A her2 / tfr1 dual-targeting magnetic ferritin nanoprobe, and a preparation method and application thereof

Abstract

The application provides a conjugated magnetic ferritin, which is obtained by biomimetic mineralization of synthetic ferritin, formation of nanoparticles in the interior of the ferritin, and finally separation and purification of the metal-containing magnetic ferritin; and the magnetic ferritin is conjugated with an affibody, a nanobody or a specific polypeptide. The conjugated magnetic ferritin of the application can specifically target specific tumors, has the characteristics of HER2 / TfR1 dual targeting, and can be used as a nuclear magnetic resonance contrast agent.

Description

Technical Field

[0001] This invention belongs to the field of biomimetic synthesis technology of magnetic nanomaterials, specifically relating to a HER2 / TfR1 dual-targeting magnetic ferritin nanoprobe and its preparation method and application. Background Technology

[0002] In the clinical diagnosis and treatment of HER2-expressing malignancies such as breast cancer and gastric cancer, pathological biopsy is currently the primary method for determining whether a patient meets the criteria for HER2-targeted therapy. However, tumor tissue itself exhibits significant spatial heterogeneity—that is, HER2 expression levels in different regions of the same tumor may be completely different, and their expression status may also dynamically change (temporal heterogeneity) as the disease progresses or metastasizes. Therefore, pathological results based on a single-point puncture only represent information from the sampled area and cannot comprehensively reflect the true HER2 expression profile of all lesions in the patient's body. Clinically, this often leads to situations where a biopsy is negative but other lesions are actually positive, resulting in some patients who could benefit from anti-HER2 therapy being misdiagnosed and missing the optimal time for effective treatment.

[0003] Magnetic ferritin (MHFn), a natural nanocage structure, has been extensively studied for its application in tumor MRI imaging due to its ability to enter cells via transferrin receptor 1 (TfR1)-mediated endocytosis. TfR1 is highly expressed on the surface of various rapidly proliferating malignant tumor cells, endowing MHFn with a certain broad-spectrum tumor targeting capability. However, for the precise imaging requirements targeting specific sites, relying solely on the TfR1 recognition mechanism has significant limitations: its enrichment in tumor tissue lacks specificity, meaning it also accumulates in normal tissues, making it difficult to meet the clinical requirements for precise molecular subtyping imaging.

[0004] To address the aforementioned issues, this technical solution proposes a dual-targeting functional integration strategy. Specifically, without compromising the TfR1 targeting ability of magnetic ferritin (MHFn), a bifunctional probe is constructed by modifying its surface with HER2-targeting molecules (such as affinity molecules, nanobodies, or specific peptides), thereby possessing both natural TfR1 recognition and active HER2 targeting capabilities. Summary of the Invention

[0005] On one hand, the present invention provides a coupled magnetic ferritin, wherein the magnetic ferritin is synthesized ferritin and subjected to biomimetic mineralization to form nanoparticles inside the ferritin, and finally separated and purified to obtain metal-containing magnetic ferritin; the magnetic ferritin is coupled with an affinity compound, nanobody or specific polypeptide.

[0006] In one embodiment, the metal includes manganese, iron, and cobalt.

[0007] In one embodiment, the metal is manganese.

[0008] In one embodiment, the magnetic ferritin is synthesized ferritin that undergoes biomimetic mineralization to form nanoparticles inside the ferritin, and is finally separated and purified to obtain manganese-containing magnetic ferritin.

[0009] On the other hand, the present invention provides a method for preparing the above-mentioned coupled magnetic ferritin, the method comprising the following steps:

[0010] 1) Using recombinant human ferritin as a template, the DNA sequence of human ferritin was cloned and constructed into a plasmid;

[0011] 2) Transform or co-transform bacteria with recombinant plasmids containing human ferritin, add isopropyl-β-D-thiogalactoside to activate the T7 promoter, and induce expression;

[0012] 3) After expression, the protein is broken down by sonication to release it;

[0013] 4) Separate and purify the protein;

[0014] 5) Add iron salt solution containing metal ions and oxidant to the ferritin solution to react, control the pH value to 8.5, control the temperature to 65℃, and form nanoparticles inside the ferritin.

[0015] 6) After purification, a metal-containing magnetic ferritin was obtained;

[0016] 7) Magnetic ferritin is conjugated with affinity molecules, nanobodies, or specific peptides.

[0017] In one embodiment, the iron salt is a ferrous salt; the oxidizing agent is hydrogen peroxide.

[0018] In one implementation, in step 5, the pH is controlled at 8.5; the temperature is controlled at 65°C.

[0019] In one embodiment, the iron salt solution used in the biomimetic mineralization process is an iron salt solution containing metal (manganese) ions.

[0020] In one embodiment, the iron salt solution containing manganese ions is prepared by mixing iron salt and manganese salt.

[0021] In one embodiment, the ferric salt is a ferrous salt. In another embodiment, the ferrous salt is a water-soluble ferrous salt, such as ferrous sulfate, ferrous ammonium sulfate, or ferrous chloride.

[0022] In one embodiment, the manganese salt is selected from one or more of manganese sulfate, manganese chloride, manganese nitrate, manganese acetate, and manganese carbonate; preferably, manganese sulfate.

[0023] Preferably, the mixed salt solution of iron and manganese ions is a mixed salt solution of ferrous ammonium sulfate and manganese sulfate. The iron salt solution containing manganese ions is a mixed salt solution of ferrous ammonium sulfate and cobalt sulfate.

[0024] In one embodiment, the concentration of the metal (manganese) ions is 5%.

[0025] In one embodiment, the ferritin is selected from human ferritin, plant ferritin, or bacterial ferritin.

[0026] In one embodiment, the ferritin is recombinant human ferritin.

[0027] In one embodiment, the recombinant human ferritin includes the H chain of recombinant human ferritin, the L chain of recombinant human ferritin, an assembly of the H chain and L chain of recombinant human ferritin, a mutant or fusion protein of the H chain of recombinant human ferritin, and a mutant or fusion protein of the L chain of recombinant human ferritin.

[0028] In one embodiment, the ferritin includes the H chain of recombinant human ferritin, the L chain of recombinant human ferritin, an assembly of the H chain and L chain of recombinant human ferritin, a mutant or fusion protein of the H chain of recombinant human ferritin, a mutant or fusion protein of the L chain of recombinant human ferritin, recombinant plant ferritin, a mutant of recombinant plant ferritin, a fusion protein of recombinant plant ferritin, bacterial ferritin, a mutant of bacterial ferritin, or a fusion protein of bacterial ferritin.

