Bimodal magnetic particle fluorescent probe and application thereof in preparation, tracing and monitoring of dendritic cell vaccine

Abstract

The invention provides a bimodal magnetic particle fluorescent probe and application thereof in preparation, tracing and monitoring of dendritic cell vaccines, and belongs to the technical field of biology. The bimodal magnetic particle fluorescent probe comprises a magnetic nanoparticle, a cell penetrating peptide, a fluorescence resonance energy transfer fluorescence reporter group and a caspase substrate. The bimodal magnetic particle fluorescent probe 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 high signal-to-background ratio and specificity, realizes in-vivo monitoring of dendritic cell vaccine behaviors in real time, including migration, positioning, apoptosis and other processes, and has good application prospects. And the efficiency and the accuracy of vaccine research and application can be improved.

Description

Technical Field

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

[0002] Although dendritic cells (DCs) are present in small numbers in tumors and lymphoid organs, they play a key role in initiating antigen-specific immunity and maintaining immune tolerance. DCs are able to 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, DCs provide the necessary signals to regulate immune responses through direct contact with T cells and secretion of cytokines.

[0003] Due to their strong 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, dendritic cell vaccines work by migrating from the injection site to the lymph nodes and maintaining activity, which is the prerequisite for ensuring that they can play the antigen presenting function.

[0004] Therefore, in order to ensure that dendritic cell vaccines can effectively exert their antigen presentation function, it is necessary to develop a method to track and monitor their migration path and activity state in the body, 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 fluorescent 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 fluorescent probe is provided, comprising: magnetic nanoparticles, cell penetrating peptides, fluorescence resonance energy transfer fluorescent reporter groups and caspase substrates.

[0007] According to an embodiment of the present invention, the magnetic nanoparticles are magnetic ferroferric oxide nanoparticles; the cell penetrating peptide includes at least one of the peptide chains shown in SEQ ID NOs: 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 connected to the surface of the magnetic nanoparticles through covalent bonds.

[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 used for a DC vaccine, including a tumor-specific antigen or a tumor-associated antigen, and the antigen is contained in the dual-modal magnetic particle fluorescent probe by physical adsorption.

[0010] According to another embodiment of the present invention, a method for preparing a dual-modal magnetic particle fluorescent probe is provided, comprising the steps of covalently linking a cell penetrating peptide, a fluorescence resonance energy transfer fluorescent reporter group and a caspase substrate to the surface of a magnetic nanoparticle.

[0011] According to another embodiment of the present invention, a composition or kit comprising a dual-modal magnetic particle fluorescent probe is provided.

[0012] According to another embodiment of the present invention, there is provided an application of a dual-modal magnetic particle fluorescent probe and / or a composition or kit comprising the dual-modal magnetic particle fluorescent probe, wherein the application includes use in 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 dendritic cells with a specific antigen having a dual-modal magnetic particle fluorescent probe adsorbed on the surface to obtain a dendritic cell vaccine labeled with the dual-modal magnetic particle fluorescent probe.

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

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

[0016] According to an embodiment of the present invention, cell-penetrating peptides are used to promote the dual-modal magnetic particle fluorescent probe to enter the cell interior and improve the delivery efficiency. Magnetic nanoparticles are used for magnetic resonance imaging (MPI) to monitor the migration of DC vaccines and realize quantitative analysis of DC vaccines in vivo. Moreover, magnetic nanoparticles can also quench the fluorescence resonance energy transfer 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 fluorescent signal. The dual-modal magnetic particle fluorescent 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 real-time in vivo monitoring of DC vaccine behavior, including migration, localization, and apoptosis, which helps to improve the efficiency and accuracy of vaccine research and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The above and other objects, features and advantages of the present invention will become more apparent through the following description of the embodiments of the present invention with reference to the accompanying drawings, in which:

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

[0019] Figure 2 This is a flow cytometry result diagram of the dual-modal magnetic particle fluorescent probe Fe3O4-DEVDTAT-Cy7 stimulating the maturation test of bone marrow-derived DC cell vaccine in an embodiment of the present invention;

