Group S (25.1%). This result is highly consistent with the in vitro experimental results, confirming PN CD47 / CRT The @S group can significantly promote DC maturation; and after effective activation of DC, it can further initiate anti-tumor T cell immune responses and enhance the intensity of downstream adaptive immune responses.

[0119] In the tumor immune microenvironment, tumor-associated macrophages (TAMs) constitute the most abundant group of infiltrating immune cells, and their polarization state directly affects tumor progression and immune response efficiency. This invention uses flow cytometry to further evaluate the regulatory effects of different treatment groups on the polarization of tumor macrophage subsets. Consistent with in vitro experimental results, PN... CD47 / CRT @S can significantly reduce the number of pro-tumorigenic and immunosuppressive M2 macrophages (phenotype CD45) in tumor tissue. + CD11b +F4 / 80 + CD206 + The proportion of M1 macrophages (phenotype CD45) was significantly increased, while the proportion of anti-tumor and pro-inflammatory macrophages was also significantly increased. + CD11b + F4 / 80 + CD86 + The proportion of ), such as Figure 13 As shown in Figures B and C. It is worth noting that PN CD47 / CRT @S increased the M1 / M2 macrophage ratio in the tumor to 2.93, which was significantly higher than that of the PBS control group (0.40), PN group (0.43), PN@S group (0.62), PNCD47 / CRT group (1.32) and PNCD47@S group (1.03).

[0120] In confirming PN CD47 / CRT @S can effectively promote DC maturation and M1 macrophage polarization. This invention further evaluated its ability to stimulate the body to produce an anti-tumor T-cell immune response. For example... Figure 13 As shown in Figure D, PN CD47 / CRT @S treatment group tumor tissue CD8 + T cells (phenotype CD45) + CD3 + CD8 + The infiltration rate of the nanoparticles was significantly increased, ranging from 1.59 to 4.24 times that of the other groups, indicating that the nanoparticles possess a strong ability to activate CD8. + The ability of T cells to mediate specific anti-tumor immune responses. Meanwhile, PN CD47 / CRT @S treatment followed by intratumoral CD8 + IFN-γ + Cells (phenotype CD45) + CD3 + CD8 + IFN-γ + The proportion of cytotoxic T cells also increased significantly, indicating that the activation level and effector function of cytotoxic T cells were effectively enhanced. Figure 13 As shown in Figure E.

[0121] Further analysis was conducted on the infiltration of Tregs within the tumor tissue, such as... Figure 13 As shown in the F diagram, PN CD47 / CRT @S treatment can significantly reduce the infiltration rate of immunosuppressive Tregs in tumors, thereby significantly increasing CD8 levels. + The ratio of T cells to Tregs can relieve the immunosuppressive effect of Tregs on effector T cells.

[0122] The above experimental results fully confirm that PN CD47 / CRT@S possesses strong immunomodulatory capabilities, which can induce the body to produce a potent and coordinated anti-tumor immune response and effectively reshape the immunosuppressive tumor microenvironment, providing important immune mechanism support for its highly efficient anti-tumor effect.

[0123] 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 of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a diagnostic and therapeutic immunomodulatory material, characterized in that, include: With probe Using the probe as a raw material, nanoparticles P containing the probe were prepared; Construct a cell line that stably co-expresses CD47 and CRT; CRT stands for calreticulin; Nanovesicles (NVs) were prepared using cell lines that stably co-express CD47 and CRT. CD47 / CRT ; Utilizing the nanovesicles NV CD47 / CRT Encapsulating the nanoparticle P yields nanoparticle PN. CD47 / CRT ; Using the coordination system of tannic acid and manganese ions, the nanoparticle PN CD47 / CRT Encapsulation yields a therapeutic immunomodulatory material.

2. The method for preparing the therapeutic immunomodulatory material as described in claim 1, characterized in that, The synthesis route of the probe is shown below: 。 3. The method for preparing the diagnostic and therapeutic immunomodulatory material as described in claim 2, characterized in that, The synthetic route is shown below: 。 4. The method for preparing the diagnostic and therapeutic immunomodulatory material as described in claim 1, characterized in that, The method for preparing the nanoparticles P includes: adding an organic solution containing poloxamer and the probe to water and subjecting it to ultrasonic treatment, followed by collecting the nanoparticles P through a membrane filter.

5. The method for preparing the diagnostic and therapeutic immunomodulatory material as described in claim 1, characterized in that, The CD47 and CRT-related plasmids were transfected into HEK293T cells using a lentiviral packaging system to construct a cell line that stably co-expresses CD47 and CRT.

6. The method for preparing the therapeutic immunomodulatory material as described in claim 5, characterized in that, The lentivirus packaging system is a third-generation plasmid lentivirus system, and the transfection reagent required for transfection is Liposome 2000. The third-generation plasmid lentivirus system includes four plasmids: psPAX2, pMD2.G, pCDH-GFP-CD47, and pCDH-RFP-CRT.

7. The method for preparing the diagnostic and therapeutic immunomodulatory material as described in claim 1, characterized in that, The nanovesicles NV were prepared by membrane extrusion. CD47 / CRT .

8. A diagnostic and therapeutic immunomodulatory material, characterized in that, Prepared using the method for preparing therapeutic immunomodulatory materials as described in any one of claims 1 to 7, wherein the nanoparticle P is the core, and the surface of the nanoparticle P is coated with the nanovesicles NV. CD47 / CRT The nanovesicles NV CD47 / CRT The outer shell covering the surface; The shell layer is composed of tannic acid and Mn. 2+ Complexes formed through coordination.

9. The application of diagnostic and therapeutic immunomodulatory materials, characterized in that, At least one of the following products can be prepared using the therapeutic immunomodulatory material as described in claim 8: Integrated tumor diagnosis and treatment preparations, anti-tumor immunomodulators, anti-tumor immunotherapeutic agents, in vivo tumor immune dynamics real-time tracking and reporting reagents, immune adjuvants or immune enhancement carriers.

10. The application of the therapeutic immunomodulatory material as described in claim 9, characterized in that, The immunomodulatory material has at least one of the following functions: It promotes the body's anti-tumor immune response, enhances the phagocytic activity of dendritic cells, strengthens the phagocytic capacity of macrophages in the tumor microenvironment, and increases the intensity of tumor imaging signals.

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