[0029] In one embodiment, the ferritin is the H chain of recombinant human ferritin.

[0030] In one embodiment, the amino acid sequence of the human ferritin is shown in SEQ ID No. 1.

[0031] In one embodiment, the affinity protein is an affinity protein that specifically recognizes HER2, and the amino acid sequence of the affinity protein is shown in SEQ ID No. 2.

[0032] In one implementation, the coupling includes the following steps:

[0033] After the carboxyl group of the affinity protein, nanobody, or specific polypeptide is activated, it undergoes a condensation reaction with the amino group of magnetic ferritin to form an amide bond.

[0034] In one implementation, the coupling includes the following steps:

[0035] (a) Amino blocking of affinity molecules, nanobodies, or specific peptides;

[0036] (b) The carboxyl group of the affinity compound, nanobody, or specific peptide is activated by EDC / NHS;

[0037] (c) When a carboxyl-activated affinity, nanobody, or specific polypeptide comes into contact with magnetic ferritin, the amino group of the magnetic ferritin undergoes a condensation reaction with the carboxyl group of the affinity, nanobody, or specific polypeptide molecule to form an amide bond.

[0038] In one embodiment, the affinity is obtained through prokaryotic recombination expression, specifically including the following steps:

[0039] The nucleic acid sequence of the affinity variant was constructed into an expression vector, transformed into host cells, and induced to express. After affinity purification, tag removal by enzyme digestion, and further purification, the affinity variant was obtained.

[0040] In one embodiment, the purification and preparation of the affinity includes the following steps:

[0041] A GST tag is introduced at the N-terminus of the protein, and a thrombin cleavage site is set between the GST tag and the affinity protein. After the fusion protein is purified by GST affinity, the GST tag is cleaved by thrombin, and the affinity protein is obtained by secondary purification.

[0042] In one embodiment, step (a) is: the affinity compound, nanobody or specific peptide is mixed with DMA, NaOH is added, the pH of the reaction system is adjusted to about 9, and the pH is continuously monitored and adjusted until the pH is stable.

[0043] On the other hand, the present invention provides a protein nanoprobe or nuclear magnetic resonance contrast agent, wherein the protein nanoprobe or nuclear magnetic resonance contrast agent contains the above-mentioned coupled magnetic ferritin, or is obtained by the above-mentioned preparation method.

[0044] In one embodiment, the contrast agent is a nuclear magnetic resonance contrast agent.

[0045] On the other hand, the present invention provides the application of the above-mentioned coupled magnetic ferritin in the preparation of magnetic contrast agents or molecular probes for diagnosing tumors.

[0046] In one embodiment, the tumor is a HER2-expressing tumor. Attached Figure Description

[0047] Figure 1 A is the characterization result of manganese-containing magnetic ferritin; B is the SDS-PAGE electrophoresis result of HFn and HFn(Mn5%); C is the hydrated particle size distribution of HFn and HFn(Mn5%); D is the transmission electron microscope image of HFn(Mn5%).

[0048] Figure 2 It is Z HER2:342 Figure 1 shows the results of affinity protein expression, purification, and identification; A represents GST-Z. HER2:342SDS-PAGE electrophoresis results of fusion protein expression, purification, and enzyme digestion processes. B represents the purified protein. HER2:342 Image of SDS-PAGE electrophoresis results of affinity proteins.

[0049] Figure 3 It is mineralized ferritin and Z HER2:342 Characterization results of the ligation products; A represents MHFn and MHFn+Z. HER2:342 The SDS-PAGE electrophoresis results of the ligation products, B represents HFn, MHFn, and MHFn+Z. HER2:342 Hydrated particle size distribution diagram.

[0050] Figure 4 It is material Z HER2:342 -Flow cytometry results of MHFn in different breast cancer cells at different incubation times; A represents material Z. HER2:342 - Flow cytometry fluorescence distribution of MHFn in MDA-MB-231 cells after incubation at 0h, 1h, 2h, and 3h. B represents material Z. HER2:342 - Flow cytometry fluorescence distribution of MHFn in SK-BR-3 cells after incubation at 0h, 1h, 2h and 3h.

[0051] Figure 5 Different cells react to material Z HER2:342 -Flow cytometry analysis and quantitative results of MHFn uptake differences; A is a comparison of flow cytometry fluorescence distribution after 1 h of incubation, B is a comparison of flow cytometry fluorescence distribution after 2 h of incubation, C is a comparison of flow cytometry fluorescence distribution after 3 h of incubation, and D is a statistical graph of cell uptake at different times.

[0052] Figure 6 The diagram shows a comparison of the NMR relaxation properties of the dual-target magnetic ferritin probes; A is the linear fitting result of the longitudinal relaxation rate r1, and B is the linear fitting result of the transverse relaxation rate r2. Detailed Implementation

[0053] To make the objectives, technical solutions, and advantages of this application clearer, the application will be further described in detail below with reference to the accompanying drawings. The described embodiments should not be regarded as limitations on this application. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0054] Example 1: Expression, extraction and desalting of ferritin

[0055] This invention relates to a human heavy chain ferritin (HFn) and its recombinant expression method. The amino acid sequence of the ferritin is as follows:

[0056] MTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES (SEQ ID No.1).

[0057] The amino acid sequence contains a total of 183 amino acid residues.

[0058] The ferritin can be obtained through recombinant expression. Based on the amino acid sequence of the ferritin, the corresponding nucleotide sequence is designed and synthesized. The gene fragment encoding the ferritin is ligated into the prokaryotic expression vector pET-22b. The recombinant pET-22b expression vector is then transformed into E. coli BL21(DE3) competent cells. After ampicillin resistance screening to obtain positive transformants, the cells are expanded for culture. When the bacterial culture reaches the logarithmic growth phase, IPTG is added to induce the expression of the target protein. After induction, the bacterial cells are collected and disrupted to obtain a protein sample containing the ferritin. If necessary, further purification can be performed using one or more methods selected from affinity chromatography, ion exchange chromatography, and molecular sieve chromatography to obtain the recombinant ferritin.

[0059] 1. Preparation of culture medium and stock solution

[0060] The LB medium formula is as follows: each liter of medium contains 5g of yeast extract, 10g of tryptone, and 5g of sodium chloride; when preparing solid medium, add 12–15g of agar and adjust the pH to 7.0. The medium is then autoclaved at 121°C for 20 minutes before use.

[0061] 2. Activation and scale-up of bacterial strains

[0062] On day 1, the frozen E. coli were inoculated onto LB agar plates for activation. After picking up the bacterial culture with an inoculation loop, the culture was sterilized by flame, then streak-coated in three zones and incubated overnight at 37°C.

