A multifunctional biomimetic ferritin nanomaterial responsive to matrix metalloproteinases and its preparation method and application

Abstract

The present invention discloses a multifunctional biomimetic ferritin nanomaterial responsive to matrix metalloproteinases and its preparation method and application. The present invention modifies melittin on the surface of ferritin nanoparticles by a genetic engineering method, connects with a linker containing a matrix metalloproteinase 9 (MMP‑2 / 9) cleavage site, and obtains recombinant ferritin (SEQ ID NO: 6) expressed in Escherichia coli and self-assembled to form enzyme-responsive functionalized biomimetic nanoparticles. The present invention utilizes this method to connect melittin in series on ferritin nanoparticles, significantly reducing the toxicity of melittin, and responding to matrix metalloproteinases in the tumor microenvironment to kill tumor cells; At the same time, intravenous administration of melittin can also be achieved, with good therapeutic effects on both in situ and metastatic tumors.

Description

Technical Field

[0001] The present invention relates to the field of anti-tumor protein drugs, and in particular to a multifunctional biomimetic ferritin nanomaterial responsive to matrix metalloproteinases, and a preparation method and application thereof. Background Art

[0002] Melittin is the main active pharmacological component of honey bee venom. It is an amphiphilic peptide with a molecular weight of 2840 Da and is composed of 26 amino acids. The N-terminus is primarily hydrophobic with a +4 charge, while the C-terminus is primarily hydrophilic with a +2 charge, resulting in an overall charge of +6 at physiological pH. Melittin can directly kill tumor cells by pore-forming: Melittin normally exists as a monomer with a random coil structure; however, when inserted into lipid membranes, it aggregates into tetramers, and its structure changes to an α-helical conformation. Because of its positive charge, melittin can bind to negatively charged membrane surfaces, forming pores that lead to leakage of cellular components and increased permeability, thereby disrupting the integrity of the phospholipid bilayer and ultimately killing the cell. Melittin causes greater damage to tumor cell membranes than to normal cells due to its greater membrane potential. Melittin's positive charge selectively binds to the relatively abundant negative charges in the membranes of target tumor cells compared to healthy cells. In addition, tumor cells are less likely to develop acquired chemotherapy resistance; for example, it targets the overall structure of the cell membrane rather than specific cellular components. On the other hand, melittin has shown synergistic effects when combined with some chemotherapy drugs.

[0003] The anti-tumor mechanisms of melittin are as follows: ① After disrupting the cell membrane, melittin enters the cytoplasm directly, interfering with various signal transduction pathways associated with tumor malignancy. ② Inducing tumor cell apoptosis (cell shrinkage, chromatin condensation, and the formation of apoptotic bodies): Melittin increases cytoplasmic calcium levels and promotes the production of oxygen free radicals, which in turn reduces mitochondrial membrane potential, leading to the release of cytochrome C and promoting apoptosis. It also promotes the expression of death receptors (DRs), further inducing caspase activation and apoptosis. ③ Inhibiting tumor invasion and migration: Rac1, a member of the Ras superfamily of small GTP (guanosine triphosphate)-binding proteins, promotes tumor cell migration and invasion in various cancers. Melittin inhibits Rac1, thereby suppressing tumor cell invasion and migration. By inhibiting NF-κB expression, bee venom suppresses the expression and activity of MMP-9, thereby inhibiting tumor invasion and migration. ④ Inhibiting tumor angiogenesis: It inhibits tumor angiogenesis by downregulating vascular endothelial growth factor (VEGF) and cyclooxygenase-2. ⑤Cell cycle arrest: It can downregulate methylation binding protein 2 to promote cell cycle arrest; it can also increase the level of tensin homologous protein (PTEN), thereby downregulating the expression of cyclin D1 and cyclin-dependent kinase 4 (CDK4) to arrest the cell cycle and inhibit the growth of tumor cells.

[0004] However, melittin is a non-selective cytolytic peptide that can cause severe toxicity upon intravenous injection. It directly binds to red blood cells and releases hemoglobin. Furthermore, it is rapidly degraded in the blood. These limitations restrict the application of melittin in cancer therapy. Therefore, a suitable carrier is needed to deliver melittin to the target.

