Dual-modal magnetic particle fluorescence probe and its application in the preparation, tracing and monitoring of dendritic cell vaccines

Abstract

The present invention provides a dual-modal magnetic particle fluorescence probe and its application in the preparation, tracing and monitoring of dendritic cell vaccines, belonging to the field of biotechnology. The dual-modal magnetic particle fluorescence probe includes: magnetic nanoparticles, cell-penetrating peptides, fluorescence resonance energy transfer fluorescence reporter groups and caspase substrates. The dual-modal magnetic particle fluorescence probe of the present invention combines the advantages of nuclear magnetic resonance imaging and fluorescence resonance energy transfer imaging, can provide real-time imaging information in different biological events, has a high signal-to-background ratio and specificity, and can realize the in vivo monitoring of the behavior of dendritic cell vaccines in real time, including processes such as migration, localization and apoptosis, which helps to improve the efficiency and accuracy of vaccine research and application.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a dual-modal magnetic particle fluorescence probe and its application in the preparation, tracing, and monitoring of dendritic cell vaccines. Background Art

[0002] Dendritic cells (DCs) are present in small numbers in tumors and lymphoid organs, but they play a key role in initiating antigen-specific immunity and maintaining immune tolerance. Dendritic cells can capture and process antigens by recognizing pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), and then present these antigens to T cells. In addition, dendritic cells provide the necessary signals to regulate the immune response through direct contact with T cells and the secretion of cytokines.

[0003] Due to their powerful antigen-presenting ability, dendritic cells have great potential in inducing effective anti-tumor immune responses. In cancers with high immunogenicity such as melanoma, prostate cancer, glioblastoma, and renal cell carcinoma, the research on dendritic cell vaccines has received extensive attention. As a live cell vaccine, the prerequisite for dendritic cell vaccines to function is to migrate from the injection site to the lymph nodes and remain active to ensure their antigen-presenting function.

[0004] Therefore, in order to ensure that dendritic cell vaccines can effectively exert their antigen-presenting function, a method is needed to trace and monitor their migration path and activity status in vivo, so as to optimize the design and application of the vaccine and improve its therapeutic effect and safety. Summary of the Invention

[0005] In view of this, in order to at least partially solve at least one of the above-mentioned technical problems, the present invention provides a dual-modal magnetic particle fluorescence probe and its application in the preparation, tracing, and monitoring of dendritic cell vaccines.

[0006] According to an embodiment of one aspect of the present invention, a dual-modal magnetic particle fluorescence probe is provided, comprising: magnetic nanoparticles, cell-penetrating peptides, fluorescence resonance energy transfer fluorescence reporter groups, and caspase substrates.

[0007] According to an embodiment of the present invention, the magnetic nanoparticles are magnetic iron oxide nanoparticles; the cell-penetrating peptide includes at least one of the peptide chains shown in SEQ ID NO: 1 to 19; the fluorescence resonance energy transfer fluorescent reporter group includes a fluorescence resonance energy transfer fluorescent reporter group that can be quenched by the magnetic nanoparticles; the caspase substrate includes at least one of DEVD, IETD, LEHD, and VAD; the cell-penetrating peptide, the fluorescence resonance energy transfer fluorescent reporter group, and the caspase substrate are covalently linked to the surface of the magnetic nanoparticles.

[0008] According to an embodiment of the present invention, the fluorescent probe further comprises an antigen.

[0009] According to an embodiment of the present invention, the antigen is an antigen for a DC vaccine, including a tumor-specific antigen or a tumor-associated antigen, and the antigen is included in the bimodal magnetic particle fluorescent probe by physical adsorption.

[0010] According to an embodiment of another aspect of the present invention, there is provided a method for preparing a bimodal magnetic particle fluorescent probe, including the step of covalently linking a cell-penetrating peptide, a fluorescence resonance energy transfer fluorescent reporter group, and a caspase substrate to the surface of magnetic nanoparticles.

[0011] According to an embodiment of still another aspect of the present invention, there is provided a composition or kit containing the bimodal magnetic particle fluorescent probe.

[0012] According to an embodiment of still another aspect of the present invention, there is provided an application of the bimodal magnetic particle fluorescent probe and / or a composition or kit containing the bimodal magnetic particle fluorescent probe, and the application includes being used for preparing, tracing, and / or monitoring dendritic cell vaccines.

[0013] According to an embodiment of the present invention, preparing a dendritic cell vaccine includes: co-culturing a specific antigen adsorbed with the bimodal magnetic particle fluorescent probe with dendritic cells to obtain a dendritic cell vaccine labeled with the bimodal magnetic particle fluorescent probe.

