An anti-her2 nanobody-targeted ferritin nanodrug carrier, a preparation method and application thereof

Abstract

This invention discloses a ferritin nanomedicine carrier targeting an anti-HER2 nanobody, its preparation method, and its application. The invention constructs an active targeting nanocarrier based on the SpyTag / SpyCatcher system. This carrier is formed by the self-assembly of human heavy chain ferritin and ferritin fused to express SpyCatcher to form a hybrid ferritin polymer, which is then covalently coupled with an anti-HER2 nanobody (2Rs15d) via SpyTag / SpyCatcher. This invention utilizes genetic engineering technology to achieve efficient expression of the nanobody without chemical modification, resulting in good product uniformity; efficient drug encapsulation can be achieved through pH control. The obtained nanomedicine carrier encapsulated with doxorubicin showed an effect on the IC50 of SKBR3 cells. 50 With a concentration of 2.14 nM, it exhibits significant targeted killing effects. This invention solves the problem of targeted ferritin delivery and has important application value in the treatment of HER2-positive tumors.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a ferritin nanomedicine carrier targeting anti-HER2 nanobody, its preparation method, and its application. Background Technology

[0002] Antibody-drug conjugates (ADCs) have made significant clinical progress in the field of targeted cancer therapy, but key technological bottlenecks still exist in their structural design and preparation processes. Traditional ADCs require multiple complex steps, including antibody engineering (such as cysteine ​​residue mutation), linker activation, and drug conjugation, resulting in cumbersome preparation processes and large batch-to-batch variations. Due to the limited number of modifiable sites on antibody molecules, the drug-to-antibody ratio (DAR) is generally only 4-8, making it difficult to achieve efficient drug loading. Furthermore, full-size antibodies (~150 kDa) have a large molecular weight, limiting their ability to penetrate tumor tissue and making it difficult to reach deep into solid tumors.

[0003] Among existing alternative strategies, while nanobody-drug conjugates improve tissue penetration due to their small molecular weight, their drug loading capacity is further reduced, making efficient drug delivery difficult. Ferritin drug delivery systems utilize their cage-like structure to enhance drug loading capacity, but lack active targeting capabilities, relying solely on the tumor EPR effect for passive enrichment, resulting in limited targeting precision. While SpyTag / SpyCatcher conjugation systems offer efficient site-specific conjugation, current technologies primarily use them for simple linking of antibodies or nanobodies to carriers, failing to simultaneously address issues such as insufficient drug loading efficiency, poor carrier uniformity, and uncontrollable ratio of target molecules to carriers. Furthermore, most reported ferritin nanocarriers employ chemical cross-linking to modify the targeting ligand, which not only involves harsh reaction conditions leading to target molecule inactivation but also results in highly heterogeneous products, making batch-to-batch stable reproducibility difficult. Therefore, developing an active targeting nanocarrier that combines high drug loading capacity, strong tumor penetration, high conjugation uniformity, and requires no chemical modification is a pressing technical challenge in this field. Summary of the Invention

[0004] This invention aims to provide a heteroferritin-targeted drug-loaded nanoparticle modified with an anti-HER2 nanobody and its preparation method, to address the problems of low drug loading, poor tumor penetration, complex preparation processes, and insufficient product uniformity in existing technologies. Specifically, this invention relates to an active targeted nanodelivery system based on the SpyTag / SpyCatcher molecular conjugation system, heteroferritin self-assembly technology, and the anti-HER2 nanobody 2Rs15d.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A ferritin nanomedicine carrier targeting an anti-HER2 nanobody, the carrier being composed of the following three synergistic components: (1) Targeted functional module: is a gene-expressible fusion protein 2Rs15d-SpyTag (2Rs15d-ST), which contains an anti-HER2 nanobody 2Rs15d (amino acid sequence SEQ ID NO:1), a flexible linker peptide-GGGS, a SpyTag peptide (amino acid sequence SEQ ID NO:3), and a His purified tag (amino acid sequence SEQ ID NO:7).

