Method and regulatory platform for cyclically activating immune function

Abstract

The application relates to the fields of tumor immunotherapy and biomedical engineering technology, and particularly relates to a method for cyclically activating immune functions and a regulation platform, the method comprising the following steps: a) delivering a composition containing magnetic response nanomotors into organelles of immune cells; b) applying a programmable rotating magnetic field with a preset frequency and magnetic field intensity to the immune cells to drive the magnetic response nanomotors to rotate and generate local shear force, and to induce reversible permeability changes of the organelle membranes; c) activating immune-related signal pathways to make the immune cells change from an inhibitory phenotype to an activated phenotype; and d) periodically repeating steps b) and c) to cyclically activate the immune-related signal pathways and maintain the activated phenotype state of the immune cells. The application controls the movement of the magnetic response nanomotors in the immune cells, induces reversible permeability changes of lysosome membranes, realizes controllable cyclic activation of cell biochemical signal pathways, and then reshapes the immune cell phenotype and continuously improves the anti-tumor response of the immune cells.

Description

Technical Field

[0001] This application relates to the fields of tumor immunotherapy and biomedical engineering technology, and in particular to a mechanical method and regulation platform for cyclically activating immune function. Background Technology

[0002] Immunotherapy, a significant breakthrough in cancer treatment, relies on intervening in the biochemical signaling pathways of immune cells to activate the body's endogenous anti-tumor capabilities. However, due to inherent tumor heterogeneity and the presence of an immunosuppressive tumor microenvironment, traditional immunomodulatory interventions, primarily using chemotherapeutic drugs or antibodies, have significant limitations in terms of the precision of signal regulation, the duration of action, and the depth of targeting. Particularly in the treatment of solid tumors, current immunomodulatory strategies have limited response rates, urgently necessitating the development of more precise and controllable novel immunomodulatory mechanisms.

[0003] Recent studies have revealed that immune cells not only possess biochemical reaction characteristics but also exhibit strong mechanosensing capabilities, enabling them to regulate their behavior and function in response to mechanical stimuli. This discovery provides a new physical regulatory pathway for overcoming traditional immune regulation methods. Furthermore, organelles have been confirmed as important receptors and amplifiers of mechanosensory signals. As intracellular biochemical signaling hubs, lysosomes have multiple functions in energy metabolism and signal transduction, while also sensing mechanical changes such as membrane deformation and intraluminal fluid shear, thereby precisely mediating downstream signal activation. However, despite the increasing emphasis on organelle-level mechanosensory regulation, mature technologies for controlling the output of mechanical stress to specific intracellular sites are still lacking. This prevents the full utilization of the lysosome's precise mechanosensing structure for immune function regulation. Simultaneously, achieving "mild and reversible" membrane permeability changes to avoid irreversible cell damage and defining the required mechanical threshold for this process remain key unresolved scientific questions. Summary of the Invention

[0004] To address the aforementioned issues, the purpose of this application is to provide a method and regulatory platform for cyclically activating immune function. This method can induce reversible changes in the permeability of lysosomal membranes by regulating the movement behavior of magnetically responsive nanomotors within immune cells, thereby achieving controllable cyclic activation of intracellular biochemical signaling pathways, further driving immune cell phenotypic remodeling and continuously enhancing their anti-tumor response capabilities.

[0005] This application discloses a method for cyclically activating immune function, comprising the following steps:

[0006] (a) Delivering a composition containing magnetically responsive nanomotors into the organelles of immune cells;

[0007] (b) A programmable rotating magnetic field with preset frequency and magnetic field strength parameters is applied to the immune cells to drive the magnetic response nanomotor to rotate and generate local shear force, thereby inducing a reversible change in the permeability of the organelle membrane;

[0008] (c) Activate immune-related signaling pathways to induce the immune cells to change from an inhibitory phenotype to an active phenotype;

[0009] (d) Repeat steps (b)-(c) periodically to achieve cyclical activation of the immune-related signaling pathways and maintain the activated phenotype of the immune cells.

[0010] In another preferred embodiment, the immune cell is a macrophage.

[0011] In another preferred embodiment, the magnetically responsive nanomotor achieves self-assembly under the influence of the magnetic field.

[0012] In another preferred embodiment, the magnetically responsive nanomotor self-assembles before or after delivery into the organelle. In another preferred embodiment, the frequency of the programmable rotating magnetic field is 0.2 Hz to 5 Hz; more preferably, the frequency of the programmable rotating magnetic field is 0.8 Hz to 2 Hz.

[0013] In another preferred embodiment, the magnetic field strength of the programmable rotating magnetic field is 10mT-800mT; more preferably, the magnetic field strength of the programmable rotating magnetic field is 20mT-500mT; even more preferably, the magnetic field strength of the programmable rotating magnetic field is 260mT.

[0014] In another preferred embodiment, the local shear force is 0.4 Pa to 3 Pa; more preferably, the local shear force generated in step (b) is 0.8 Pa to 2 Pa.

[0015] In another preferred embodiment, the activation of the immune-related signaling pathway includes:

[0016] Prior to performing step (a), the immune cells are given additional cytokines to polarize them to the M2 type.

[0017] In step (c), the membrane damage sites of the organelles specifically recruit lectin-like proteins to the membrane surface of the organelles;

[0018] It recruits and activates downstream metabolic sensing and immune inflammatory pathways.

[0019] In another preferred embodiment, the membrane damage site of the organelle specifically recruits Galectin-9, which then sequentially activates AMPK phosphorylation and NF-κB signaling cascade amplification.

[0020] In another preferred embodiment, the activation of the immune-related signaling pathway leads to the upregulation of metabolites related to the immune cell glycolysis pathway, accompanied by the downregulation of metabolites related to the oxidative phosphorylation pathway.

