Magnetic field activated genetic memory bio-microrobot system, preparation method and application

Abstract

The application relates to the technical field of biomedical engineering, and discloses a magnetic field activated genetic memory biological micro-robot system, a preparation method and application. The system comprises a microorganism carrier and a magnetic heat transduction material associated with the microorganism carrier. The magnetic heat transduction material is configured to generate heat under the action of an external magnetic field, so that the microorganism carrier is in a heating state. The microorganism carrier is an engineered microorganism, and contains a genetic circuit. The genetic circuit at least comprises a temperature response regulation unit, a genetic memory unit and a functional output unit. The genetic memory unit is configured to switch the genetic circuit from a first expression state to a second expression state under the triggering of the temperature response regulation unit, and maintain the second expression state after the heating state is removed, so that the functional output unit continuously expresses a functional molecule.

Description

Technical Field

[0001] This application relates to the field of biomedical engineering technology, specifically to a magnetic field activated genetic memory bio-microrobot system, its preparation method, and its application. Background Technology

[0002] Medical microrobots can perform targeted delivery, local treatment, and minimally invasive procedures in complex, narrow, and hard-to-reach areas within the human body, showing promising applications in precision medicine. Existing medical microrobots typically achieve wireless control through methods such as magnetic fields, light, ultrasound, chemical gradients, or bioenergy. Among these, magnetic fields have advantages such as strong penetration, non-contact operation, and ease of remote control, thus attracting widespread attention in the field of medical microrobots.

[0003] However, existing medical microrobots generally rely on the continuous presence of external stimuli to maintain their working state and functional output in practical applications. In other words, external stimuli only serve as a momentary trigger, and the system itself usually lacks the ability to record the stimulus and maintain it as a stable working state for the future. Once the external stimulus is removed, the microrobot often struggles to maintain its intended function, resulting in unsustainable therapeutic effects, poor system autonomy, and thus limiting its application in complex internal environments.

[0004] To endow microsystems with a certain degree of state retention capability, existing technologies have also introduced memory-related components to achieve state continuation after stimulation. However, such solutions typically suffer from the following problems: first, the related memory components are relatively large, which is not conducive to system miniaturization; second, the integration method is relatively complex, increasing the difficulty of system construction and manufacturing; third, biocompatibility is insufficient, making it difficult to meet the needs of in vivo applications; and fourth, it is difficult to effectively couple with micron-scale medical microrobots, limiting their practicality in biomedical scenarios.

[0005] On the other hand, in tumors, fibrosis, pathological scars, and other tissue sclerosis-related diseases, the lesion area is often accompanied by abnormal extracellular matrix deposition, fibrin enrichment, and increased tissue stiffness, thus forming an abnormal mechanical microenvironment. This abnormal mechanical microenvironment not only hinders the deep penetration of therapeutic molecules but also weakens the local therapeutic effect. Existing interventions targeting lesion softening or microenvironment improvement often suffer from problems such as short duration of action, the need for repeated treatments, and insufficient persistence, making it difficult to achieve long-term, stable regulation of the lesion microenvironment.

[0006] Therefore, existing technologies have at least the following drawbacks: microrobots are highly dependent on external stimuli and have difficulty maintaining their intended functional state after the stimuli are removed; existing memory construction methods are not conducive to achieving efficient integration in micrometer-scale systems; existing solutions cannot simultaneously achieve miniaturization, biocompatibility, memory retention, and continuous functional output; and the regulation of abnormal mechanical microenvironment of lesions lacks sustainability and is difficult to meet long-term treatment needs.

[0007] Therefore, there is an urgent need to provide a biological microrobot system that can achieve state memory after being triggered by an external magnetic field and can continue to output functional molecules after the stimulus is removed, so as to improve the system's autonomy and meet the long-term regulatory needs of diseases related to abnormal microenvironments of mechanics. Summary of the Invention

[0008] The purpose of this application is to provide a magnetic field activated genetic memory bio-microrobot system, its preparation method and application. By triggering the writing of genetic memory into engineered microorganisms through magnetothermal transduction, the bio-microrobot can continue to output functional molecules even after the external magnetic field is removed, thereby realizing the transition from a transient external stimulus to a long-term autonomous functional state.

[0009] This application discloses a magnetic field activated genetic memory biological microrobot system, which includes at least a microbial carrier and a magnetothermal transconducting material associated with the microbial carrier; The magnetocaloric transducer is configured to generate heat under the action of an external magnetic field, so that the microbial carrier is in a heated state. The microbial carrier is an engineered microorganism containing a genetic circuit, which includes at least a temperature response regulation unit, a genetic memory unit, and a functional output unit. The genetic memory unit is configured to switch the genetic circuit from a first expression state to a second expression state when triggered by the temperature response regulation unit, and to maintain the second expression state after the heating state is lifted, so that the functional output unit continuously expresses functional molecules.

[0010] In a preferred embodiment, the genetic memory unit includes a site-specific recombinase and its corresponding first recombination recognition sequence and second recombination recognition sequence, wherein the first recombination recognition sequence and the second recombination recognition sequence are positioned on both sides of the transcriptional regulatory sequence; The temperature response regulation unit triggers the expression or activation of the site-specific recombinase under the heating state, causing the site-specific recombinase to mediate a conformational change in the transcriptional regulatory sequence, thereby switching the genetic circuit from a first expression state to a second expression state, and maintaining the second expression state after the heating state is lifted, so that the functional output unit continuously expresses functional molecules.

[0011] In a preferred embodiment, the magnetocaloric transducer material includes magnetically responsive nanomaterials, magnetic composite materials, coated magnetic particles, or combinations thereof, preferably ferrite materials, metallic magnetic materials, doped magnetic materials, or core-shell magnetic materials.

[0012] In a preferred embodiment, the magnetocaloric transducer material comprises one or more of Fe3O4, γ-Fe2O3, MnFe2O4, CoFe2O4, ZnFe2O4, FeCo, or composites thereof.

[0013] In a preferred embodiment, the magnetocaloric transducer material is spindle-shaped, rod-shaped, ellipsoidal, cubic, sheet-shaped, nanoflower-shaped, or core-shell-shaped.

[0014] In a preferred embodiment, the aspect ratio of the magnetic nanoparticles is approximately 3.15 ± 0.18.