[0020] Figure 3 This is a confocal image showing changes in the mouse bone marrow-derived dendritic cells after they have taken up the Fe3O4-DEVDTAT-FITC probe and before and after the addition of the cell apoptosis-inducing agent staurosporine;

[0021] Figure 4 This is a flow cytometry comparison diagram of the mouse bone marrow-derived dendritic cells taking up Fe3O4-DEVDTAT-FITC probe and then adding the cell apoptosis-inducing agent staurosporine;

[0022] Figure 5 This is a flow cytometry comparison statistical diagram of the mouse bone marrow-derived dendritic cells taking up Fe3O4-DEVDTAT-FITC probe and then adding the cell apoptosis-inducing agent staurosporine;

[0023] Figure 6This is a bar chart showing the quantitative analysis of apoptosis detection by flow cytometry after the mouse bone marrow-derived dendritic cells have taken up Fe3O4-DEVDTAT-FITC probe and then added with the cell apoptosis-inducing agent staurosporine caspase substrate-3;

[0024] Figure 7 This is a comparison chart of flow cytometry images of Annexin V / propidium iodide apoptosis detection of mouse bone marrow-derived dendritic cells in an embodiment of the present invention;

[0025] Figure 8 This is a bar chart showing the quantitative analysis of the Annexin V / propidium iodide apoptosis detection flow cytometric graph of mouse bone marrow-derived dendritic cells in an embodiment of the present invention;

[0026] Fig. 9 This is a live fluorescence imaging image of the in vivo migration of a DC vaccine loaded with Fe3O4-DEVDTAT-FITC probe according to an embodiment of the present invention;

[0027] Fig.10 This is a graph showing the change in fluorescence signal intensity of the DC vaccine in vivo migration loaded with Fe3O4-DEVDTAT-FITC probe according to an embodiment of the present invention;

[0028] Fig.11 This is an imaging diagram of the in vivo migration of magnetic particles of DC vaccine loaded with Fe3O4-DEVDTAT-FITC probes according to an embodiment of the present invention;

[0029] Fig.12 This is a graph showing the signal intensity changes of the in vivo migration magnetic particles of the DC vaccine loaded with Fe3O4-DEVDTAT-FITC probe according to an embodiment of the present invention;

[0030] Fig.13 This is a lymph node pathology image after in vivo migration of the DC vaccine loaded with Fe3O4-DEVDTAT-FITC probe according to an embodiment of the present invention;

[0031] Fig.14 This is a comparison chart of in vivo fluorescence imaging of the DC vaccine loaded with Fe3O4-DEVDTAT-Cy7 probe in an embodiment of the present invention and tested in combination with chemotherapy drugs. DETAILED DESCRIPTION

[0032] Below, embodiments of the present invention will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present invention. In the following detailed description, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of embodiments of the present invention. However, it is apparent that one or more embodiments may also be implemented without these specific details. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion of concepts of the present invention.

[0033] The terms used herein are only for describing specific embodiments and are not intended to limit the present invention. The term "comprising" used herein indicates the existence of features, steps, operations, but does not exclude the existence 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.", it should generally be interpreted in accordance with the meaning of the expression generally understood by those skilled in the art (for example, "a system having at least one of A, B, and C" should include but is not limited to a system having A alone, B alone, C alone, A and B, A and C, B and C, and / or A, B, C, etc.). In the case of using expressions such as "at least one of A, B, or C, etc.", it should generally be interpreted in accordance with the meaning of the expression generally understood by those skilled in the art (for example, "a system having at least one of A, B, or C" should include but is not limited to a system having A alone, B alone, C alone, A and B, A and C, B and C, and / or A, B, C, etc.).