[0005] Ferritin is an iron-storage protein that plays a key role in the body's iron homeostasis and cellular antioxidant processes. Its primary function is to transport iron in a redox-inactive form to the tissues or cells where it is needed. Ferritin enters cells through interaction with the ferritin receptor (TfR1), found on the surface of most cells. This biological property makes ferritin highly targeted. TfR1 is highly expressed on the surface of tumor cells and has long been considered a potential target for tumor diagnosis and treatment. Ferritin can spontaneously assemble into hollow cage structures with an outer diameter of approximately 12 nm and an inner diameter of approximately 8 nm, making it a natural drug-carrying nanomaterial. The outer shell of natural ferritin is a mixture of light and heavy chains, with only the heavy chains capable of recognizing tumors. Engineered ferritin nanoparticles, engineered to be pure heavy chain, can more effectively target tumor cells, binding to the TfR1 membrane protein and mediating entry into tumor cells, leading to their accumulation in lysosomes.

[0006] Currently, most research is limited to utilizing ferritin nanocarriers to encapsulate chemotherapeutic drugs and deliver them to tumor cell lysosomes for release and tumor killing, but this remains ineffective against drug-resistant tumors. In recent years, peptide anti-tumor drugs have garnered increasing attention due to their low molecular weight, simple structure, high anticancer activity, high selectivity, minimal side effects, versatile administration routes, and resistance to multidrug resistance. Among them, melittin, the primary active component of bee venom, possesses diverse pharmacological activities, including anti-inflammatory, analgesic, antibacterial, anti-HIV, and anti-tumor activities. It has been reported to exhibit broad anti-tumor activity against various cancers, including hepatocellular carcinoma, leukemia, and breast cancer. During aggregation, it forms ion channels that penetrate the plasma membrane, disrupting membrane structures such as cell membranes and lysosomes. However, due to its strong nonspecific membrane-permeability, aggregation can easily induce thrombotic and other toxic side effects, severely limiting its application.

[0007] Matrix metalloproteinases (MMPs) regulate extracellular matrix degradation, cell proliferation, adhesion, and migration, thereby affecting cell activity and function. MMPs are expressed at low levels in normal tissues but at high levels in tumor tissues. Matrix metalloproteinases 2 / 9 (MMP-2 / 9) promote tumor cell growth by promoting angiogenesis and, at the same time, facilitate tumor cell infiltration and invasion by degrading and disrupting the basement membrane. Numerous studies have demonstrated that MMP2 / 9 levels are significantly elevated in malignant tumors such as colon, gastric, lung, and cervical cancer, making it a target for anti-tumor drugs. However, to date, there have been no reports of MMP-responsive cell death for tumor treatment. Summary of the Invention

[0008] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a multifunctional biomimetic ferritin nanomaterial that responds to matrix metalloproteinases.

[0009] Another object of the present invention is to provide a method for preparing the multifunctional biomimetic ferritin nanomaterial responsive to matrix metalloproteinases.

[0010] Another object of the present invention is to provide an application of the multifunctional biomimetic ferritin nanomaterial responsive to matrix metalloproteinases.

[0011] The purpose of the present invention is achieved through the following technical solutions:

[0012] A multifunctional biomimetic ferritin nanomaterial responsive to matrix metalloproteinases, wherein the active ingredient is recombinant ferritin (MMFn protein), and the amino acid sequence is shown in SEQ ID NO:6.

[0013] The nucleotide sequence of the gene encoding the recombinant ferritin (MMFn protein) is shown in SEQ ID NO: 5.

[0014] The method for preparing the multifunctional biomimetic ferritin nanomaterial responsive to matrix metalloproteinases comprises the following steps:

[0015] (1) The gene sequence of recombinant ferritin (MMFn protein) as shown in SEQ ID NO: 5 was ligated into the pET30a(+) vector to obtain a ferritin expression vector;

[0016] (2) The ferritin expression vector is transformed into Escherichia coli competent cells, and the multifunctional biomimetic ferritin nanomaterial responsive to matrix metalloproteinase is obtained through induction expression and purification.

