A magnetic heat immune closed loop system for in vivo tumor microenvironment and control method

Abstract

The application discloses a kind of for in-vivo tumor microenvironment magnetic heat immune closed loop system and control method, belong to biomedical engineering and nanotechnology cross field.For the existing magnetic heat therapy technology cannot carry out real-time self-adaptive regulation according to the dynamic change of GAL-1, IFN-gamma, IL-4 and Th1 / Th2 ratio in tumor microenvironment, easy to cause the problem that immune marker deviates from window, a kind of magnetic heat immune closed loop regulation system is proposed.Firstly, baseline value is obtained and target concentration window is set;Control alternating magnetic field to activate vortex magnetic iron oxide nanoparticles to generate heat and active oxygen;Real-time acquisition of immune marker concentration fed back by implantable electrochemical sensor, dynamically adjust field strength and / or frequency;With single effective activation window, maximum single time length, temperature as stop criterion;After stop, standby and pulse reset.Real-time self-adaptive regulation of in-vivo immune state is realized, immune activation deficiency or immune marker deviation from window is avoided, and normal tissue thermal damage is prevented by multi-level thermal safety constraint.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of biomedical engineering and nanotechnology, and specifically relates to a magnetothermal immune closed-loop system and control method for in vivo tumor microenvironment. Background Technology

[0002] In recent years, magnetothermal techniques based on vortex magnetic iron oxide nanoparticles (FVIO) have been shown to downregulate the expression of various immunosuppressive molecules by inducing the accumulation of intracellular reactive oxygen species (ROS). However, existing magnetothermal control technologies employ an open-loop control mode, executing the entire process after pre-setting fixed magnetic field parameters. This approach cannot adjust in real time according to the dynamic changes of galectin-1 (GAL-1) and the Th1 / Th2 ratio of helper T cells (Th) in the in vivo tumor microenvironment (TME), which can easily lead to insufficient or excessive immune activation.

[0003] Existing technologies primarily utilize the magnetocaloric effect for in vitro cell treatment, treating isolated cell suspensions in a relatively closed and homogeneous environment. However, this approach cannot be applied to in vivo environments because the in vivo TME exhibits highly heterogeneous and dynamically evolving immune network characteristics. Feedback cannot characterize the actual changes in the immunosuppressive molecule GAL-1 and the effector cytokine interferon-gamma (IFN-γ). Furthermore, the delayed response characterization of immunosuppressive molecules and / or effector cytokines employed in in vitro treatment cannot address the nonlinear fluctuations and overactivation risks inherent in in vivo immune responses.

[0004] Therefore, there is a need to develop a closed-loop control method and system that can receive GAL-1 and Th1 / Th2 key cytokines in the TME in real time, dynamically regulate the alternating magnetic field generator and control unit, and dynamically adjust the magnetic field parameters. Summary of the Invention

[0005] This invention aims to address the technical problem of existing magnetothermal therapy technologies being unable to adaptively regulate immunosuppressive molecules and Th1 / Th2 cytokines in real time based on dynamic changes in the in vivo tumor microenvironment, thus avoiding deviations of immune markers from the target window caused by open-loop control. Furthermore, unlike ex vivo cell processing methods, this invention is specifically designed for in vivo applications, using immune marker concentrations as feedback signals and maintaining immune homeostasis and a single effective activation window as control targets. The single effective activation window refers to the dynamic state formed during a single magnetic field control process when the real-time received concentrations of GAL-1, IFN-γ, IL-4, and the Th1 / Th2 ratio are all simultaneously within the target concentration window.

[0006] A magnetothermal immune closed-loop system for use in the in vivo tumor microenvironment, comprising: A responsive nanothermal unit comprising vortex magnetic iron oxide nanoparticles, which can generate heat and induce an increase in intracellular reactive oxygen species levels under the action of an alternating magnetic field. An implantable electrochemical sensing unit is used to detect the concentrations of GAL-1, IFN-γ, and IL-4 in the tumor microenvironment in real time and output electrical signals. An alternating magnetic field generation and control unit is connected to the implanted electrochemical sensing unit, which can receive the electrical signal and dynamically adjust the field strength and / or frequency of the alternating magnetic field according to the deviation between the real-time detected concentration and the preset target concentration window. The preset target concentration window is as follows: the concentration of GAL-1 does not exceed 50% of the baseline level, the concentration of IFN-γ is 200%-400% of the baseline level, the concentration of IL-4 does not exceed 80% of the baseline level, and the Th1 / Th2 ratio is 1.5-3.0, where the Th1 / Th2 ratio is the ratio of the IFN-γ concentration to the IL-4 concentration.