[0014] According to an embodiment of the present invention, the dendritic cell vaccine is subjected to migration and localization tracing and monitoring of the vaccine by magnetic particle imaging technology; the dendritic cell vaccine is subjected to apoptosis monitoring of the vaccine by fluorescence resonance energy transfer technology.

[0015] According to an embodiment of the present invention, the bimodal magnetic particle fluorescent probe is used to activate the maturation and / or migration process of dendritic cell vaccines.

[0016] According to an embodiment of the present invention, a cell-penetrating peptide is used to promote the entry of a dual-modal magnetic particle fluorescence probe into cells and improve the delivery efficiency. Magnetic nanoparticles for magnetic particle imaging (MPI) can monitor the migration of DC vaccines and achieve quantitative analysis of DC vaccines in vivo. Moreover, the magnetic nanoparticles can also quench the fluorescence resonance energy transfer (FRET) fluorescent reporter group in the "off" state through fluorescence resonance energy transfer (FRET). When the DC vaccine undergoes apoptosis, the caspase substrate is cleaved, resulting in the separation of the caspase substrate and the magnetic nanoparticles, releasing the fluorescence resonance energy transfer fluorescent reporter group in the "on" state and emitting a fluorescence signal. The dual-modal magnetic particle fluorescence probe combines the advantages of MPI and FRET, can provide real-time imaging information in different biological events, has a high signal-to-background ratio and specificity, and can realize in vivo monitoring of the behavior of DC vaccines in real time, including processes such as migration, localization, and apoptosis, which helps to improve the efficiency and accuracy of vaccine research and application. Description of the Drawings

[0017] Through the following description of the embodiments of the present invention with reference to the drawings, the above and other objects, features, and advantages of the present invention will become clearer. In the drawings:

[0018] Figure 1 It is a graph showing the test results of caspase substrate response of Fe3O4-DEVDTAT-Cy7 according to an embodiment of the present invention;

[0019] Figure 2 It is a flow cytometry result graph of the test for stimulating the maturation of bone marrow-derived DC cell vaccines with the dual-modal magnetic particle fluorescence probe Fe3O4-DEVDTAT-Cy7 according to an embodiment of the present invention;

[0020] Figure 3 It is a confocal change graph before and after adding the cell apoptosis inducer staurosporine after mouse bone marrow-derived dendritic cells uptake the Fe3O4-DEVDTAT-FITC probe according to an embodiment of the present invention;

[0021] Figure 4 It is a flow cytometry comparison graph after adding the cell apoptosis inducer staurosporine after mouse bone marrow-derived dendritic cells uptake the Fe3O4-DEVDTAT-FITC probe according to an embodiment of the present invention;

[0022] Figure 5 It is a statistical graph of the flow cytometry comparison after adding the cell apoptosis inducer staurosporine after mouse bone marrow-derived dendritic cells uptake the Fe3O4-DEVDTAT-FITC probe according to an embodiment of the present invention;

[0023] Figure 6Bar chart of flow cytometry quantitative analysis of apoptosis detection of mouse bone marrow-derived dendritic cells after uptake of Fe3O4-DEVDTAT-FITC probe and addition of the cell apoptosis inducer staurosporine caspase substrate-3 in the embodiments of the present invention;

[0024] Figure 7 Comparison chart of flow cytometry diagrams of Annexin V / propidium iodide apoptosis detection of mouse bone marrow-derived dendritic cells in the embodiments of the present invention;

[0025] Figure 8 Bar chart of quantitative analysis of flow cytometry diagrams of Annexin V / propidium iodide apoptosis detection of mouse bone marrow-derived dendritic cells in the embodiments of the present invention;

[0026] Figure 9 In vivo migration live fluorescence imaging diagram of DC vaccine loaded with Fe3O4-DEVDTAT-FITC probe in the embodiments of the present invention;

[0027] Figure 10 Graph of change in fluorescence signal intensity of in vivo migration of DC vaccine loaded with Fe3O4-DEVDTAT-FITC probe in the embodiments of the present invention;

[0028] Figure 11 In vivo migration magnetic particle imaging diagram of DC vaccine loaded with Fe3O4-DEVDTAT-FITC probe in the embodiments of the present invention;

[0029] Figure 12 Graph of change in magnetic particle signal intensity of in vivo migration of DC vaccine loaded with Fe3O4-DEVDTAT-FITC probe in the embodiments of the present invention;

[0030] Figure 13 Pathological diagram of lymph nodes after in vivo migration of DC vaccine loaded with Fe3O4-DEVDTAT-FITC probe in the embodiments of the present invention;

[0031] Figure 14 Comparison chart of live fluorescence imaging of DC vaccine loaded with Fe3O4-DEVDTAT-Cy7 probe and combined with chemotherapy drugs in the embodiments of the present invention. Detailed implementation manners

[0032] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary and are not intended to limit the scope of the present invention. In the following detailed description, for the sake of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present invention. However, obviously, one or more embodiments can also be implemented without these specific details. In addition, in the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present invention.