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

[0036] In the process of realizing the concept of the present invention, it was found that magnetic vaccination is an innovative alternative method that can simultaneously capture tumor antigens and magnetic resonance (MR) contrast agents and label dendritic cells in situ 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 to guide the development of cancer vaccines. In related technologies, the effect of tumor vaccination and anti-tumor response can be predicted by simply observing the changes in MRI signals in early lymph nodes within 1 hour.

[0037] Magnetic particle imaging (MPI) is an emerging non-invasive imaging technology. It has the advantages of positive contrast, low tissue background, unlimited tissue penetration depth and no ionizing radiation, and has shown great superiority in in vivo cell tracking, and can achieve cell tracking for more than 3 months. Among related technologies, MPI can quantitatively monitor the migration, distribution and clearance of mesenchymal stem cells in vivo.

[0038] Researchers are increasingly interested in the homeostasis of dendritic cells in vivo, and fluorescent molecular imaging has been shown to provide highly sensitive and specific information on molecular and cellular activities such as apoptotic events. Related technologies have been used to track the survival of stem cells and adoptive natural killer cells in vivo. Therefore, the development of fluorescent molecular imaging / magnetic particle imaging (FMI / MPI) dual-modality imaging probes will be able to quantitatively track the migration of adoptive dendritic cell vaccines using MPI and monitor apoptosis using fluorescent 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 fluorescent probe is provided, comprising: magnetic nanoparticles, cell penetrating peptides, fluorescence resonance energy transfer fluorescent reporter groups and caspase substrates.

[0040] According to an embodiment of the present invention, cell-penetrating peptides are used to promote the dual-modal magnetic particle fluorescent probe to enter the cell interior and improve the delivery efficiency. Magnetic nanoparticles are used for magnetic resonance imaging (MPI) to monitor the migration of DC vaccines and realize quantitative analysis of DC vaccines in vivo. Moreover, magnetic nanoparticles can also quench the fluorescence resonance energy transfer 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 fluorescent signal. The dual-modal magnetic particle fluorescent 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 real-time in vivo monitoring of DC vaccine behavior, including 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 ferroferric oxide nanoparticles; the cell penetrating peptide includes at least one of the peptide chains shown in SEQ ID NOs: 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, 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 connected to the surface of the magnetic nanoparticles through covalent bonds.

[0042] According to an embodiment of the present invention, the cell penetrating peptide may 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 fluorescent reporter group includes a fluorescence resonance energy transfer fluorescent reporter group that can be quenched by magnetic nanoparticles, such as a near-infrared fluorescent molecule, such as at least one of the near-infrared fluorescent donor cyanine dye Cy7 (Cy7), fluorescein isothiocyanate (FITC), IR800 succinimidyl ester (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 dual-modal magnetic particle fluorescent probes can be Fe3O4-DEVDTAT-Cy7, wherein the magnetic nanoparticles are ferroferric oxide (Fe3O4), the cell penetrating peptide is a transcription transactivator (TAT), the sequence of which is shown in SEQ ID No. 1, the fluorescence resonance energy transfer fluorescent reporter group is Cy7, and the caspase substrate is a DEVD peptide substrate that can be cleaved by caspase-3 / 7. For example, the combination can also be Fe3O4- IETDTAT-Cy7, Fe3O4-LEHDTAT- IRDye 800CW, Fe3O4- IETDTAT-FITC, etc.

[0050] According to an embodiment of the present invention, cell penetrating peptides, fluorescence resonance energy transfer fluorescent reporter groups and caspase substrates are covalently linked to the surface of magnetic nanoparticles. Through covalent bond connection, cell penetrating peptides, fluorescent reporter groups and caspase substrates can work closely together, and can also accurately control the spatial distance and relative position between the groups and the magnetic nanoparticles to avoid functional interference caused by spatial hindrance; covalent bond connection allows these groups to form a stable chemical structure with magnetic nanoparticles, which is not easy to fall off or decompose in a complex biological environment. For example, during the in vivo circulation process, covalent bonds can resist enzymatic hydrolysis and metabolic processes in the blood, ensuring the integrity of the probe and the continuity of its function; covalent bonds have higher bond energy and can withstand certain temperature changes. Covalent bond connection allows cell penetrating peptides, fluorescence resonance energy transfer fluorescent reporter groups and caspase substrates to work synchronously, realizing accurate monitoring and analysis of intracellular events.