[0007] Furthermore, the alternating magnetic field generating and control unit is configured to perform the following control steps: S1, Target setting: Obtain the initial concentration data of GAL-1, IFN-γ and IL-4 detected by the implanted electrochemical sensing unit, and set the target concentration window; S2, initial activation, controlling the alternating magnetic field generating unit to output initial alternating magnetic field parameters, so as to drive the responsive nanothermal generating unit to generate heat and induce an increase in intracellular reactive oxygen species levels; S3, Dynamic feedback maintenance, receiving GAL-1, IFN-γ and IL-4 concentration signals from the implanted electrochemical sensing unit in real time, and dynamically adjusting the field strength and / or frequency of the alternating magnetic field according to the deviation between the concentration and the target concentration window. S4, Single valid activation window judgment: When at least one of the following conditions is met, the output stop signal controls the alternating magnetic field generating unit to stop outputting: Condition A is that the concentration received in real time is within the target concentration window; Condition B is that the single continuous output time reaches the preset maximum single duration; Condition C is that the received temperature signal exceeds the preset safety threshold. S5, pulse reset, after outputting a stop signal, enters standby mode, waiting for an external trigger signal or timer signal to re-execute S2.

[0008] Furthermore, the initial alternating magnetic field parameters mentioned in S2 are: field strength 180-350 Oe, frequency 300-400 kHz.

[0009] Furthermore, the method for dynamically adjusting the field strength and / or frequency of the alternating magnetic field described in S3 is any one of the proportional-integral-derivative control algorithm, hysteresis comparison control algorithm, fuzzy logic control algorithm, or neural network control algorithm.

[0010] Furthermore, the proportional-integral-derivative control algorithm of S3 satisfies: (1) in, , , , The concentrations of GAL-1, IFN-γ, and IL-4 were respectively... The deviation between the ratio and the target value , , , For the concentrations of GAL-1, IFN-γ, and IL-4 and The weighting coefficient of the ratio, and These are the adjusted field strength and frequency, respectively. , , These are the field strength ratio, field strength integral, and field strength differential coefficients. , , These are the frequency proportional, frequency integral, and frequency differential coefficients. The initial field strength, This is the initial frequency.

[0011] Furthermore, the control unit in S3 outputs control signals according to the following control rules: S301, when the concentration of GAL-1 is greater than the upper limit of the target window, the concentration of IFN-γ is less than the lower limit of the target window, and the concentration of IL-4 is greater than the upper limit of the target window, increase the field strength or frequency of the alternating magnetic field. S302, when the GAL-1 concentration is not greater than the upper limit of the target window, the IFN-γ concentration is between the lower and upper limits of the target window, and the IL-4 concentration is not greater than the upper limit of the target window, maintain the field strength and frequency of the current alternating magnetic field; S303: When the concentration of GAL-1 is not greater than the upper limit of the target window, the concentration of IFN-γ is greater than the upper limit of the target window, and the concentration of IL-4 is less than the lower limit of the target window, reduce the field strength or frequency of the alternating magnetic field, or pause the output.

[0012] Furthermore, the dynamic feedback maintenance of S3 also includes at least one of the following safety protection mechanisms: upper and lower limits of magnetic field parameters, field strength 180-350 Oe, frequency 300-400 kHz; single adjustment amplitude not exceeding 20% ​​of the current value; automatic switching to a preset safe output mode when the sensing unit fails; automatic suspension of magnetic field output and issuance of a warning signal when the IFN-γ concentration exceeds the preset upper limit; limiting the single increase of field strength not to 5% of the current value when the local temperature of the tumor exceeds 43 ℃; and forcibly reducing the field strength to below 200 Oe or suspending the output when the temperature exceeds 45 ℃, until the temperature drops below 42 ℃.

[0013] Furthermore, the preset safety threshold in condition C includes: local tumor temperature ≥ 46 ℃, or a heating rate ≥ 2 ℃ / min within 5 consecutive seconds.

[0014] Furthermore, in the responsive nanothermal unit, the vortex magnetic iron oxide nanoparticles have a particle size of 50-70 nm, a hydrated particle size of 70-80 nm, a saturation magnetization of 58-72 emu / g, and a specific absorptivity of 2000-5000 W / gFe. The surface of the vortex magnetic iron oxide nanoparticles is modified with tumor-targeting molecules, which are selected from one or more of RGD peptides, folic acid, transferrin, antibodies, or their antigen-binding fragments.