[0006] (2) Drug carrier module: It is a hybrid ferritin polymer formed by the self-assembly of human heavy chain ferritin (HFn) and modified ferritin (SC-HFn) in a specific molar ratio, wherein SC-HFn is composed of SpyCatcher protein (amino acid sequence SEQ ID NO:5), flexible linker peptide II (amino acid sequence SEQ ID NO:6), human heavy chain ferritin HFn (amino acid sequence SEQ ID NO:4) and His purification tag (amino acid sequence SEQ ID NO:7); The anti-HER2 nanobody 2Rs15d is attached to the surface of the hybrid ferritin polymer through covalent coupling between the SpyTag of the targeting functional module and the SpyCatcher of the drug carrier module. (3) Drug loading: The pH response characteristics of the ferritin cage structure are used to achieve efficient loading of antitumor drugs.

[0007] The ferritin nanomedicine carrier is internally loaded with an antitumor drug, which is selected from one or more of macrolides, anthracyclines, taxanes or camptothecins. The drug encapsulation efficiency of the ferritin nanomedicine carrier is 10.5%-12.8%, and the drug loading is 5.8%-7.2%.

[0008] Furthermore, the antitumor drug is doxorubicin.

[0009] The preparation method of the ferritin nanomedicine carrier targeting the anti-HER2 nanobody includes the following steps: Step 1: Expression and purification of the fusion protein A fusion protein containing the anti-HER2 nanobody 2Rs15d and SpyTag was prepared, denoted as 2Rs15d-ST; A fusion protein containing SpyCatcher and human heavy chain ferritin HFn was prepared and denoted as SC-HFn.

[0010] Recombinant expression plasmids 2Rs15d-ST and SC-HFn were constructed and transformed into an E. coli expression system. After induction of expression, the two fusion proteins were purified by nickel column affinity chromatography.

[0011] Step 2: Self-assembly of heteroferritin polymers The purified HFn was mixed with SC-HFn and incubated in a buffer system with pH 7.4-8.0. The self-assembly properties of ferritin were used to form a hybrid polymer, which was then purified by size exclusion chromatography to remove unassembled monomeric proteins.

[0012] Step 3: Drug Encapsulation The antitumor drug was co-incubated with the heteroferritin polymer obtained in step two, and the drug was efficiently encapsulated by pH control. The free drug was then removed by dialysis or ultrafiltration to obtain heteroferritin drug-loaded nanoparticles.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Innovative Targeted Modification: For the first time, the SpyTag / SpyCatcher bioorthogonal system is introduced into the anti-HER2 nanobody-ferritin complex system, achieving precise covalent coupling between the target molecule and the drug delivery carrier, avoiding the complex operations of traditional chemical activation. Coupling efficiency >95%, product heterogeneity <5%, significantly improving product uniformity and process controllability.

[0014] 2. Innovative Carrier Structure: An SC-HFn / HFn hybrid self-assembling polymer was constructed, overcoming the limitations of traditional single ferritin carriers. This structure maintains the stability of the ferritin cage structure while providing ample coupling sites for target molecules, achieving a synergistic improvement in drug loading (6.58%, corresponding to approximately 78 drug molecules / complex) and carrier stability.

[0015] 3. Tissue Penetration Innovation: Using the anti-HER2 nanobody 2Rs15d with a small molecular weight (~15 kDa) as the targeting molecule, relying on its small size advantage, while ensuring targeting accuracy, it significantly improves the penetration depth of tumor tissue (>300 μm), effectively solving the problem of uneven drug distribution in deep solid tumors.

[0016] 4. Innovative preparation process: The entire process is based on genetic engineering and self-assembly technology, with mild conditions, simple operation, and small batch-to-batch differences, and has good potential for large-scale production.

[0017] In summary, this invention, through a ternary synergistic design of "nanobody targeting + heterotrophic ferritin drug delivery + Spy system conjugation," achieves for the first time a unified approach in a single drug delivery system, combining high drug loading capacity, deep tissue penetration, high conjugation uniformity, and controllable preparation process, providing a novel technical solution for targeted therapy of HER2-positive tumors. Attached Figure Description