[0051] According to an embodiment of the present invention, the dual-modal magnetic particle fluorescent 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 the tumor cells by binding to the specific antibodies on the surface of the tumor cells. Through the specific binding of the antigen-antibody, the nonspecific binding of the probe in normal tissues can be reduced, the background signal can be reduced, and the signal-to-background ratio of the imaging can be improved.

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

[0054] According to the embodiments of the present invention, tumor-specific antigens (TSA) and tumor-associated antigens (TAA) can be specifically recognized and presented by dendritic cells (DCs), thereby activating antigen-specific T cell responses. The antigens physically adsorbed on the probes can be effectively taken up and processed by DCs, thereby activating CD8 + T cells and CD4 + T cells produce a strong immune response; physical adsorption allows the antigen to be stably present on the probe, ensuring its effective delivery and presentation in the body, thereby monitoring the cellular behavior of the DC vaccine through a dual-modal magnetic particle fluorescent probe.

[0055] According to an embodiment of the present invention, the method for preparing a dual-modal magnetic particle fluorescent probe includes 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 a magnetic nanoparticle.

[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 neutral pH 6.8-7 phosphate buffer solution (PBS), performing a dehydration condensation reaction to obtain a mixture, and then purifying the mixture by column chromatography to obtain a dual-modal magnetic particle fluorescent probe.

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

[0058] According to an embodiment of the present invention, the kit may further include other reagents, such as auxiliary reagents for cell culture, staining or labeling, to enhance the function of the probe or improve the experimental effect.

[0059] According to another embodiment of the present invention, there is also disclosed an application of a composition or kit of a dual-modal magnetic particle fluorescent probe, which includes use in preparing, tracing and / or monitoring dendritic cell vaccines.

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

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

[0062] According to the embodiments of the present invention, DC vaccines can be prepared simply and quickly through the specific binding of DC cells and antigens and the process of physical adsorption of fluorescent probes.

[0063] According to an embodiment of the present invention, the migration and location tracking monitoring of the dendritic cell vaccine is performed by magnetic particle imaging technology; and the apoptosis monitoring of the dendritic cell vaccine is performed by fluorescence resonance energy transfer technology.

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

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

[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: 5 mL of Fe3O4 (4 mg / mL) was mixed with 1 mL of DT (Cy7) (1 mg / mL), and then stirred at room temperature for 8 h, and then ultrafiltered (3 kDa, 3500 g) at 4 °C for 30 min to remove unreacted DT (Cy7). Then 0.5 mL of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (10 mg / mL) was added, stirred, and incubated on a rotator for 2 h, and then 0.5 mL of n-hydroxysuccinamide (10 mg / mL) and 1 mL of OVA (2 mg / mL) were gently added to the mixture. After 17 h, ultrafiltration (50 kDa, 3500 g) was performed at 4 °C for 1 h to remove unreacted OVA. The resulting product was 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 traces and monitors the activity status of DC vaccine by magnetic particle imaging and fluorescent molecular imaging.

[0071] Example 2 Caspase substrate response test of dual-modal magnetic particle fluorescent probe Fe3O4-DEVDTAT-Cy7

[0072] One hour after adding Caspase-3 to Fe3O4-DEVDTAT-Cy7 at room temperature, the fluorescence spectrum changes as shown in the following figure: Figure 1 shown.

[0073] Figure 1 This is a graph showing the caspase substrate response test results of Fe3O4-DEVDTAT-Cy7 in an embodiment of the present invention.

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

[0075] Example 3 Dual-modal magnetic particle fluorescent probe Fe3O4-DEVDTAT-FITC stimulates bone marrow-derived dendritic cells (BMDC) vaccine maturation test

[0076] Primary bone marrow cells were obtained and induced to differentiate into BMDCs after 6 days by adding cytokines interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-GSF).