[0015] Furthermore, the implantable electrochemical sensing unit includes: a needle probe body, a working electrode array, a reference electrode, a counter electrode, an electromagnetic shielding layer, and a miniature temperature sensor. The needle probe body comprises a tip and a rod; the working electrode array, disposed at the tip of the needle probe body, includes a first working electrode, a second working electrode, and a third working electrode; the miniature temperature sensor, disposed at the tip of the needle probe body, is adjacent to the working electrode array; the reference electrode is disposed on the needle probe body; the counter electrode is disposed on the needle probe body; and the electromagnetic shielding layer is disposed on the needle probe body. The first working electrode surface is provided with a GAL-1 specific nucleic acid aptamer modification layer, the second working electrode surface is provided with an IFN-γ specific nucleic acid aptamer modification layer, and the third working electrode surface is provided with an IL-4 specific nucleic acid aptamer modification layer.

[0016] Compared with the prior art, the present invention has the following beneficial technical effects: 1. Real-time adaptive regulation of in vivo immune status: By receiving the concentrations of GAL-1, IFN-γ, and IL-4 and the Th1 / Th2 ratio in real time through an implanted electrochemical sensor, and using immune markers as direct feedback signals, dynamic closed-loop regulation of magnetocaloric parameters in the in vivo tumor microenvironment is realized, solving the problem that open-loop control cannot adapt to dynamic changes in immune status.

[0017] 2. Precise control mechanism driven by a single effective activation window: This invention defines and utilizes a single effective activation window as the core criterion for stopping the output of the alternating magnetic field. Once the immune marker enters the target window, the current control is stopped, avoiding the possibility of the immune marker deviating from the window due to traditional fixed-duration magnetothermal methods.

[0018] 3. The pulse reset strategy achieves long-term immune homeostasis maintenance. Through a pulsed working mode that stops and then stands, waits for a trigger or timed signal to restart, the immune response can be repeatedly activated on a timescale of several days to several weeks to maintain the Th1 / Th2 balance and avoid immune exhaustion.

[0019] 4. Adaptive individualized tumor microenvironment: In vivo baseline acquisition allows the target window to vary from person to person, and the weight coefficients in the control algorithm can be dynamically adjusted according to different stages, achieving true individualized magnetothermal immune regulation of the tumor microenvironment.

[0020] 5. Precise control under thermal safety constraints: By introducing a temperature safety threshold as an independent stop criterion and incorporating a multi-level thermal protection mechanism, it effectively prevents thermal damage to normal tissues and non-specific inflammatory reactions, ensuring safety. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Flowchart of a control method for a magnetothermal immune closed-loop system for in vivo tumor microenvironment according to the present invention; Figure 2 a. Comparison of tumor-bearing mice using the magnetothermal immunoassay open-loop method (Example 1); Figure 2 b. Comparison of tumor-bearing mice using the magnetocaloric immunological closed-loop method in Example 1 of the present invention; Figure 3 a. Comparison of tumor-bearing mice without a single effective activation window in Example 2; Figure 3 b. Comparison of tumor-bearing mice with a single effective activation window in Embodiment 1 of the present invention; Figure 4 a. Comparative Example 3: Tumor-bearing mice without pulse reset.

[0023] Figure 4 b. Comparative image of pulse-reset tumor-bearing mice in Embodiment 1 of the present invention; Figure 5 a. Comparison of tumor-bearing mice without safe temperature constraints (Example 4); Figure 5 b. Comparison of tumor-bearing mice with safe temperature constraint in Embodiment 1 of the present invention; Figure 6a The curve of GAL-1 changing over time in Example 1; Figure 6b The curve of IFN-γ changing with time in Example 1; Figure 6c The curve of IL-4 changing over time in Example 1; Figure 6d The curves showing the change of Th1 / Th2 over time in Example 1. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1 according to Figure 1 , Figure 2 b、 Figure 3 b、 Figure 4 b、 Figure 5 b、 Figure 6a , Figure 6b , Figure 6c , Figure 6d In the responsive nanothermal unit, the vortex magnetic iron oxide nanoparticles FVIO have an average particle size of 60 nm, a hydrated particle size of 75 nm, a saturation magnetization of 65 emu / g, a specific absorption rate (SAR) of 3500 W / g Fe, and are surface-modified with RGD peptides.

