Intelligent extracellular drug release ferritin drug carrier and application thereof

Abstract

The invention discloses an intelligent extracellular drug release ferritin carrier and application thereof, and belongs to the technical field of biological medicine. The intelligent extracellular drug-release ferritin prepared by the invention is based on the structure of a human heavy chain ferritin protein cage, the protein cage tends to be everted through E spiral mutation, a focus tissue targeting peptide is fused at a C terminal, a focus environment overexpression protease recognition sequence is inserted into a DE ring, and the intelligent ferritin which can target focus tissue and respond to the environment of the focus tissue is created. Extracellular release of loaded drugs in focus tissues is achieved, and an efficient treatment effect is achieved through targeting and controllable release.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an intelligent extracellular drug-releasing ferritin carrier and an application thereof. Background Art

[0002] The rapid development of nanomedicine has injected new vitality into the field of drug delivery. Its significant advantages such as precise targeting, efficient bioavailability and controlled drug release have greatly promoted the innovation of treatment models. However, despite the remarkable achievements, how to achieve precise delivery and efficient release of drugs at the lesion tissue and even at the specific cell level is still a technical problem that needs to be overcome.

[0003] Compared with synthetic nanomaterials, natural protein cages stand out in biomedical applications with their unique advantages of low biotoxicity and high biocompatibility. Among them, human heavy chain ferritin (HFn), as an outstanding representative of protein cages, has become a popular candidate for anti-tumor drug delivery because it can specifically bind to transferrin receptor 1 (TfR1) overexpressed in tumor tissues, showing natural tumor targeting potential. The HFn subunit consists of a 4-helix bundle and a short C-terminal helix (E helix). 24 HFn subunits spontaneously assemble a uniform protein cage with an outer diameter of 12 nm and an inner diameter of 8 nm. Compared with conventional nanomaterials, the smaller outer diameter makes it easier to pass through the matrix barrier, while the 8 nm inner diameter allows it to effectively load small molecule drugs. Currently, HFn is used to deliver a variety of clinical drugs including doxorubicin and cisplatin.

[0004] However, since TfR1 is not specifically expressed in tumor cells, HFn does not have very specific tumor targeting. At the same time, HFn does not have targeting for other diseases such as thrombotic diseases, and its lysosomal-dependent drug-downregulation method not only reduces drug utilization, but may also have adverse effects on specific drugs. Through protein engineering, the lesion tissue targeting sequence and the recognition sequence of the lesion environment overexpressed enzyme are fused to specific parts of the outer surface of HFn, and a smart HFn protein cage (Smart extracellular drug-releasingHFn, Sedr-HFn) is designed that can both target the lesion tissue and respond to its environment to release drugs. This will enable the release of drugs outside the lesion tissue, achieve lysosomal escape, and improve drug efficacy. Summary of the invention

[0005] The present invention aims at the problem of HFn in the drug transportation process, and provides an intelligent extracellular drug-releasing ferritin carrier and its application. The extracellular drug-releasing ferritin carrier can both target the lesion tissue and respond to its environment to release the drug, thereby achieving accurate delivery and efficient release of the drug.

[0006] A type of intelligent extracellular drug-releasing ferritin drug carrier, in which a polypeptide sequence targeting the lesion tissue is fused to the everted C-terminus of ferritin, and a recognition sequence of an enzyme overexpressed in the lesion is fused to the DE loop of ferritin; both the polypeptide targeting the lesion tissue and the recognition sequence of the enzyme overexpressed in the lesion are exposed on the outer surface of the ferritin protein cage, which can target the lesion and respond to the corresponding protease in the lesion environment to release the drug extracellularly.

[0007] Furthermore, the above-mentioned outward-turned C-terminus of ferritin is to change the hydrophilicity of the amino acids in the middle of the ferritin E helix to tend to be everted outward.

[0008] Furthermore, the above-mentioned outward-turned C-terminus of ferritin is achieved by mutating the leucine at position 169 in the middle of the ferritin E helix to arginine to turn the ferritin C-terminus outward.

[0009] Furthermore, the above-mentioned lesion tissue targeting sequences include: urokinase receptor targeting sequence CVSYKYFSRIRQC, P-selectin targeting sequence SDAEWVDVS; the recognition sequences of the lesion overexpressed enzymes include: matrix metalloproteinase recognition sequence PLGLAG; thrombin recognition sequence LVPRGS.

[0010] Furthermore, the above-mentioned intelligent extracellular drug-releasing ferritin drug carrier is a urokinase receptor-targeted intelligent extracellular drug-releasing ferritin drug carrier UTSedr-HFn, and its amino acid sequence is shown in SEQ ID NO.1: MHHHHHHTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKEPDCDDWESGQNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPGGGPLGLAGGGSESGLAEYRFDKHTLGDSDNESCVSYKYFSRIRQC.

[0011] Furthermore, the above-mentioned intelligent extracellular drug-releasing ferritin drug carrier is a P-selectin targeted intelligent extracellular drug-releasing ferritin drug carrier PTSedr-HFn, and its amino acid sequence is shown in SEQ ID NO.2: MHHHHHHTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKEPDCDDWESGQNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPGGLVPRGSGGSESGLAEYRFDKHTLGDSDNESGGGGSDAEWVDVS.

[0012] The above-mentioned intelligent extracellular ferritin drug carrier can be used for targeted drug delivery.

[0013] The inventive idea of ​​the present invention: A type of intelligent extracellular drug-releasing ferritin drug carrier is the HFn variant, which expands its drug release channel by targeting the lesion and responding to the corresponding proteases in the lesion environment, releasing the loaded drug outside the lesion tissue to improve the therapeutic effect ( Figure 1 ).

[0014] The invention involves introducing a mutation site that tends to turn outward into the E helix of HFn, fusing a lesion tissue targeting peptide to its C-terminus, and inserting a lesion environment overexpression enzyme recognition sequence at the DE Loop position near its 4-fold symmetry axis channel. The HFn variant thus modified can be enriched in lesion tissues, and the recognition sequence is cut under the action of proteases in the lesion environment, resulting in the release of the E helix and the expansion of the channel, thereby promoting the release of the drug. These designs have the following advantages: (1) The peptide fused to the C-terminus is exposed on the outer surface of HFn and can effectively target lesion tissues; (2) The protease recognition sequence is exposed on the outer surface of HFn and is easily recognized by proteases; (3) After cutting the E helix, the 4-fold symmetry axis channel of HFn can be opened, which is conducive to the controlled release of the drug.

[0015] The significant advantage of the present invention is that the lesion tissue targeting sequence and the recognition sequence of the lesion environment overexpressed enzyme are simultaneously introduced into different parts of the outer surface of HFn, and the inserted sequence does not affect the expression of such variants in Escherichia coli and the formation of protein cages, and the cavity thereof can load anti-tumor drugs, target lesion tissues and respond to their microenvironment, release drugs extracellularly, and achieve the purpose of efficient disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Diagram of the mechanism of extracellular drug release of intelligent extracellular drug release ferritin.