[0015] In a preferred embodiment, the magnetocaloric transconducting material is associated with the microbial carrier through surface coupling, electrostatic adsorption, bioaffinity binding, self-assembly, encapsulation, or a combination thereof.

[0016] In a preferred embodiment, the magnetocaloric transducer is surface-functionalized and then linked to the microbial carrier. The surface functionalization includes amination, carboxylation, silanization, dopamine modification, polysaccharide coating, or a combination thereof. Preferably, the association is achieved through amide condensation, biotin-avidin binding, or click chemistry.

[0017] In a preferred embodiment, the magnetocaloric transducer is an amino-functionalized magnetic nanoparticle, preferably an amino-functionalized Fe3O4 magnetic nanoparticle.

[0018] In a preferred embodiment, the concentration of the engineered microbial suspension is 10. 8 CFU / mL.

[0019] In a preferred embodiment, every 10 8 Surface coupling was achieved by adding 500 μg magnetic nanoparticles, 0.55 mg EDC, and 0.65 mg NHS to an engineered microbial suspension with a concentration of CFU / mL.

[0020] In a preferred embodiment, the coupling process is carried out at room temperature with stirring at 100 rpm for 3 hours.

[0021] In a preferred embodiment, the microbial carrier is a probiotic, a non-pathogenic bacterium, a virus-attenuated bacterium, yeast, engineered algae, or other engineerable live microorganisms.

[0022] In a preferred embodiment, the microbial carrier comprises Escherichia coli Nissle 1917, lactic acid bacteria, Bifidobacterium, Bacillus subtilis, attenuated Salmonella, or a combination thereof.

[0023] In a preferred embodiment, the microbial vector is Escherichia coli Nissle 1917.

[0024] In a preferred embodiment, the genetic circuit further includes a feedback unit for outputting a detectable signal in the first expression state and / or the second expression state to characterize the state of the genetic circuit.

[0025] In a preferred embodiment, the feedback unit includes a fluorescent protein expression module, a luminescent protein expression module, a near-infrared imaging biomarker expression module, or a combination thereof, preferably including one or more of mCherry, GFP, RFP, YFP, luciferase, or near-infrared fluorescent proteins.

[0026] In a preferred embodiment, the temperature response regulation unit includes a thermosensitive repressor protein regulatory element, a thermosensitive promoter, an RNA thermosensitive switch, a heat shock protein promoter, or a combination thereof.

[0027] In a preferred embodiment, the temperature response regulation unit includes TcI, cI857, or a thermosensitive regulation module of equivalent function; preferably, the site-specific recombinase is regulated by a pR / pL thermo-inducible promoter.

[0028] In a preferred embodiment, the site-specific recombinase includes serine integrase, tyrosine recombinase, or other recombinases capable of mediating stable rearrangements of the transcriptional regulatory sequence.

[0029] In a preferred embodiment, the site-specific recombinase is a Bxb1 serine integrase, the first recombination recognition sequence is the attB site, and the second recombination recognition sequence is the attP site.

[0030] In a preferred embodiment, the transcriptional regulatory sequence is a promoter, enhancer, operator, repressor, or a combination thereof, preferably a constitutive promoter, and more preferably a P7 promoter.

[0031] In a preferred embodiment, the conformational change of the transcriptional regulatory sequence includes inversion, excision, insertion, rearrangement, or a combination thereof; preferably, the conformational change is a transcriptional regulatory sequence inversion mediated by the site-specific recombinase.

[0032] In a preferred embodiment, the first recombination recognition sequence and the second recombination recognition sequence are transformed into sequence states that are not easily recognized again by the site-specific recombinase after the conformational change occurs, thereby stabilizing the second expression state.

[0033] In a preferred embodiment, the expression cassette corresponding to the site-specific recombinase further includes a ribosome binding site, a start codon optimization element, and / or a protein degradation tag, so that the genetic circuit maintains low baseline activity at physiological temperature and achieves enhanced expression under the elevated temperature state.

[0034] In a preferred embodiment, the protein degradation tag includes an ssrA degradation tag.

[0035] In a preferred embodiment, the first expression state is the function output unit in a closed state or a low expression state, and the second expression state is the function output unit in a closed state or a high expression state.

[0036] In a preferred embodiment, the functional output unit is used to express therapeutic molecules, diagnostic molecules, imaging molecules, regulatory molecules, or combinations thereof.

[0037] In a preferred embodiment, the functional molecule includes proteases, fibrin-degrading enzymes, extracellular matrix-degrading enzymes, antifibrotic factors, immunomodulatory factors, antitumor proteins, nucleic acid drug expression products, lesion softening factors, or combinations thereof.

[0038] In a preferred embodiment, the functional molecule includes nattokinase or a functionally equivalent molecule thereof, and preferably, the functional output unit includes the nattokinase gene aprN.

[0039] In a preferred embodiment, the functional molecules include collagenase, hyaluronidase, matrix metalloproteinase, fibrinolytic enzyme, antifibrotic peptide, cytokine, or a combination thereof.

[0040] In a preferred embodiment, the external magnetic field is an alternating magnetic field, a rotating magnetic field, a pulsed magnetic field, a gradient magnetic field, or a combination thereof, preferably an alternating magnetic field.

[0041] In a preferred embodiment, after the external magnetic field acts on the magnetocaloric transducer, the temperature of the microbial carrier rises to a threshold temperature that can activate the temperature response control unit; preferably, the threshold temperature is above 37°C.

[0042] In a preferred embodiment, the threshold temperature is 39°C-45°C, preferably about 42°C.

[0043] In a preferred embodiment, the operating parameters of the alternating magnetic field are 375 kHz and 330 Oe.

[0044] In a preferred embodiment, the magnetocaloric transconducting material can raise the system temperature by 18.3°C within 20 minutes under the action of the alternating magnetic field.

[0045] In a preferred embodiment, the magnetothermal transconducting material is stably distributed on the surface of the microbial carrier to form a biohybrid microrobot.

[0046] In a preferred embodiment, the functional molecule is characterized in that it is used to regulate the mechanical properties of lesion tissue, reduce tissue stiffness, degrade abnormally deposited extracellular matrix, promote lesion softening, or improve the deformability of lesion tissue.

[0047] In a preferred embodiment, the lesion tissue is tumor sclerotic tissue, fibrotic tissue, scarred tissue, abnormal matrix deposition tissue, sclerotic lesion, thrombosis-related lesion, or lesion with enhanced matrix barrier leading to treatment resistance.