[0033] The terms used herein are merely for describing specific embodiments and are not intended to limit the present invention. The term "comprising" used herein indicates the presence of features, steps, operations, but does not exclude the presence or addition of one or more other features.

[0034] In the case of using expressions such as "at least one of A, B, and C, etc.", generally it should be interpreted according to the meaning that those skilled in the art usually understand this expression (for example, "a system having at least one of A, B, and C" should include but not be limited to a system having only A, only B, only C, having A and B, having A and C, having B and C, and / or having A, B, and C, etc.). In the case of using expressions such as "at least one of A, B, or C, etc.", generally it should be interpreted according to the meaning that those skilled in the art usually understand this expression (for example, "a system having at least one of A, B, or C" should include but not be limited to a system having only A, only B, only C, having A and B, having A and C, having B and C, and / or having A, B, and C, etc.).

[0035] In the present invention, the term "DC vaccine" refers to a dendritic cell vaccine.

[0036] In the process of implementing the inventive concept of the present invention, it is found that magnetic vaccination is an innovative alternative method, which can simultaneously capture tumor antigens and magnetic resonance (MR) contrast agents and in-situ label dendritic cells in vivo. This method can accurately and quantitatively monitor the antigen presentation of T cells in lymph nodes. Therefore, magnetic resonance imaging (MRI) is considered to be one of the most comprehensive tools for guiding cancer vaccine development. In the related art, the effect of tumor vaccination and the anti-tumor response can be predicted by observing only the change of the MRI signal in the early lymph nodes within 1 hour.

[0037] Magnetic particle imaging (MPI) is an emerging non-invasive imaging technology. Because its signal has a long-term linear relationship with the tracer mass, it has advantages such as positive contrast, low tissue background, infinite tissue penetration depth, and no ionizing radiation, and shows extremely high superiority in in-vivo cell tracing, and can achieve cell tracking for more than 3 months. In the related art, MPI can quantitatively monitor the migration, distribution, and clearance of mesenchymal stem cells in vivo.

[0038] Researchers have paid increasing attention to the homeostasis of dendritic cells in vivo. Fluorescent molecular imaging has been proven to be able to provide highly sensitive and specific information for molecular and cellular activities such as apoptosis events. Related techniques have been applied to track the survival rate of stem cells and adoptive natural killer cells in vivo. Therefore, the development of a fluorescence molecular imaging / magnetic particle imaging (FMI / MPI) dual-modal imaging probe will be able to quantitatively track the migration of adoptive dendritic cell vaccines using MPI and monitor apoptosis using fluorescence molecular imaging (FMI), which will provide more comprehensive information for the guidance of dendritic cell vaccine immunotherapy.

[0039] Specifically, according to an embodiment of one aspect of the present invention, a dual-modal magnetic particle fluorescence probe is provided, comprising: magnetic nanoparticles, a cell-penetrating peptide, a fluorescence resonance energy transfer fluorescent reporter group, and a caspase substrate.

[0040] According to an embodiment of the present invention, the cell-penetrating peptide is used to promote the entry of the dual-modal magnetic particle fluorescence probe into the cell interior and improve the delivery efficiency. The magnetic nanoparticles are used for magnetic resonance imaging (MPI) to monitor the migration of DC vaccines and achieve quantitative analysis of in vivo DC vaccines. Moreover, the magnetic nanoparticles can also quench the fluorescence resonance energy transfer fluorescent reporter group in the "off" state through fluorescence resonance energy transfer (FRET). When apoptosis occurs in the DC vaccine, the caspase substrate is cleaved, resulting in the separation of the caspase substrate and the magnetic nanoparticles, releasing the fluorescence resonance energy transfer fluorescent reporter group in the "on" state and emitting a fluorescent signal. The dual-modal magnetic particle fluorescence probe combines the advantages of MPI and FRET, can provide real-time imaging information in different biological events, has a high signal-to-background ratio and specificity, and can realize in vivo monitoring of the behavior of DC vaccines in real time, including processes such as migration, localization, and apoptosis, which helps to improve the efficiency and accuracy of vaccine research and application.

[0041] According to an embodiment of the present invention, the magnetic nanoparticles are magnetic iron oxide nanoparticles; the cell-penetrating peptide includes at least one of the peptide chains shown in SEQ ID NO: 1-19; the fluorescence resonance energy transfer fluorescent reporter group includes a fluorescence resonance energy transfer fluorescent reporter group that can be quenched by the magnetic nanoparticles, and the caspase substrate includes at least one of DEVD, IETD, LEHD, and VAD; the cell-penetrating peptide, the fluorescence resonance energy transfer fluorescent reporter group, and the caspase substrate are covalently linked to the surface of the magnetic nanoparticles.