[0063] On the second day, a single colony was picked from the plate and inoculated into 200 mL of LB liquid medium. Amp antibiotic was added at a ratio of 1000:1, i.e., 200 μL of antibiotic solution was added. The culture was shaken at 37°C and 200 rpm for 10–12 h to complete the seed culture expansion.

[0064] 3. Induced expression

[0065] On the third day, 20 mL of seed culture was inoculated into 500 mL of LB medium, and Amp antibiotic was added at a ratio of 1000:1, i.e., 500 μL of Amp antibiotic was added per 500 mL of medium. The culture was incubated at 37°C and 200 rpm with shaking for 2–3 hours. When the OD600 of the bacterial culture reached 0.4–0.6, IPTG was added to induce expression, bringing the final concentration to 0.5 mM. Induction was then continued overnight at 30°C and 200 rpm.

[0066] The 0.5 M I PTG stock solution is prepared fresh for immediate use: based on the relative molecular mass of IPTG of 238.31, weigh 1.1916 g of IPTG and add deionized water to bring the volume to 5 mL.

[0067] 4. Bacterial cell collection, lysis, and soluble protein extraction

[0068] On the fourth day, the induced bacterial culture was first centrifuged at 8000 rpm for 6 minutes. After discarding the supernatant, the bacterial cells were washed twice with PBS buffer. Then, PBS was added to thoroughly resuspend the bacterial cells by pipetting.

[0069] During lysis, add EDTA to the bacterial suspension to a final concentration of 1 mM, for example, add 100 μL of 1 M EDTA to 50 mL of bacterial suspension; then add lysozyme to a final concentration of 100 μg / mL, for example, add 100 μL of 50 mg / mL lysozyme to 50 mL of bacterial suspension. After mixing, incubate at 37°C for 1 h to promote cell wall disruption.

[0070] After incubation, the samples were placed in an ice-water bath and cell walls were disrupted using an ultrasonic cell disruptor with the following parameters: total duration 15 min, pulse on 10 s, off 5 s, and amplitude 80%. Subsequently, protein heat treatment was performed by heating the samples at 75°C for 20 min. Afterward, the samples were centrifuged at 4°C and 20,000 g for 20 min, and the supernatant was collected as the soluble protein fraction and filtered through a 0.22 μm filter membrane for later use.

[0071] 5. Protein purification

[0072] Protein purification was performed using an AKTA protein liquid chromatography system. Before system operation, the pump was placed in PBS buffer, the program was set to a flow rate of 5 mL / min, and the total elution volume was 1600 mL. Before loading the sample, the loading loop was cleaned, the residual liquid was drained, and the loop was rinsed with pure water and drained again. Then, 8 mL of crude protein was aspirated and injected into the loading loop, and the program was started for chromatographic separation. After the program was completed, the corresponding protein fractions were collected according to the elution peak shape. Based on experimental experience, the target peak of ferritin is mainly located in the 600–900 mL elution range. The collected target protein was concentrated using a concentration tube, for example, 10 mL of sample was aspirated into the concentration tube, centrifuged at 3200 g for 6 min, and the supernatant was collected. After all samples were concentrated, they were filtered through a 0.22 μm filter membrane.

[0073] 6. Desalting and buffer replacement

[0074] Desalting was performed using an AKTA protein liquid chromatography system. The program was set to a flow rate of 4 mL / min and a total elution volume of 120 mL, ensuring the column was connected as a desalting column. During the run, the pump was placed in 0.1 M NaCl solution. Before desalting, the system and desalting column were fully equilibrated with 0.1 M NaCl solution; simultaneously, the loading loop was cleaned by first draining the residual liquid, then rinsing with pure water, and then draining again to reduce cross-contamination. The concentrated and filtered sample was added to the loading loop and the program was started. During the run, the UV absorption peak and conductivity changes were observed in real time. The target protein fraction was collected based on the peak shape, and the salt-containing and small molecule impurity fractions in the initial stage were discarded. The collected target fraction could be further concentrated as needed, filtered through a 0.22 μm filter membrane, and stored for later use. This step is used to remove impurities and complete buffer replacement, providing suitable sample conditions for subsequent protein quantification and mineralization experiments.

[0075] 7. Protein concentration determination

[0076] The concentration of desalted protein samples was determined using the BCA method. The assay reagent was prepared at a ratio of solution A to solution B of 50:1, with 200 μL of working solution added to each well. Two to three parallel wells were prepared for each sample. 10 μL of appropriately diluted protein sample was added to each well, thoroughly mixed, and incubated in the dark for 30 min. The absorbance was then measured at 562 nm using a microplate reader to calculate the protein concentration.

[0077] Example 2: Preparation of biomimetic mineralization for the synthesis of magnetic ferritin

[0078] 1. Pretreatment of reactor vessels

[0079] Beforehand, immerse and clean the 100mL blue-capped bottle, 250mL blue-capped bottle, 100mL four-necked flask, 500mL four-necked flask, titrator, and stirring rotor separately in acid solution. The rotor should be immersed in 10% hydrochloric acid. Before the experiment, remove them and rinse thoroughly with water at least three times, then dry them in a drying oven. Prepare all necessary tape and waste container beforehand.

[0080] 2. Calculation of reaction parameters

[0081] The total reaction volume of this system is 250 mL, with a planned iron input of 3000 mg. The iron solution used is a mixed iron-manganese solution. The manganese content in the mixed iron-manganese solution is 5%. The ferrous ammonium sulfate hexahydrate solution and the manganese sulfate solution have the same concentration, and are therefore mixed at a volume ratio of 95:5. In the 250 mL reaction system with an iron input of 3000 mg, the volume of the metal salt mixed solution added, Vmetal = 14.881 mL; of which the volume of the ferrous ammonium sulfate hexahydrate solution is 14.137 mL, and the volume of the manganese sulfate solution is 0.744 mL. The volume of hydrogen peroxide added is the same as the volume of the metal salt mixed solution added, which is 14.881 mL. The addition rate is calculated using the formula v = Vmetal / (planned iron input / 40), therefore the addition rate of this system is 0.2 mL / min. This part is a pre-experiment parameter confirmation step and should be completed before the formal reaction.

[0082] 3. Solution deoxygenation treatment

[0083] Double-distilled water (ddH2O) and 0.1M NaCl solution were placed in rotor-equipped flasks, sealed, and deoxygenated by alternating vacuuming and argon gas introduction. Vacuuming was performed for 20 minutes, followed by multiple cycles of argon introduction and vacuuming to minimize the oxygen content in the system.