[0048] This application also discloses a method for preparing a magnetic field-activated genetic memory bio-microrobot system, including: S1. Provide a microbial carrier and engineer the microbial carrier to contain a genetic circuit, wherein the genetic circuit includes at least a temperature response regulation unit, a genetic memory unit, and a functional output unit; S2. Provide a magnetocaloric transducer material, which is configured to generate heat under the action of an external magnetic field, thereby placing the microbial carrier in a heated state; S3. Associate the magnetocaloric transconducting material with the engineered microbial carrier to obtain the magnetic field-activated genetic memory biological microrobot system; The genetic memory unit is configured to switch the genetic circuit from a first expression state to a second expression state when triggered by the temperature response regulation unit, and to maintain the second expression state after the heating state is lifted, so that the functional output unit continuously expresses functional molecules.

[0049] In a preferred embodiment, the genetic memory unit includes a site-specific recombinase and its corresponding first recombination recognition sequence and second recombination recognition sequence, wherein the first recombination recognition sequence and the second recombination recognition sequence are positioned on both sides of the transcriptional regulatory sequence; When the magnetocaloric transduction material generates heat and the microbial vector is in a heated state, the temperature response regulation unit triggers the expression or activation of the site-specific recombinase, causing the site-specific recombinase to mediate a conformational change in the transcriptional regulatory sequence, thereby switching the genetic circuit from a first expression state to a second expression state, and maintaining the second expression state after the heated state is removed, so that the functional output unit continuously expresses functional molecules.

[0050] In a preferred embodiment, the engineered microorganism is constructed by first culturing and activating a microbial vector and preparing competent cells suitable for genetic engineering operations; then constructing a genetic circuit containing a temperature response regulation unit, a genetic memory unit, and a functional output unit, and introducing the genetic circuit into the microbial vector, thereby obtaining an engineered microorganism capable of state switching under heat stimulation and continuously expressing functional molecules.

[0051] In a preferred embodiment, the magnetocaloric transducer material is prepared by the following process: first, magnetic precursor particles are prepared; then, the magnetic precursor particles are coated and heat-treated; then, the coating layer is removed to obtain magnetic nanoparticles; finally, the magnetic nanoparticles are surface-functionalized to obtain a magnetocaloric transducer material suitable for association with microbial carriers.

[0052] In a preferred embodiment, the surface-functionalized magnetocaloric transducer material prepared in S2 is coupled to the engineered microbial surface prepared in S1 under the action of a coupling agent.

[0053] This application also discloses the use of the system as described above in the preparation of pharmaceutical compositions for treating diseases related to abnormal microenvironments of mechanical processes.

[0054] In a preferred embodiment, the diseases associated with abnormal mechanical microenvironment include one or more of fibrosis, scarring, abnormal matrix deposition, tissue sclerosis, thrombosis-related lesions, and treatment resistance diseases resulting from enhanced matrix barrier.

[0055] In this embodiment, the external magnetic field is first converted into a thermal stimulus using a magnetocaloric transduction material. Then, the temperature response regulation mechanism inside the engineered microorganism is used to convert this short-term stimulus into a state switch of the genetic circuit. This state is then stably preserved using a genetic memory unit. In this way, the external magnetic field no longer needs to be present all the time; it only serves to trigger the process. The subsequent functional output can be maintained by the internal expression state that has already been switched. In other words, this application does not simply increase the strength of external control, but solves the problems of insufficient autonomy and poor sustainability in the prior art by introducing a sustainable internal state change into the microbial carrier. At the same time, relying on the microscale, biocompatibility, and functional expression capabilities of the engineered microorganism itself, it also avoids the shortcomings of traditional physical memory modules in terms of size, integration, and applicability.

[0056] Furthermore, site-specific recombinases are used to rearrange the transcriptional regulatory sequences between specific recombination recognition sequences, enabling the genetic circuit to stably switch from one expression state to another after temperature triggering. Further limiting the recombination sequence state to prevent it from being re-recognized by the original recombinase or to make it difficult to be re-recognized, and regulating translation efficiency and protein stability, are essentially aimed at improving the unidirectionality, stability, and controllability of this switch. The resulting technical effects are mainly to make the writing and retention of genetic memory more reliable, reduce background expression or false triggering under non-triggered conditions, and thus more effectively address the problem of difficulty in maintaining the state after stimulus withdrawal.

[0057] The specification of this application contains numerous technical features distributed across various technical solutions. Listing all possible combinations of these technical features (i.e., technical solutions) would make the specification excessively lengthy. To avoid this problem, the various technical features disclosed in the above-described invention, the various technical features disclosed in the following embodiments and examples, and the various technical features disclosed in the accompanying drawings can be freely combined to form various new technical solutions (all of which are considered to have been described in this specification), unless such a combination of technical features is technically infeasible. For example, one example discloses feature A+B+C, and another example discloses feature A+B+D+E. Features C and D are equivalent technical means that serve the same function, and technically only one needs to be used; they cannot be used simultaneously. Feature E can technically be combined with feature C. Therefore, the solution A+B+C+D should not be considered as described because it is technically infeasible, while the solution A+B+C+E should be considered as described. Attached Figure Description

[0058] Figure 1 This is a schematic diagram of the structure of a magnetic field-activated genetic memory bio-microrobot system according to one embodiment of this application.

[0059] Figure 2 This is a schematic diagram of the fabrication process of a magnetic sensor MNP according to one embodiment of this application.

[0060] Figure 3 This is an analysis of the average aspect ratio of magnetic sensor MNPs according to one embodiment of this application (n = 100; mean ± standard deviation).

[0061] Figure 4 The temperature change stability of magnetic sensors MNPs according to one embodiment of this application was measured during five cycles of alternating magnetic field treatment (ON; 375 kHz, 330 Oe) and natural cooling (OFF).

[0062] Figure 5 This is a schematic diagram of a magnetic sensing memory coding circuit design according to one embodiment of this application.

[0063] Figure 6 This is a feasibility assessment of the optimized nattokinase sequence codons in a magnetic sensing memory coding circuit according to one embodiment of this application.

[0064] Figure 7 This is a sequencing of key gene sequences of a magnetic sensing memory coding circuit according to one embodiment of this application.