[0026] The implantable electrochemical sensor uses a needle-shaped probe body made of biocompatible polyetheretherketone (PEEK), with a diameter of 0.8 mm and a length of 8 mm. It includes a tip and a stem; the tip is the sensing area, and the stem is covered with an insulating layer. An array of working electrodes is disposed at the tip of the needle-shaped probe body, including a first working electrode, a second working electrode, and a third working electrode, each with an area of ​​0.03 mm². 2 The probe is made of gold. The first working electrode has a GAL-1 specific nucleic acid aptamer modification layer, the second working electrode has an IFN-γ specific nucleic acid aptamer modification layer, and the third working electrode has an IL-4 specific nucleic acid aptamer modification layer. The nucleic acid aptamers are fixed to the gold electrode surface via thiol self-assembly. A miniature temperature sensor, a platinum resistance temperature sensor with an accuracy of ±0.1℃, is located at the tip of the needle probe body adjacent to the working electrode array. It is used to receive the ambient temperature of the electrodes in real time and to perform temperature compensation for the electrochemical signal. A reference electrode, an Ag / AgCl electrode, is located on the rod of the needle probe body, providing a stable potential reference. A counter electrode, a platinum wire electrode, is located on the rod of the needle probe body and is adjacent to the reference electrode. An electromagnetic shielding layer, a permalloy layer with a thickness of 50 μm, is located on the outer surface of the rod of the needle probe body and is used to attenuate the induced electromotive force generated by the alternating magnetic field in the electrode circuit.

[0027] Animal model: Female C57BL / 6 mice, weighing 20-22 g, subcutaneously injected with 2×10⁶ Hepa1-6 mouse liver cancer cells in the right axilla. 6 One, when the tumor volume reaches 100-150 mm. 3 The experiment will begin at that time.

[0028] The alternating magnetic field generator and control unit employs a water-cooled helical coil magnetic field generator with an inner diameter of 40 mm, a length of 60 mm, an adjustable frequency range of 300-400 kHz, and an adjustable field strength range of 180-350 Oe. The control unit is a real-time controller based on a field-programmable gate array (FPGA), with a sampling period of 1 second. It communicates wirelessly with the implanted electrochemical sensing unit and executes a proportional-integral-derivative (PID) control algorithm.

[0029] Transmission module: Electrically connected to the working electrode, reference electrode, counter electrode and temperature sensor, wirelessly transmits electrochemical detection signals and temperature data to the alternating magnetic field generator and control unit.

[0030] Animal model: Female C57BL / 6 mice, weighing 20-22 g, subcutaneously injected with 2×10⁶ Hepa1-6 mouse liver cancer cells in the right axilla. 6 One. When the tumor volume reaches 100-150 mm. 3 The experiment will begin at that time.

[0031] S1, Target setting: Obtain initial concentration data detected by the implanted electrochemical sensing unit. Measurement results: GAL- 18.1 ng / mL, IFN-γ 23 pg / mL, IL-4 37 pg / mL. Calculate the Th1 / Th2 ratio using the following formula. The target concentration windows are set as follows: GAL-1 ≤ 4.05 ng / mL, 50% of the baseline value; IFN-γ 46-92 pg / mL, 200%-400% of the baseline value; IL-4 ≤ 29.6 pg / mL, 80% of the baseline value; Th1 / Th2 ratio 1.5-3.0. S2, initial activation, controls the alternating magnetic field generating unit to output initial alternating magnetic field parameters, field strength 300 Oe, frequency 365 KHz; S3, Dynamic Feedback Maintenance: During a single alternating magnetic field control process, the control unit receives real-time concentration signals of GAL-1, IFN-γ, and IL-4 from the implanted electrochemical sensor. Based on the deviation between these concentrations and the target concentration window, the control unit dynamically adjusts the field strength of the alternating magnetic field. The control unit employs a proportional-integral-derivative control algorithm to satisfy: (2) in, , , , The concentrations of GAL-1, IFN-γ, and IL-4 were respectively... The deviation between the ratio and the target value , , , For the concentrations of GAL-1, IFN-γ, and IL-4 and The weighting coefficient of the ratio, The adjusted field strength and frequency, field strength scaling factor Field strength integral coefficient Differential coefficients of electric field strength , The initial field strength and the real-time received data are shown in Table 1 below: Table 1