[0042] According to an embodiment of the present invention, the cell-penetrating peptide can be selected from at least one of the following Table 1.

[0043] Table 1.

[0044]

[0045] According to an embodiment of the present invention, the fluorescence resonance energy transfer (FRET) fluorescent reporter group includes a FRET fluorescent reporter group that can be quenched by magnetic nanoparticles, such as a near-infrared fluorescent molecule, such as at least one of near-infrared fluorescent donor cyanine dye Cy7 (Cy7), fluorescein isothiocyanate (FITC), IRDye 800CW, and iFluor 750.

[0046] According to an embodiment of the present invention, the caspase substrate includes at least one of DEVD, IETD, LEHD, and VAD, wherein the amino acid sequences of DEVD, IETD, LEHD, and VAD are shown in Table 2.

[0047] Table 2.

[0048]

[0049] According to an embodiment of the present invention, the combination of the dual-modal magnetic particle fluorescence probe can be Fe3O4-DEVDTAT-Cy7, wherein the magnetic nanoparticles are iron oxide (Fe3O4), the cell-penetrating peptide is the transcriptional transactivator (TAT), and its sequence is as shown in SEQ ID No.1, the fluorescence resonance energy transfer fluorescent reporter group is Cy7, and the caspase substrate is the DEVD peptide substrate that can be cleaved by caspase-3 / 7. For example, it can also be combined as Fe3O4-IETDTAT-Cy7, Fe3O4-LEHDTAT-IRDye 800CW, Fe3O4-IETDTAT-FITC, etc.

[0050] According to an embodiment of the present invention, the cell-penetrating peptide, the fluorescence resonance energy transfer fluorescent reporter group, and the caspase substrate are covalently linked to the surface of the magnetic nanoparticles. Through covalent bonding, the cell-penetrating peptide, the fluorescent reporter group, and the caspase substrate can work closely together, and the spatial distance and relative position between the groups and the magnetic nanoparticles can also be precisely controlled to avoid functional interference caused by steric hindrance; covalent bonding enables these groups to form a stable chemical structure with the magnetic nanoparticles and is not easily detached or decomposed in a complex biological environment. For example, during in vivo circulation, the covalent bond can resist enzymatic hydrolysis and metabolic processes in the blood, ensuring the integrity of the probe and the persistence of its function; the covalent bond has a relatively high bond energy and can tolerate certain temperature changes. Covalent bonding enables the cell-penetrating peptide, the fluorescence resonance energy transfer fluorescent reporter group, and the caspase substrate to play their roles synchronously, realizing precise monitoring and analysis of intracellular events.

[0051] According to an embodiment of the present invention, the dual-modal magnetic particle fluorescence probe further comprises an antigen.

[0052] According to an embodiment of the present invention, the antigen can be recognized by a specific antibody. When the probe contains the antigen, it can achieve precise targeting of tumor cells by binding to the specific antibody on the surface of tumor cells. Through the specific binding of antigen-antibody, the non-specific binding of the probe in normal tissues can be reduced, the background signal can be lowered, and the signal-to-background ratio of imaging can be improved.

[0053] According to an embodiment of the present invention, the antigen is an antigen for DC vaccine, including tumor-specific antigen (TSA) or tumor-associated antigen. The antigen is included in the dual-modal magnetic particle fluorescence probe by physical adsorption.

[0054] According to an embodiment of the present invention, tumor-specific antigen (TSA) and tumor-associated antigen (TAA) can be specifically recognized and presented by dendritic cells (DC), thereby activating antigen-specific T cell responses. The antigen physically adsorbed on the probe can be effectively taken up and processed by DC, and then activate CD8 + T cells and CD4 + T cells, generating a strong immune response; the physical adsorption method enables the antigen to stably exist on the probe, ensuring its effective delivery and presentation in vivo, so as to monitor the cellular behavior of DC vaccine through the dual-modal magnetic particle fluorescence probe.

[0055] According to an embodiment of the present invention, the preparation method of the dual-modal magnetic particle fluorescence probe includes the step of covalently connecting a cell-penetrating peptide, a fluorescence resonance energy transfer fluorescent reporter group, and a caspase substrate to the surface of magnetic nanoparticles.

[0056] According to an embodiment of the present invention, the preparation process may include: adding a cell-penetrating peptide, a fluorescence resonance energy transfer fluorescent reporter group, a caspase substrate, and magnetic nanoparticles to a mild phosphate buffer solution (PBS) with a neutral pH of 6.8 - 7, performing a dehydration condensation reaction to obtain a mixture, and then purifying the mixture by column chromatography to obtain the dual-modal magnetic particle fluorescence probe.