[0065] Figure 8 This is a stability test of a magnetic sensing memory coding circuit according to one embodiment of this application.

[0066] Figure 9 This is a schematic diagram of a magnetic sensor integrated with a probiotic having a magnetic sensing memory coding circuit according to one embodiment of this application.

[0067] Figure 10 This invention relates to the determination of saturation magnetization intensity of a bio-microrobot based on magnetic sensing memory encoding under different magnetic sensor feeding conditions, according to one embodiment of this application.

[0068] Figure 11 This invention relates to the determination of the bioactivity of a microrobot based on magnetic sensing memory encoding under different magnetic sensor feeding conditions, according to one embodiment of this application.

[0069] Figure 12 The coupling efficiency between a magnetic sensor and a probiotic with a magnetic sensing memory coding circuit is described in one embodiment of the present application in a bio-microrobot system based on an embodiment of the present application.

[0070] Figure 13 This is a scanning electron microscope image of a bio-microrobot system with magnetic sensing memory encoding according to one embodiment of this application.

[0071] Figure 14 It is the saturation magnetization intensity of a bio-microrobot system with magnetic sensing memory encoding according to one embodiment of this application.

[0072] Figure 15 The magnetocaloric conversion performance of a bio-microrobot system with magnetic sensing memory encoding according to one embodiment of this application was measured under an alternating magnetic field (375 kHz, 330 Oe, 20 min).

[0073] Figure 16 The response of a magnetically sensing memory-encoded bio-microrobot system according to one embodiment of this application is shown at different temperatures, with activation occurring at approximately 42°C (n = 3; mean ± standard deviation).

[0074] Figure 17 This is a comparison of the synthesis of red fluorescent protein mCherry in a bio-microrobot system with magnetic sensing memory encoding according to one embodiment of this application and a bio-microrobot without memory encoding.

[0075] Figure 18 This is a comparison of nattokinase release between a bio-microrobot system with magnetic sensing memory encoding according to one embodiment of this application and a bio-microrobot without memory encoding.

[0076] Figure 19 This invention relates to the determination of the fibrin degradation effect in subcutaneous 4T1 breast cancer tumor tissue of mice using a magnetic sensing memory-encoded bio-microrobot system according to one embodiment of the present application.

[0077] Figure 20 This invention relates to the determination of the softening effect of a magnetically sensing memory-encoded bio-microrobot system on subcutaneous 4T1 breast cancer tumor tissue in mice, according to one embodiment of this application.

[0078] Figure 21 This invention relates to the determination of the softening effect of a magnetically sensing memory-encoded bio-microrobot system on subcutaneous Panc02 pancreatic cancer tumor tissue in mice, according to one embodiment of this application.

[0079] Figure 22 This application describes the determination of the duration of memory retention in vivo for a magnetically sensed memory-encoded bio-microrobot system according to one embodiment of the present application.

[0080] Figure 23 This is a comparative analysis of the onboard integration level (Ni / Lc) and production volume of microrobots reported in the literature. Ni represents the number of integrated onboard functions; Lc represents the feature length of the microrobot. Detailed Implementation

[0081] 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.

[0082] the term It should be understood that the following terms are used only to explain the technical solutions of this application, and unless otherwise expressly defined in this application, they should not be construed as limiting the scope of protection of this application.

[0083] "Bio-microrobots" refer to microscale systems formed by coupling microorganisms, cells or other biological materials with functional nanomaterials, which can perform functions such as sensing, movement, delivery and / or treatment.

[0084] "Magnetic sensing" refers to the ability of a system to produce a physical response to an external magnetic field, an alternating magnetic field, and / or a magnetic field gradient.

[0085] "Memory encoding" refers to the process or ability of a system to transform instantaneous external stimuli into an internal state that can be maintained stably over a long period of time.

[0086] "Genetic memory module" refers to a biological memory unit constructed using DNA recombination, DNA flipping, or other heritable state switching mechanisms.

[0087] "Bxb1" refers to a serine integrase that catalyzes site-specific recombination between the attB and attP sites.

[0088] "TcI" refers to a temperature-sensitive inhibition module that can inhibit the expression of downstream genes at low temperatures and relieve the inhibition when the temperature rises.

[0089] "P7 promoter" refers to a constitutive bacterial promoter used in this application to drive the continuous expression of downstream genes after a genetic state switch.

[0090] "Nattokinase" or "NK" refers to a serine protease that has the potential to degrade fibrin and regulate the biomechanical microenvironment of lesions.

[0091] "Mechanical abnormality disease" refers to a disease caused or aggravated by abnormal extracellular matrix deposition, tissue sclerosis, fibrosis and / or abnormal mechanical properties of lesions.

[0092] "Magnetic-thermal conversion" refers to the process by which magnetic materials convert magnetic energy into thermal energy under the action of an alternating magnetic field.

[0093] This application has at least the following advantages: Compared to existing bacterial microrobots that only have magnetic drive capabilities, this invention not only responds to magnetic fields but also activates internal genetic memory circuits through magnetocaloric conversion, writing a one-time external stimulus into a long-term stable therapeutic state. This significantly enhances the system's autonomy and continuous therapeutic capability. Most existing technologies can only maintain therapeutic output while the external field is continuously present, whereas this invention continues to express therapeutic proteins even after the stimulus is removed, thereby reducing the need for repeated stimulation and lowering the in vivo therapeutic burden.

[0094] Compared to engineered bacterial systems that only have a thermal response but no memory, this invention introduces a permanent Bxb1-attB / attP flipping module, enabling the system to possess true state storage capabilities, rather than transient induction of expression. Compared to simple genetic memory systems, this invention further achieves external, non-contact, spatially controllable activation through magnetic nanoparticles. Combined with the continuous secretion of lesion-softening factors such as nattokinase, this invention can be used to treat mechanically abnormal diseases such as tumor sclerosis, fibrosis, and abnormal extracellular matrix deposition, forming a complete technological chain of physical input, biological information processing, and continuous therapeutic output.

[0095] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings.