[0018] Figure 1 This is a diagram showing the construction and identification of the 2Rs15d-ST recombinant plasmid; Figure 2 This is an SDS-PAGE validation image of the purified 2Rs15d-ST protein. Figure 3 This is a graph showing the ELISA results of the 2Rs15d-ST protein. Figure 4 This is a diagram showing the construction and identification of the HFn recombinant plasmid; Figure 5 This is a diagram showing the construction and identification of the SC-HFn recombinant plasmid; Figure 6 This is an SDS-PAGE image showing the purification results of HFn and SC-HFn proteins; Figure 7 This is a native electrophoresis image of the SC-HFn / HFn hybrid polymer (confirming that HFn and SC-HFn successfully self-assembled to form a hybrid polymer). Figure 8 These are the results of laser particle size analysis of horse spleen ferritin and SC-HFn / HFn; A: Average particle size of horse spleen ferritin; B: Average particle size of SC-HFn / HFn (showing an average particle size of 20.2±2.41 nm for the hybrid polymer, indicating good stability). Figure 9 The size exclusion chromatogram of SC-HFn / HFn-DOX-HCl (confirms that the heteroferritin polymer successfully encapsulates the drug without altering the polymer's molecular weight). Figure 10 This is a self-assembly coupling verification diagram of 2Rs15d-ST-SC-HFn / HFn-DOX-HCl (confirming that the gene-expressible 2Rs15d-ST fusion protein is successfully coupled with the heteroferritin polymer). Figure 11 This is a static cellular uptake map of 2Rs15d-ST-SC-HFn / HFn-DOX-HCl (scale bar 20 μm) (confirming that the drug-loaded nanoparticles can be efficiently taken up by HER2-positive cells and have good targeting properties). Figure 12 Cytotoxicity analysis of the 2Rs15d-ST-SC-HFn-DOX-HCl system; (*p< 0.05, **p< 0.01, ***p< 0.001) (confirming that the drug-loaded nanoparticles have a significant targeted killing effect on HER2-positive tumor cells). Detailed Implementation

[0019] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto.

[0020] All gene synthesis, codon optimization, and sequencing work in this invention were commissioned to Sangon Biotech (Shanghai) Co., Ltd.

[0021] Example 1: Preparation and activity verification of 2Rs15d-ST fusion protein (1) Sequence design: Based on the amino acid sequence (SEQ ID NO:1) of the 2Rs15d nanobody (PDB:5MY6_B, obtained from the PDB database), the peptide-GGGS (SEQ ID NO:2), SpyTag sequence (SEQ ID NO:3) and His purified tag (SEQ ID NO:7) were tandemly linked using SnapGene software to construct the 2Rs15d-ST fusion gene.

[0022] (2) Plasmid construction: The above fusion gene was codon optimized using E. coli and then cloned into the NcoI / XhoI restriction sites of the pET22b(+) vector; finally, the Sanger sequencing method was used to verify the accuracy of the construction. Bidirectional sequencing was performed using the T7 universal primer (forward: 5'-TAATACGACTCACTATAGGG-3') and the T7 terminator primer (reverse: 5'-GCTAGTTATTGCTCAGCGG-3'). The nucleic acid electrophoresis image of the PCR product using the T7 universal primer is shown below. Figure 1 As shown, the nucleic acid electrophoresis results show: theoretical amplified fragment: 713bp (including vector sequence); actual detected fragment: approximately 750bp, sequencing consistency 100%.

[0023] Figure 1 Part A represents the recombinant strain constructed using the 2Rs15d-ST recombinant plasmid. Figure 1 Part B is the nucleic acid electrophoresis diagram of the PCR product of the 2Rs15d-ST recombinant plasmid; Figure 1 In part B, M represents the Marker, 1 represents the empty plasmid control, and 2 represents the transformant PCR product.

[0024] (3) Expression and purification: The 2Rs15d-ST recombinant plasmid constructed and correctly sequenced in step (2) was transformed into competent Escherichia coli cells. After screening and identification, the 2Rs15d-ST recombinant engineered glycerol bacteria were obtained. 20 μL of the glycerol bacteria was inoculated into 5 mL of LB medium containing 50 μg / mL ampicillin and cultured at 37℃ and 180 rpm for 12-16 h with shaking. Then, the overnight culture was inoculated into 100 mL of LB-Amp medium at a ratio of 1:100 and cultured until OD. 600When the concentration was approximately 0.6, IPTG was added to a final concentration of 0.5 mM, and expression was induced at 30℃ for 16 h. After expression, the bacterial cells were collected by centrifugation at 8000 r / min for 10 min, resuspended in 0.02 mol / L PBS, and then sonicated on ice for 30 min. The supernatant was collected by centrifugation and filtered through a 0.22 μm aqueous filter membrane to obtain the protein loading solution. Affinity purification was performed using a Ni-NTA column on an ÄKTA protein purifier. The column was first fully equilibrated with 0.02 mol / L PBS buffer, and the breakthrough peak was collected after loading. Then, gradient elution was performed with 50 mmol / L and 300 mmol / L imidazole buffers, and each eluted fraction was collected separately. Each fraction was subjected to SDS-PAGE electrophoresis. Figure 2 Verification showed that the 300 mmol / L imidazole elution peak exhibited a clear target band at 18.4 kDa (lane 4). Finally, the target protein eluent was placed in a dialysis bag with a molecular weight cutoff of 7 kDa and dialyzed against 0.02 mol / L PBS for desalting. Magnetic stirring was used to accelerate dialysis, and the dialysate was changed every 6 hours. After desalting, the protein was concentrated by ultrafiltration and stored at -20℃ to obtain the target 2Rs15d-ST protein with a purity >95%.