[0032] S4, Single Effective Activation Window Judgment: During a single continuous alternating magnetic field control process, the control unit stops the alternating magnetic field output when at least one of the following conditions is met: Condition A is that the single effective activation window has been established; Condition B is that the single continuous alternating magnetic field control time reaches the preset maximum single duration of 30 minutes; Condition C is that the real-time received tumor microenvironment temperature exceeds the preset safety threshold, i.e., the local tumor temperature ≥ 46 ℃. Specifically, at the 15th minute, GAL-1, IFN-γ, IL-4, and the Th1 / Th2 ratio are all within the target concentration window, the single effective activation window is established, and the control unit immediately stops the alternating magnetic field output. The duration of this single continuous control is 15 minutes, which does not reach the preset maximum single duration, and the tumor microenvironment temperature does not exceed the preset safety threshold. S5, pulse reset. After stopping the alternating magnetic field output, the control unit enters standby mode, waiting for an external trigger signal or timer signal to start the next alternating magnetic field control. Specifically, a timer is set to automatically trigger the next alternating magnetic field control every 48 hours. From day 2 to day 7, a timer triggers once every other day. Before each trigger, the implanted electrochemical sensor automatically detects the concentration of immunomarkers. If GAL-1 > 4.05 ng / mL, or Th1 / Th2 < 1.5, magnetic field control is automatically started; otherwise, the control for that day is skipped. The duration and reason for stopping each control are recorded as shown in Table 2. Table 2

[0033] After the window for dynamic changes in immune markers was established on day 1, GAL-1 remained below 4.0 ng / mL, IFN-γ fluctuated between 45-85 pg / mL, IL-4 was between 20-30 pg / mL, and the Th1 / Th2 ratio was between 1.6-3.0, maintaining good immune homeostasis for 7 consecutive days. On day 5, due to a drop in the Th1 / Th2 ratio to 1.4, an automatic pulse control was triggered, and the window was re-established 16 minutes later.

[0034] Tumor suppression effect: Mice were sacrificed on day 7, and tumors were removed and weighed. The average tumor volume in Example 1 was 45 ± 12 mm. 3The inhibition rate reached 82.5% compared with the control group without magnetothermal immunotherapy.

[0035] Immune cell infiltration analysis: Tumor tissue was collected on day 7 to prepare a single-cell suspension, and flow cytometry was used to detect: CD8+ in the immune activation group. + T cells account for CD3 + The proportion of T cells was 18.7% ± 2.1%, significantly higher than that of the control group (5.2% ± 0.8%); Treg (CD4+) + FoxP3 + The proportion was 2.1% ± 0.3%, significantly lower than the control group's 9.8% ± 1.2%, CD8 + The / Treg ratio increased from 0.53 before immune activation to 8.9.

[0036] A closed-loop control method, employing an implanted electrochemical sensor to receive real-time concentrations of GAL-1, IFN-γ, and IL-4, as well as the Th1 / Th2 ratio, and using a single effective activation window as the stopping criterion, can precisely establish an immune activation window within a single control cycle. A pulse reset strategy maintains long-term immune homeostasis, significantly inhibiting tumor growth while preventing immune markers from deviating from the window. In this embodiment, the preset maximum single-cycle duration described in condition B is set to 30 minutes. This setting is based on the temperature rise characteristics of FVIO at a minimum safe operating field strength of 180 Oe and a frequency of 300 kHz. The time required to rise from 36.5°C to 46°C in vitro is approximately 30.9 minutes. Considering in vivo heat dissipation and safety margin, the maximum single-cycle duration is set to 30 minutes to ensure protection is still provided even if the temperature safety threshold fails. This duration can be adjusted according to actual applications and does not constitute a limitation of the invention.

[0037] Example 2 The only difference between this embodiment and Embodiment 1 is that the method for dynamically adjusting the alternating magnetic field strength and / or frequency in S3 uses a hysteresis comparison control algorithm instead of a PID control algorithm. All other experimental materials, including the properties of vortex magnetic iron oxide nanoparticles (FVIO), the implantable electrochemical sensor, the animal model, the alternating magnetic field generating unit, and steps S1, S2, S4, and S5, are exactly the same as in Embodiment 1.

[0038] The hysteresis comparison control algorithm outputs a switch-type control signal based on the comparison result of the weighted deviation e calculated in real time and the preset threshold, which increases the magnetic field, decreases the magnetic field, or keeps it unchanged.