[0096] The first embodiment of this application relates to a magnetic field-activated genetic memory bio-microrobot system, the structure of which is shown in the figure below. Figure 1 As shown, it includes at least a microbial carrier and a magnetocaloric transducer associated with the microbial carrier; The magnetocaloric transducer is configured to generate heat under the influence of an external magnetic field, thus warming the microbial carrier. The microbial vector is an engineered microorganism containing a genetic circuit, which includes at least a temperature response regulation unit, a genetic memory unit, and a functional output unit. The genetic memory unit is configured to switch the genetic circuit from a first expression state to a second expression state when triggered by the temperature response regulation unit, and to maintain the second expression state after the temperature rise state is lifted, so that the functional output unit can continuously express functional molecules.

[0097] To achieve the effective conversion of the aforementioned physical signal into a thermal signal, the magnetocaloric transconducting material can be a magnetically responsive material capable of generating a temperature rise under a preset magnetic field. Available materials include magnetically responsive nanomaterials, magnetic composite materials, coated magnetic particles, or combinations thereof; further comprising ferrite materials, metallic magnetic materials, doped magnetic materials, and core-shell magnetic materials. The magnetocaloric transconducting material can be, for example, one or more of Fe3O4, γ-Fe2O3, MnFe2O4, CoFe2O4, ZnFe2O4, FeCo, or composite materials thereof.

[0098] Furthermore, the microstructure of the magnetocaloric transducer can be designed as needed, such as spindle-shaped, rod-shaped, ellipsoidal, cubic, sheet-like, nanoflower-like, or core-shell-like. This is because the morphology of the magnetocaloric transducer affects its magnetic response performance, thermal conversion capacity, surface modification efficiency, and binding state with the microbial carrier. This application does not limit this; any morphology that meets the heat generation requirements under magnetic field triggering and is suitable for coupling with the microbial carrier to form the aforementioned bio-microrobot system can be used.

[0099] After identifying the magnetocaloric transducer (MTT), it is necessary to establish a stable association between it and the microbial carrier to ensure that the heat generated by the external magnetic field can be efficiently transferred to the biological carrier within a microscale range. Based on this consideration, the MTT can be associated with the microbial carrier through surface coupling, electrostatic adsorption, bioaffinity binding, self-assembly, coating, or a combination thereof. To further improve the connection stability and interfacial compatibility, the MTT can be pre-functionalized, such as through amination, carboxylation, silanization, dopamine modification, polysaccharide coating, or a combination thereof. Then, the two can be linked through amide condensation, biotin-avidin binding, or click chemistry. This approach not only improves the binding strength of the material on the bacterial cell surface but also enhances the effectiveness of magnetocaloric stimulation transmission and the overall stability of the system.

[0100] The preferred microbial carrier is a live microorganism that can be engineered, such as probiotics, non-pathogenic bacteria, attenuated bacteria, yeast, or engineered algae. This type of microorganism is chosen because it possesses good biocompatibility and programmable modification capabilities, and because it can simultaneously perform environmental sensing, genetic information processing, and functional molecule expression within a microscale system, thus making the entire system a living microrobot platform with autonomous processing capabilities.

[0101] Building upon the aforementioned vector, the system further includes a genetic circuit. This genetic circuit comprises at least a temperature-response regulation unit, a genetic memory unit, and a functional output unit, and may include a feedback unit as needed. The temperature-response regulation unit primarily senses thermal stimuli and can employ thermosensitive repressor protein regulatory elements, thermosensitive promoters, RNA thermosensitive switches, heat shock protein promoters, or combinations thereof. When the magnetocaloric transduction material experiences a localized temperature rise under the influence of an external magnetic field, the temperature-response regulation unit responds to this temperature rise and initiates downstream genetic events.

[0102] In some embodiments, the genetic memory unit further transforms transient thermal stimulation into a stable state-switching event. In a typical implementation, the genetic memory unit is constructed based on a site-specific recombinase and its corresponding recombination recognition sequence, flanking the transcriptional regulatory sequence. When the temperature-responsive regulatory unit is activated, the recombinase is expressed or activated, mediating conformational changes in the transcriptional regulatory sequence, such as flipping, excision, insertion, or rearrangement. Through this process, the genetic circuit switches from a first expression state to a second expression state. More importantly, after the state switch, the site state of the recombination product changes, preventing the system from easily returning to its initial state, thus endowing the circuit with memory characteristics. In other words, thermal stimulation here is not merely a temporary induction of expression, but is written into a sustainable genetic state.

[0103] The first expression state refers to the expression state of the genetic circuit before a state switch occurs, in which the functional output unit is in a closed, repressed, or low-expression state. The second expression state refers to the expression state of the genetic circuit after a state switch occurs, in which the functional output unit is in an open, de-repressed, or high-expression state, and can continuously express functional molecules. More specifically, the first expression state refers to the expression state corresponding to the transcriptional regulatory sequence before the conformational change, in which the functional output unit does not express or expresses functional molecules at a low level; the second expression state refers to the expression state corresponding to the transcriptional regulatory sequence after the conformational change, in which the functional output unit continuously expresses or expresses functional molecules at a higher level.

[0104] Once the genetic circuit completes its state switch, the functional output unit enters a sustained expression state. This functional output unit can be used to express therapeutic molecules, diagnostic molecules, imaging molecules, regulatory molecules, or combinations thereof. For therapeutic applications, the expressed functional molecules may include proteases, fibrin-degrading enzymes, extracellular matrix-degrading enzymes, anti-fibrotic factors, and immunomodulatory factors. For lesion environments with fibrin accumulation or matrix densification, proteases with fibrin-degrading activity can be preferentially selected as output molecules to continuously regulate the abnormal mechanical microenvironment of the lesion tissue.

[0105] In some embodiments, the system may also include a feedback unit to monitor whether the genetic circuit has completed switching. This feedback unit can provide detectable signals using fluorescent proteins, luminescent proteins, near-infrared imaging markers, etc., thereby characterizing the current genetic state of the system. This module not only allows for verification of effectiveness during the experimental phase but also facilitates dynamic observation of the system's operational status in subsequent applications.

[0106] Based on the above structure, the system's startup depends on an external magnetic field input. The external magnetic field can be an alternating magnetic field, a rotating magnetic field, a pulsed magnetic field, a gradient magnetic field, or a combination thereof, among which an alternating magnetic field is particularly suitable as an excitation method for driving the heat generation of the magnetocaloric transconducting material.

[0107] In summary, the core of this implementation is not merely the parallel arrangement of magnetothermal materials, biological carriers, and genetic circuits, but rather the establishment of a bio-microrobot system capable of incorporating external magnetic field stimuli into genetic memory and continuously outputting functional molecules even after the stimulus is removed, through a clear division of functions and sequential connection. This structure lays the foundation for its subsequent application in the regulation of the lesion microenvironment.