[0084] 4. Transfer the material to the anaerobic chamber.

[0085] The prepared reactor dishes, deoxygenated 0.1M NaCl and deionized water, and weighed reagents were transferred into the anaerobic chamber through the transition chamber. During the transfer, attention should be paid to sealing and vacuuming procedures to prevent dust or oxygen from entering the reaction system. Protein samples were transferred to the anaerobic chamber via a small compartment according to regulations.

[0086] Example 3: Biomimetic Mineralization Reaction

[0087] The purpose of the biomimetic mineralization reaction is to use the natural protein cage of HFn as a template to construct a manganese-containing magnetic iron oxide core in situ within its lumen, thereby preparing magnetic ferritin that combines the structural integrity of the protein cage with magnetic properties. The final product obtained through this reaction is magnetic ferritin MHFn containing 5% manganese, which can serve as a basic material for subsequent targeted coupling and MRI imaging evaluation.

[0088] 1. Preparation of the reaction stock solution

[0089] In an anaerobic chamber, stock solutions of ferrous ammonium sulfate hexahydrate, manganese sulfate, hydrogen peroxide, and sodium hydroxide were prepared using deoxygenated water. The specific concentrations were: 200 mL of 32.33 mM hydrogen peroxide; 250 mL of 200 mM sodium hydroxide; 200 mL of 100 mM ferrous ammonium sulfate hexahydrate; and 50 mL of 100 mM manganese sulfate. During preparation, a portion of deoxygenated water was added first to fully dissolve the chemicals, then transferred to the corresponding containers. The final volume was made up, mixed thoroughly, and clearly labeled. Ferrous ammonium sulfate hexahydrate and manganese sulfate were prepared using a premixing method to form a metal salt mixture, with a manganese content of 5%. The specific preparation method was as follows: First, ferrous ammonium sulfate hexahydrate and manganese sulfate solutions of the same concentration were prepared separately. Then, they were mixed at a volume ratio of 95:5, i.e., 95 parts by volume of ferrous ammonium sulfate hexahydrate solution were mixed with 5 parts by volume of manganese sulfate solution, and this mixture was used as the metal salt mixture for subsequent addition. Solutions such as hydrogen peroxide should be sealed promptly after opening to minimize their impact on the anaerobic environment.

[0090] 2. Preparation of the reaction system

[0091] According to the experimental design, the protein solution was mixed with 0.1M NaCl to prepare the reaction system. The standard total volume of the system is 250 mL, but some experiments use a 50 mL system. The protein concentration should be greater than 10 mg / mL to meet the requirements of subsequent mineralization.

[0092] 3. Reaction temperature and stirring conditions settings

[0093] Place the thermometer below the surface of the reaction solution and adjust the heating base and temperature control parameters to maintain a stable reaction solution temperature of approximately 65°C. Adjust the magnetic stirring speed according to the system volume to ensure uniform mixing and stable rotor operation. Initially, the base temperature can be appropriately increased, then reduced to a suitable range once the target temperature is approached.

[0094] 4. Titration pump cleaning and preparation

[0095] Remove the pretreated diffuser from the titration system, rinse it repeatedly with oxygen-free water, and then install it onto the titration head. After connecting the waste liquid tank, follow the software operation procedure to complete the following steps in sequence: emptying, rinsing with oxygen-free water, adding a fixed volume of liquid, emptying again, and aspirating the reaction stock solution, to ensure that the titration system is clean, unobstructed, and free of residue.

[0096] 5. pH meter calibration

[0097] The pH electrode was calibrated at two points using a standard buffer solution. After calibration, the electrode sensitivity was checked by measuring the pH value of the standard solution, and the slope in the calibration report was verified to meet the requirements. Once calibration was successful, the pH electrode was placed in the reaction system for later use.

[0098] 6. Reaction program check and setup

[0099] Retrieve the corresponding experimental method in the software and check and confirm that the parameters such as the added volume of ferrous ions and hydrogen peroxide, and the addition rate are set correctly. In a 250ml system, the volume of ferrous ions is 14.881ml, the volume of hydrogen peroxide is 14.881ml, and the addition rate is 0.2ml / min. After saving the method, check the program parameters again before the actual run.

[0100] 7. Initiate the mineralization reaction

[0101] Initiate heating and set the reaction temperature to 65℃. Simultaneously, adjust the pH of the protein solution to 8.5 using a pH titrator with 200mM sodium hydroxide solution. Once the total reaction volume is 250mL and the temperature has stabilized at 65℃, measure and record the initial pH value (approximately 8.5). Then, start the reaction program. During the reaction, continuously monitor the system temperature to ensure it remains at 65℃ and the pH remains stable around 8.5. Check the titration system, injection tubing, and reaction apparatus connections for leaks and ensure the instrument and computer program are functioning correctly. If the reaction is unexpectedly interrupted, immediately record the volumes of metal salt mixture, hydrogen peroxide, and sodium hydroxide added at the time of interruption. Adjust the program parameters based on the remaining volume to be added and the predetermined addition rate before restarting the experiment.

[0102] 8. Reaction termination and post-treatment

[0103] After the reaction procedure is completed, continue timing for 10 minutes to ensure that the mineralization reaction is fully completed. Then record the volume of sodium hydroxide consumed, stop heating but keep stirring, remove the titrator from the system, and add the specified volume (300 μL per 50 ml reaction volume) of 0.3 M trisodium citrate. Mix well and measure and record the pH value again. Then turn off the stirring and carefully remove the reaction solution.

[0104] 9. Cooling, centrifugation and concentration

[0105] Place the removed reaction flask in an ice bath to cool it down. Once the temperature has dropped to approximately 40°C or below, transfer it to a centrifuge tube for centrifugation. Recommended centrifugation parameters: 19000g, 20min. After centrifugation, carefully discard the supernatant, retain the sample as required, and concentrate the remaining sample to below the specified volume. After concentration, filter the sample sterilely and store it for later use.

[0106] Example 4: Molecular sieve separation and purification

[0107] 1. Pretreatment before column placement

[0108] After the mineralization reaction is complete, the obtained sample is first filtered (using a 0.22 μm filter membrane) to remove any particulate impurities and insoluble aggregates that may be present in the system, preventing them from clogging the molecular sieve column or affecting the separation effect after entering the chromatography system. The filtered sample is then used as the sample to be purified.