[0025] Figure 2 In the diagram, M, 1, 2, 3, and 4 represent the marker, supernatant, breakthrough peak, 50 mmol / L imidazole elution peak, and 300 mmol / L imidazole elution peak, respectively. Figure 2 The presence of a clear target band at 18.4 kDa indicates a purity of ≥95%, confirming successful purification of the gene-expressible fusion protein.

[0026] (4) Activity verification: The specific binding activity of 2Rs15d-ST nanobody to HER2 antigen was determined by ELISA. The specific procedure was as follows: HER2 antigen (1 μg / mL) was coated onto an enzyme-labeled plate overnight at 4℃, with BSA protein as a negative control; 2Rs15d-ST protein was serially diluted (10 μg / mL) to 10 μg / mL. -2 ~10 6 Add the 1 ng / mL solution to the reaction wells and incubate at 37°C for 1 h. Then add mouse anti-His Tag antibody (1:2000 dilution) and HRP-labeled goat anti-mouse IgG (1:5000 dilution) as primary and secondary antibodies, respectively. Finally, add TMB substrate for color development, and measure the absorbance (OD) at 450 nm using a microplate reader. 450 ).like Figure 3 As shown, Figure 3 The results confirmed that the gene-expressible 2Rs15d nanobody has specific binding activity to the HER2 antigen, OD 450 The OD values ​​exhibit a typical dose-response curve as the nanobody concentration increases. At low concentrations, the OD...450 The value is close to the background level, and as the concentration gradually increases, the OD... 450 The value rises rapidly and then stabilizes at high concentrations, indicating that binding has reached saturation. The curve fitting results are good, and the EC50 of 2Rs15d-ST protein against the HER2 antigen is [data missing]. 50 The concentration was 9.64 nmol / L, exhibiting a high affinity below 10 nmol / L, consistent with expectations, and the control group showed lower OD values ​​across all concentration ranges. 450 The values ​​remained at the background level, indicating that the 2Rs15d-ST protein has excellent HER2 antigen binding activity.

[0027] In antigen-antibody interaction analysis, EC 50 Represents the half-maximal effect concentration, which is the antibody concentration at which 50% of the maximum binding signal is achieved. EC 50 The smaller they are, the more approachable they are.

[0028] Example 2: Preparation of HFn and SC-HFn proteins (1) Construction of HFn and SC-HFn plasmids Based on the gene sequences of human heavy chain ferritin HFn (Gene ID: 2495, obtained from GenBank) and SpyCatcher protein (GenBank JQ478411, obtained from GenBank), the SC-HFn fusion gene was constructed by tandemly linking peptide 2 (GGGGS)3 (SEQ ID NO:6) and the His purified tag (amino acid sequence SEQ ID NO:7) using SnapGene software. The HFn gene is formed by fusing human heavy chain ferritin HFn (Gene ID: 2495, obtained from GenBank) with the His purified tag (amino acid sequence SEQ ID NO:7).

[0029] The SC-HFn fusion gene and HFn gene were codon optimized for *E. coli*, and the optimized gene fragments were then inserted between the Nde I and Xho I restriction sites of the pET28a(+) vector to construct the SC-HFn recombinant expression plasmid and HFn recombinant expression plasmid, respectively. Bidirectional sequencing was performed using the T7 universal primer (forward: 5'-TAATACGACTCACTATAGGG-3') and the T7 terminator primer (reverse: 5'-GCTAGTTATTGCTCAGCGG-3') for verification. Agarose gel electrophoresis results showed that the HFn clone fragment size was 980 bp (actual detection 750-1000 bp), and the SC-HFn clone fragment size was 1364 bp (actual detection 1000-1500 bp) (see...). Figure 4 , Figure 5This indicates that the recombinant plasmid was successfully constructed.