[0108] like Figure 23 As shown, the system of this application exhibits excellent airborne integration (Ni / Lc = 5.33; where Ni refers to the number of functions integrated on the microrobot, and Lc refers to the feature length of the microrobot) and high throughput (3.32 × 10⁻⁶). 7 h -1 With a biohybridization rate as high as 98.93 ± 0.68%, it achieves efficient manufacturing and enhanced autonomous functions, representing a significant advancement compared to traditional mechanical and hybrid microrobots.

[0109] The second embodiment of this application relates to a method for preparing a magnetic field-activated genetic memory bio-microrobot system. It should be noted that the sequence numbers used in describing the method steps do not inherently limit the order of these steps. For example, a step with a higher sequence number does not necessarily have to be executed after a step with a lower sequence number; the higher-numbered step can be executed first, followed by the lower-numbered step, or they can be executed in parallel, as long as this execution order is reasonable to those skilled in the art. Furthermore, multiple steps with consecutively numbered sequences (e.g., step 101, step 102, step 103, etc.) do not restrict other steps from being executed between them; for example, there can be other steps between step 101 and step 102. The method includes the following process: S1. Provide a microbial carrier and engineer the microbial carrier to contain a genetic circuit, wherein the genetic circuit includes at least a temperature response regulation unit, a genetic memory unit, and a functional output unit; S2. Provide a magnetocaloric transducer material, which is configured to generate heat under the action of an external magnetic field, thereby placing the microbial carrier in a heated state; S3. Associate the magnetocaloric transconducting material with the engineered microbial carrier to obtain the magnetic field-activated genetic memory biological microrobot system; The genetic memory unit is configured to switch the genetic circuit from a first expression state to a second expression state when triggered by the temperature response regulation unit, and to maintain the second expression state after the heating state is lifted, so that the functional output unit continuously expresses functional molecules.

[0110] Based on the above preparation method, this application further explains the implementation of each step.

[0111] In S1, in some embodiments, engineered microorganisms can be constructed by first culturing and activating a microbial vector and preparing competent cells suitable for genetic engineering; then constructing a genetic circuit comprising a temperature-response regulation unit, a genetic memory unit, and a functional output unit, and introducing the genetic circuit into the microbial vector, thereby obtaining engineered microorganisms capable of state switching under heat stimulation and continuously expressing functional molecules. If necessary, the genetic circuit may further include a feedback unit for characterizing the state of the genetic circuit.

[0112] In S2, in some embodiments, the magnetocaloric transducer material can be prepared by the following process: first, magnetic precursor particles are prepared; then, the magnetic precursor particles are coated and heat-treated; then, the coating layer is removed to obtain magnetic nanoparticles; finally, the magnetic nanoparticles are surface-functionalized to obtain a magnetocaloric transducer material suitable for association with microbial carriers.

[0113] In S3, in some embodiments, the surface-functionalized magnetocaloric transconducting material prepared in S2 can be coupled to the engineered microbial surface prepared in S1 under the action of a coupling agent.

[0114] To better understand this preparation method, a specific example will be used for illustration below. The details listed in this example are mainly for ease of understanding and are not intended to limit the scope of protection of this application.

[0115] Preparation of magnetocaloric transduction materials 1) Synthesis of spindle-shaped nuclei (α-Fe₂O₃): Fe(ClO₄)₃·6H₂O (6.5 mmol), NaH₂PO₄ (0.15 mmol), and NH₂CONH₂ (5 mmol) were thoroughly dispersed in 50 mL of deionized water (ddH₂O). The solution was placed in an Erlenmeyer flask and heated in an oil bath at 100°C for 24 hours. After the solution cooled naturally, α-Fe₂O₃ was separated from the dispersion by centrifugation (9500 rpm).

[0116] 2) Synthesis of α-Fe₂O₃@SiO₂: α-Fe₂O₃ (320 mg), TEA (68 mg), CTAB (380 mg), and NaSal (126 mg) were added to 25 mL of ddH₂O. The mixture was stirred vigorously at 80°C for 3 hours. Then, TEOS (3.6 mL) was added to the above solution, and the mixture was stirred at 80°C for 45 minutes. α-Fe₂O₃@SiO₂ was collected by centrifugation (9500 rpm, 15 minutes) and washed 6 times with anhydrous ethanol.

[0117] 3) Synthesis of Fe3O4@SiO2: α-Fe2O3@SiO2 was calcined in air at 550℃ for 5 hours to remove residual surfactants. Then, α-Fe2O3@SiO2 was annealed in H2 / Ar atmosphere at 450℃ for 3 hours to obtain Fe3O4@SiO2.

[0118] 4) Synthesis of Fe3O4 (MNPs): 20 mg of Fe3O4@SiO2 was added to 50 mL of NaOH (0.1 mol), and sonicated for 5 hours to remove the silica shell. Synthesis of MNPs@APTES: 10 mg of MNPs was uniformly dispersed in 20 mL of isopropanol. 500 μL of ammonia and 200 μL of silane coupling agent (APTES) were added to the solution. The solution was vigorously stirred for 3 hours at 35°C under argon. Then, the solution was vigorously stirred for 8 hours at 80°C under argon. MNPs@APTES were collected by magnetic separation and washed three times with ddH2O.

[0119] Construction of genetic circuits 1) Probiotic culture: *Escherichia coli* Nissle 1917 (EcN) was cultured in Lysogeny Broth (LB) medium containing 1 g tryptone, 0.5 g yeast extract powder, and 1 g NaCl per liter of double-distilled water (ddH2O). Preparation of highly competent cells: EcN cells stored in an ultra-low temperature freezer were streaked onto LB plates and incubated at 37°C for 12 hours. Single colonies were picked and inoculated into 5 mL of LB medium and incubated at 37°C and 220 rpm for 12 hours.

[0120] 2) Preparation of probiotic competent cells: Activated bacterial culture (500 μL) was inoculated into 50 mL LB medium and cultured with shaking (37℃, 220 rpm) until OD reached. 600 = 0.4. Centrifuge (4000 rpm, 10 minutes) to collect competent bacteria, and wash three times with glycerol.