[0039] (3) in, , , , The concentrations of GAL-1, IFN-γ, and IL-4 were respectively... The deviation between the ratio and the target value , , , For the concentrations of GAL-1, IFN-γ, and IL-4 and The weighting coefficient for the ratio is set, and a hysteresis interval is defined. The upper threshold of the hysteresis interval is +0.15, and the lower threshold is -0.10. Keeping the current magnetic field parameters unchanged, If the positive deviation is too large, the immune activation is insufficient, and a control signal to increase the magnetic field is output. If the negative deviation is too large, the immune system will be over-activated, and a control signal to reduce the magnetic field will be output. The magnetic field is increased by stepping +10 Oe. If the field strength is close to the upper limit of 350 Oe, the frequency step is changed to +10 kHz. The magnetic field is decreased by stepping -15 Oe. If the field strength is close to the lower limit of 180 Oe, the frequency step is changed to -10 kHz. The initial magnetic field parameters are 300 Oe and 365 kHz, which are the same as in Example 1.

[0040] After placing the mouse at the center of the alternating magnetic field coil, magnetic field control was activated, and the concentrations of GAL-1, IFN-γ, and IL-4 were acquired in real time with a sampling period of 1 second. The weighted deviation e was calculated, and the magnetic field was adjusted according to the hysteresis comparison rule described above. Data at key time points are shown in Table 3. Table 3

[0041] S4, Single-use effective activation window assessment: At the 12-minute mark, GAL-1 = 3.9 ng / mL, IFN-γ = 74 pg / mL, IL-4 = 27 pg / mL, Th1 / Th2 = 2.74. All four indicators have entered the target window, and the single-use effective activation window has been established. The control unit immediately stops the alternating magnetic field output. The duration of this single continuous application was 12 minutes, which did not reach the preset maximum single-use duration.

[0042] S5, pulse reset, is exactly the same as in Example 1. After stopping, the system enters standby mode, and the timer automatically triggers the next control every 48 hours.

[0043] After the 12-minute window was established, GAL-1 remained below 4.0 ng / mL, IFN-γ fluctuated between 45-85 pg / mL, IL-4 was between 20-30 pg / mL, and the Th1 / Th2 ratio was between 1.6-3.0. Immunological homeostasis was maintained well for 7 consecutive days. The magnetic hysteresis comparison was controlled by reducing the field strength in advance when the deviation was close to the lower limit of the threshold, which effectively avoided overshoot.

[0044] Example 3 The difference between this embodiment and Embodiment 1 is that condition 3 is introduced in S4, and its protective effect is verified. Experimental setup: The same animal model, FVIO, and implantable sensor as in Embodiment 1 are used. An initial magnetic field strength of 350 Oe and a frequency of 400 kHz are applied, with both the field strength and frequency at the upper limit of parameter S2. During magnetic field control, the local tumor temperature is received in real time. When the temperature rises to 45.8℃ and the heating rate reaches 2.1℃ / min, the control unit determines that condition C is met. The threshold of temperature ≥46℃ has not yet been reached, but the heating rate has exceeded 2℃ / min, and the alternating magnetic field output is immediately stopped. At this time, the immune markers have not yet fully entered the target window: GAL-1 = 4.5 ng / mL, IFN-γ = 68 pg / mL, IL-4 = 29 pg / mL, Th1 / Th2 = 2.34. However, to avoid thermal damage, the system prioritizes a safe stop. After stopping, the temperature quickly drops back to 39℃. Subsequent pulse resets are performed as planned. On day 7, the tumor inhibition rate was 76.8%, slightly lower than in Example 1. However, tumor tissue sections showed no areas of thermal coagulation necrosis, the surrounding normal tissue was intact, and the mice exhibited no heat stress behavior. This example demonstrates that condition 3, as an independent termination criterion, can promptly terminate the magnetic field when the immune window has not been reached but thermal safety is threatened.

[0045] Comparative Example 1 according to Figure 2 a) Open-loop fixed-parameter magnetocaloric immunoassay: Except for not using closed-loop feedback, all other conditions were the same as in Example 1. The fixed magnetic field was controlled at 300 Oe and 365 kHz for 30 minutes continuously. Results: At the 20th minute, IFN-γ rose to 186 pg / mL, exceeding the upper limit of the target window concentration by 4.2 times. Mice showed adverse reactions such as piloerection, curling up, and decreased appetite. On day 7, the tumor inhibition rate was only 41.3%, significantly lower than in Example 1. Flow cytometry detection: CD8 + The T-cell infiltration rate was 11.2%, and the Treg rate was 5.8%, indicating poor immune activation and excessive inflammation.