[0017] Figure 2 Screening of smart extracellular drug-releasing ferritin. (A) SDS-PAGE identified the sensitivity of MMPs-responsive HFn to MMPs; (B) SDS-PAGE identified the sensitivity of thrombin-responsive HFn to thrombin; (C) UTSedr-HFn has the ability to target uPAR at the protein level; (D) UTSedr-HFn has the ability to target uPAR at the cellular level; (E) PTSedr-HFn has the ability to target activated platelets.

[0018] Figure 3 Assembly characterization of smart extracellular drug-releasing ferritin. (A) Dynamic light scattering characteristics of UTSedr-HFn; (B) Dynamic light scattering characteristics of PTSedr-HFn; (C) Transmission electron microscopy image of negatively stained sample of UTSedr-HFn; (D) Transmission electron microscopy image of negatively stained sample of PTSedr-HFn.

[0019] Figure 4 Characterization of NPs@UTSedr-HFn. (A) UV-visible absorption spectrum of NPs@UTSedr-HFn; (B) Fluorescence emission spectrum of NPs@UTSedr-HFn under 610 nm excitation; (C) Fluorescence emission spectrum of NPs@UTSedr-HFn under 294 nm excitation; (D) Dynamic light scattering results of NPs@UTSedr-HFn; (E) Transmission electron microscopy of negatively stained samples of NPs@UTSedr-HFn; (F) Responsive release curve of NPs@UTSedr-HFn in the presence of collagenase IV.

[0020] Figure 5 Tumor cell uptake and toxicity of NPs@UTSedr-HFn. (A) CPZ cell uptake in different drug treatment groups; (B) Cytotoxicity of different drug treatment groups under normoxic and hypoxic conditions; (C) Cytotoxicity of TPZ and protein alone; (D) Dark toxicity of NPs@UTSedr-HFn.

[0021] Figure 6 Effects of NPs@UTSedr-HFn on tumor cell colony formation. (A) Imaging of cell colonies after treatment with different drugs; (B) Qualitative analysis of Figure A.

[0022] Figure 7 Effect of NPs@UTSedr-HFn on tumor cell migration.

[0023] Figure 8 Tumor 3D spheroid permeability of NPs@UTSedr-HFn. (A) 2 hours; (B) 4 hours; (C) 8 hours; (D) 12 hours.

[0024] Fig. 9 NPs@UTSedr-HFn can effectively target tumors in mice.

[0025] Fig.10 In vivo anti-tumor evaluation of NPs@UTSedr-HFn in mice. (A) Monitoring of tumor volume of mice in each drug-loaded protein cage group; (B) Tumor tissues peeled off from mice in each drug-loaded protein cage group on the last day of monitoring; (C) Tumor weight of mice in each group after treatment; (D) Weight changes of mice in each group at different times after administration.

[0026] Fig.11 Characterization of Rutin@PTSedr-HFn. (A) Dynamic light scattering results of Rutin@PTSedr-HFn; (B) UV / visible absorption spectrum of Rutin@PTSedr-HFn; (C) Stability of Rutin@PTSedr-HFn.

[0027] Fig.12 Rutin@PTSedr-HFn can inhibit carotid artery thrombosis in mice. (A) Effects of different treatment groups on the vascular occlusion time of the carotid artery thrombosis model induced by direct current stimulation in mice; (B) Effects of different treatment groups on the vascular occlusion time of the carotid artery thrombosis model induced by ferric chloride in mice.

[0028] Fig.13 Bleeding risk assessment of Rutin@PTSedr-HFn. (A) Bleeding time after tail amputation of mice; (B) Analysis of hemoglobin loss. DETAILED DESCRIPTION

[0029] The following is a detailed description of the construction process of the intelligent extracellular drug-releasing ferritin Sedr-HFn of the present invention and the research on drug delivery applications by way of examples in conjunction with the accompanying drawings, but the scope of the present invention is not limited in any way. Without departing from the scope of the main features of the present invention, the present invention may have various changes and improvements, which all fall within the scope of protection of the present invention.

[0030] In the embodiments, conventional plasmid construction involves experiments such as PCR and ligation, and protein expression involves experiments such as transformation, bacterial culture and induction, and cell culture, which are familiar to researchers in the field, and the specific experimental details are not described in detail in the present invention.

[0031] Example 1 Screening and characterization of intelligent extracellular drug-releasing ferritin This study mainly screened two types of intelligent extracellular drug-releasing ferritins: uPAR-targeted Sedr-HFn (UTSedr-HFn) and P-selectin targeted Sedr-HFn (PTSedr-HFn), which are used for the delivery of anti-tumor and anti-thrombotic drugs, respectively. uPAR (urokinase-type plasminogen activator receptor) is a cell membrane surface receptor. The expression of uPAR is elevated in tumor tissues, while the expression is low or absent in surrounding normal tissues, making it a potential target for tumor diagnosis and treatment. The uPAR-targeting peptide and the protease recognition sequence that responds to the tumor microenvironment are fused to the outer surface of HFn to construct UTSedr-HFn that can target tumors and respond to their microenvironment. P-selectin is the first glycoprotein that is upregulated after platelet and endothelial cell activation. During thrombosis, it can be translocated to the surface of endothelial cells and platelets through exocytosis, making it an excellent thrombotic target. The P-selectin targeting peptide and the protease recognition sequence that responds to the thrombus environment are fused to the outer surface of HFn to construct PTSedr-HFn that can target thrombus and respond to its environment.

[0032] 1. Cloning of ferritin variants The HFn target gene with 6his-tag at the N-terminus was synthesized by Shanghai Yonglian Company and cloned into the pET22 plasmid. After that, oligonucleotides encoding protease recognition sequences and different connectors were inserted into Pro161 of HFn using non-ligase-dependent cloning to obtain responsive ferritin; on this basis, the lesion tissue targeting sequence was fused to the N-terminus or C-terminus of HFn respectively; for the C-terminal fusion variant, a mutant in which leucine at position 169 in the middle of the E helix was mutated to arginine was also constructed. The constructed recombinant plasmid was transformed into DH5α and screened on LB agar plates containing 100 μg / mL Amp. Positive colonies were verified by colony PCR and DNA sequencing.

[0033] The above-mentioned protease recognition sequence includes: matrix metalloproteinase recognition sequence such as PLGLAG; thrombin recognition sequence such as LVPRGS.

[0034] The different connectors mentioned above include: GG, GGG, GGS, GGGGSGG.

[0035] The above-mentioned lesion tissue targeting sequences include: urokinase receptor targeting sequences such as CVSYKYFSRIRQC; P-selectin targeting sequences such as SDAEWVDVS.

[0036] 2. Preparation of recombinant protein The recombinant plasmid was transformed into E. coli BL21 (DE3) for protein expression. The transformed E. coli BL21 (DE3) was cultured at 37°C until OD 600 The pH value was 0.6-0.8, and then IPTG was added at a final concentration of 0.5 mM for induction at 18 °C and 150 rpm for 16 hours.