[0109] 2. Molecular sieve column equilibration

[0110] The AKTA protein liquid chromatography system was used for molecular sieve separation and purification. Before sample loading, the molecular sieve column was fully equilibrated with PBS buffer to ensure that the internal environment of the column was consistent with the sample system, thereby improving the stability and repeatability of the separation process. The equilibration method of the molecular sieve column was consistent with the purification steps before mineralization.

[0111] 3. Sample loading and chromatographic separation

[0112] Pretreated mineralized samples were loaded in 8 mL increments. Before loading, the loading loop was cleaned, drained of residual liquid, rinsed with pure water, and then emptied again to reduce cross-contamination and ensure injection accuracy. The sample was then injected into the loading loop, and the chromatography program was started for separation. During chromatography, PBS buffer was used as the mobile phase, the flow rate was set to 5 mL / min, and the total elution volume was set to 1600 mL. These operating conditions were consistent with the purification steps before mineralization.

[0113] 4. Collection of target components

[0114] During the chromatographic separation process, each eluted component is identified based on the shape of the chromatographic elution peaks, and the elution peak corresponding to the target magnetic ferritin is manually identified. After chromatography, the eluted components identified as the target product are collected and combined separately, serving as the target sample for subsequent concentration processing.

[0115] 5. Sample Concentration

[0116] The combined target components were added to a concentration tube with a molecular weight cutoff of 10,000 for concentration. The concentration conditions were consistent with the purification steps before mineralization, i.e., centrifugation at 3200g for 6 min, and the concentration operation was repeated according to the sample volume until the target volume requirement was reached. Specifically, samples from a 250mL mineralization system were concentrated to less than 8mL, and samples from a 50mL mineralization system were concentrated to less than 2mL.

[0117] 6. Post-concentration processing and preservation

[0118] After concentration, the sample was filtered again to further remove any possible particles and ensure sample cleanliness. The purified magnetic ferritin sample was then labeled and stored at 4°C for later use.

[0119] 7. Molecular sieve column cleaning and preservation

[0120] After molecular sieve separation and purification, the chromatography system and molecular sieve column are cleaned and stored. The specific operation method is consistent with the purification steps before mineralization to ensure the stability of the chromatography column performance and meet the requirements for subsequent reuse.

[0121] 8. Product characterization and result analysis

[0122] To verify whether the above-mentioned biomimetic mineralization reaction and subsequent purification process successfully obtained the magnetic ferritin product, the purified sample was analyzed by polyacrylamide gel electrophoresis, particle size analysis, and transmission electron microscopy. The results are as follows: Figure 1 As shown. Among them, Figure 1 A represents the electrophoresis result. (From...) Figure 1 As can be seen from A, both the samples before and after mineralization showed obvious protein bands at the corresponding positions, indicating that the main protein structure of ferritin still exists after biomimetic mineralization and subsequent purification. Figure 1 B represents the hydrated particle size distribution. Figure 1 As can be seen from B, the particle size distribution of both HFn and HFn(Mn5%) is relatively concentrated, mainly distributed in the range of about 10 to 20 nm. Moreover, the mineralized HFn(Mn5%) did not show a significant abnormal increase or broad peak distribution compared with the unmineralized HFn, indicating that the magnetic ferritin sample obtained after mineralization has good dispersibility and no obvious aggregation. It also suggests that the protein cage structure remains stable after mineralization. Figure 1 C represents the results from transmission electron microscopy. (From...) Figure 1 C-ray fluorescence (C-FR) reveals well-dispersed nanoparticles with high electron density in the mineralized sample, indicating the successful formation of a magnetic core within the ferritin protein cage. Combined with the introduction of manganese into the aforementioned mineralization system, this demonstrates the successful preparation of a magnetic ferritin product with a manganese-containing magnetic mineralized core.

[0123] The above results demonstrate that the biomimetic mineralization reaction and purification method described in this embodiment can successfully construct a magnetic nanocore within the ferritin shell while maintaining the basic integrity of the ferritin protein shell structure and good sample dispersibility, thereby obtaining the target magnetic ferritin sample. This result proves the feasibility of the method of the present invention and its ability to stably prepare magnetic ferritin products with intact structure, uniform particle size, and mineralized cores.

[0124] Example 5, Z HER2:342 Recombinant construction, expression and purification of affinity proteins

[0125] 1. Design of target protein and recombinant expression framework

[0126] This section provides a Z-type implementation method. HER2:342A method for prokaryotic recombinant expression and purification of the affinity protein. The target protein is an affinity protein that specifically recognizes HER2, and its amino acid sequence is as follows:

[0127] VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQAPK(SEQ IDNo.2);

[0128] To facilitate efficient expression and subsequent affinity purification in prokaryotic systems, a glutathione S-transferase (GST) tag was introduced at the N-terminus of the target protein, and the GST tag was then linked to a Z-linked platelet-binding polymerase (Z-L). HER2:342 A thrombin recognition cleavage site is established between the affinity proteins to form a fusion expression framework that can be enzymatically cleaved and detagged. Preferably, the amino acid sequence of the recombinant fusion protein is as follows:

[0129] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYL NGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKLVPRGSVDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQAPK* (SEQ ID No.3);

[0130] The first segment is a GST tag sequence, the middle segment contains a thrombin cleavage site, and the last segment is a Z-type tag sequence. HER2:342 Affinity protein sequence. This design allows for efficient affinity purification of the fusion protein using a GST tag, followed by thrombin digestion to obtain the tag-free Z-type protein. HER2:342 Target protein.

[0131] 2. Construction of encoding nucleic acid sequences and recombinant vectors

[0132] Encoding the GST-thrombin cleavage site-Z HER2:342 The nucleic acid sequence of the fusion protein is shown in SEQ ID NO:4:

[0133] ;

[0134] The above coding sequence was inserted into the pET22b expression vector to obtain the recombinant expression plasmid. Construction methods can employ conventional molecular cloning techniques in the field, including but not limited to enzyme digestion and ligation, homologous recombination, or seamless cloning. After obtaining the recombinant plasmid, sequencing verification confirmed that the inserted fragment sequence was correct, the reading frame was accurate, and it was consistent with the expression elements of the vector. It is then ready for subsequent transformation and expression.

[0135] 3. Construction of engineered bacteria and seed culture

[0136] The recombinant expression plasmid, which was confirmed to be correct by sequencing, was transformed into BL21 Escherichia coli competent cells, plated on LB solid medium plates containing ampicillin (Amp), and incubated overnight at 37°C. Positive single colonies were obtained by screening.

[0137] Positive single colonies were picked and inoculated into LB liquid medium containing Amp, and seed culture was carried out according to the aforementioned E. coli shake culture conditions. The type and final concentration of Amp used were consistent with the aforementioned E. coli shake culture expression steps. The seed culture was obtained by shaking culture at 37°C and 200 rpm for 10–12 h.