[0046] Comparative Example 2 according to Figure 3 a) Magnetothermic immunoassay without a single effective activation window determination: Except for S4, which only uses condition B with a maximum single duration of 30 minutes, the rest is the same as in Example 1. That is, even if the immune marker has entered the target window, the magnetic field is still applied for 30 minutes. The window is established at 12 minutes, but it is controlled for 30 minutes, which causes IFN-γ to eventually rise to 1.6 times the upper limit of the target window concentration of 142 pg / mL. Mice showed a mild stress response, and the tumor inhibition rate was 69.5% on day 7. Although it was better than the open-ring comparative example 1, it was inferior to Example 1 and there was a risk of overactivation.

[0047] Comparative Example 3 according to Figure 4 In the control group (a), without pulse reset of S5, and without further control after a single pulse, immune markers entered the window on day 1, and the tumor shrank. By day 3, GAL-1 rose to 5.6 ng / mL, IFN-γ decreased to 35 pg / mL, and the Th1 / Th2 ratio dropped to 0.9, indicating tumor recurrence. By day 7, the tumor volume was not significantly different from the control group. This demonstrates that a single activation cannot maintain immune homeostasis long-term, and the pulse reset strategy is essential for sustained tumor suppression.

[0048] Comparative Example 4 according to Figure 5 a. The difference between this comparative example and Example 1 is that condition 3 is not included in S4, and no temperature limit is set in the safety protection mechanism. All other conditions are the same as in Example 1. During the magnetic field control process, due to individual differences, the tumor had poor blood supply and weak heat dissipation capacity. The local temperature had risen to 47.2°C by the 11th minute of magnetic field control, but the immune markers had not yet fully entered the window. GAL-1 = 4.2 ng / mL, slightly higher than 4.05, and the control unit continued to output the magnetic field. At the 13th minute, the temperature rose to 48.5°C, and the mice showed agitation and local skin reddening. After stopping, tissue sections showed small-scale thermal coagulation necrosis of normal muscle tissue at the tumor edge. On day 7, the tumor inhibition rate was 73.1%, but with mild thermal damage. This comparative example shows that the lack of thermal safety constraints may lead to thermal damage to normal tissue.

[0049] Those skilled in the art can select appropriate PID, hysteresis comparison, fuzzy logic, or neural network control algorithms based on actual application scenarios, all of which fall within the protection scope of this invention.

Claims

1. A magnetothermal immunological closed-loop system for use in the in vivo tumor microenvironment, characterized in that, include: A responsive nanothermal unit comprising vortex magnetic iron oxide nanoparticles, which can generate heat and induce an increase in intracellular reactive oxygen species levels under the action of an alternating magnetic field. An implantable electrochemical sensing unit is used to detect the concentrations of GAL-1, IFN-γ, and IL-4 in the tumor microenvironment in real time and output electrical signals. An alternating magnetic field generation and control unit is connected to the implanted electrochemical sensing unit, which can receive the electrical signal and dynamically adjust the field strength and / or frequency of the alternating magnetic field according to the deviation between the real-time detected concentration and the preset target concentration window. The preset target concentration window is as follows: the concentration of GAL-1 does not exceed 50% of the baseline level, the concentration of IFN-γ is 200%-400% of the baseline level, the concentration of IL-4 does not exceed 80% of the baseline level, and the Th1 / Th2 ratio is 1.5-3.0, where the Th1 / Th2 ratio is the ratio of the IFN-γ concentration to the IL-4 concentration.

2. The magnetothermal immunological closed-loop system for in vivo tumor microenvironment according to claim 1, characterized in that, The vortex magnetic iron oxide nanoparticles have a particle size of 50-70 nm, a hydrated particle size of 70-80 nm, a saturation magnetization of 58-72 emu / g, and a specific absorption rate of 2000-5000 W / g Fe. The surface of the vortex magnetic iron oxide nanoparticles is modified with tumor-targeting molecules, which are selected from one or more of RGD peptides, folic acid, transferrin, antibodies, or their antigen-binding fragments.

3. The magnetothermal immunological closed-loop system for in vivo tumor microenvironment according to claim 1, characterized in that, The alternating magnetic field generator and control unit uses any one of the following algorithms to achieve the dynamic adjustment: proportional-integral-derivative control algorithm, hysteresis comparison control algorithm, fuzzy logic control algorithm, or neural network control algorithm.