[0021] In another preferred embodiment, the periodic repetition step (b) includes using a pulsed rotating magnetic field, with each magnetic field action lasting 10 to 30 minutes and an interval of 30 to 1440 minutes.

[0022] In another preferred embodiment, the immune cells include one or more of macrophages, dendritic cells, T cells, or natural killer cells; preferably, the immune cells are macrophages.

[0023] In another preferred embodiment, the organelle is a lysosome.

[0024] This application also discloses a regulatory platform for cyclically activating immune function, the platform comprising:

[0025] Magnetic-responsive components, including a magnetic-responsive nanomotor composition capable of being phagocytosed by immune cells and localized within organelles;

[0026] A magnetic field-driven component generates a rotating magnetic field at a preset frequency and magnetic field strength parameters to drive the magnetically responsive nanomotor to rotate within the organelle and generate localized shear force; and

[0027] The control component, electrically connected to the magnetic field driving component, is configured to periodically start and stop the rotating magnetic field according to a preset pulse program, so as to cyclically activate the immune-related signaling pathways of the immune cells and cause the immune cells to change from an inhibitory phenotype to an activated phenotype.

[0028] The control platform is configured to implement the method described above.

[0029] In another preferred embodiment, the magnetic field driving component includes:

[0030] A rotating rotor that carries a permanent magnet;

[0031] An electric motor used to drive the rotating rotor to generate a rotating magnetic field.

[0032] In another preferred embodiment, a magnetic field simulation component is also included, comprising a coil assembly used in conjunction with a high-resolution microscope to simulate the operating conditions of a rotating permanent magnet when the magnetically responsive nanomotor motion behavior within the immune cells is observed and recorded through the microscope.

[0033] In another preferred embodiment, the permanent magnet is one or more of neodymium iron boron, samarium cobalt, alnico, or hard magnetic ferrite; more preferably, the permanent magnet is neodymium iron boron.

[0034] In another preferred embodiment, the two permanent magnets are arranged opposite each other.

[0035] In the embodiments of this application, a dynamic micromagnetic modulation platform formed by combining magnetically responsive nanomotors and rotating magnetic fields is used to achieve programmable activation of immune cell function. Under the action of the rotating magnetic field, the magnetically responsive nanomotors can generate stable microscale shear forces within immune cells, inducing reversible changes in lysosomal membrane permeability, activating key immune signaling pathways, thereby achieving controllable repolarization of cell phenotype and cyclic activation of signaling pathways, maintaining the continuous activation state of immune cells. This application breaks through the limitations of traditional immune activation methods that rely on unidirectional stimulation or chemical regulation, and has the advantages of programmability, reversibility and high safety, showing broad application prospects in tumor immunotherapy. Attached Figure Description

[0036] Figure 1 This is a schematic diagram illustrating the principle of one embodiment of this application.

[0037] Figure 2 This image shows the fabrication and characterization of a magnetically responsive nanomotor MNM according to one embodiment of this application.

[0038] Figure 3 This is a graph showing the range of intracellular fluid velocity and shear force threshold under different frequency magnetic fields according to one embodiment of this application.

[0039] Figure 4 This is a verification diagram of RMF (Regenerative Membrane Fiber) in vitro for lysosomal vesicle membrane damage according to one embodiment of this application.

[0040] Figure 5 This is a schematic diagram illustrating the co-localization effect of MNM and lysosomes according to one embodiment of this application.

[0041] Figure 6 This is a verification diagram of the frequency-dependent RMF regulation of reversible lysosomal membrane permeability according to one embodiment of this application.

[0042] Figure 7 This is a schematic diagram of the RMF-induced macrophage immune activation results according to one embodiment of this application.

[0043] Figure 8 This is a WB diagram verifying the RMF-induced Gal9-AMPK-NF-κB mechanism according to one embodiment of this application.

[0044] Figure 9 This is a schematic diagram showing the results of RMF-regulated changes in macrophage metabolic levels according to one embodiment of this application.

[0045] Figure 10This is a schematic diagram of WB results for dynamic RMF-induced cyclic activation of biochemical signals according to one embodiment of this application.

[0046] Figure 11 This is a schematic diagram illustrating the antitumor efficacy of dynamic RMF in three tumor models according to one embodiment of this application.

[0047] Figure 12 This is a schematic diagram illustrating the therapeutic effects of dynamic RMF in an intravenous injection model and a lung carcinoma in situ model according to one embodiment of this application.

[0048] Figure 13 This diagram illustrates the mechanism of RMF-induced biochemical signal activation in vivo according to one embodiment of this application. Detailed Implementation

[0049] In the following description, many technical details are presented to help the reader better understand this application. However, those skilled in the art will understand that the technical solutions claimed in this application can be implemented even without these technical details and various variations and modifications based on the following embodiments.

[0050] Compared with the prior art, this application has the following beneficial effects:

[0051] 1. A dynamic micromagnetic regulation platform within lysosomes was constructed for the first time. By remotely applying a rotating magnetic field of 20-500 mT and 0.8-2 Hz, magnetically responsive nanomotors (MNMs) were driven to generate fluid vortices within lysosomes and act on the lysosomal membrane in the form of shear force. This enabled non-contact, controllable, and reversible regulation of membrane permeability, providing a novel pathway for mechanical signal intervention at the organelle level.

[0052] 2. The shear force threshold was clearly defined and frequency-dependent reversible membrane regulation was achieved. For the first time, simulation and experiments confirmed that shear force in the range of 0.8-2 Pa can enhance and restore lysosomal membrane permeability without causing irreversible rupture of the lysosomal membrane, thus establishing a "reversible safety window" for the mechanical regulation of organelle membranes.