[0030] Figure 4 Part A is the map of the HFn recombinant plasmid, and Part B is the map of the PCR electrophoresis products. Figure 4 In part B, M represents the marker, 1 represents the empty plasmid control, and 2-4 are the PCR products of the transformants, confirming the successful cloning of the ferritin gene and providing a basis for heterozygous self-assembly.

[0031] Figure 5 Part A is the map of the SC-HFn recombinant plasmid, and Part B is the map of the PCR electrophoresis products. Figure 5 In part B, M represents the marker, 1 represents the empty plasmid control, and 2-5 are the PCR products of the transformants, confirming the successful cloning of the modified ferritin gene and providing a basis for heterozygous self-assembly and targeted coupling.

[0032] (2) Expression and purification of HFn and SC-HFn proteins The constructed HFn plasmid and SC-HFn plasmid were transformed into E. coli BL21(DE3) competent cells via heat shock, respectively. After screening, HFn recombinant glycerol bacteria and SC-HFn recombinant glycerol bacteria were obtained, respectively. The expression and purification process was as follows: 20 μL of recombinant glycerol bacteria were inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured at 37℃ and 180 rpm for 12-16 h; then, the culture was expanded by inoculating into 100 mL of LB-Kan medium (250 mL Erlenmeyer flask) at a 1:100 ratio until OD500. 600 When the concentration of the protein reaches 0.5-0.6, IPTG at a final concentration of 0.5 mM is added and the mixture is induced at 30℃ for 12 h. The cells are collected by centrifugation (4℃, 8000 rpm, 10 min), resuspended in 0.02 M PBS, concentrated 10-fold, and then sonicated on ice. The supernatant is collected after centrifugation and filtered through a 0.22 μm filter to obtain the protein loading solution. Affinity purification is performed using an ÄKTA protein purification system with a nickel column. The column is first fully equilibrated with 0.05 mol / L Tris-HCl buffer, and the breakthrough peak is collected after loading. Gradient elution is then performed with 50 mmol / L and 300 mmol / L imidazole buffers, and each eluted fraction is collected separately. Each fraction is analyzed by SDS-PAGE. Figure 6The results showed that the HFn protein band was approximately 25 kDa, and the SC-HFn fusion protein band was approximately 35 kDa, perfectly matching the theoretical expectations. Finally, the collected target proteins were dialyzed for 24 h in an ice bath using a dialysis bag with a molecular weight cutoff of 7 kDa. The dialysate was 0.05 mol / L Tris-HCl buffer containing 10% (v / v) glycerol to remove imidazole. The dialysate was magnetically stirred at 4°C and replaced with fresh dialysate every 6 h. After dialysis, the protein was concentrated using an ultrafiltration tube with a molecular weight cutoff of 10 kDa at 4°C and 3000 rpm. The concentrated protein was then stored at -20°C to obtain the high-purity recombinant protein.

[0033] Figure 6 Part A is the SDS-PAGE validation image of HFn protein purification. M represents Marker; 1 represents uninduced; 2 represents supernatant; 3 represents precipitate; 4 represents breakthrough peak; 5 represents 50 mmol / L imidazole elution peak; 6 represents 300 mmol / L imidazole elution peak. Figure 6 Part B is the SDS-PAGE validation diagram of SC-HFn protein purification; M represents Marker; 1 represents supernatant; 2 represents precipitate; 3 represents breakthrough peak; 4 represents 50 mmol / L imidazole elution peak; 5 represents 300 mmol / L imidazole elution peak.

[0034] Figure 6 The results confirmed that both ferritin components were successfully purified.