[0037] The cells were collected by centrifugation, and the cell pellet was resuspended in a buffer containing 40 mM Tris-HCl pH 8.0 and 150 mM NaCl (1 g of cells / 10 mL of buffer) and disrupted with a high-pressure cell disruptor. After centrifugation at 10,000 g for 20 min to remove cell debris, the target protein in the supernatant was captured with Ni Sepharose excel filler, washed with a buffer containing 40 mM Tris-HCl pH 8.0, 150 mM NaCl and 20 mM imidazole, and eluted with a buffer containing 40 mM Tris-HCl pH 8.0, 150 mM NaCl and 300 mM imidazole.

[0038] During protein expression, the recombinant protein Sedr-HFn with the targeting sequence fused to the N-terminus of HFn was expressed in the form of inclusion bodies, and the recombinant protein Sedr-HFn with the targeting sequence fused to the C-terminus of HFn was expressed in a soluble form. Therefore, the subsequent experiments on targeted proteins only used proteins with the targeting sequence fused to the C-terminus.

[0039] 3. Screening of intelligent extracellular ferritin 3.1 Identification of matrix metalloproteinase-responsive ferritin by enzyme digestion: The protein cage (0.5 mL, 2 mg / mL) and 2 U collagenase IV (200 U / mL, containing MMP2 / 9) were added to 5 mM CaCl2 and 3 μM ZnCl2 and incubated at 37 °C for 2 hours, followed by SDS-PAGE analysis. After collagenase IV treatment, the band position of HFn on SDS-PAGE did not change, while MR-HFn with different linkers treated with collagenase IV showed bands with smaller molecular weights ( Figure 2 A), the reduced molecular weight is close to the sum of the molecular weights of the amino acid sequence after the C-terminal response sequence, indicating that collagenase IV can effectively cut the inserted recognition sequence. fle1 and MR-HFn fle2 The more cleavage bands were generated, the more likely it was that the recognition sequence was more exposed after adding the flexible linker, making it easier to be recognized by collagenase IV. fle2It is easier to precipitate, so MR-HFn is selected fle1 Carry out further targeted transformation.

[0040] 3.2 Identification of thrombin-responsive ferritin by enzyme digestion: Thrombin responsive ferritin TR-HFn and TR-HFn fle 30 μg of each protein was added with different amounts of thrombin (0.1 U, 0.2 U, 0.5 U, 1 U) and incubated at 37°C for 3 hours. Samples were taken for 15% SDS-PAGE analysis to evaluate the response efficiency of different recombinant proteins to thrombin. After thrombin treatment, TR-HFn containing flexible linkers on both sides of the thrombin recognition sequence fle A new band with a smaller molecular weight was generated, indicating that it could be effectively cleaved by thrombin (Figure 2B), and the cleavage efficiency was dependent on the thrombin concentration. When 1 U of thrombin was added, the original band completely disappeared, and only the new band was generated in the lane. In contrast, the molecular weight of TR-HFn without flexible linkers on both sides of the thrombin recognition sequence did not change after thrombin cleavage, which shows the importance of adding flexible linkers on both sides of the recognition sequence to the response of this ferritin variant.

[0041] 3.3 Determination of uPAR targeting ability of UTSedr-HFn: Human uPAR (huPAR) and mouse uPAR (muPAR) are species-specific. To facilitate evaluation at the animal level, the uPAR targeting peptide we selected was a sequence that was designed and modified in the early stage to bind to huPAR and muPAR at the same time, and the targeting ability of UTSedr-HFn to uPAR of different species was detected at the protein level and the cell level. MicroScale Thermophoresis (MST) was used to determine the targeting ability at the protein level. First, huPAR or muPAR was labeled with a fluorescent dye. Specifically, huPAR or muPAR was first dialyzed into an amino-free buffer, 100 μL of 20 μM huPAR or muPAR was taken and mixed with 12.7 μL of 470 μM dye, the molar ratio of dye to protein was 3:1, incubated at room temperature for 2 hours, and purified through a desalting column to remove excess free dye. In a 0.2 mL EP tube, UTSedr-HFn was diluted in a two-fold concentration gradient, with a solution volume of 10 μL per tube. The highest concentration of UTSedr-HFn was set to 50 μM, and 10 μL of dye-labeled uPAR was added to each tube, with a uPAR concentration of 50 nM. UTSedr-HFn and uPAR were incubated at room temperature for 30 min and detected using a microthermophoresis instrument. The results (Figure 2C) showed that the Kd value of UTSedr-HFn for human uPAR was approximately 123 ± 39 nM, and the Kd value for mouse uPAR was approximately 12 ± 2 nM, indicating that UTSedr-HFn can target both huPAR and muPAR, which is convenient for preclinical studies at the mouse level and can also be used in clinical practice later.

[0042] The targeting ability of UTSedr-HFn to uPAR was further detected at the cellular level. First, FITC (Fluorescein Isothiocyanate) was used to label UTSedr-HFn. Specifically, 250 μL UTSedr-HFn (3 mg / mL) was mixed with 6.5 μL FITC (10 mg / mL), incubated at 4°C for 2 hours, and the excess free FITC was purified by desalting column. 293T cells overexpressing uPAR were used to detect the amount of uPAR bound to them. huPAR-293T, muPAR-293T and 293T cells in the logarithmic growth phase were inoculated in 96-well plates, with 5,000 cells per well, cultured overnight in a 37°C, 5% CO2 incubator, and replaced with 100 μL DMEM-diluted UTSedr-HFn at a concentration of 1 μM. After culturing for 2 h, the culture medium was removed, PBS was washed twice, and finally 100 μL PBS was added. The fluorescence intensity of the Alexa488 channel was detected by high-content imaging. The experimental results (Figure 2D) showed that huPAR-293T and muPAR-293T cells had obvious uptake effects on UTSedr-HFn and MR-HFn, mainly because there were HFn receptors on the surface of 293T cells. However, it can be seen that the uptake of UTSedr-HFn with uPAR targeting by these cells was higher than that of MR-HFn without uPAR targeting. Among them, the uptake of UTSedr-HFn by muPAR-293T was significantly different from that of MR-HFn (P<0.001). This result is consistent with the affinity detection results at the protein level.