[0053] 3. Establish the Gal9–AMPK–NF-κB signaling cycle activation pathway, continuously induce lysosomal membrane stress response through dynamic micromagnetic force, drive Galectin-9 to recruit and activate downstream pathways of AMPK and NF-κB, maintain macrophage M1 polarization and metabolic reprogramming, and achieve magnetically driven periodic regulation of immune phenotype.

[0054] 4. The system validated the in vivo therapeutic potential and immune memory effect of the strategy, achieving immune activation and tumor suppression in three subcutaneous tumor models, completing remote enrichment and local shear regulation in the intravenous injection model, and demonstrating stable and durable immune response and good biosafety in about one-third of the mice in the orthotopic lung adenocarcinoma model.

[0055] The present application will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0056] The first embodiment of this application discloses a method for cyclically activating immune function, comprising the following steps:

[0057] (a) Delivering magnetically responsive nanomotors into organelles of immune cells and enabling them to self-assemble under the influence of a magnetic field;

[0058] (b) Apply a programmable rotating magnetic field with preset frequency and magnetic field strength parameters to immune cells to drive magnetically responsive nanomotors to rotate and generate local shear force, thereby inducing reversible changes in the permeability of organelle membranes.

[0059] (c) Activate immune-related signaling pathways, prompting immune cells to change from an inhibitory phenotype to an active phenotype;

[0060] (d) Repeat steps (b)-(c) periodically to achieve cyclical activation of immune-related signaling pathways and maintain the activated phenotype of immune cells.

[0061] In an optional embodiment, the immune cells may be macrophages.

[0062] In one alternative embodiment, the magnetically responsive nanomotors self-assemble under the influence of a magnetic field. For example, under the influence of a rotating magnetic field, significant magnetic dipole interactions occur between individual nanomotors, driving them to align and aggregate along the magnetic field direction within the lysosomal cavity, thereby spontaneously forming chain-like, ring-like, or other regularly ordered assembly structures. This assembly process can be achieved without additional chemical cross-linking agents, relying solely on the control of an external magnetic field.

[0063] In one optional embodiment, the magnetically responsive nanomotors self-assemble before or after delivery to organelles. Specifically, in the case of post-delivery self-assembly, dispersed magnetically responsive nanomotors are delivered into the organelles of immune cells. Under the influence of an applied rotating magnetic field, the nanomotors align and aggregate in an orderly manner within the local space of the organelle cavity through magnetic dipole interactions, forming stable assemblies. Alternatively, in the case of pre-delivery self-assembly, dispersed magnetically responsive nanomotors can be suspended in a physiological buffer solution, and a rotating magnetic field of a specific frequency and strength can be applied, causing them to align and aggregate in the solution through magnetic dipole interactions, forming chain-like, ring-like, or other regular and orderly assembly structures. After self-assembly, the resulting nanomotor assemblies are directly injected into the target immune cells, allowing them to maintain their original structure and exert their mechanical effects within the organelles.

[0064] In one optional embodiment, the frequency of the programmable rotating magnetic field can be 0.2Hz-5Hz; more preferably, the frequency of the programmable rotating magnetic field can be 0.8Hz-2Hz.

[0065] In one optional embodiment, the magnetic field strength of the programmable rotating magnetic field can be 10mT-800mT; more preferably, the magnetic field strength of the programmable rotating magnetic field can be 20mT-500mT; even more preferably, the magnetic field strength of the programmable rotating magnetic field is 260mT.

[0066] In an optional embodiment, the local shear force can be 0.4 Pa to 3 Pa; preferably, the local shear force generated in step (b) can be 0.8 Pa to 2 Pa.

[0067] In an optional embodiment, activating immune-related signaling pathways may include:

[0068] Before step (a), immune cells are given additional cytokines to polarize them to the M2 type;

[0069] In step (c), the membrane damage sites of organelles specifically recruit lectin-like proteins to the membrane surface of the organelles;

[0070] It recruits and activates downstream metabolic sensing and immune inflammatory pathways.

[0071] In an alternative embodiment, the membrane damage site of the organelle can specifically recruit Galectin-9, which in turn activates AMPK phosphorylation and NF-κB signaling cascade amplification.

[0072] In an alternative embodiment, activation of immune-related signaling pathways can induce upregulation of metabolites associated with immune cell glycolysis pathways, accompanied by downregulation of metabolites associated with oxidative phosphorylation pathways.

[0073] In an optional embodiment, the periodic repetition of step (b) may include using a pulsed rotating magnetic field, with each magnetic field application lasting 10 to 30 minutes and an interval of 30 to 1440 minutes.

[0074] In theory, lysosomal membranes can repair damage caused by external forces in about 30 minutes. Therefore, in short-interval mode, the next magnetic field stimulation can be applied immediately after the 30-minute repair period to achieve high-frequency signal pathway cyclic activation. In long-interval mode, the interval can be extended to several hours or even 24 hours depending on experimental needs to achieve low-frequency but long-cycle immune cell regulation, which is suitable for applications requiring lower stimulation frequencies or avoiding excessive stress.

[0075] In an optional embodiment, the immune cells may include one or more of macrophages, dendritic cells, T cells, or natural killer cells; preferably, the immune cells may be macrophages.

[0076] In an alternative embodiment, the organelle may be a lysosome.