[0138] 4. Induced expression

[0139] The seed culture was inoculated into LB liquid medium containing Amp using the aforementioned expansion culture method, and cultured with shaking at 37°C and 200 rpm. When the bacterial culture reached the logarithmic growth phase, i.e., OD600 of 0.4–0.6, IPTG was added to induce expression. The final IPTG concentration was 0.5 mM, and the induction temperature was 16°C. Induction was continued overnight with shaking at this temperature.

[0140] 5. Bacterial cell collection and lysis

[0141] After induction, the bacterial cells were collected according to the conditions described in the previous E. coli shaker culture expression steps. The bacterial culture was centrifuged, the supernatant was discarded, and the cells were washed twice with PBS buffer and resuspended.

[0142] The bacterial cells were then lysed using the aforementioned lysis method. EDTA and lysozyme were added to the bacterial suspension, and the cells were lysed using ultrasonication after incubation at 37°C. After lysis, the cells were centrifuged at 20,000g for 20 minutes at 4°C, and the supernatant was collected as the soluble protein fraction. Unlike the ferritin purification described above, this embodiment does not perform heating to remove impurities, in order to avoid affecting Z. HER2:342 The conformational stability of the fusion protein and its subsequent GST affinity binding were adversely affected. Finally, the supernatant was filtered through a 0.22 μm filter membrane and used for later use.

[0143] 6. GST affinity purification

[0144] The clarified and filtered soluble protein supernatant was loaded into a GST affinity chromatography system for purification. The chromatography column used was a pre-packed GST affinity column.

[0145] The equilibration buffer consisted of 25 mM Tris and 0.1 M NaCl. The column was thoroughly equilibrated with this buffer before sample loading to establish a stable binding environment. After sample loading, the GST tag specifically binds to the column medium, and contaminating proteins are removed via flow-through.

[0146] After washing, the fusion protein was eluted using an elution buffer containing reduced glutathione. The elution buffer was a 10 mM reduced glutathione solution. The GST-tagged fusion protein could be obtained through competitive elution with reduced glutathione.

[0147] 7. Thrombin removal of GST tag

[0148] To obtain Z without GST tags HER2:342 Affinity protein, for the eluted GST-Z HER2:342 The fusion protein was digested with thrombin.

[0149] The GST elution fraction was first collected and then subjected to enzymatic digestion. Thrombin was added at a ratio of approximately 1 U thrombin / 1 mg fusion protein; when it is necessary to improve the digestion efficiency, the amount of enzyme can be appropriately increased, and the optimization should be combined with substrate concentration, buffer composition and digestion time.

[0150] The enzyme digestion temperature was 4℃, and the digestion time was overnight. Using low-temperature, long-duration enzyme digestion helps to achieve more complete tag removal while maintaining the stability of the target protein.

[0151] 8. Buffer replacement after enzyme digestion and secondary GST purification

[0152] After thrombin digestion, the sample was concentrated using ultrafiltration and the buffer was replaced to convert the system to Tris-NaCl buffer. Ultrafiltration centrifuge tubes with a molecular weight cutoff of 3000 Da were used for centrifugation and concentration at 3200g, followed by buffer replacement.

[0153] After buffer replacement, the digestion product was passed through the GST affinity column again. This was due to the presence of free GST tags and uncut GST-Z. HER2:342 The fusion protein can still bind to the GST column, while the Z-column with the GST tag removed... HER2:342 The target protein no longer binds specifically to the column medium, so the target protein can be effectively separated from the GST tag and uncleaved substrate by passing it through the column again, thereby improving the purity of the final product.

[0154] 9. Final concentration, buffer replacement and preservation

[0155] The flow-through fraction containing the target protein obtained from the second GST purification was collected and concentrated again using an ultrafiltration centrifuge tube with a molecular weight cutoff of 3000 Da. The buffer system was then replaced with PBS. The ultrafiltration centrifugation conditions were 3200 g.

[0156] After concentration and liquid replacement, purified Z was obtained. HER2:342 Affinity protein sample. The protein sample was stored at 4°C for later use.

[0157] 10. GST-Z HER2:342 Protein identification

[0158] like Figure 2 As shown, SDS-PAGE analysis was performed on samples obtained at different stages of expression, purification, and enzyme digestion to evaluate GST-Z. HER2:342 Expression of the fusion protein, GST tag excision effect, and purity of the final product. Figure 2 A shows that a clear target protein band appeared in the sample after induced expression, indicating that GST-Z HER2:342 The fusion protein has been successfully expressed; after GST affinity purification, the target band was significantly enriched, and new bands with smaller molecular weights were observed after enzyme digestion, indicating that the GST tag has been effectively removed. Figure 2 B shows that after subsequent purification, a single and clear low molecular weight band was obtained, corresponding to Z. HER2:342 The presence of affinity proteins indicates that the final product has high purity. These results demonstrate that the prokaryotic expression, GST affinity purification, enzyme cleavage detacking, and subsequent purification processes employed in this embodiment can effectively obtain the target Z. HER2:342 Affinity proteins provide reliable materials for subsequent coupling and functional evaluation.

[0159] Example 6: Mineralized ferritin and Z HER2:342 connection

[0160] Under the influence of EDC / NHS, Z HER2:342 The surface carboxyl groups can be activated to form an active ester intermediate, which then undergoes a condensation reaction with the amino groups on the surface of magnetic ferritin to form a stable amide bond, thereby achieving covalent linkage between the two. This linkage method features mild reaction conditions, high coupling efficiency, and good product stability. Since the mineralization core is located in the lumen of the ferritin, the coupling activity of the outer surface is not significantly lost due to mineralization, thus ensuring that the ferritin can still further bind with Z-type magnets after mineralization. HER2:342 connect.

[0161] 1. Closed Z HER2:342 Amino

[0162] Measure 10mgZ HER2:342Add 11 mg DMA and 500 μL water, and stir to mix well. Then add 0.2 M NaOH to adjust the pH of the reaction system to approximately 9, and continuously monitor and adjust the pH until it stabilizes. After the system stabilizes, the reaction solution should be clear and transparent, without obvious turbidity. Continue stirring overnight to obtain the Z-blocked amino group. HER2:342 Reaction solution.

[0163] 2. Z HER2:342 Activation of the carboxyl group

[0164] Add 4.5 mg EDC-HCl and 3 mg NHS to the above reaction solution to adjust the pH of the reaction system to approximately 7. After adjustment, continue stirring for 30 min to obtain the activated Z. HER2:342 Solution.