[0043] 3.4 Determination of thrombus targeting ability of PTSedr-HFn The thrombus targeting ability of PTSedr-HFn was evaluated by activated platelets. First, the protein was labeled with FITC. Specifically, 1 mL of protein (concentration of 1 mg / mL) was dialyzed in phosphate buffer (pH=9) for 2-3 hours three times to make the solution alkaline and remove Tris for subsequent labeling. FITC was prepared as a 1 mg / mL solution (dissolved in DMSO) and stored at 4 °C in the dark. The dialyzed protein was mixed with FITC at a molar ratio of 1:20. FITC was added slowly and stirred continuously, and the reaction was carried out at 4 °C in the dark for 12 h. After the reaction, NH4Cl was added to a final concentration of 50 mM, and the reaction was terminated at 4 °C for 2 h. Finally, the excess free dye was purified with a desalting column. 200 μL collagen I (3 mg / mL) was added to a black 96-well plate, with three replicate wells set up in each group, and incubated at 37°C overnight to allow the collagen to fully cover the wells to facilitate subsequent platelet adhesion; fresh human whole blood was centrifuged at 150×g for 15 min at room temperature to obtain platelet-rich plasma (PRP), and ADP (final concentration 1 mM) was added to PRP to activate platelets; the uncovered collagen was gently washed with PBS, and 200 μL of activated and unactivated platelets were added respectively and incubated at 37°C for 1 h. Gently wash the uncovered platelets with PBS, then add 200 μL FITC-labeled PTSedr-HFn (3.5 mg / mL), and use wild-type HFn and ferritin PTR-HFn without inversion modification but also fused with P-selectin targeting peptide and thrombin recognition sequence as controls, and incubate at 37°C for one hour; finally, gently wash the unbound proteins with PBS, add 200 μL PBS, and measure the fluorescence intensity of each well with a microplate reader, setting the excitation wavelength to 490 nm and the emission wavelength to 520 nm. The results (Figure 2E) showed that compared with wild-type HFn, PTSedr-HFn can efficiently target activated platelets, showing extremely significant differences compared with the control group and PTR-HFn group (P<0.0001), while the corresponding control PTR-HFn (without the point mutation at L169, and theoretically the targeting peptide is located inside the protein cage) has only weak targeting to activated platelets relative to wild-type HFn. This may be because the fusion of the polypeptide at the C-terminus has a certain effect on the assembly of HFn, causing its E helix to turn outward slightly.

[0044] From the above screening, it can be seen that inserting a protease recognition sequence after P161 in the DE loop of HFn, with the presence of short flexible linkers on both sides of the recognition sequence, can enhance the responsiveness to proteases. Fusion of the targeting peptide at the C-terminus is more advantageous than fusion at the N-terminus in expressing soluble proteins, but it is necessary to change the hydrophilicity of the amino acids in the middle of its E helix so that its C-terminus tends to be turned outward and exposed to the outer surface to increase targeting. The following is the determined preferred amino acid sequence uPAR targets Sedr-HFn (SEQ ID NO.1):MHHHHHHTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKEPDCDDWESGQ NAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPGGGPLGLAGGGSESGLAEYRFDKHTLGDSDNESCVSYKYFSRIRQC P-selectin targets Sedr-HFn (SEQ ID NO.2):MHHHHHHTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKEPDCDDWESG QNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPGGLVPRGSGGSESGLAEYRFDKHTLGDSDNESGGGGSDAEWVDVS 4. Assembly and characterization of smart extracellular drug release ferritin Dynamic light scattering (DLS): Dynamic light scattering was used to measure the uniformity and particle size of the protein cage. The sample with a concentration of 1 mg / mL was filtered through a 0.22 μm filter membrane, and 50 μL of the sample was placed in a quartz colorimetric tube for particle size measurement. The measurement was repeated three times at 25 °C. The DLS results showed that the particle sizes of UTSedr-HFn and PTSedr-HFn were 14.8 nm ( Figure 3 A) and 14.6 nm (Figure 3B). Compared with the hydrated particle size of HFn reported in the literature of 12 nm, the larger particle size is presumably caused by the inverted E helix and the targeting peptide on the surface of the protein cage.

[0045] Transmission electron microscopy: The protein cage electron microscopy samples were prepared by negative staining. The protein cage was diluted to 50-100 μg / mL with Tis-HCl buffer without sodium chloride. The copper mesh was placed with the front side facing up. 10 μL of the sample was drawn and dropped onto the copper mesh. The sample was allowed to stand for 10 min. The sample was slowly dried from the edge of the droplet with a filter paper cut into strips. 10 μL of 2 vol% phosphotungstic acid negative staining solution was drawn and dropped onto the copper mesh. The sample was allowed to stand for 5 min. The negative staining solution was removed with filter paper. The sample was allowed to stand at room temperature until the surface was dry. The sample was detected using a HF7700 transmission electron microscope with a voltage set to 100 KV. The electron microscopy results (Figures 3C and 3D) showed that the particle sizes of the two protein cages were between 13-17 nm. This heterogeneity compared to the wild-type HFn indicates the structural flexibility of its everted part.

[0046] Example 2 Application of uPAR-targeted Sedr-HFn in anti-tumor drug delivery Photosensitizers CPZ and tirapazamine TPZ were selected as loading drugs for cancer treatment. Photosensitizers produce photochemical reactions under the irradiation of light of a specific wavelength, consuming oxygen in tumor tissues and producing reactive oxygen species, thereby destroying tumor cells. However, the hypoxic characteristics of solid tumors limit the photodynamic therapy effect of photosensitizers. As a hypoxia-activated drug, tirapazamine TPZ can play a cell-killing role under hypoxic conditions, making up for the limitations of photosensitizers in hypoxic environments. Photosensitizers consume oxygen, making the tumor microenvironment more hypoxic. This aggravated hypoxia can further enhance the therapeutic effect of TPZ.

[0047] 1. Preparation and characterization of uPAR-targeted Sedr-HFn (NPs@UTSedr-HFn) 1.1 Preparation of NPs@UTSedr-HFn protein cages NPs@UTSedr-HFn was prepared by the urea method. The specific method is to add 9.6 μL 10 mM TPZ and 9.6 μL 10 mM CPZ to the EP tube, add 2.5 μL Tween80 (final concentration 1 vol%), pipette evenly, add 42 μL 9 M urea (final concentration 1.5 M), add 200 μL UTSedr-HFn, the protein cage / CPZ / TPZ molar ratio is 1:240:240, then incubate at room temperature in the dark for 2 hours, then load the mixture into a 15 kDa dialysis bag and dialyze into PBS solution for 3 times, each time for 4 hours to remove urea and free compounds. The TPZ and CPZ that are not encapsulated in the protein cage are further removed by a desalting column to obtain a green, transparent solution. UV-visible absorption spectrum ( Figure 4A) shows that the protein cage has the characteristic absorption peak of CPZ monomer at 680 nm and the characteristic absorption peak of TPZ at 460 nm. Fluorescence spectroscopy measurement ( Figure 4 BC) shows that under excitation at 610 nm, the protein cage exhibits maximum fluorescence at 680 nm ( Figure 4 B) is the fluorescence spectrum characteristic of CPZ monomer; under 294 nm excitation, the protein cage shows maximum fluorescence at 597 nm ( Figure 4 C) is the fluorescence spectrum characteristic of TPZ. The above results all indicate that CPZ and TPZ are successfully encapsulated in ferritin.

[0048] 1.2 CPZ and TPZ loading determination The concentration of carrier protein in the protein cage drug was determined by BCA protein detection kit according to the manufacturer's instructions and protocol. The concentration of drug-loaded compounds was determined by spectroscopy. Specifically, the protein cage drug was dissolved in dimethyl sulfoxide (DMSO) and the concentration of CPZ was determined by ex610 / em680 and the concentration of TPZ was determined by ex294 / em597 using a microplate reader. The encapsulation efficiency and loading capacity were calculated as follows: Where C represents mass concentration. The results show that when the initial molar ratio of TPZ / CPZ / protein cage is fixed at 240:240:1, one protein cage can encapsulate about 135 TPZ molecules with an encapsulation rate of about 56% (w / w); and about 170 CPZ molecules with an encapsulation rate of about 71% (w / w).