[0077] The second embodiment of this application also discloses a regulatory platform for cyclically activating immune function, the platform comprising:

[0078] Magnetic-responsive components, including a magnetic-responsive nanomotor composition capable of being phagocytosed by immune cells and localized within organelles;

[0079] A magnetic field-driven component generates a rotating magnetic field at a preset frequency and magnetic field strength parameters to drive a magnetically responsive nanomotor to rotate within an organelle and generate localized shear forces; and

[0080] The control component, electrically connected to the magnetic field drive component, is configured to periodically start and stop the rotating magnetic field according to a preset pulse program, so as to cyclically activate the immune-related signaling pathways of immune cells and promote the transformation of immune cells from an inhibitory phenotype to an activated phenotype.

[0081] The control platform is configured to implement the methods described above.

[0082] In an optional embodiment, the magnetic field driving component may include:

[0083] A rotating rotor that carries a permanent magnet;

[0084] An electric motor used to drive a rotating rotor to generate a rotating magnetic field.

[0085] In an optional embodiment, a magnetic field simulation component may also be included, which may include a coil assembly used in conjunction with a high-resolution microscope to simulate the operating conditions of a rotating permanent magnet when observing and recording the motion behavior of magnetically responsive nanomotors within immune cells through a microscope.

[0086] In an optional embodiment, the permanent magnet may be one or more of neodymium iron boron, samarium cobalt, alnico, or hard magnetic ferrite; preferably, the permanent magnet is neodymium iron boron.

[0087] In an optional embodiment, the two permanent magnets can be arranged opposite each other. For example, the two permanent magnets are 180° apart, forming an opposite arrangement. The magnetization directions of the two permanent magnets are opposite to each other (e.g., the N pole of the upper magnet faces outward and the S pole of the lower magnet faces outward). When the rotor rotates at a constant speed around the central axis, a uniformly rotating magnetic field vector in the plane can be generated at the center of the sample cavity, realizing stable and preset phase micro-magnetic force drive.

[0088] The mechanism of action of this application is briefly described below. In this application, a regulatory platform formed by the combination of magnetically responsive nanomotors and rotating magnetic fields is used to achieve programmable activation of immune cell function. After being delivered by injection, the magnetically responsive nanomotors rotate within immune cells such as macrophages under the action of an external rotating magnetic field, generating local shear force and inducing reversible permeability changes in lysosomal membranes. This, in turn, promotes the recruitment of Galectin-9 to the lysosomal membrane surface, activating key biochemical signaling pathways such as AMPK and NF-κB. By periodically applying the rotating magnetic field, the biochemical signaling pathways within immune cells are cyclically activated, thereby maintaining their continuous activated phenotype state and achieving a dynamically programmable immune regulation effect, thus realizing safe, controllable, and durable anti-tumor immunotherapy.

[0089] To facilitate understanding of the above structures and their mechanisms in tumor immunotherapy, the following section combines... Figure 1 To explain, Figure 1A programmable macrophage repolarization mechanism for tumor immunotherapy is illustrated. In an embodiment of this application, a magnetically responsive nanomotor is delivered to the lysosome of a macrophage and rotates intracellularly under the drive of an external rotating magnetic field (RMF), generating local shear forces that act on the lysosomal membrane, thereby inducing increased lysosomal membrane permeability and triggering the LMP (lysosomal membrane permeabilization) process. After LMP occurs, Galectin-9 (Gal9) is specifically recruited to the damaged lysosomal membrane surface, further recruiting and activating downstream signaling molecules AMPK and IKKβ, initiating metabolic sensing and immune-inflammatory pathways, and driving the macrophage to transform from an M2-like immunosuppressive phenotype to an M1-like immunoactivating phenotype. Activated M1 macrophages can secrete pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, thereby enhancing their anti-tumor immune function. Furthermore, by applying periodic pulse control to the RMF, the "on-off" cycle regulation of the shear force driven by the magnetically responsive nanomotor within the cell is achieved, thereby periodically activating intracellular signaling pathways and maintaining the continuous immune activation state of macrophages, exhibiting good controllability and programmability.

[0090] The objective of this application can be achieved through the following technical solutions:

[0091] First aspect: Providing compositions for tumor immunomodulatory therapy.

[0092] The composition includes a magnetically responsive nanomotor as a therapeutic component. The magnetically responsive nanomotor includes three-dimensional magnetic nanoparticles and a functionalized layer coated on the surface of the three-dimensional magnetic nanoparticles. The functionalized layer contains an amino acid polymer.

[0093] The three-dimensional magnetic nanoparticles in this application include, but are not limited to, Fe3O4, CoFe2O4, NiFe2O4, NdFeB, or combinations thereof. Preferably, the three-dimensional magnetic nanoparticles are zinc-doped iron oxide nanoparticles. Amino acid polymers include, but are not limited to, poly-L-lysine, poly-L-ornithine, or poly-L-arginine.

[0094] The three-dimensional magnetic nanoparticles have an average particle size of 10–50 nm, preferably 15–30 nm. The magnetically responsive nanomotors form chain-like self-assembled structures under the influence of a rotating magnetic field, and generate localized fluid vortices in a liquid environment under the same magnetic field.

[0095] The second aspect: providing a method for fabricating magnetically responsive nanomotors.

[0096] a) Synthesis of 25 nm zinc-doped iron-based magnetically responsive nanomotors (MNPs): Iron acetylacetonate (0.8 mmol, 282.5 mg), zinc acetylacetonate (1.2 mmol, 316.3 mg), and tetraphenylbenzoic acid (400 mg) were dissolved in a mixed solvent of diphenyl ether (10.4 mL) and oleic acid (1.2 mL) in a three-necked flask. The mixture was ultrasonically dispersed for 10 minutes, then argon gas was introduced and the mixture was stirred (400 rpm) for 30 minutes. The solution was then heated to 290 °C and maintained at this temperature for 30 minutes. The prepared solution was dispersed in ethanol for later use.