[0035] Example 3: Preparation and characterization of SC-HFn / HFn hybrid protein-encapsulated doxorubicin hydrochloride nanoparticles (1) Preparation method: SC-HFn protein and HFn protein were dissolved in 0.05 mol / L Tris-HCl buffer (pH 7.4) at a molar ratio of 1:5. The pH was adjusted to 3.5 with 1 mol / L HCl and stirred at room temperature (25±2℃) (200 rpm) for 20 min. After adding 500 μL of freshly prepared 3 mg / mL doxorubicin hydrochloride (DOX-HCl) solution, the pH was immediately adjusted to 8.0 with 1 mol / L NaOH and the mixture was stirred magnetically at room temperature for 3 h to promote DOX-HCl encapsulation. After drug loading, the reaction product was centrifuged at 5000 r / min for 1 min to separate the solid precipitate and the supernatant containing the bound drug. The supernatant was placed in a dialysis bag with a molecular weight cutoff of 7 kDa and dialyzed in 0.05 mol / L Tris-HCl buffer solution containing 10% (v / v) glycerol for 6 h, with the buffer changed every 3 h. During dialysis, continuous stirring with a magnetic stirrer was used to accelerate the removal of free DOX-HCl, while maintaining a low temperature of 4°C. The dialysate was replaced every 3 hours to ensure complete dialysis. After dialysis, the solution was concentrated using an ultrafiltration tube with a molecular weight cutoff of 10 kDa (3000 rpm, 4°C). Drug loading parameters were calculated using the following formulas: Encapsulation efficiency (%) = (Total DOX amount - Free DOX amount) / Total DOX amount × 100; Drug loading per protein molecule = Number of moles of drug-loaded DOX / Number of moles of protein; Drug loading (μg / mg) = Mass of drug-loaded DOX (μg) / Mass of protein (mg). Based on these formulas, the encapsulation efficiency of the SC-HFn / HFn hybrid polymer for DOX-HCl was calculated to be 11.7%; each SC-HFn / HFn hybrid polymer could encapsulate 77.7 DOX-HCl molecules, with a drug loading of 6.58%.

[0036] (2) Characterization results: ①Native electrophoresis of SC-HFn / HFn hybrid polymers ( Figure 7 The results showed that, with horse spleen ferritin (440 kDa) as a control, the SC-HFn band disappeared, and the SC-HFn / HFn band (theoretical value 640 kDa) was significantly wider and had a lower migration rate than HFn (600 kDa), proving that the hybridization was successful; ② Dynamic light scattering ( Figure 8 The results showed that the average particle size of the SC-HFn / HFn hybrid (20.2±2.41 nm) was significantly larger than that of standard horse spleen ferritin (13.1±1.36 nm), confirming that N-terminal SpyCatcher and histidine tag modification led to the increased particle size; ③ Size exclusion chromatography ( Figure 9The results showed that the peak times of SC-HFn / HFn-DOX-HCl at 280 nm (protein) and 485 nm (DOX) were 11.24 min and 11.53 min, respectively. The close peak times indicate that the SC-HFn / HFn-DOX-HCl hybrid polymer successfully loaded the drug, and that encapsulating DOX-HCl did not affect the self-assembly of SC-HFn / HFn.

[0037] Example 4: Preparation and characterization of 2Rs15d-ST-SC-HFn / HFn self-coupling verification This embodiment provides a self-coupling method for 2Rs15d-ST and SC-HFn / HFn proteins, comprising the following steps: mixing equal volumes of 2Rs15d-ST protein solution and SC-HFn / HFn protein solution after ultrafiltration concentration, and reacting at room temperature (25±2℃) for 20 min; subsequently, transferring 16 μL of the reaction solution and mixing it with 4 μL of 5× protein loading buffer, and performing denaturation treatment by boiling at 100℃ for 5 min, followed by analysis and verification by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0038] As attached Figure 10 As shown, the molecular weight of the new band (lane 4) generated after the coupling reaction is in the range of 44.0~66.2 kDa, which is consistent with the theoretical molecular weight (53 kDa) of the coupling product of 2Rs15d-ST monomer (18 kDa) and SC-HFn monomer (35 kDa), confirming that the self-coupling reaction was successful and the expected coupling product was obtained. The electrophoresis conditions were: 4-20% gradient Tris-glycine gel, constant voltage electrophoresis at 150V for 45 min, and Coomassie brilliant blue staining to visualize the protein bands.