4. A magnetothermal immunological closed-loop system for in vivo tumor microenvironment according to claim 1, characterized in that, The alternating magnetic field generator and control unit outputs control signals according to the following control rules: When the received GAL-1 concentration is greater than the upper limit of the target window, the IFN-γ concentration is less than the lower limit of the target window, and the IL-4 concentration is greater than the upper limit of the target window, increase the field strength or frequency of the alternating magnetic field. When the received GAL-1 concentration is not greater than the upper limit of the target window, the IFN-γ concentration is between the lower and upper limits of the target window, and the IL-4 concentration is not greater than the upper limit of the target window, the field strength and frequency of the current alternating magnetic field are maintained. When the received GAL-1 concentration is not greater than the upper limit of the target window, the IFN-γ concentration is greater than the upper limit of the target window, and the IL-4 concentration is less than the lower limit of the target window, reduce the field strength or frequency of the alternating magnetic field, or pause the output.

5. A magnetothermal immunological closed-loop system for in vivo tumor microenvironment according to claim 1, characterized in that, The alternating magnetic field generator and control unit also has a built-in safety protection mechanism, which includes at least one of the following: upper and lower limit constraints on magnetic field parameters, with a field strength of 180-350 Oe and a frequency of 300-400 kHz; when the local temperature of the tumor exceeds 43°C, limiting the single increase in field strength to no more than 5% of the current value; when the temperature exceeds 45°C, forcibly reducing the field strength to below 200 Oe or pausing the output until the temperature drops back to below 42°C.

6. A magnetothermal immunological closed-loop system for in vivo tumor microenvironment according to claim 1, characterized in that, The alternating magnetic field generator and control unit is further configured to stop outputting the alternating magnetic field when at least one of the following conditions is met during a single continuous alternating magnetic field application: Condition A is that the concentrations of GAL-1, IFN-γ, and IL-4 and the Th1 / Th2 ratio detected in real time are all within the preset target concentration window; Condition B is that the application time of the single continuous alternating magnetic field reaches the preset maximum single duration. Condition C is when the real-time monitored tumor microenvironment temperature exceeds a preset safety threshold.

7. A magnetothermal immunological closed-loop system for in vivo tumor microenvironment according to claim 1, characterized in that, After the alternating magnetic field generator and control unit stops outputting, it enters standby mode and waits for an external trigger signal or timing signal to start the next application of the alternating magnetic field.

8. A magnetothermal immunological closed-loop system for in vivo tumor microenvironment according to claim 6, characterized in that, The preset safety thresholds in condition C include: local tumor temperature ≥ 46 ℃, or a heating rate ≥ 2 ℃ / min within 5 consecutive seconds.

9. A control method for a magnetothermal immunological closed-loop system for an in vivo tumor microenvironment according to any one of claims 1 to 8, characterized in that, Includes the following steps: S1, Target setting: Obtain the initial concentration data of GAL-1, IFN-γ and IL-4 detected by the implanted electrochemical sensing unit, and set the preset target concentration window; S2, Initial activation, controlling the alternating magnetic field generating unit to output initial alternating magnetic field parameters to drive the responsive nanothermal generating unit to generate heat; S3, Dynamic feedback maintenance, receiving GAL-1, IFN-γ and IL-4 concentration signals from the implanted electrochemical sensing unit in real time, calculating the deviation from the preset target concentration window, and dynamically adjusting the field strength and / or frequency of the alternating magnetic field according to the deviation. S4, Single valid activation window judgment: When at least one of the following conditions is met, the output stop signal controls the alternating magnetic field generating unit to stop outputting: Condition A is that the concentration received in real time is all within the preset target concentration window; Condition B is that the single continuous output time reaches the preset maximum single duration. Condition C is that the received temperature signal exceeds a preset safety threshold; S5, pulse reset, after outputting a stop signal, enters standby mode, waiting for an external trigger signal or timer signal to re-execute S2.

10. The control method according to claim 9, characterized in that: The dynamic adjustment described in S3 employs a proportional-integral-derivative control algorithm, satisfying the following: （1） in, , , , The concentrations of GAL-1, IFN-γ, and IL-4 were respectively... The deviation between the ratio and the target value , , , For the concentrations of GAL-1, IFN-γ, and IL-4 and The weighting coefficient of the ratio, and These are the adjusted field strength and frequency, respectively. , , These are the field strength ratio, field strength integral, and field strength differential coefficients. , , These are the frequency proportional, frequency integral, and frequency differential coefficients. The initial field strength, This is the initial frequency.

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