[0097] b) MNP Surface Modification with PLL (MNMs): To improve their water solubility and cellular uptake capacity, MNPs were surface modified with poly-L-lysine (PLL). 100 mg of PLL was dissolved in 4 mL of ultrapure water and mixed with 8 mL of an ethanol solution containing 10 mg of MNP. The mixture was sonicated for 2 hours at 40 W (5 s working time, 2 s interval) to coat the MNP surface with PLL. After sonication, the product was washed three times with ultrapure water and finally dispersed in water to obtain amino-terminated MNMs.

[0098] The magnetically responsive nanomotors prepared by the above method possess excellent magnetic response capability, dispersion, and structural uniformity. Figure 2 The image shows the fabrication and characterization of the magnetically responsive nanomotor MNM. Transmission electron microscopy (TEM) images reveal that the MNMs exhibit a regular nanostructure with a side length of approximately 25 nm, demonstrating good geometric consistency and controllability. The hydration particle size and Zeta potential (~+30 mV) in the water-dispersed state were determined using dynamic light scattering and electrophoretic light scattering. The dried MNMs were then used for testing with a vibrating sample magnetometer, and the saturation magnetization value (69 emu / g) was recorded.

[0099] Third aspect: Providing a rotating magnetic field device

[0100] The rotating magnetic field device of this application uses two rotating NdFeB permanent magnets to construct the magnetic field source. The magnets are driven by a motor to achieve frequency-adjustable rotational motion, thereby generating a controllable rotating magnetic field. The maximum magnetic field strength can reach 500mT, and the operating frequency range is 1-30Hz, meeting the mechanical control experimental needs at the cellular level and even in vivo animal level.

[0101] A rotating magnetic field device (magnetic field drive assembly) may include: a rotating rotor carrying permanent magnets, and a motor for driving the rotating rotor to generate a rotating magnetic field. The permanent magnets are fixed to the rotating rotor and can be uniformly distributed along the rotor's circumference to form a uniform two-dimensional planar rotating magnetic field when the rotor rotates. This magnetic field is constructed by the magnetism of the permanent magnets themselves and has a fixed magnetic field strength, for example, approximately 260 mT. The motor's output shaft can be directly connected to the rotating rotor via a coupling, or via a transmission mechanism such as a belt or gears, to drive the rotor to rotate smoothly. The magnetic field acts uniformly on the target area, thereby achieving the drive of a magnetically responsive nanomotor.

[0102] Fourth aspect: Design of shear force parameters and cell-level validation

[0103] Based on the above-mentioned rotating magnetic field device and composition, this application also needs to further determine a suitable fluid shear force to achieve reversible regulation of the permeability of immune cell lysosomal membranes.

[0104] By combining mechanical modeling and finite element simulation of the rotational behavior of MNMs within lysosomes, this study systematically reveals the evolution of fluid shear force within lysosomes under the influence of magnetic fields of different frequencies. A mechanical simulation model was constructed to simulate the rotational-induced fluid vortex of MNMs within a closed lysosome space. The local shear stress distribution generated at different frequencies (0.2-5 Hz) was calculated, thus determining the critical shear force threshold for reversible regulation of lysosomal membrane permeability to be 0.8-2 Pa. Furthermore, to verify the accuracy of the simulated mechanical response prediction, this application also constructed a biomimetic lysosomal lipid vesicle system in vitro, simulating the lysosomal membrane structure, and used a magnetic field to control the controlled rotation of MNMs within the vesicles.

[0105] To verify the rationality of the above theoretical model, such as Figure 3 As shown, numerical simulations of the flow field evolution within a lysosome induced by rotation of MNMs were conducted using the ANSYS Fluent module. The MNM assembly was considered a rigid body, placed at the center of a biomimetic lysosome space with radii of 359.2 nm and 453.0 nm, and was set to rotate about its minor axis. In the simulation, the liquid within the lysosome was assumed to be an incompressible viscous fluid, with a viscosity of 471.1 cp (measured average) and a density of 1000 kg / m³. 3 Dynamic meshing technology was employed to ensure mesh quality and computational convergence, with a time step set to 0.001 s. After calculating the three-dimensional flow field velocity distribution induced by MNMs assembly at different frequencies, the fluid shear force tensor and total shear stress were further calculated, and the results were imported into the CFD-Post module for visualization analysis. Simulation results show that the shear force distribution under low-frequency RMF driving falls within the range of 0.8–2 Pa.

[0106] Based on the critical shear force threshold determined by the above simulation, in order to further verify the ability of micromagnetic force to regulate LMP, this application further conducted verification experiments in an in vitro biomimetic lysosome model, as follows: Figure 4 As shown, a lysosome-like model was constructed—cationic small liposomes (SUVs) encapsulating a high concentration of the fluorescent dye Rhodamine B (SRB) and its magnetically responsive version (SUVs-MNM). Liposomes were synthesized via a membrane rehydration method. Liposome membranes were prepared using a mixture of DOPC and DOPE, and subsequently rehydrated in PBS containing or without 200 μg / mL MNMs and 50 mM SRB to form SUVs-MNM and SUVs. After purification through freeze-thaw cycles and 200 nm membrane extrusion, the liposomes were stored at 4°C in the dark. To assess the RMF-induced SRB release, fluorescence intensity was measured using a multi-mode microplate reader at an excitation wavelength of 565 nm and an emission wavelength of 586 nm. Due to the self-quenching effect of the high concentration of SRB inside the SUVs, the fluorescence intensity was low in the unreleased state. When RMF stimulation disrupted the liposome membrane, SRB was released and diluted in the external solution, resulting in enhanced fluorescence signal. SUVs and SUVs-MNM were stimulated for 15 minutes at RMF frequencies of 0.2, 0.8, 1, 2, and 5 Hz, respectively. Fluorescence intensity changes were detected in three states: before release, after release, and at maximum release (after a second freeze-thaw cycle). The results showed that the SRB fluorescence signal gradually increased with increasing RMF frequency, indicating enhanced membrane permeability. SUVs-MNM exhibited mild and controllable SRB release in the 0.8-2 Hz frequency range, consistent with the shear force threshold obtained from the aforementioned finite element simulation.