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

[0078] After 24 h of incubation, BMDCs were subjected to flow cytometry testing, and FITC-CD11c, APC-CD80, and PE-CD86 antibodies were added in sequence to obtain flow cytometric images of BMDC maturation. The results are shown in Figure 2. Figure 2 shown.

[0079] Figure 2 This is a flow cytometry result diagram of the maturation test of bone marrow-derived DC cell vaccine stimulated by the dual-modal magnetic particle fluorescent probe Fe3O4-DEVDTAT-Cy7 according to an 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 BMDC vaccine, and the expression of CD80 and CD86 is higher than that of the OVA group and the blank group.

[0081] Example 4 Dual-modal magnetic particle fluorescent probe Fe3O4-DEVDTAT-FITC test on DC vaccine apoptosis response

[0082] Mouse bone marrow-derived dendritic cells (DC2.4) were cultured in RPMI-1640 medium in a glass-bottomed culture dish to a density of 70% and then incubated with Fe3O4-DEVDTAT-FITC probe for 24 hours.

[0083] After adding 1 µM staurosporine (STS) for 1 hour to induce apoptosis of DC2.4 cells, the supernatant was discarded, the nuclei were stained with DAPI, the supernatant was discarded, the cells were fixed with paraformaldehyde, and the FITC signal with and without STS was observed using a laser confocal microscope. The results are shown in Figure 3 shown.

[0084] DC2.4 cells were cultured in RPMI-1640 medium to 70% density and incubated with Fe3O4-DEVDTAT-FITC probe for 24 hours.

[0085] After adding PBS or 1 µM STS for 1 hour, the cells were centrifuged and PE-labeled Caspase-3 antibody and APC-labeled Annexin V were added. The fluorescence signals of fluorescein isothiocyanate (FITC), propidium iodide (PI), phycoerythrin (PE) and allophycocyanin (APC) were detected by flow cytometry. The results are shown in Figure 4~Figure 8 shown.

[0086] Figure 3This is a confocal image showing changes in the mouse bone marrow-derived dendritic cells after they have taken up the Fe3O4-DEVDTAT-FITC probe and before and after the addition of the cell apoptosis-inducing agent staurosporine; Figure 4 This is a flow cytometry comparison diagram of the mouse bone marrow-derived dendritic cells taking up Fe3O4-DEVDTAT-FITC probe and then adding the cell apoptosis-inducing agent staurosporine; Figure 5 This is a flow cytometry comparison statistical diagram of the mouse bone marrow-derived dendritic cells taking up Fe3O4-DEVDTAT-FITC probe and then adding the cell apoptosis-inducing agent staurosporine in the example of the present invention.

[0087] Figure 6 This is a bar chart showing quantitative flow cytometric analysis of apoptosis detection after the mouse bone marrow-derived dendritic cells took up Fe3O4-DEVDTAT-FITC probe and then added the cell apoptosis-inducing agent staurosporine caspase substrate-3.

[0088] Figure 7 This is a comparison chart of flow cytometry images of Annexin V / propidium iodide apoptosis detection of mouse bone marrow-derived dendritic cells in an embodiment of the present invention; Figure 8 This is a bar chart showing quantitative analysis of the Annexin V / propidium iodide apoptosis detection flow cytometric graph of mouse bone marrow-derived dendritic cells in an embodiment of the present invention.

[0089] according to Figure 3~Figure 8 It can be seen that after the DC2.4 cells to which the Fe3O4-DEVDTAT-FITC probe was added induced apoptosis by STS, the FITC fluorescence intensity accumulated more and more in the cytoplasm under laser confocal microscopy, while it was negligible in living cells; flow cytometry analysis showed that compared with the control group, the FITC-positive cell expression of STS-treated BMDC increased by 50%, which can effectively release the FITC reporter group in the "on" state by cutting caspase substrate-3, resulting in the separation of caspase substrate-3 and Fe3O4, thereby emitting an obvious FITC signal, proving that the dual-modal magnetic particle fluorescent probe of the present invention can significantly and effectively monitor the apoptotic behavior of cells.