[0049] 1.3 Particle size and morphology of NPs@UTSedr-HFn Dynamic light scattering method: The sample with a concentration of 0.5 mg / mL was filtered through a 0.22 μm filter membrane, and 50 μL of the sample was placed in a quartz colorimetric tube for particle size measurement, and the measurement was repeated three times at 25 °C. The DLS results (Figure 4D) showed that the hydration kinetic diameter of the drug-loaded protein cage was 15.0 nm, which was similar to the hydration particle size of the protein cage without drug.

[0050] Transmission electron microscopy imaging: UTSedr-HFn (10 μL, 0.1 mg / mL) was dropped into a carbon-coated copper grid and allowed to stand for 10 min. The sample was slowly dried from the edge of the droplet with filter paper cut into strips, and negatively stained with 2 vol% phosphotungstic acid for 5 min. The negative staining solution was removed with filter paper, and the surface was allowed to stand at room temperature until it was dry. The transmission electron microscopy image was scanned at 100 kV by transmission electron microscopy. The results (Figure 4E) showed that NPs@UTSedr-HFn had a spherical structure and particle size similar to UTSedr-HFn.

[0051] 1.4 Responsive release of NPs@UTSedr-HFn The drug-loaded protein cage was co-incubated with collagenase IV containing a mixture of various matrix metalloproteinases, and the fluorescence intensity change was detected in real time to reflect the responsive release of the drug-loaded protein cage to matrix metalloproteinases. The protein cage released CPZ into the aqueous solution after enzyme cleavage, and the hydrophobic CPZ aggregated and precipitated, and the fluorescence intensity decreased. The concentration of the drug-loaded protein cage in the enzyme reaction system was 0.5 mg / mL (calculated based on protein content), and the concentration of collagenase IV was 0.1 mg / mL. Results ( Figure 4 F) shows that after incubation with the enzyme for 0.5 hours, the CPZ release is about 50%, 1 hour later, the release is about 80%, and 1.5 hours later, the release is 90%; while the control protein cage without collagenase IV has no obvious drug release. The above results show that NPs@UTSedr-HFn can quickly respond to matrix metalloproteinases, which is conducive to its enzyme-responsive release of drugs in the tumor microenvironment without drug leakage in normal tissues.

[0052] 2. Antitumor evaluation of NPs@UTSedr-HFn at the cellular level 2.1 Cellular uptake assay of NPs@UTSedr-HFn The cellular uptake of CPZ was quantified by detecting its fluorescence. 3 Cells / well) were inoculated in 96-well plates with equal portions (200 μL) and incubated overnight. After incubation of cells with NPs@UTSedr-HFn, NPs@MR-HFn, NPs@HFn and free CPZ containing 1 μM CPZ for different time periods (2, 4, 6, 8, 12, 24, 36, 48 hours), the cells were washed with sterile PBS buffer, and then the CPZ fluorescence intensity was detected by high content detection. Each condition was repeated in 4 wells, and each experiment was repeated 3 times. Statistical results of fluorescence analysis ( Figure 5A) shows that the uptake of CPZ in the NPs@UTSedr-HFn group by 4T1 cells is time-dependent. As the incubation time of the drug-loaded protein cage and cells increases, the fluorescence intensity gradually increases and reaches the maximum value at 24 h. When the uptake time is extended, the uptake amount no longer increases, indicating that the uptake has reached saturation, and there is a very significant difference compared with the free CPZ group at each time point (P<0.001). The uptake of CPZ in the NPs@MR-HFn group by 4T1 cells is also time-dependent, and there is a significant difference compared with the free CPZ group (12-36 hours P<0.01). In comparison, the uptake of CPZ in the NPs@HFn group by 4T1 cells is only slightly higher than that in the free CPZ group. These results indicate that due to the ferritin receptor-mediated endocytosis on the surface of 4T1 cells, 4T1 cells have a certain dependence on the uptake of NPs@HFn. NPs@MR-HFn containing enzyme recognition sequences can further increase the uptake according to the controlled drug release mechanism of the response, and the uPAR targeting peptide on the surface of NPs@UTSedr-HFn further promotes its targeted binding to the uPAR receptor on the cell surface and promotes its uptake. In summary, NPs@UTSedr-HFn can increase cell uptake of drugs through multiple mechanisms and is a targeted drug delivery system with excellent properties.

[0053] 2.2 Cytotoxicity assay of NPs@UTSedr-HFn CCK8 kit was used to detect the photodynamic toxicity of NPs@UTSedr-HFn to 4T1. 4 Cells in the exponential growth phase were seeded in 96-well plates and incubated overnight. After the incubation, the culture medium was removed and the cells were incubated with different concentrations of NPs@UTSedr-HFn (4, 1.3, 0.4, 0.15, 0.05, 0.015, 0.005, 0.0018 μM, quantified by CPZ in the protein cage) diluted in the culture medium for 12 hours. The control group cells were treated with culture medium only. The supernatant was then removed, the cells were washed three times with PBS solution, and then treated with 1.5 J / cm 2 The cells were irradiated with an LED light source (680 nm) and continued to be cultured under hypoxic conditions for 12 hours, and then CCK8 diluted 10 times with DMEM was added. The light absorption was then measured at a wavelength of 450 nm. The relative cell survival rate was calculated according to the formula: Cell survival rate = (As-Ab) / (Ac-Ab)*100%, As: absorbance of the experimental wells (culture medium containing cells, CCK-8, and test drugs), Ac: absorbance of the control wells (culture medium containing cells, CCK-8, and no test drugs), Ab: absorbance of the blank wells (culture medium without cells and test drugs, CCK-8). Four replicate wells were set up for each cell line at each concentration, and each experiment was repeated three times. Results ( Figure 5B) The protein cages loaded with CPZ and TPZ showed a strong photodynamic therapy effect on 4T1 tumor cells. The cytotoxicity of the protein cages under hypoxia (IC 50 =0.15 μM) is stronger than that under normoxic conditions (IC 50 =0.3 μM) and was stronger than the direct mixture of free CPZ and TPZ (IC 50 =0.79 μM), the single protein and the current experimental concentration of TPZ had no obvious toxicity to cells ( Figure 5 C), indicating that TPZ in the protein cage can enhance the photodynamic toxicity of CPZ. The protein cage has no obvious dark toxicity ( Figure 5 D), indicating its safety for cells under light-protected conditions.