[0121] 3) Construction of magnetosensory memory genetic coding circuits (genetic circuits): Memory circuits with independent outputs of nattokinase and red fluorescent protein mCherry were constructed. From Bacillus subtilis (… Bacillus subtilis Nattokinase gene obtained aprN ( pre-pro-nk (NCBI GenBank: S51909); A codon optimization strategy was employed to enhance the expression of exogenous proteins. Specifically, to achieve stable heat-induced switching function, the expression of the serine integrase Bxb1 was placed under the regulation of the heat-inducible promoter of pL / pR phage λ, and its activity was regulated by the TcI42 repressor protein. The serine integrase Bxb1, initially discovered in bacteriophages, is an enzyme that targets DNA sequences at attP and attB sites. In the natural environment, these sites serve as hubs for phage DNA integration into the target cell genome. However, these sites can be reused to flank arbitrary DNA sequences and mediate their inversions, thereby achieving stable switching function. This design combines the temperature sensitivity of TcI42 with the permanent effector function of the Bxb1 integrase. At a physiological temperature of approximately 37°C, constitutive expression of the TcI42 repressor protein driven by the pLacI promoter inhibits Bxb1 expression. Upon heat stimulation, the TcI42 repression is released, leading to a surge in Bxb1 gene expression. Following heat-induced deinhibition, the expressed Bxb1 protein catalyzes the inversion of the minimal recognition sites attP and attB flanking the P7 promoter, leading to the expression of a fluorescent reporter gene for monitoring the circuit status. Because the attP and attB recognition sites are modified by the Bxb1 enzyme after inversion, subsequent Bxb1 interactions cannot recognize the inverted DNA sequence, making this inversion permanent. Subsequently, even after temperature stimulation ceases, the P7 promoter continues to drive the expression of its protein product. The P7 promoter was chosen because it is derived from a synthetic constitutive bacterial promoter library and can strongly drive gene product expression without placing excessive stress on the cell. Ideally, this circuit should maintain low baseline activity at physiological temperatures while providing strong and sustained induction after heat stimulation. To achieve this, three key sequence elements affecting Bxb1 translation and stability were regulated: the Bxb1 ribosome-binding sequence (RBS), the start codon, and the ssrA degradation tag. The ssrA tag is a short peptide that is naturally added to the C-terminus of proteins with translational arrest. Fusion proteins carrying this sequence are targeted and degraded by endogenous bacterial proteases.

[0122] Integration of bio-microrobot systems based on magnetic sensing and memory encoding Magnetoresistive memory coding circuits were transferred into EcNs to construct engineered EcNs. Amino-functionalized MNPs (MNPs@APTES) were coupled to the EcN surface via an amide condensation reaction. The engineered EcNs were dispersed in PBS solution at a concentration of 10. 8 CFU mL -1 500 μg of MNPs@APTES, 0.55 mg of EDC, and 0.65 mg of NHS were added to an engineered EcN suspension. The mixture was then stirred at 100 rpm for 3 hours at room temperature. The microrobots were separated by differential centrifugation (3000 rpm, 5 min) and further separated by magnetic separation. The separated microrobots were washed three times with PBS and resuspended in PBS.

[0123] Characterization of bio-microrobot systems based on magnetic sensing memory encoding First, spindle-shaped Fe3O4 magnetic nanoparticles (MNPs) with high magnetic responsiveness and good magnetocaloric properties were prepared. Figure 2 The obtained magnetic nanoparticles have an aspect ratio of approximately 3.15 ± 0.18. Figure 3 Furthermore, the temperature rise curves during multiple cycles of heating and cooling largely overlap, indicating that it possesses good magnetocaloric stability. Figure 4 ).

[0124] Furthermore, a genetic memory coding circuit capable of being activated by a magnetocaloric signal is constructed. This genetic circuit includes: a thermal sensor module TcI, an execution module pR-pL, a memory module Bxb1-ssrA-attB-P7-attP, a treatment output module aprN, and a feedback module mCherry (…). Figure 5 Among them, the treatment output module is derived from Bacillus subtilis (…). Bacillus subtilis Nattokinase gene obtained aprN ( pre-pro-nk (NCBI GenBank: S51909); A codon optimization strategy was used to improve the expression of exogenous proteins, and the rationality of the design was verified by molecular docking. Figure 6 Key fragments were validated using Sanger sequencing. Figure 7 (Table 1). In the 120th generation of the magnetically sensed memory-encoded bio-microrobot system, approximately 85.48 ± 8.08% of the cells still retained genetic memory. Figure 8 ). Table 1 Preferably, this embodiment employs an amide condensation reaction coupled with a magnetic sensor and probiotics possessing a magnetic sensing memory coding circuit. Figure 9 By exploring different magnetic sensor feed ratios, the optimal saturation magnetization of the magnetically sensing memory-encoded bio-microrobot system was determined to be 31.01 emu g. -1 ( Figure 10 ), and maintain good biological activity and growth capacity ( Figure 11 The system biointegration rate in this embodiment can reach 98.93 ± 0.68% ( ). Figure 12 ).

[0125] Subsequently, scanning electron microscopy (SEM) showed that the magnetic sensors were stably distributed on the bacterial surface. Figure 13 The saturation magnetization is 31.01 emu g. -1 ( Figure 14 Under the influence of an alternating magnetic field (375 kHz, 330 Oe), the temperature increased by 18.3°C within 20 minutes. Figure 15 When the system temperature reached 42°C, the mCherry fluorescence intensity peaked, indicating that TcI can stably translate the magnetocaloric signal into a transcriptional response. Figure 16 Comparing the long-term performance of memoryless encoded microrobots after a single magnetothermal intervention, 120 h after intervention, the mCherry fluorescence of the magnetically sensed memory-encoded biorobot system was approximately 7.8 times higher than that of the memoryless encoded microrobot. Figure 17 The memory writing process enabled the transition from transient input to a persistent state. The magnetically sensed memory-encoded bio-microrobot system continuously released nattokinase for up to 72 hours, reaching 211.77 ± 26.83 U / mL. -1 It was 2.13 times that of the memoryless group. Figure 18 ).

[0126] The third embodiment of this application also relates to its use in the preparation of a pharmaceutical composition for treating diseases associated with abnormal microenvironments of mechanical processes.