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

[0091] The in vivo fluorescence imager (IVIS) was used to verify whether the loaded Fe3O4-DEVDTAT-FITC probe affected the migration efficiency of the DC vaccine. The MPI was used 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 for 24 hours.

[0093] BMDCs were incubated with Fe3O4-DEVDTAT-FITC probe for 12 hours, centrifuged and washed with PBS to obtain the vaccine test group loaded with fluorescent probes. BMDCs not loaded with fluorescent probes were used as the vaccine control group.

[0094] The experimental group and the control group were incubated with 10 μM cell membrane fluorescent probe DiR (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate) in PBS for 15 min at 37 °C. After centrifugation and PBS washing, 1 × 10 6 The BMDC vaccine was injected into the left and right foot pads, with the right foot as the experimental group and the left foot as the control group. IVIS imaging was then performed 0, 2, 24, 48, and 72 hours later. The results are shown in the figure. Fig. 9 and Fig.10 shown.

[0095] 1×10 6 The BMDC vaccine was injected into the left and right foot pads, with the right foot as the experimental group and the left foot as the control group. MPI imaging was performed 0, 2, 24, 48, and 72 hours later. The results are shown in Figure 2. Fig.11 and Fig.12 shown.

[0096] After imaging, the mice were killed, and the popliteal lymph nodes on both 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 follows Fig.13 shown.

[0097] Fig. 9 This is a live fluorescence imaging image of the in vivo migration of a DC vaccine loaded with Fe3O4-DEVDTAT-FITC probe according to an embodiment of the present invention; Fig.10 This is a graph showing the change in fluorescence signal intensity of the DC vaccine in vivo migration loaded with Fe3O4-DEVDTAT-FITC probe according to an embodiment of the present invention; Fig.11 This is an imaging diagram of the in vivo migration of magnetic particles of DC vaccine loaded with Fe3O4-DEVDTAT-FITC probes according to an embodiment of the present invention; Fig.12 This is a graph showing the signal intensity changes of the in vivo migration magnetic particles of the DC vaccine loaded with Fe3O4-DEVDTAT-FITC probe according to an embodiment of the present invention; Fig.13 This is a lymph node pathology image after in vivo migration of the DC vaccine loaded with Fe3O4-DEVDTAT-FITC probe according to an embodiment of the present invention.

[0098] according to Figure 9~Figure 13It can be seen that the experimental group BMDC and the control group BMDC showed similar DiR fluorescence signals only in the mouse foot pad after injection. After 24 hours, the right popliteal lymph node (PLN) area showed more DiR fluorescence signals than the left PLN area, indicating that the Fe3O4-DEVDTAT-FITC probe can 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 within the right PLN could be shown. In vitro Prussian blue staining of bilateral PLNs collected immediately 24 hours after imaging confirmed that the presence of Fe3O4-DEVDTAT-FITC probe-labeled BMDC was positively stained in the right PLN, and no corresponding staining was observed in the left PLN. It is proved that the fluorescent probe of the present invention can effectively trace and monitor the behavior of DC vaccines under the dual modality of fluorescence imaging and magnetic particle imaging, and promote the migration and maturation process of DC vaccines.

[0099] Example 6 Testing of DC vaccine loaded with Fe3O4-DEVDTAT-Cy7 probes and combined with chemotherapy drugs

[0100] A small animal in vivo fluorescence imager was used to verify whether the DC vaccine loaded with Fe3O4-DEVDTAT-Cy7 probe could display the status of the DC vaccine when used in combination with chemotherapy drugs. The specific operation is as follows.

[0101] TNF-α was injected into the left and right footpads of mice 24 hours later.