[0057] According to an embodiment of another aspect of the present invention, a composition or kit of the dual-modal magnetic particle fluorescence probe is also disclosed.

[0058] According to an embodiment of the present invention, other reagents may also be included in the kit, for example, it may include auxiliary reagents for cell culture, staining, or labeling to enhance the function of the probe or improve the experimental effect.

[0059] According to an embodiment of another aspect of the present invention, an application of a composition or kit of a dual-modal magnetic particle fluorescence probe is also disclosed, and the application includes being used for preparing, tracing and / or monitoring dendritic cell vaccines.

[0060] According to an embodiment of the present invention, the dual-modal magnetic particle fluorescence probe can be used to prepare DC vaccines. By using the fluorescence characteristics of the probe, DC cells can be labeled during the preparation process to facilitate the observation and analysis of cell growth and differentiation, trace and monitor cell behaviors such as apoptosis of DC vaccines, and can also be used to evaluate the immune response induced by DC vaccines, such as the activation and proliferation of T cells.

[0061] According to an embodiment of the present invention, preparing a dendritic cell vaccine includes: co-culturing a specific antigen adsorbed with a dual-modal magnetic particle fluorescence probe on its surface with dendritic cells to obtain a dendritic cell vaccine labeled with the dual-modal magnetic particle fluorescence probe.

[0062] According to an embodiment of the present invention, through the specific binding of DC cells and antigens and the process of physically adsorbing fluorescence probes, DC vaccines can be prepared simply and quickly.

[0063] According to an embodiment of the present invention, the dendritic cell vaccine is traced and monitored for vaccine migration and localization through magnetic particle imaging technology; the dendritic cell vaccine is monitored for apoptosis through fluorescence resonance energy transfer technology.

[0064] According to an embodiment of the present invention, through magnetic particle imaging and fluorescence resonance energy transfer technology for dual-modal intelligent imaging, in-vivo monitoring of DC vaccine behavior, including migration, localization and apoptosis, can be realized in real time, providing guidance for optimizing the administration sequence of DC vaccines and chemotherapy, and can also be used to improve the efficacy of combined clinical immunotherapy of DC vaccines and chemotherapy.

[0065] According to an embodiment of the present invention, the dual-modal magnetic particle fluorescence probe is used to activate the maturation and / or migration process of dendritic cell vaccines.

[0066] According to an embodiment of the present invention, the dual-modal magnetic particle fluorescent probe can transmit signals and promote the maturation of dendritic cells by interacting with receptors on the surface of dendritic cells. For example, the magnetic nanoparticles in the probe can be taken up by dendritic cells, thereby activating the signal pathways in the cells, leading to the upregulation of mature markers such as CD80, CD86, etc. Mature dendritic cells have stronger antigen presentation capabilities and can more effectively activate T cells. The dual-modal magnetic particle fluorescent probe can improve the maturity of dendritic cells, thereby enhancing their antigen presentation function, and can also stimulate the migration process of DC vaccines, which can make them more effectively migrate from peripheral tissues to immune organs such as lymph nodes, thereby enhancing the efficiency of antigen presentation, increasing the contact opportunities between DC and T cells, thereby more effectively activating T cells, enhancing immune responses, and can also make them work better with other immune cells to form a more effective immune response.

[0067] The test materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. Specific techniques or conditions not specified in the examples are conventional methods and can be carried out according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0068] Example 1 Method for preparing probe

[0069] (1) Materials: Fe3O4 was purchased from Micromod Partikeltechnologie GmbH (Rostock, Germany). Mercaptopropionic acid-DEVDK(Cy7 or FITC)-RKKRRQRRR (purity 98%, also known as DT(Cy7 or FITC)) and TAT were purchased from Taopute Biotechnology Co., Ltd. (Shanghai, China). OVA257-264 was purchased from Invivogen (France). Cy5-labeled OVA257-264 was purchased from St. Louis Company (Sigma-Aldrich, USA).

[0070] (2) The synthesis method is as follows: Mix 5 mL of Fe3O4 (4 mg / mL) with 1 mL of DT (Cy7) (1 mg / mL), then stir at room temperature for 8 h, and perform ultrafiltration (3 kDa, 3500 g) at 4 °C for 30 min to remove the unreacted DT (Cy7). Then add 0.5 mL of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (10 mg / mL), stir and incubate on a rotator for 2 h, and gently add 0.5 mL of n-hydroxysuccinimide (10 mg / mL) and 1 mL of OVA (2 mg / mL) to the mixture. After 17 h, perform ultrafiltration (50 kDa, 3500 g) at 4 °C for 1 h to remove the unreacted OVA. The resulting product is named Fe@O-DT (Cy7). When the peptide is DT (FITC) or OVA (Cy5), Fe@O-DT (FITC) and Fe@O(Cy5)-DT are synthesized using a similar method. The probe is used for magnetic particle imaging and fluorescence molecular imaging to trace and monitor the active state of the DC vaccine.