[0054] 2.3 Effect of NPs@UTSedr-HFn on cell colony formation 4T1 cells in the logarithmic growth phase were inoculated in 6-well plates, 700 cells / well, 2 mL DMEM was added to each well, and 5 groups were set up, with 2 replicates in each group. After 24 h of culture, the drug was administered by changing the medium containing the drug-loaded protein cage, and the drug concentration was 0.1 μM (quantified by CPZ). After incubation for 24 h, the medium was changed to fresh DMEM. The cells were illuminated for 3 min using a 680 nm wavelength light source with a light power of 1.5 J / cm 2 . Continue to culture for about 5 to 7 days until the blank control group grows uniformly dispersed and dense cell colonies, and the number of cells in most single clones is greater than 50. During this period, fresh DMEM is replaced every 2 days. Wash the cells once with PBS, fix them with 4% paraformaldehyde for 30 min, wash them twice with distilled water, and soak them in distilled water for 2 min each time. Then stain them with crystal violet for 20 min, wash them with distilled water twice, and dry them at 37 ℃. Fix the digital camera on the bracket to take pictures of each well, and turn the 6-well plate upside down on A4 paper. Use 30% acetic acid to dissolve crystal violet, and detect the absorbance value at 570 nm with an enzyme marker, which can also indirectly reflect the number of cell colonies. The results (Figure 6) showed that the number of cell colonies in the NPs@UTSedr-HFn wells was the least, and the cell viability after crystal violet staining was 12% compared with the blank control group. The significant inhibition of cell colony formation was due to the cytotoxic effect mediated by the cell uptake of NPs@UTSedr-HFn; the cell viability of the NPs@MR-HFn wells without uPAR targeting function was 30%, and the cell viability of the protein cage NPs@HFn wells after wild-type ferritin encapsulation was 60%. The significant inhibition of cell colony formation by NPs@UTSedr-HFn indicates that it can inhibit the formation of tumor masses by single cancer cells.

[0055] 2.4 Effect of NPs@UTSedr-HFn on cell migration The effect of NPs@UTSedr-HFn on 4T1 cell migration was detected by scratch test. Before inoculating cells, the 6-well plate was turned upside down and a horizontal line was drawn along the X-axis at the bottom with a marker pen for partitioning when taking pictures. Cells in the logarithmic growth phase were inoculated in the 6-well plate at 3×10 5 Cells / well / 2 mL culture medium. Place in a cell thermostat incubator at 37 °C and 5% CO2 for 24 h. Replace each well with 2 mL of culture medium containing different test substances, with a test substance concentration of 0.05 μM (quantified by CPZ in the drug-loaded protein cage). After incubation for 12 h, draw a vertical line along the Y axis in each well with a 10 μL white pipette tip, remove the culture medium, and wash once with culture medium to remove cells floating on the scratch. Subsequently, take photos under an optical microscope at 0, 6, 12, and 24 h after scratching to record changes in the scratch area. The scratch recording results (Figure 7) show that after 6 hours, the scratch area of ​​each treatment group has no obvious trend of decreasing; after 12 hours, the scratch area of ​​the blank control group and the free compound group has been significantly reduced; after 24 hours, the scratches of the blank control group and the free compound group have been completely closed, the scratch of NPs@HFn is close to closed, the scratch area of ​​NPs@MR-HFn is slightly larger than that of the NPs@HFn group, and the scratch area of ​​the NPs@UTSedr-HFn group is still about 50% unclosed. The scratch area is ranked as NPs@UTSedr-HFn>NPs@MR-HFn>NPs@HFn, indicating that NPs@UTSedr-HFn has a stronger ability to inhibit tumor cell migration than other drug-loaded protein cages.

[0056] 2.5 Tumor 3D cell spheroid permeability of NPs@UTSedr-HFn 3D cell spheroids were prepared using a round-bottom 96-well plate with ultra-low attachment properties. 4T1 cells were plated at 1×10 4Cells were seeded onto the plate at a density of 10 cells / well and incubated for 2 days. Then, the cell spheroids were treated with 1 µM NPs@UTSedr-HFn or NPs@HFn for different times (2, 4, 8, and 12 h), followed by gentle washing of the cell spheroids three times with PBS. Then, the cell spheroids were observed under a confocal microscope. Z-stack images were acquired at 20 μm intervals. The mean fluorescence intensity was plotted as a function of the distance from the periphery. The NPs@UTSedr-HFn-treated group showed significant penetration enhancement compared with the NPs@HFn-treated group and displayed stronger fluorescence intensity (Figure 8). The enhanced penetration of CPZ in NPs@UTSedr-HFn is mainly attributed to the tumor microenvironment-responsive extracellular drug release properties of this protein cage. Initially, NPs@UTSedr-HFn accumulated around the tumor due to uPAR receptor-mediated binding and TfR1 receptor-mediated enhanced penetration and retention effects specific to tumors. Subsequently, NPs@UTSedr-HFn was cleaved by MMPs, promoting the release of CPZ. Due to its lipophilic nature, CPZ diffused relatively easily across the cell membrane and matrix barriers, thereby penetrating deep into the tumor; the NPs@HFn group could only undergo TfR1 receptor-mediated endocytosis, and its targeting mechanism pathway and uptake pathway were less than those of the NPs@UTSedr-HFn group.

[0057] 3. In vivo antitumor evaluation of NPs@UTSedr-HFn 3.1 Establishment of tumor-bearing mouse model Subcutaneous tumor models were established in 6-8 week old female Balb / c mice (about 20 g). After depilation, 200 μL of 4T1 cells (1×10 7 The mice injected with tumor cells were returned to the cage for further feeding. The bulge formed by the injection gradually hardened and formed a tumor at the injection site. After 1 week, the tumor size was about 60-70 mm 3 The localization of CPZ and tumor proliferation inhibition experiments were performed.

[0058] 3.2 Tumor enrichment of NPs@UTSedr-HFn in tumor-bearing mice Fluorescence Molecular Tomography (FMT) was used to determine the tumor enrichment of CPZ: NPs@UTSedr-HFn (0.2 mg CPZ / kg mouse body weight) was injected into tumor-bearing mice via the tail vein. The mice were imaged at different time points (2, 4, 6, 8, 12, 24, 36, 48, 72, and 96 h) using the FMT 2500TM LX fluorescence molecular tomography (Figure 9). The recorded images were reconstructed in three dimensions using TrueQuant v3.0 software, and regions of interest (ROIs) were established at the tumor and non-tumor sites of tumor-bearing mice. The autofluorescence of tumor-bearing mice treated with saline was subtracted to obtain quantitative information on the average concentration of CPZ.

[0059] The results are as follows Fig. 9 , CPZ in the NPs@UTSedr-HFn group was more highly enriched in the tumor site than that in the free CPZ and NPs@HFn groups. The average drug concentration change trend at the tumor site of the NPs@UTSedr-HFn group was as follows: the drug concentration gradually increased after administration and reached a maximum value of 230 nM at around 6 h, which was 1.5 times that of the NP@HFn group and 2.3 times that of the free CPZ group; the drug concentration in the free CPZ group reached its maximum at 4 h and then began to decrease. The retention time of free CPZ at the tumor site was shorter, and the drug concentration dropped to a low plateau at 8 h, while the two drug-loaded protein cage groups could still maintain a high drug concentration until 24 h. The higher peak concentration and longer retention time of the drug-loaded protein cage at the tumor site than that of free CPZ indicated that ferritin had excellent drug carrier performance, which may be due to the high permeability and long retention (EPR) effect of the drug-loaded protein cage at the tumor site, and the ferritin receptors highly expressed by tumor cells can bind to the drug-loaded protein cage. NPs@UTSedr-HFn further fused with tumor-specific targeting peptides and their microenvironment response sequences can better target tumors and increase drug release.