[0102] BMDCs were incubated with Fe3O4-DEVDTAT-Cy7 probes for 12 h, centrifuged and washed with PBS, and then injected with oxaliplatin (OXA) at a dose of 10 mg / kg body weight into the tail vein. 6 The above-mentioned BMDC vaccine loaded with Fe3O4-DEVDTAT-Cy7 probe was used as the experimental group, and the BMDC vaccine without the probe was used as the control group and injected into the left and right foot pads respectively, of which the right foot was the experimental group; the left foot was the control group, and IVIS imaging was performed 24 hours later. The results are shown in the figure. Fig.14 shown.

[0103] Fig.14 This is a comparison chart of in vivo fluorescence imaging of the DC vaccine loaded with Fe3O4-DEVDTAT-Cy7 probe in an embodiment of the present invention and tested in combination with chemotherapy drugs.

[0104] according to Fig.14It can be seen that during the period of 0 to 8 days, the right popliteal lymph nodes (PLN) of the mice in the experimental group showed Cy7 fluorescence signals. The bilateral popliteal lymph nodes (PLN) were further dissected and confirmed by in vitro imaging that the Cy7 fluorescence signal indeed came from the DC vaccine injected into the right draining lymph node, proving that the fluorescent probe of the present invention can still effectively track and monitor the DC vaccine when used in combination with chemotherapy drugs.

[0105] The above specific embodiments further illustrate the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A dual-mode magnetic particle fluorescent probe, characterized in that: include: Magnetic nanoparticles, cell-penetrating peptides, fluorescence resonance energy transfer fluorescent reporters, and caspase substrates.

2. The dual-mode magnetic particle fluorescent probe according to claim 1, characterized in that: The magnetic nanoparticles are magnetic ferroferric oxide nanoparticles; The cell penetrating peptide comprises at least one of the peptide chains shown in SEQ ID NOs: 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 comprises at least one of DEVD, IETD, LEHD and VAD; and / or The cell penetrating peptide, the fluorescence resonance energy transfer fluorescent reporter group and the caspase substrate are linked to the surface of the magnetic nanoparticles through covalent bonds.

3. The dual-modal magnetic particle fluorescent probe according to claim 1 or 2, characterized in that: The dual-modal magnetic particle fluorescent probe further comprises an antigen.

4. The dual-modal magnetic particle fluorescent probe according to claim 3, characterized in that: The antigen is an antigen for dendritic cell vaccine, including a tumor-specific antigen or a tumor-associated antigen, and / or The antigen is contained in the dual-modal magnetic particle fluorescent probe by physical adsorption.

5. A method for preparing a dual-modal magnetic particle fluorescent probe according to any one of claims 1 to 4, characterized in that: The method comprises the steps of covalently linking the cell penetrating peptide, the fluorescence resonance energy transfer fluorescent reporter group and the caspase substrate to the surface of the magnetic nanoparticle.

6. A composition or kit comprising the dual-modal magnetic particle fluorescent probe according to any one of claims 1 to 4.

7. Use of the dual-modal magnetic particle fluorescent probe according to any one of claims 1 to 4, the dual-modal magnetic particle fluorescent probe prepared by the method of claim 5, and / or the composition or kit according to claim 6, characterized in that: The applications include preparation, tracing and / or monitoring of dendritic cell vaccines.

8. The use according to claim 7, characterized in that: Preparation of dendritic cell vaccine includes: The antigen with the dual-modal magnetic particle fluorescent probe adsorbed on the surface is co-cultured with dendritic cells to obtain a dendritic cell vaccine marked with the dual-modal magnetic particle fluorescent probe.

9. The use according to claim 8, characterized in that: The dendritic cell vaccine is monitored for migration and location tracking by magnetic particle imaging technology; or The dendritic cell vaccine is used to monitor the apoptosis of the vaccine by fluorescence resonance energy transfer technology.

10. The use according to claim 7, characterized in that: The dual-modal magnetic particle fluorescent probe is used to activate the maturation and / or migration process of the dendritic cell vaccine.

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