[0071] Example 2 Caspase Substrate Response Test of Bimodal Magnetic Particle Fluorescent Probe Fe3O4-DEVDTAT-Cy7

[0072] One hour after adding Caspase-3 to Fe3O4-DEVDTAT-Cy7 at room temperature, the results of the fluorescence spectrum change are as Figure 1 shown.

[0073] Figure 1 This is the result graph of the Caspase substrate response test of Fe3O4-DEVDTAT-Cy7 in the embodiment of the present invention.

[0074] According to Figure 1 It can be seen that after reacting with Caspase-3, Cy7 becomes free to emit light, indicating that the Fe3O4-DEVDTAT-Cy7 probe can be used for the responsive detection of Caspase-3.

[0075] Example 3 Maturation Test of Bimodal Magnetic Particle Fluorescent Probe Fe3O4-DEVDTAT-FITC on Bone Marrow-Derived Dendritic Cells (BMDC) Vaccine

[0076] Obtain primary bone marrow cells, add cytokines interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-GSF), and induce differentiation into BMDC after 6 days.

[0077] Add the relevant blank group PBS, the control group of tumor-specific antigen - chicken ovalbumin (OVA), and the experimental group of Fe3O4-DEVDTAT-FITC probe.

[0078] After incubating for 24 h, flow cytometry was performed on BMDC. The antibodies FITC-CD11c, APC-CD80, and PE-CD86 were added sequentially to obtain the flow cytometry plot of BMDC maturation. The results are as Figure 2 shown.

[0079] Figure 2 This is the flow cytometry result plot of the maturation test of the dual-modal magnetic particle fluorescence probe Fe3O4-DEVDTAT-Cy7-stimulated bone marrow-derived DC cell vaccine in the embodiment of the present invention.

[0080] According to Figure 2 it can be seen that the Fe3O4-DEVDTAT-FITC probe can significantly activate the maturation of the BMDC vaccine, and the expressions of CD80 and CD86 are higher than those in the group using OVA alone and the blank group.

[0081] Example 4 Apoptosis response test of the dual-modal magnetic particle fluorescence probe Fe3O4-DEVDTAT-FITC on DC vaccine

[0082] When culturing mouse bone marrow-derived dendritic (DC2.4) cells in a bottom glass culture dish with RPMI-1640 medium to 70% density, incubate the Fe3O4-DEVDTAT-FITC probe for 24 hours.

[0083] After adding 1 µM staurosporine (STS) for 1 hour to induce apoptosis of DC2.4 cells, discard the supernatant, stain the cell nuclei with DAPI, discard the supernatant, fix with paraformaldehyde, and observe the FITC signal with or without STS using a laser confocal microscope. The results are as Figure 3 shown.

[0084] When culturing DC2.4 cells in RPMI-1640 medium to 70% density, incubate the Fe3O4-DEVDTAT-FITC probe for 24 hours.

[0085] After adding PBS or 1 µM STS for 1 hour respectively, centrifuge, add the PE-labeled Caspase-3 antibody and the APC-labeled Annexin V, and use flow cytometry to detect the fluorescence signals of fluorescein isothiocyanate (FITC), propidium iodide (PI), phycoerythrin (PE), and allophycocyanin (APC). The results are as Figures 4 to 8 shown.

[0086] Figure 3Confocal change diagrams of mouse bone marrow-derived dendritic cells in the embodiments of the present invention before and after adding the cell apoptosis inducer staurosporine after taking up the Fe3O4-DEVDTAT-FITC probe; Figure 4 Flow cytometry comparison diagrams of mouse bone marrow-derived dendritic cells in the embodiments of the present invention after taking up the Fe3O4-DEVDTAT-FITC probe and adding the cell apoptosis inducer staurosporine; Figure 5 Flow cytometry comparison statistical diagrams of mouse bone marrow-derived dendritic cells in the embodiments of the present invention after taking up the Fe3O4-DEVDTAT-FITC probe and adding the cell apoptosis inducer staurosporine.

[0087] Figure 6 Histogram statistical diagrams of flow cytometry quantitative analysis of caspase substrate-3 apoptosis detection of mouse bone marrow-derived dendritic cells in the embodiments of the present invention after taking up the Fe3O4-DEVDTAT-FITC probe and adding the cell apoptosis inducer staurosporine.