[0165] 3. Mineralized ferritin and activated Z-10 HER2:342 connection

[0166] Take the mineralized ferritin solution, dilute it with an appropriate amount of water, and adjust the pH of the system to approximately 8.5. Then add the activated Z... HER2:342 The solution was stirred at room temperature for 2 hours to complete mineralization, after which ferritin and Z were reacted. HER2:342 The connection forms mineralized ferritin-Z. HER2:342 Coupled object (i.e. Z) HER2:342 -MHFn). After the ligation reaction is complete, it can be used directly for subsequent detection.

[0167] 4. SDS-PAGE detection of the ligation products

[0168] Mineralized ferritin (MHFn) and its relationship with Z HER2:342 The connection product (Z) HER2:342 SDS-PAGE analysis was performed on MHFn, and the results are as follows: Figure 3 As shown in Figure A. As can be seen from the figure, the connecting product ( Figure 3 MHFn+HER2 in A and mineralized ferritin ( Figure 3 Compared to MHFn in A, its band position shifts slightly upward. Because Z HER2:342 Attachment to the surface of mineralized ferritin increases the molecular weight of the complex, thus reducing electrophoretic mobility and resulting in an upward shift of the band. These results indicate that Z... HER2:342 It has been successfully attached to the surface of mineralized ferritin to form mHFn-Z HER2:342 Coupling product.

[0169] 5. Detection of hydrated particle size of the bonding products

[0170] Take HFn, mineralized ferritin (MHFn), and the conjugation product (MHFn-Z). HER2:342 The hydrated particle size was measured, and the results are as follows: Figure 3 As shown in Figure B, the hydrated particle size distribution of HFn and MHFn is relatively concentrated, with the particle size mainly distributed within a small range; while the fusion product MHFn-Z... HER2:342 The hydrated particle size increased significantly, and the particle size distribution range widened. This result indicates that Z-linked ferritin particles are attached to the surface of the mineralized ferritin. HER2:342 Subsequently, the hydrated size of the sample increased, indicating that Z HER2:342 It has been successfully modified on the surface of mineralized ferritin to form a coupling complex with a particle size larger than that of the mineralized ferritin itself.

[0171] Example 7: Evaluation of Material Targeting Effect

[0172] 1. Preparation of fluorescent labeling materials

[0173] To evaluate Z HER2:342 To assess the targeted uptake effect of MHFn, the test material was first fluorescently labeled. Using PBS as a buffer, 1 mL of protein solution (protein concentration 10 mg / mL) was added to 100 μL of fluorescent dye solution (dye concentration 1 mg / mL). The mixture was incubated for 8 hours on a rotary mixer to allow the fluorescent dye to fully bind to the protein material. After incubation, the sample was purified and concentrated using ultrafiltration. The sample was washed twice with PBS to remove unbound free dye, and finally concentrated to 1 mL to obtain the fluorescently labeled material solution.

[0174] 2. Cell Model and Plating

[0175] HER2-positive breast cancer cells SK-BR-3 and HER2-negative breast cancer cells MDA-MB-231 were selected as cell models to evaluate the differences in uptake of cells with different HER2 expression levels. SK-BR-3 cells are a human breast cancer cell line with high HER2 expression and are commonly used as a research model for HER2-positive breast cancer; MDA-MB-231 cells are a human breast cancer cell line with low or no HER2 expression and strong invasiveness, and are commonly used as a research model for HER2-negative breast cancer. Both cell lines showed high levels of TfR1 expression. One to two days before the experiment, both cell lines were seeded into 6-well plates for culture. After the cells adhered well and showed stable growth, they were used for subsequent incubation experiments.

[0176] 3. Cell incubation experiment design

[0177] The fluorescently labeled material was diluted to 0.5 mg / mL with complete culture medium and added to SK-BR-3 and MDA-MB-231 cells after plating, respectively, and incubated at 37°C in the dark. Three time points (1 h, 2 h, and 3 h) were set to evaluate cell uptake and enrichment of the material under different incubation durations.

[0178] 4. Cell processing before flow cytometry detection

[0179] After the predetermined incubation time, cell processing was performed uniformly. First, the culture medium containing the material was aspirated; then, PBS was added to wash the cells, and after standing for 1-2 minutes, the medium was aspirated. This washing process was repeated 3 times to remove unbound or untaken free material. After washing, trypsin was added to digest the cells, and the digestion time was controlled to avoid over-digestion affecting the cell state. The digested cells were collected by centrifugation, washed once more with PBS, and finally resuspended in 500 μL of PBS to obtain cell samples ready for analysis.

[0180] 5. Flow cytometry detection

[0181] The treated cell samples were analyzed by flow cytometry, and the uptake and enrichment of the material by cells were analyzed by changes in fluorescence signal. The fluorescence signal differences between the HER2-positive and HER2-negative cell groups at the same incubation time were compared, as were the fluorescence signal changes of the same cell group at different incubation times, to assess the time-dependent uptake behavior of the material in cells and its targeted enrichment ability on HER2-positive cells.

[0182] 6. Result Judgment Method

[0183] First, the changes in the position and intensity of the fluorescence peaks of the cell population were compared under different incubation times. If the overall fluorescence peak shifted towards the high fluorescence region with prolonged incubation time, it indicates that the material could be gradually bound or endocytosed by cells. Second, the differences in fluorescence distribution and quantitative results between HER2-positive and HER2-negative cells were compared. If the fluorescence signal and corresponding cellular uptake in the HER2-positive cell group were higher than those in the HER2-negative cell group, it indicates that the material was transported via Z-cell incubation. HER2:342 It has a specific recognition of HER2 and a stronger binding and endocytic capacity for HER2-positive cells.

[0184] Simultaneously, since the material uses ferritin as a carrier, and ferritin can recognize transferrin receptor 1 (TfR1) on the cell surface, the material simultaneously possesses TfR1-targeting and HER2-targeting properties. When the material exhibits a certain uptake capacity in both types of cell types, but with stronger uptake in HER2-positive cells, it indicates that the material possesses both ferritin-mediated basal uptake capacity and Z-cell uptake capacity. HER2:342 The enhanced HER2 recognition capability is thus demonstrated, exhibiting the dual-targeting characteristics of TfR1 / HER2.