[0060] 3.3 In vivo antitumor evaluation of NPs@UTSedr-HFn The steps of tumor inoculation are the same as described in 3.1. About 5-7 days after tumor inoculation, the mouse tumor grows to 150-200 mm 3 The 4T1-Balb / c tumor-bearing mice were randomly divided into 4 groups, including NPs@UTSedr-HFn, CPZ@UTSedr-HFn, NPs@HFn, and saline groups. The drug was injected into the tail vein at a dose of 0.2 mg CPZ / kg mouse body weight, that is, each mouse was injected with approximately 200 μL of 40μM test substance (quantified by CPZ in the drug-loaded protein cage). Six hours after administration, the dorsal tumor of the mouse was locally illuminated at a light dose of 50 J / cm 2Tumor size was measured with a vernier caliper every two days, and the weight changes of mice were recorded. Fig.10 A) shows that the three drug-loaded protein cages can effectively inhibit the proliferation of subcutaneous tumors in mice. The size of the subcutaneous tumors on day 6 was different from that in the saline group. On day 22, the average tumor volume in the saline group was close to 2000 mm 3 , while the average tumor volume of the NPs@UTSedr-HFn group did not exceed 800 mm 3 There was a significant statistical difference between the CPZ@UTSedr-HFn group and the saline group (P<0.001). The average tumor volume in the CPZ@UTSedr-HFn group was approximately 1000 mm 3 There was a significant statistical difference compared with the saline group (P < 0.01). The average tumor volume in the NPs@HFn group was about 1500 mm 3 The difference was statistically significant compared with the saline group (P<0.05). After the subcutaneous tumor was peeled off, the tumor weight was measured and photographed ( Fig.10 BC), there were significant differences between the CPZ@UTSedr-HFn group and the NPs@HFn group compared with the saline group (P<0.01), and the tumor inhibition degree of the CPZ@UTSedr-HFn group was stronger than that of the NPs@HFn group, indicating that uPAR-targeted intelligent ferritin has better targeted delivery and drug release ability than wild-type ferritin; NPs@UTSedr-HFn was significantly different from the saline group (P<0.001), indicating that NPs@UTSedr-HFn has a strong anti-cancer effect; NPs@UTSedr-HFn was statistically significantly different from the CPZ@UTSedr-HFn group (P<0.05), indicating that TPZ in the drug-loaded protein cage can enhance the anti-cancer effect. During the current detection period, the weight of the mice did not change significantly ( Fig.10 D), indicating that the drug-loaded protein cage has no obvious toxic side effects on mice.

[0061] Example 3 Application of P-selectin-targeted Sedr-HFn in antithrombotic drug delivery Rutin was selected as an antithrombotic drug. Rutin is a flavonol glycoside that can inhibit thrombosis by specifically binding to disulfide isomerase PDI.

[0062] 1. Preparation and characterization of P-selectin-targeted Rutin@Sedr-HFn (Rutin@PTSedr-HFn) 1.1 Preparation of Rutin@PTSedr-HFn Rutin@PTSedr-HFn was prepared by pH method. Specifically, ferritin (45 μM, 1000 μL) was adjusted to pH=12 with 0.5 M NaOH; then, Rutin was added to the above solution at a protein subunit / Rutin molar ratio of 1:100, and then placed in a silent mixer and slowly rotated for 25 min, and then adjusted to a neutral pH with 0.5 M HCl. It was left to stand at 4 °C for 2 h to allow the protein to renature. Unbound Rutin was removed by a desalting column, and finally a yellow, transparent, clear solution was obtained.

[0063] 1.2 Rutin loading determination The protein concentration in the drug-loaded protein cage was determined by the BCA protein assay kit according to the manufacturer's instructions and protocol. The drug-loaded protein cage was dissolved in a balanced buffer containing 40 mM Tris-HCl pH 8.0 and 150 mM NaCl, and the UV / visible absorption spectrum was analyzed by a microplate reader to determine the Rutin content at a wavelength of 350 nm. The encapsulation efficiency and loading capacity were calculated as follows: Where C represents mass concentration. The results show that when the molar ratio of Rutin / ferritin protein cage is fixed at 2400:1, one protein cage can encapsulate about 100 molecules, and the encapsulation rate is about 4.17% (w / w).

[0064] 1.3 Determination of particle size and morphology of drug-loaded protein cages Dynamic light scattering (DLS) was used to measure the uniformity and particle size of the drug-loaded protein cage. The sample with a concentration of 1 mg / mL was filtered through a 0.22 μm filter membrane, and 50 μL of the sample was placed in a quartz colorimetric tube for particle size measurement. The measurement was repeated three times at 25°C. DLS results ( Fig.11 A) shows that the particle size of the drug-loaded protein cage is 15.2 nm, which is similar to the particle size of the protein without drug.

[0065] 1.4 UV / Vis absorption spectra of drug-loaded protein cages The drug-loaded protein cage, protein and rutin solutions dissolved in 40 mM Tris-HCl pH8.0 and 150 mM NaCl were taken separately, and the UV / visible spectra at 300 nm-400 nm were measured with a microplate reader. The results (Figure 11B) showed that the protein cage did not show a characteristic absorption peak at 300 nm-400 nm, and free rutin showed its characteristic absorption peak at 360 nm. In contrast, Rutin@PTSedr-HFn showed an absorption peak at 345 nm. This may be because when Rutin@PTSedr-HFn encapsulated rutin, the protein cage and the groups of Rutin interacted, affecting the ultraviolet absorption of Rutin, resulting in a blue shift in the ultraviolet absorption spectrum of Rutin@PTSedr-HFn, so the ultraviolet absorption peak of Rutin shifted from 360 nm to 345 nm.

[0066] 1.5 Stability evaluation of drug-loaded protein cages The prepared drug-loaded protein cage was placed at 4 °C, and its particle size was measured by DLS every day to evaluate its stability. The results (Figure 11C) showed that the particle size of Rutin@PTSedr-HFn remained at about 15 nm within 12 days, and the particle size of the control group Rutin@HFn remained at about 12 nm within 12 days, both of which were consistent with the particle size of the empty protein cage. After 16 days, the particle sizes of both became larger, indicating that the drug-loaded protein cage aggregated. The above results show that the drug-loaded protein cage can maintain its structural stability within 12 days.