[0088] Figure 7 Comparison diagrams of flow cytometry diagrams of Annexin V / propidium iodide apoptosis detection of mouse bone marrow-derived dendritic cells in the embodiments of the present invention; Figure 8 Histogram statistical diagrams of quantitative analysis of flow cytometry diagrams of Annexin V / propidium iodide apoptosis detection of mouse bone marrow-derived dendritic cells in the embodiments of the present invention.

[0089] According to Figures 3 to 8 It can be seen that after the DC2.4 cells taking up the Fe3O4-DEVDTAT-FITC probe are induced to apoptosis by STS, the FITC fluorescence intensity accumulates more and more in the cytoplasm under laser confocal microscopy, while it is negligible in live cells; flow cytometry analysis shows that compared with the control group, the expression of FITC-positive cells in BMDC treated with STS increases by 50%, and it can effectively cleave caspase substrate-3, resulting in the separation and release of caspase substrate-3 and Fe3O4, and the release of the "on" state FITC reporter group, so that an obvious FITC signal can be emitted, proving that the dual-modal magnetic particle fluorescence probe of the present invention can significantly and effectively monitor the apoptosis behavior of cells.

[0090] Example 5 In vivo tracing test of loading Fe3O4-DEVDTAT-FITC probe

[0091] Use a small animal in vivo fluorescence imager (IVIS) to verify whether the Fe3O4-DEVDTAT-FITC probe affects the migration efficiency of DC vaccine, and use MPI to verify the in vivo migration of the DC vaccine loaded with the Fe3O4-DEVDTAT-FITC probe at different time points. The specific operations are as follows.

[0092] First, tumor necrosis factor (TNF-α) was injected into the left and right footpads of mice respectively for 24 hours.

[0093] BMDC was co-incubated with Fe3O4-DEVDTAT-FITC probe for 12 hours, centrifuged and washed with PBS to obtain the vaccine test group loaded with the fluorescent probe. BMDC without the loaded fluorescent probe was used as the vaccine control group.

[0094] 10 μM cell membrane fluorescent probe DiR (1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindocarbocyanine Perchlorate) was incubated with the above test group and control group respectively in PBS at 37 °C for 15 minutes. After centrifugation and washing with PBS, 1×10 6 BMDC vaccines were injected into the left and right footpads, with the right foot as the test group and the left foot as the control group. Then, IVIS imaging was performed at 0, 2, 24, 48, and 72 h. The results are as Figure 9 and Figure 10 shown.

[0095] 1×10 6 BMDC vaccines were injected into the left and right footpads, with the right foot as the test group and the left foot as the control group. Then, MPI imaging was performed at 0, 2, 24, 48, and 72 h. The results are as Figure 11 and Figure 12 shown.

[0096] After imaging, the mice were sacrificed, and the popliteal lymph nodes on both the left and right sides of the mice were dissected. Prussian blue iron staining was used to locate and confirm the DC vaccine in the lymph nodes. The results are as Figure 13 shown.

[0097] Figure 9 This is the in vivo migration live fluorescence imaging map of the DC vaccine loaded with Fe3O4-DEVDTAT-FITC probe in the embodiment of the present invention; Figure 10 This is the fluorescence signal intensity change map of the in vivo migration of the DC vaccine loaded with Fe3O4-DEVDTAT-FITC probe in the embodiment of the present invention; Figure 11 This is the magnetic particle imaging map of the in vivo migration of the DC vaccine loaded with Fe3O4-DEVDTAT-FITC probe in the embodiment of the present invention; Figure 12 This is the magnetic particle signal intensity change map of the in vivo migration of the DC vaccine loaded with Fe3O4-DEVDTAT-FITC probe in the embodiment of the present invention; Figure 13 This is the lymph node pathology map after the in vivo migration of the DC vaccine loaded with Fe3O4-DEVDTAT-FITC probe in the embodiment of the present invention.

[0098] According to Figures 9 to 13It can be seen that only similar DiR fluorescence signals were shown in the mouse footpads after injection of BMDC in the experimental group and BMDC in the control group. After 24 hours, more DiR fluorescence signals were shown in the right popliteal lymph node (PLN) region than in the left PLN region, indicating that the Fe3O4-DEVDTAT-FITC probe could promote the migration of BMDC. The MPI migration results showed that there was no change in the MPI signal intensity in the left PLN, but the MPI signal intensity could be shown in the right PLN. In vitro Prussian blue staining of bilateral PLNs collected immediately 24 hours after imaging confirmed that there were BMDCs labeled with the Fe3O4-DEVDTAT-FITC probe showing positive staining in the right PLN, and no corresponding staining was observed in the left PLN. It was proved that the fluorescent probe of the present invention could effectively trace and monitor the behavior of DC vaccines under the dual modalities of fluorescence imaging and magnetic particle imaging, and promote the migration and maturation process of DC vaccines.