[0185] 7. Results Analysis

[0186] like Figure 4As shown, in MDA-MB-231 and SK-BR-3 cells, as the incubation time increased from 0 h to 1 h, 2 h, and 3 h, the cell fluorescence distribution gradually shifted towards the high fluorescence intensity region, indicating that the material Z... HER2:342 -MHFn can be recognized and taken up by both types of cells, and this process is time-dependent. MDA-MB-231 cells, although HER2-negative, still exhibited some material uptake capacity, indicating that in addition to HER2-mediated action, the material can also enter cells through the recognition and binding of ferritin to transferrin receptor 1 (TfR1) on the cell surface. Because ferritin itself has the property of binding to TfR1, material Z... HER2:342 -MHFn can still bind and endocytose via a TfR1-mediated pathway.

[0187] Further comparison revealed that, under the same incubation time, the fluorescence signal of SK-BR-3 cells was generally higher than that of MDA-MB-231 cells, suggesting that HER2-positive cells had a stronger uptake capacity for the material. This indicates that, in addition to the basal uptake of TfR1 mediated by ferritin, the ZHER2:342 on the surface of the material can further enhance the enrichment and endocytosis of the material in HER2-positive cells through specific binding to HER2.

[0188] like Figure 5 As shown, at 1h, 2h, and 3h time points, the flow cytometry fluorescence peaks of SK-BR-3 cells were significantly shifted to the right compared to MDA-MB-231 cells, indicating that SK-BR-3 cells bind to and endocytose material Z. HER2:342 -MHFn is more; quantitative statistical results also show that the cell uptake of the SK-BR-3 cell group was higher than that of the MDA-MB-231 cell group at all time points, and the difference was more obvious at 3h. The above results indicate that the material Z of the present invention HER2:342 -MHFn can achieve universal recognition and uptake of cells through the interaction between ferritin and TfR1. On the other hand, it can enhance the enrichment of HER2 in HER2-positive cells through the specific binding of ZHER2:342 to HER2. Therefore, this material belongs to a TfR1 / HER2 dual-targeting material and shows better targeting recognition and uptake ability for HER2-positive breast cancer cells.

[0189] Example 8: Detection of magnetic resonance relaxation properties of dual-targeted magnetic ferritin probes

[0190] To evaluate the magnetic resonance imaging potential of the prepared dual-targeting magnetic ferritin probe, Z... HER2:342 The longitudinal and transverse relaxation rates of the MHFn sample were measured, and MHFn was used as a control sample for comparison.

[0191] A series of samples with total metal concentrations of 0.8 mM, 0.4 mM, 0.2 mM, 0.1 mM and 0.05 mM were prepared by gradient dilution using PBS as solvent for subsequent NMR relaxation performance testing.

[0192] The T1 relaxation time and T2 relaxation time of each concentration sample were measured separately. A linear fit was then performed with sample concentration as the x-axis and 1 / T1 or 1 / T2 as the y-axis to obtain the longitudinal relaxation rate r1 and the transverse relaxation rate r2 of the sample. The results are as follows: Figure 6 As shown, the relaxation rate of the sample exhibits a good linear relationship with increasing concentration, indicating that the Z-type material... HER2:342 -MHFn exhibits relatively stable magnetic resonance response characteristics.

[0193] According to the linear fitting results, the longitudinal relaxation rates r1 of the dual-targeting magnetic ferritin probe and magnetic ferritin are 2.886 mM. -1 ·s -1 and 2.114 mM -1 ·s - The transverse relaxation rate r2 was 39.905 mM. -1 ·s -1 and 36.737mM -1 ·s -1 The above results demonstrate that the aforementioned biomimetic mineralization process and Z... HER2:342 The coupling process did not significantly weaken the magnetic response performance of the material, and the resulting dual-targeting magnetic ferritin probe maintained good magnetic resonance relaxation characteristics. This material combines ferritin-mediated TfR1 targeting capability with Z-targeting capability. HER2:342 The HER2-targeting recognition capability imparted by this invention indicates that the material constructed in this invention is a protein nanoprobe that combines TfR1 / HER2 dual-targeting characteristics with magnetic resonance imaging potential, and can be used for subsequent tumor-targeted imaging research.

Claims

1. A coupled magnetic ferritin, characterized in that, The magnetic ferritin is synthesized ferritin and then subjected to biomimetic mineralization to form nanoparticles inside the ferritin. Finally, it is separated and purified to obtain metal-containing magnetic ferritin. The magnetic ferritin is conjugated with affinity molecules, nanobodies, or specific peptides.

2. The coupled magnetic ferritin according to claim 1, characterized in that, The affinity protein is a specific HER2-recognizing affinity protein, and the amino acid sequence of the affinity protein is shown in SEQ ID No.

2.

3. The coupled magnetic ferritin according to claim 1, characterized in that, The metal is manganese.

4. The method for preparing the coupled magnetic ferritin according to any one of claims 1-3, characterized in that, The method includes the following steps: 1) Using recombinant human ferritin as a template, the DNA sequence of human ferritin was cloned and constructed into a plasmid; 2) Transform or co-transform bacteria with recombinant plasmids containing human ferritin, add isopropyl-β-D-thiogalactoside, and induce expression; 3) After expression, the protein is broken down by sonication to release it; 4) Separate and purify the protein; 5) Add iron salt solution containing metal ions and oxidant to the ferritin solution to react, control the pH value to 7-11, control the temperature to 25-90℃, and form nanoparticles inside the ferritin. 6) After purification, a metal-containing magnetic ferritin was obtained; 7) Magnetic ferritin is conjugated with affinity molecules, nanobodies, or specific peptides.

5. The method according to claim 4, characterized in that, The coupling includes the following steps: After the carboxyl group of the affinity group is activated, it undergoes a condensation reaction with the amino group of the magnetic ferritin to form an amide bond.

6. The method according to claim 4, characterized in that, The coupling includes the following steps: (a) Amino blocking of the affinity group; (b) Activation of the carboxyl group in the affinity group; (c) When the carboxyl-activated affinity comes into contact with magnetic ferritin, the carboxyl group of the affinity reacts with the amino group of the magnetic ferritin to form an amide bond.

7. A protein nanoprobe or nuclear magnetic resonance contrast agent, characterized in that, The protein nanoprobe or nuclear magnetic resonance contrast agent contains the coupled magnetic ferritin as described in any one of claims 1-3, or is obtained by the method described in any one of claims 4-6.

8. The use of the coupled magnetic ferritin according to any one of claims 1-3 in the preparation of magnetic contrast agents or molecular probes for diagnosing tumors.

9. The application according to claim 8, characterized in that, The tumor is a HER2-expressing tumor.

10. The application according to claim 8, characterized in that, The magnetic contrast agent is a nuclear magnetic resonance contrast agent.

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