[0107] Fifth aspect: Application of the composition in tumor immunomodulatory therapy

[0108] The administration route can be intratumoral, subcutaneous, or intravenous injection of a dispersion containing magnetically responsive nanomotors.

[0109] Furthermore, the immune cells are macrophages, dendritic cells, T cells, or natural killer cells; preferably, the immune cells are macrophages.

[0110] Furthermore, the tumor cells are solid tumor cells, which can be lung adenocarcinoma cells, breast cancer cells, or melanoma cells.

[0111] The magnetically responsive nanomotor of this application can be delivered as an injectable formulation to various solid tumor models, including mouse breast cancer, lung adenocarcinoma, and melanoma, via intratumoral, subcutaneous, or intravenous routes. Under the action of an applied low-frequency rotating magnetic field (0.8–2 Hz, 10–30 mT), the nanomotor rotates within the lysosomes of tumor-infiltrating macrophages, generating microscale shear forces that induce reversible enhancement of lysosomal membrane permeability, thereby initiating the Galectin-9-AMPK-NF-κB signaling cascade. This signaling pathway drives tumor-associated macrophages to transform from immunosuppressive (M2) to immunoactivated (M1) states, accompanied by upregulation of glycolysis and increased secretion of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), significantly improving the tumor immune microenvironment. By periodically applying the rotating magnetic field, the signaling pathway can be cyclically activated, continuously maintaining macrophage activation, ultimately achieving significant tumor suppression and prolonged survival benefits in various mouse tumor models, validating the broad application potential of this composition in tumor immunomodulatory therapy.

[0112] To make the objectives, technical solutions, beneficial effects, and significant advancements of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the embodiments of this application. Obviously, all the described embodiments are only some embodiments of this application, and not all embodiments; based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0113] Example 1: Reversible Regulation of Lysosomal Membrane Permeability Based on Magnetic Response Nanomotors

[0114] This embodiment constructs a magnetically responsive nanomotor with a side length of approximately 25 nm, possessing extremely high saturation magnetization and self-assembly characteristics under magnetic fields. It is paired with a highly controllable in vitro rotating magnetic field control device, with a frequency reaching 30 Hz and a magnetic field strength reaching 500 mT. This device can precisely mediate the rotational motion of the nanomotor under a rotating magnetic field. Cell biology experiments were used to verify the high degree of co-localization between the nanomotor and lysosomes, and the varying degrees of change in lysosomal membrane permeability mediated by micromagnetic forces under different magnetic field frequencies. This establishes the range of reversible membrane permeability regulation dependent on magnetic field frequency.

[0115] (1) Nanomotor synthesis and magnetic field device parameters:

[0116] Synthesis of 25 nm zinc-doped iron-based magnetically responsive nanomotors (MNPs): Iron acetylacetonate (0.8 mmol, 282.5 mg), zinc acetylacetonate (1.2 mmol, 316.3 mg), and tetraphenylbenzoic acid (400 mg) were dissolved in a mixed solvent of diphenyl ether (10.4 mL) and oleic acid (1.2 mL) in a three-necked flask. The mixture was ultrasonically dispersed for 10 min, then argon gas was introduced and the mixture was stirred (400 rpm) for 30 min. The solution was then heated to 290 °C and maintained at this temperature for 30 min. The prepared solution was dispersed in ethanol for later use.

[0117] MNPs surface-modified with PLL (MNMs): To improve their water solubility and cellular uptake capacity, MNPs were surface-modified with poly-L-lysine (PLL). 100 mg of PLL was dissolved in 4 mL of ultrapure water and mixed with 8 mL of an ethanol solution containing 10 mg of MNP. The mixture was sonicated for 2 hours at 40 W (5 s operation time, 2 s interval) to coat the MNP surface with PLL. After sonication, the product was washed three times with ultrapure water and finally dispersed in water to obtain amino-terminated MNMs.

[0118] To verify the lysosomal targeting ability of the magnetically responsive nanomotors (MNMs) of this application within cells and their regulatory effect on lysosomal membrane permeability (LMP), such as... Figure 5 As shown, MNMs and their fluorescently labeled forms (MNMs-FITC and MNMs-Cy5) were co-incubated with macrophages and tumor cells for 24 h to observe their cellular uptake efficiency and subcellular localization. Macrophages were induced for 48 h with 20 μg / mL IL-4 and 20 μg / mL IL-13, polarized to the M2 type, and then co-incubated with MNMs for 4 h, 8 h, 12 h, and 24 h, respectively. Lysosomes and nuclei were then labeled with LysoTracker Red and Hoechst, respectively, and imaged using confocal laser microscopy. The results showed that the co-localization efficiency of MNMs with lysosomes reached 79.6% within 24 hours, indicating their good lysosomal targeting ability. Further three-dimensional Z-stack imaging revealed the spatial aggregation behavior of MNMs within macrophage lysosomes after 2 h and 24 h of co-incubation.