[0099] Example 6 Test of combination with chemotherapeutic drugs after inoculation with DC vaccine loaded with Fe3O4-DEVDTAT-Cy7 probe

[0100] A small animal in vivo fluorescence imager was used to verify whether the status of the DC vaccine loaded with the Fe3O4-DEVDTAT-Cy7 probe could be shown when combined with chemotherapeutic drugs. The specific operations were as follows.

[0101] 24 h after TNF-α was injected into the left and right footpads of mice respectively.

[0102] BMDC was co-incubated with the Fe3O4-DEVDTAT-Cy7 probe for 12 hours. After centrifugation and washing with PBS, the chemotherapeutic drug oxaliplatin (OXA) was injected via the tail vein at a dose of 10 mg / kg body weight. After different time points, 1×10 6 such DC vaccines loaded with the Fe3O4-DEVDTAT-Cy7 probe were used as the experimental group, and the BMDC vaccine without the probe was used as the control group. They were respectively injected into the right and left footpads, with the right foot being the experimental group and the left foot being the control group. Then, IVIS imaging was performed 24 h later, and the results are as Figure 14 shown.

[0103] Figure 14 This is a comparative in vivo fluorescence imaging diagram of the test of combination with chemotherapeutic drugs after inoculation with the DC vaccine loaded with the Fe3O4-DEVDTAT-Cy7 probe in the embodiment of the present invention.

[0104] According to Figure 14It can be seen that during the period from 0 to 8 days, the right popliteal lymph node (PLN) of the mice in the experimental group showed Cy7 fluorescence signal. Further dissect the bilateral popliteal lymph nodes (PLN) and confirm by ex vivo imaging that the Cy7 fluorescence signal indeed comes from the DC vaccine injected into the right draining lymph node, proving that the fluorescent probe of the present invention can still effectively trace and monitor the DC vaccine in the case of combined use of chemotherapeutic drugs.

[0105] In the above specific embodiments, the purpose, technical solutions and beneficial effects of the present invention have been further described in detail. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A dual-modal magnetic particle fluorescence probe, characterized in that, Comprising: Magnetic nanoparticles, cell-penetrating peptides, fluorescence resonance energy transfer (FRET) fluorescent reporters, caspase substrates, and antigens for dendritic cell vaccines; Wherein, the fluorescence resonance energy transfer fluorescent reporter comprises a fluorescence resonance energy transfer fluorescent reporter that can be quenched by the magnetic nanoparticles; wherein, The connection order is magnetic nanoparticles - caspase substrate and cell-penetrating peptide - fluorescence resonance energy transfer fluorescent reporter; and The cell-penetrating peptide, the fluorescence resonance energy transfer fluorescent reporter, and the caspase substrate are covalently linked to the surface of the magnetic nanoparticles; The antigen is included in the bimodal magnetic particle fluorescence probe by physical adsorption.

2. The bimodal magnetic particle fluorescence probe according to claim 1, wherein The magnetic nanoparticles are magnetic iron oxide nanoparticles; The cell-penetrating peptide comprises at least one of the peptide chains shown in SEQ ID NO: 1-19; The caspase substrate comprises at least one of DEVD, IETD, LEHD, and VAD.

3. The bimodal magnetic particle fluorescence probe according to claim 1, wherein The antigen comprises a tumor-specific antigen or a tumor-associated antigen.

4. A preparation method of the dual-modal magnetic particle fluorescence probe according to any one of claims 1 to 3, characterized in that, Including the step of covalently linking the cell-penetrating peptide, the fluorescence resonance energy transfer fluorescent reporter, and the caspase substrate to the surface of the magnetic nanoparticles.

5. A composition or kit comprising the bimodal magnetic particle fluorescence probe according to any one of claims 1-3.

6. Use of a dual-modal magnetic particle fluorescence probe according to any one of claims 1 to 3, a dual-modal magnetic particle fluorescence probe prepared by the method of claim 4, and / or the composition or kit according to claim 5, characterized in that The applications include being used for preparing, tracing, and / or monitoring dendritic cell vaccines.

7. The application according to claim 6, wherein Preparing a dendritic cell vaccine comprises: Co-culturing the antigen adsorbed with the bimodal magnetic particle fluorescence probe on its surface with dendritic cells to obtain a dendritic cell vaccine labeled with the bimodal magnetic particle fluorescence probe.

8. The application according to claim 7, wherein The migration and localization tracing and monitoring of the dendritic cell vaccine are carried out by magnetic particle imaging technology; or The apoptosis monitoring of the dendritic cell vaccine is carried out by fluorescence resonance energy transfer technology.

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