[0067] 2. In vivo antithrombotic evaluation of Rutin@PTSedr-HFn 2.1 Direct current stimulation-induced common carotid artery embolism model in mice ICR mice (weighing about 25 g) were randomly divided into 6 groups (6 mice / group), namely, saline group, free rutin group, Rutin@HFn group, Rutin@TR-HFn group and Rutin@PTSedr-HFn group. Except for the marked ones, the rutin dosage in each group was 0.5 mg / kg. The mice were fixed in a mouse fixer, and the drug was injected through the tail vein. Then, 10% chloral hydrate was injected intraperitoneally and anesthetized. The mice were supine on the operating table and their limbs were fixed. The skin in the middle of the neck was cut open, and the glandular tissue was carefully peeled off with forceps to expose the left common carotid artery. Pay attention to the separation of the vagus nerve of the mice. Carefully pick up the left common carotid artery and hook it into the groove of the infrared blood flow detection probe of the thrombus generator to ensure that the blood vessel is in good contact with the metal sheet in the groove. After the thrombus generator detects a stable pulsation signal and prompts "Ready", click on the measurement (set the current to 0.5mA), monitor the blockage rate, and record the time when the blockage rate reaches 95% (Figure 12A). The results showed that the vascular occlusion time (time to occlusion, TTO) of the control group was 143.8±14.0 s. The TTO of the free rutin group was extended to 225.8±14.6 s, which was not significantly different from the control group. The arterial TTO of mice in the Rutin@PTSedr-HFn (rutin content 0.5mg / kg) group was extended to about 463.3±50.0 s, which was twice that of the free rutin group. Even at a lower dose (rutin content 0.25 mg / kg), Rutin@PTSedr-HFn still showed an antithrombotic effect that was superior to free rutin (0.5 mg / kg) (TTO was 306.7±28.8 s). The TTO of Rutin@TR-HFn, which has no P-selectin targeting but can respond to thrombin, is about 222.5±9.2 s, which is almost the same as the TTO of the free rutin group. This is probably because in the absence of targeting peptides, rutin encapsulated in the protein cage cannot be released in a concentrated and precise manner at the site of thrombosis. The TTO of Rutin@HFn is about 149.2±13.2 s, which is almost the same as the control group. It is speculated that this is because its E helix cannot be effectively cut by thrombin to release the drug, and it cannot play an anti-thrombotic role.

[0068] 2.2 Ferric chloride-induced common carotid artery embolism model in mice The laser speckle contrast imaging (LSCI) was used to monitor blood flow in real time and image. The first half of the operation was consistent with the direct current stimulation-induced carotid artery embolism model. Thrombosis modeling was performed immediately after the tail vein injection. After the left common carotid artery was stripped, a filter paper with a width of about 2 mm was placed underneath it, and then placed under the laser speckle blood flow imaging system. After 15% FeCl3 was added, the blood flow velocity was immediately monitored (monitored every 5 s) until the blood vessels were completely blocked, and the blood vessel blockage time was recorded (Figure 12B). The results showed that the TTO of mice in the control group was approximately 295.8±18.1 s, and the TTO of mice in the free rutin group was approximately extended to 324.2±20.1 s, but there was no statistical difference between the two. The TTO of mice in the Rutin@PTSedr-HFn (0.5 mg / kg) group was 548.3±45.0 s, which was approximately 1.7 times that of the free rutin group. When the dose was reduced (rutin content 0.25 mg / kg), the TTO of the Rutin@PTSedr-HFn group mice was 438.3±17.1 s, which was about 1.3 times that of the free rutin group. The TTO of the Rutin@TR-HFn group mice with only thrombin responsiveness and the unmodified Rutin@HFn group mice were 334.2±23.9 s and 307.5±25.0 s, respectively, which were not significantly different from the free rutin group. These results are similar to those of direct current-induced carotid artery thrombosis, which fully demonstrates that Rutin@PTSedr-HFn can effectively inhibit FeCl3-induced arterial thrombosis, indicating the importance of smart ferritin with both targeting and response for drug delivery in lesion tissues.

[0069] 3. Evaluation of Bleeding Risk of Rutin@PTSedr-HFn ICR mice were randomly divided into 3 groups (6 mice / group): saline group, Rutin group and Rutin@PTSedr-HFn group. According to the dosing standard of 0.5 mg / kg rutin, the mice were injected through the tail vein. The mice were placed in a fixture, their tails were kept horizontal, and the tails were transected 0.5 cm away from the tip of the tail. They were immediately immersed in 10 mL of saline preheated to 37 °C. The time was counted from the spontaneous outflow of blood until the tip of the broken tail stopped bleeding continuously for 10 s, which was the tail bleeding time (Figure 13A), and the amount of mouse bleeding was quantified by measuring the hemoglobin content collected in the saline. The specific operation is as follows: The above solution was centrifuged at 2000 g for 10 min, the red blood cell sediment in the lower layer was collected, and 2 mL of lysis buffer (8.3 g / L NH4Cl, 1.0 g / L KHCO3, 0.037 g / L EDTA) was added and resuspended. After the red blood cells were completely ruptured, the absorbance at 575 nm was measured ( Fig.13 B). The results showed that the bleeding time of the control group mice after tail amputation was 113.0±4.4 s, and the bleeding time of the Rutin@PTSedr-HFn group mice was 106.3±4.7 s, which was not statistically different from the control group; the amount of bleeding after tail amputation of the control group mice was 0.3±0.05, and the amount of bleeding in the Rutin@PTSedr-HFn group was 0.33±0.05, and there was no significant difference in the amount of hemoglobin loss compared with the control group. The above results show that Rutin@PTSedr-HFn does not show hemorrhage risk in mice.

Claims

1. A smart extracellular ferritin drug carrier, characterized in that: The polypeptide sequence targeting the lesion tissue is fused to the everted C-terminus of ferritin, and the recognition sequence of the lesion overexpressed enzyme is fused to the DE loop of ferritin; the lesion tissue targeting polypeptide and the recognition sequence of the lesion overexpressed enzyme are both exposed on the outer surface of the ferritin protein cage, which can both target the lesion and respond to the corresponding protease in the lesion environment to release the drug extracellularly.

2. The intelligent extracellular ferritin drug carrier according to claim 1, characterized in that: The ferritin everted C-terminus is to change the hydrophilicity of the amino acids in the middle of the ferritin E helix so that they tend to be everted.

3. The intelligent extracellular ferritin drug carrier according to claim 2, characterized in that: The outward-turned C-terminus of ferritin is achieved by mutating the leucine at position 169 in the middle of the ferritin E helix to arginine to turn the ferritin C-terminus outward.

4. The intelligent extracellular ferritin drug carrier according to claim 1, characterized in that: The lesion tissue targeting sequences include: urokinase receptor targeting sequence CVSYKYFSRIRQC, P-selectin targeting sequence SDAEWVDVS; the recognition sequences of the lesion overexpressed enzymes include: matrix metalloproteinase recognition sequence PLGLAG; thrombin recognition sequence LVPRGS.

5. The intelligent extracellular ferritin drug carrier according to claim 1, characterized in that: The intelligent extracellular drug-releasing ferritin drug carrier is a urokinase receptor-targeted intelligent extracellular drug-releasing ferritin drug carrier UTSedr-HFn, and its amino acid sequence is shown in SEQ ID NO.1: MHHHHHHTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKEPDCDDWESGQNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPGGGPLGLAGGGSESGLAEYRFDKHTLGDSDNESCVSYKYFSRIRQC.

6. The intelligent extracellular ferritin drug carrier according to claim 1, characterized in that: The intelligent extracellular drug-releasing ferritin drug carrier is a P-selectin-targeted intelligent extracellular drug-releasing ferritin drug carrier PTSedr-HFn, and its amino acid sequence is shown in SEQ ID NO.2: MHHHHHHTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKEPDCDDWESGQNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPGGLVPRGSGGSESGLAEYRFDKHTLGDSDNESGGGGSDAEWVDVS.

7. Use of the intelligent extracellular ferritin drug carrier as claimed in claim 1 in targeted drug delivery.

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