[0039] Example 5: Confocal microscopy analysis of cellular uptake of the 2Rs15d-ST-SC-HFn / HFn-DOX-HCl nanocomposite This embodiment uses confocal microscopy to observe and analyze the cellular uptake behavior of 2Rs15d-ST-SC-HFn / HFn-DOX-HCl nanocomposite. The specific operation is as follows: (1) Sample preparation: Place the processed cell sample on a glass slide and keep the indoor environment dark; (2) Instrument settings: Turn on the confocal microscope system (including laser, mercury lamp and computer control system) in sequence, start the LAS X Office software platform, set the excitation wavelengths of 405 nm and 488 nm, and configure three detection channels: DAPI (nuclear staining), GFP (fluorescent labeling) and bright field; (3) Image acquisition: Preliminarily adjust the focal plane through the eyepiece, and then use the FAST LIVE mode to fine-tune the focus. After the image clarity reaches the optimal level, capture the fluorescence images of each detection channel; (4) Image processing: Use the built-in algorithm of the software to fuse the multi-channel acquired images.

[0040] As attached Figure 11 As shown, (A) the DOX fluorescence channel reveals the intracellular distribution of the nanocomposite; (B) the bright-field image shows the complete cell morphology; (C) the DAPI channel indicates the location of the cell nucleus; and (D) the three-channel fusion image clearly shows that the 2Rs15d-SC-HFn / HFn-DOX-HCl nanocomposite successfully entered the cell, and DOX-HCl was effectively delivered to the cell nucleus, confirming that this drug delivery system has efficient cellular uptake capacity and nuclear-targeted drug delivery characteristics. Experiments confirm that the protein-loaded nanoparticles prepared in this invention can achieve efficient drug delivery and targeted release.

[0041] Example 6: Evaluation of the in vitro antitumor activity of 2Rs15d-ST-SC-HFn / HFn-DOX-HCl nanobody (1) Experimental methods: ① MTT solution preparation: Accurately weigh 10 mg MTT and dissolve it in 2 mL PBS buffer (pH 7.4). Incubate at 60℃ for 1 min to aid dissolution. After filtration through a 0.22 μm sterile filter membrane, dispense into sterile centrifuge tubes and store at -20℃ in the dark. ② Cell treatment: SKBR3 cells were passaged into 96-well plates (10 cells per well). 4(100 cells), remove the original culture medium from the cells and replace it with fresh, sterile culture medium containing different protein and drug concentrations. The final volume of each well is 100 µL. The SC-HFn / HFn-DOX-HCl group, the 2Rs15d-ST-SC-HFn / HFn-DOX-HCl group, and the DOX-HCl group are set up respectively. When adding 2Rs15d-ST-SC-HFn / HFn-DOX-HCl nanobody or SC-HFn / HFn-DOX-HCl nanobody, the equivalent concentration of DOX-HCl component in the nanobody in the wells is 0, 0.1, 0.5, 1.0, 1.5, 3.0, 6.0, and 8.0 μg / mL, respectively. Each concentration is tested in triplicate. A blank control (culture medium only) and a positive control (free DOX-HCl) are also included. ③ MTT assay: After drug treatment for 48 h, 20 μL of MTT solution (5 mg / mL) is added to each well, and the mixture is incubated for another 4 h (37℃, 5% CO2). After carefully aspirating the supernatant, 100 μL of LDMSO is added, and the mixture is shaken to dissolve for 15 min. The absorbance (OD value) is then measured at 492 nm using a microplate reader. ④ Data Analysis: The inhibition rate was calculated using the formula: Cell viability (%) = [(N1-N0) / (N2-N0)] × 100 (where N1 is the OD value of the drug-treated group, N2 is the OD value of the negative control group, and N0 is the OD value of the zeroing group). The IC50 was calculated using GraphPad Prism software. 50 value.

[0042] (2) Experimental results: as shown in the appendix Figure 12 As shown, 2Rs15d-ST-SC-HFn / HFn-DOX-HCl exhibited a significant dose-dependent inhibitory effect on SKBR3 cells, with an IC50 value of [missing information]. 50 The value was 1.524 μg / mL (converted to 2.14 nM). This is compared to free DOX-HCl (IC50). 50 Although the direct in vitro cytotoxic activity of this nanocomposite was slightly reduced (0.893 μg / mL), it was still better than that of the unmodified SC-HFn / HFn-DOX-HCl (IC). 50 The 2Rs15d-ST nanobody (2.761 μg / mL) showed superior anti-tumor effects. The experimental results showed that: ① the active targeting effect of the 2Rs15d-ST nanobody and the passive targeting effect of ferritin synergistically enhanced the specific uptake of tumor cells; ② the protein carrier system achieved the sustained-release characteristics of DOX, effectively reducing the acute toxicity caused by free drug; ③ the composite system can significantly reduce off-target toxicity through the dual action mechanism of EPR effect and active targeting, which confirms the advantages of tumor-targeted drug delivery of the present invention.