[0119] To further evaluate the modulatory effect of MNMs on LMP under the action of a rotating magnetic field (RMF), such as Figure 6As shown, RAW 264.7 cells and bone marrow-derived macrophages (BMDMs) were transfected using EGFP-Gal3 and YFP-Gal9 reporter systems, respectively, to visualize changes in lysosomal macrophages (LMP). After transfection, cells were co-incubated with MNMs-Cy5 for 24 h and then stimulated with refractive indexing (RMF) for 15 min. Results showed that Gal3 bright spots were clearly observed in cells after 10-30 min of RMF stimulation at a frequency range of 0.8-5 Hz, indicating increased lysosomal membrane permeability. Further observation revealed that under low-frequency (0.8-2 Hz) RMF stimulation, the Gal3 fluorescent bright spots gradually decreased and disappeared after 20-40 min, suggesting that LMP can recover within a certain timescale and is reversible. This result was further confirmed by real-time dynamic tracking using a high-content imaging system and a self-made magnetron sputtering high-resolution fluorescence imaging microscope. The above experimental results fully demonstrate that MNMs can achieve reversible and controllable intervention on lysosomal membrane permeability under RMF regulation, and have the potential to achieve mechanical function regulation at the organelle level.

[0120] Example 2: Dynamic Micromagnetic Cyclic Regulation of Immunobiochemical Signaling Mechanism and its Application in Activating Immune Function

[0121] This application further systematically verifies the effect of reversibly regulating lysosomal membrane permeability based on an intracellular dynamic micromagnetic regulation platform, thereby achieving the cyclic activation of intracellular biochemical signaling pathways and the sustained activation of immune cells such as macrophages. Firstly, as... Figure 7 As shown, a series of cell biology experiments confirmed that micromagnetic forces can effectively induce immune activation of macrophages. Mechanistic studies revealed that micromagnetic forces induce increased lysosomal membrane permeability, prompting the specific recruitment of Galectin-9 (Gal9) to the lysosomal membrane surface at damage sites. This, in turn, recruits and activates downstream key signaling pathways, including the AMPK metabolic sensing pathway and the NF-κB immune-inflammatory pathway, thereby driving a shift in macrophage phenotype from inhibitory to activating, enhancing their immune response capabilities. Figure 8 As shown. Simultaneously, metabolomics analysis revealed that macrophage glycolysis pathway-related metabolites were significantly upregulated after magnetic regulation, suggesting a significant reprogramming of their energy metabolism state, further supporting the shift in cellular functional activation state, such as... Figure 9 As shown. Based on this, periodic dynamic micromagnetic stimulation experiments verified that this regulatory strategy can achieve cyclical activation of the aforementioned biochemical signaling pathways, continuously maintaining the activated state of macrophages, revealing the platform's broad application potential in immune regulation and anti-tumor immunotherapy, such as... Figure 10 As shown.

[0122] Micromagnetic-induced immune activation: This application provides a method for regulating macrophage immune function based on a micromagnetic regulation strategy. First, a macrophage immune activation model is constructed. After co-incubating cells with MNMs, RMF is applied, and cytoskeleton markers are used to detect cell morphological changes to determine their activation state. Subsequently, flow cytometry is used to detect the expression levels of immune phenotype-related proteins, analyzing the effect of magnetic stimulation on macrophage phenotypic transformation.

[0123] To assess the repolarization effect of micromagnetic forces on M2 phenotype macrophages, macrophages were pretreated to induce an M2-like state before RMF stimulation was applied. RNA sequencing was then used to analyze changes in gene transcription levels under magnetic stimulation. Comparative analysis revealed an activation trend in multiple typical inflammation-related signaling pathways. To further validate the reliability of these gene expression changes, real-time quantitative PCR was performed on M1 and M2 phenotype-related genes in BMDMs and RAW 264.7 cells.

[0124] (2) Verification of dynamic micromagnetic force-induced activation of macrophage biochemical signal cycle

[0125] This application provides a method for macrophage functional remodeling by reversibly stimulating intracellular signaling pathways through lysosomal membrane mechanical stimulation. Co-immunoprecipitation experiments confirmed that magnetic stimulation enhanced the interaction between Gal9 and AMPK, while simultaneously detecting increased AMPK phosphorylation levels and enhanced expression of the M1-type marker protein iNOS. Further analysis of NF-κB pathway-related protein expression changes revealed that magnetic stimulation significantly increased the phosphorylation levels of IKKβ and p65 while inhibiting IκBα expression, suggesting that the NF-κB pathway is involved in this repolarization regulation process, and that this process is driven by AMPK activation. At the metabolic level, metabolomics analysis revealed a significant increase in macrophage glycolysis after micromagnetic stimulation, accompanied by a decrease in aerobic phosphorylation levels, indicating a clear trend of metabolic pathway reprogramming, further supporting the transition to the M1 phenotype.

[0126] Furthermore, to maintain the M1 phenotype of macrophages, a programmable dynamic RMF stimulation model was constructed. Lysosomal acidic fluorescent probes were used to detect changes in membrane stability. A decrease in fluorescence intensity was observed after a single RMF stimulation, which gradually recovered within 48 hours. This decrease was repetitive after subsequent RMF stimulation, indicating that lysosomal membrane damage is reversible. At the signaling pathway level, changes in the expression of AMPK and NF-κB-related phosphorylated proteins were detected, verifying that this strategy can achieve periodic activation of intracellular biochemical signals. Compared to single stimulation, repeated RMF treatment also induced the re-upregulation of key proteins such as AMPK and iNOS, sustained phosphorylation of IKKβ and p65, and a decrease in IκBα expression, further confirming that this regulatory system possesses programmable and repeatable immune activation capabilities.