[0127] Among them, diseases associated with abnormal microenvironments of fibrosis, scarring, abnormal matrix deposition, tissue sclerosis, thrombosis-related lesions, and treatment resistance diseases caused by enhanced matrix barrier are one or more of these.

[0128] Among the aforementioned diseases related to abnormal microenvironments, solid tumors, often accompanied by abnormal extracellular matrix deposition, fibronectin enrichment, and increased tissue stiffness, can serve as a representative application scenario for this type of disease. Based on this, the tissue softening effect and sustained regulatory ability of the system described in this application are validated using a subcutaneous tumor-bearing mouse model: To evaluate the softening effect of a magnetically sensed memory-encoded bio-microrobot system on tumor tissue in tumor-bearing mice, subcutaneous 4T1 breast cancer tumor mouse models and subcutaneous Panc02 pancreatic cancer tumor mouse models were established. The magnetically sensed memory-encoded bio-microrobot system was injected into the tumor via intratumoral injection. Before tumor injection, the magnetically sensed memory-encoded bio-microrobot system (10 μL) was activated under an alternating magnetic field (AMF, 375 kHz, 330 Oe, for 6 min). 7 CFU, 100 μL). First, the clearance effect on the abundant fibronectin in the tumor stroma was assessed on day 7. Figure 19 Secondly, ultrasound shear wave elastography was performed on days 0, 3, and 7. The tissue matrix regulation mediated by a magnetically sensing memory-encoded bio-microrobot system can reverse and inhibit the tumor sclerosis process. Figure 20 Finally, the magnetically sensing memory-encoded bio-microrobot system was extended to a mouse pancreatic cancer model with abnormally rich extracellular matrix. It continued to autonomously and effectively reduce tumor stiffness and improve tissue deformability over a long period, validating the applicability of this system's autonomous and continuous regulation of tissue softening strategies in different types and degrees of fibrosis of tumors. Figure 21 In vivo real-time dynamic imaging results indicate that genetic memory remains active and continuously outputs detectable signals within an observation window of at least 12 days, providing direct functional evidence for its medium- to long-term therapeutic effects. Figure 22 ).

[0129] 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.

[0130] 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 magnetic field-activated genetic memory biological microrobot system, characterized in that, It includes at least a microbial carrier and a magnetocaloric transducer associated with the microbial carrier; The magnetocaloric transducer is configured to generate heat under the action of an external magnetic field, so that the microbial carrier is in a heated state. The microbial carrier is an engineered microorganism containing a genetic circuit, which includes at least a temperature response regulation unit, a genetic memory unit, and a functional output unit. The genetic memory unit is configured to switch the genetic circuit from a first expression state to a second expression state when triggered by the temperature response regulation unit, and to maintain the second expression state after the heating state is lifted, so that the functional output unit continuously expresses functional molecules.

2. The magnetic field-activated genetic memory bio-microrobot system as described in claim 1, characterized in that, The genetic memory unit includes a site-specific recombinase and its corresponding first recombination recognition sequence and second recombination recognition sequence, wherein the first recombination recognition sequence and the second recombination recognition sequence are positioned on both sides of the transcriptional regulatory sequence. The temperature response regulation unit triggers the expression or activation of the site-specific recombinase under the heating state, causing the site-specific recombinase to mediate a conformational change in the transcriptional regulatory sequence, thereby switching the genetic circuit from a first expression state to a second expression state, and maintaining the second expression state after the heating state is lifted, so that the functional output unit continuously expresses functional molecules.

3. The magnetic field-activated genetic memory bio-microrobot system as described in claim 1, characterized in that, The magnetocaloric transduction material includes magnetically responsive nanomaterials, magnetic composite materials, coated magnetic particles, or combinations thereof.

4. The magnetic field-activated genetic memory bio-microrobot system as described in claim 1, characterized in that, The genetic circuit further includes a feedback unit, which is used to output a detectable signal in the first expression state and / or the second expression state to characterize the state of the genetic circuit.

5. The magnetic field-activated genetic memory bio-microrobot system as described in claim 4, characterized in that, The feedback unit includes a fluorescent protein expression module, a luminescent protein expression module, a near-infrared imaging biomarker expression module, or a combination thereof.

6. The magnetic field-activated genetic memory bio-microrobot system as described in claim 2, characterized in that, After the conformational change occurs, the first recombination recognition sequence and the second recombination recognition sequence are transformed into sequence states that are not easily recognized again by the site-specific recombinase, thereby stabilizing the second expression state.

7. The magnetic field-activated genetic memory bio-microrobot system as described in claim 2, characterized in that, The expression cassette corresponding to the site-specific recombinase also includes a ribosome binding site, a start codon optimization element, and / or a protein degradation tag, so that the genetic circuit maintains low baseline activity at physiological temperature and achieves enhanced expression under the elevated temperature state.

8. A method for preparing a magnetic field-activated genetic memory bio-microrobot system, characterized in that, include: S1. Provide a microbial carrier and engineer the microbial carrier to contain a genetic circuit, wherein the genetic circuit includes at least a temperature response regulation unit, a genetic memory unit, and a functional output unit; S2. Provide a magnetocaloric transducer material, which is configured to generate heat under the action of an external magnetic field, thereby placing the microbial carrier in a heated state; S3. Associate the magnetocaloric transconducting material with the engineered microbial carrier to obtain the magnetic field-activated genetic memory biological microrobot system; The genetic memory unit is configured to switch the genetic circuit from a first expression state to a second expression state when triggered by the temperature response regulation unit, and to maintain the second expression state after the heating state is lifted, so that the functional output unit continuously expresses functional molecules.

9. The method as described in claim 8, characterized in that, The genetic memory unit includes a site-specific recombinase and its corresponding first recombination recognition sequence and second recombination recognition sequence, wherein the first recombination recognition sequence and the second recombination recognition sequence are positioned on both sides of the transcriptional regulatory sequence. When the magnetocaloric transduction material generates heat and the microbial vector is in a heated state, the temperature response regulation unit triggers the expression or activation of the site-specific recombinase, causing the site-specific recombinase to mediate a conformational change in the transcriptional regulatory sequence, thereby switching the genetic circuit from a first expression state to a second expression state, and maintaining the second expression state after the heated state is removed, so that the functional output unit continuously expresses functional molecules.

10. The use of the system according to any one of claims 1-7 in the preparation of a pharmaceutical composition for treating diseases related to abnormal microenvironments of mechanical processes.

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