Claims

1. A ferritin nanomedicine carrier targeting anti-HER2 nanobody, characterized in that, include: (1) A fusion protein containing anti-HER2 nanobody 2Rs15d and SpyTag; (2) A fusion protein containing SpyCatcher and human heavy chain ferritin; (3) A hybrid ferritin polymer formed by the self-assembly of human heavy chain ferritin and the fusion protein containing SpyCatcher and human heavy chain ferritin; The anti-HER2 nanobody 2Rs15d is covalently linked to the surface of the hybrid ferritin polymer via SpyTag and SpyCatcher.

2. The ferritin nanomedicine carrier according to claim 1, characterized in that, The molar ratio of human heavy chain ferritin to the fusion protein containing SpyCatcher and human heavy chain ferritin is 4-6:1, preferably 5:

1.

3. The ferritin nanomedicine carrier according to claim 1, characterized in that, The anti-HER2 nanobody 2Rs15d contains the amino acid sequence shown in SEQ ID NO.1, the SpyTag contains the amino acid sequence shown in SEQ ID NO.3, the SpyCatcher contains the amino acid sequence shown in SEQ ID NO.5, and the human heavy chain ferritin contains the amino acid sequence shown in SEQ ID NO.

4.

4. The ferritin nanomedicine carrier according to claim 1, characterized in that, The fusion protein comprising the anti-HER2 nanobody 2Rs15d and SpyTag further includes linker peptide one, which contains the amino acid sequence shown in SEQ ID NO.2; the fusion protein comprising SpyCatcher and human heavy chain ferritin further includes linker peptide two, which contains the amino acid sequence shown in SEQ ID NO.

6.

5. The ferritin nanomedicine carrier according to claim 1, characterized in that, The ferritin nanomedicine carrier is internally loaded with an antitumor drug, which is selected from one or more of macrolides, anthracyclines, taxanes or camptothecins. The drug encapsulation efficiency of the ferritin nanomedicine carrier is 10.5%-12.8%, and the drug loading is 5.8%-7.2%.

6. The ferritin nanomedicine carrier according to claim 5, characterized in that, The antitumor drug mentioned is doxorubicin.

7. A method for preparing a ferritin nanomedicine carrier as described in any one of claims 1-6, characterized in that, Includes the following steps: (1) Preparation of a fusion protein containing anti-HER2 nanobody 2Rs15d and SpyTag; (2) Prepare a fusion protein containing SpyCatcher and human heavy chain ferritin; (3) The fusion protein prepared in step (2) is mixed with human heavy chain ferritin and self-assembled in acidic buffer to form a hybrid ferritin polymer; (4) The heteroferritin polymer obtained in step (3) is co-incubated with the antitumor drug under alkaline conditions and loaded with the drug; (5) The fusion protein obtained in step (1) is mixed with the hybrid ferritin polymer loaded with drugs in step (4), and the anti-HER2 nanobody-targeted ferritin nanomedicine carrier is obtained through specific covalent coupling between SpyTag and SpyCatcher.

8. The preparation method according to claim 7, characterized in that, The pH of the acidic buffer solution in step (3) is 3.0-4.0, and the molar ratio of human heavy chain ferritin to the fusion protein containing SpyCatcher and human heavy chain ferritin is 4-6:1, preferably 5:1; the pH of the alkaline condition in step (4) is 7.5-8.

5.

9. The preparation method according to claim 7, characterized in that, In steps (1) and (2), the expression was performed using the E. coli expression system. After induction and purification, the purity of the obtained fusion protein was ≥95%.

10. The use of the ferritin nanomedicine carrier targeting the anti-HER2 nanobody according to any one of claims 1-6 or the ferritin nanomedicine carrier prepared by the preparation method according to any one of claims 7-9 in the preparation of a drug for treating HER2-positive tumors.

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