[0127] Example 3: Dynamic Micromagnetic Cyclic Regulation of Immunobiochemical Signaling Mechanism and its Application in Activating Immune Function

[0128] To evaluate the potential of micromagnetic therapy strategies in in vivo tumor treatment, three mouse subcutaneous xenograft models were established, including Lewis lung adenocarcinoma (LLC), breast cancer (4T1), and melanoma (B16). MNMs were injected directly into the tumor site or via tail vein injection combined with local static magnetic field guidance to achieve their accumulation in the tumor region, followed by RMF stimulation, such as... Figure 11 As shown, a significant tumor-suppressing effect was observed under magnetic modulation conditions in different models. Flow cytometry analysis showed that magnetic modulation effectively enhanced the activation state of macrophages in tumor tissue, suggesting that MagLMP has the ability to regulate the tumor immune microenvironment. Further long-term survival tracking experiments were conducted in an LLC orthotopic lung cancer model. Some test animals showed prolonged survival after RMF treatment, with a few mice surviving for more than 300 days, demonstrating the potential of this strategy in the treatment of orthotopic lung cancer. Figure 12 As shown.

[0129] To verify the anti-tumor mechanism mediated by micromagnetic forces, a macrophage removal-replenishment model was constructed. After removing host macrophages via tail vein injection of liposomes, Gal9-knockout bone marrow-derived macrophages (Gal9-KO BMDMs) and Gal9-KO BMDMs transfected with AMPK-siRNA were locally injected back into the tumor region. The anti-tumor effect and signal transduction changes were observed after MagLMP regulation. The results showed that the anti-tumor effect was significantly weakened under Gal9 deficiency or AMPK signaling blockade, suggesting that the Gal9-AMPK-NF-κB signaling axis plays a crucial role in MagLMP-mediated immune activation and tumor therapy. Figure 13 As shown.

[0130] It should be noted that in this patent application, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one" does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this patent application, if it refers to performing an action according to an element, it means performing the action at least according to that element, including two cases: performing the action only according to that element, and performing the action according to that element and other elements. Expressions such as "multiple," "repeatedly," and "various" include two, two times, two kinds, and more than two, more than two times, and more than two kinds.

[0131] It should be understood that the numbering used in describing the steps of a method does not inherently limit the order of these steps. For example, a step with a higher number does not necessarily have to be executed after a step with a lower number; it can be executed first and then second, or even in parallel, as long as this execution order is reasonable to those skilled in the art. Similarly, multiple steps with consecutively numbered sequences (e.g., step a, step b, step c, etc.) do not restrict other steps from being executed between them; for example, there can be other steps between step a and step b.

[0132] All documents mentioned in this application are considered to be incorporated in their entirety into the disclosure of this application so that they can serve as a basis for modifications if necessary. Furthermore, it should be understood that after reading the foregoing disclosure of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope of protection claimed in this application.

Claims

1. A method for cyclically activating immune function, characterized in that, Includes the following steps: (a) Delivering a composition containing magnetically responsive nanomotors into the organelles of immune cells; (b) A programmable rotating magnetic field with preset frequency and magnetic field strength parameters is applied to the immune cells to drive the magnetic response nanomotor to rotate and generate local shear force, thereby inducing a reversible change in the permeability of the organelle membrane; (c) Activate immune-related signaling pathways to induce the immune cells to change from an inhibitory phenotype to an active phenotype; (d) Repeat steps (b)-(c) periodically to achieve cyclical activation of the immune-related signaling pathways and maintain the activated phenotype of the immune cells.

2. The method as described in claim 1, characterized in that, The frequency of the programmable rotating magnetic field is 0.2Hz-5Hz; preferably, the frequency of the programmable rotating magnetic field is 0.8Hz-2Hz.

3. The method as described in claim 1, characterized in that, The magnetic field strength of the programmable rotating magnetic field is 10mT-800mT; preferably, the magnetic field strength of the programmable rotating magnetic field is 20mT-500mT; more preferably, the magnetic field strength of the programmable rotating magnetic field is 260mT.

4. The method as described in claim 1, characterized in that, The local shear force is 0.4 Pa to 3 Pa; preferably, the local shear force generated in step (b) is 0.8 Pa to 2 Pa.

5. The method as described in claim 1, characterized in that, The activation of immune-related signaling pathways includes: Prior to performing step (a), the immune cells are given additional cytokines to polarize them to the M2 type. In step (c), the membrane damage sites of the organelles specifically recruit lectin-like proteins to the membrane surface of the organelles; It recruits and activates downstream metabolic sensing and immune inflammatory pathways.

6. The method as described in claim 1, characterized in that, The periodic repetition step (b) includes using a pulsed rotating magnetic field, with each magnetic field action lasting 10 to 30 minutes and an interval of 30 to 1440 minutes.

7. The method as described in claim 1, characterized in that, The immune cells include one or more of macrophages, dendritic cells, T cells, or natural killer cells; preferably, the immune cells are macrophages.

8. The method as described in claim 1, characterized in that, The organelles mentioned are lysosomes.

9. A regulatory platform for cyclically activating immune function, characterized in that, The platform includes: Magnetic-responsive components, including a magnetic-responsive nanomotor composition capable of being phagocytosed by immune cells and localized within organelles; A magnetic field-driven component generates a rotating magnetic field at a preset frequency and magnetic field strength parameters to drive the magnetically responsive nanomotor to rotate within the organelle and generate localized shear force; and The control component, electrically connected to the magnetic field driving component, is configured to periodically start and stop the rotating magnetic field according to a preset pulse program, so as to cyclically activate the immune-related signaling pathways of the immune cells and cause the immune cells to change from an inhibitory phenotype to an activated phenotype. The control platform is configured to implement the method as described in any one of claims 1-8.

10. The control platform as described in claim 9, characterized in that, The magnetic field driving component includes: A rotating rotor that carries a permanent magnet; An electric motor used to drive the rotating rotor to generate a rotating magnetic field.

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