[0017] In step (1), the pET30a(+) vector is modified with the sequences of matrix metalloproteinase and melittin at the N-terminus of ferritin by PCR, and then the sticky ends are exposed by enzyme cleavage, and finally the target protein is connected to the vector by enzyme ligation; wherein the restriction endonucleases used are BamHI and HindIII.

[0018] In step (1), the molar ratio of the pET30a(+) vector and the gene sequence of the MMFn protein in the ligation system is preferably 1:5.

[0019] The E. coli competent cells described in step (2) are preferably BL21 / DE3 E. coli competent cells.

[0020] The induction conditions described in step (2) are preferably: Escherichia coli cell OD600 is 0.8, the induction concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) is 1 mmol / L, the induction temperature is 18° C., and the induction time is 4 hours.

[0021] The purification in step (2) is preferably performed by Ni column affinity chromatography.

[0022] The application of the multifunctional biomimetic ferritin nanomaterial responsive to matrix metalloproteinases in the preparation of anti-tumor drugs and / or tumor vaccines.

[0023] The multifunctional biomimetic ferritin nanomaterial responsive to matrix metalloproteinases can target tumor cells, induce tumor cell apoptosis, and inhibit tumor cell proliferation, thereby achieving the purpose of anti-tumor.

[0024] The tumor includes conventional malignant tumors in the art; preferably at least one of pancreatic cancer, breast cancer and colon cancer.

[0025] The anti-tumor drug is administered intravenously, preferably by intravenous injection.

[0026] The present invention has the following advantages and effects compared to the prior art:

[0027] (1) The present invention designs a nanoparticle Melittin-MMP-HFn based on human heavy-chain ferritin (HFn) for tumor microenvironment-responsive release of melittin, a nonspecific membrane-permeating peptide. In the present invention, melittin is modified on the surface of ferritin nanoparticles by genetic engineering methods and connected with a linker containing a matrix metalloproteinase 9 (MMP-2 / 9) cleavage site. The recombinant ferritin is expressed in Escherichia coli and self-assembled to form enzyme-responsive functionalized biomimetic nanoparticles MMFn. The biomimetic nanoparticles MMFn specifically target the tumor site through active targeting mediated by the enhanced permeability and retention effect (EPR) and transferrin receptor 1 (TfR1). MMP-2 / 9 highly expressed in tumor cells cleaves the particles, thereby releasing melittin, which binds to the negatively charged cell membrane, forming pores in the cell membrane and destroying the integrity of the phospholipid bilayer, allowing the cell contents to flow out. After breaking through the membrane, melittin enters the cytoplasm and induces apoptosis by activating caspases, thereby further killing tumor cells.

[0028] (2) The present invention is based on ferritin nanoparticles and uses genetic engineering methods to carry out genetic modification on the pET30a(+)-HFn plasmid that induces the expression of ferritin to construct a plasmid that can express ferritin, an enzyme response element and a melittin fusion protein in series. The MMP-2 / 9 specific enzyme cleavage site and melittin with membrane lysis function after aggregation are sequentially added to the N-terminus to construct an enzyme-responsive multifunctional biomimetic ferritin nanomaterial MMFn; in addition, the enzyme response site is replaced by a linker to modify melittin and the linker on ferritin (melittin and the linker are fused and expressed at the N-terminus of ferritin), that is, by replacing the MMP-2 / 9 enzyme response sequence with an equal-length flexible linker (GGSGGS), a mutant MFn is constructed as a control. The present invention utilizes this system to connect melittin in series to ferritin nanoparticles and respond to matrix metalloproteinases in the tumor microenvironment to kill tumor cells. At the same time, the intravenous administration of melittin can also be achieved, which has a good therapeutic effect on both in situ and metastatic tumors.

[0029] (3) The present invention connects bee venom peptide to the N-terminus of ferritin through the matrix metalloproteinase cleavage site (enzyme response site), and constructs an enzyme-responsive multifunctional biomimetic ferritin nanomaterial MMFn. The MMFn can selectively kill tumor cells in vitro, and MMFn can be specifically enriched in the tumor site of mice after tail vein injection, and has a certain tumor targeting ability; at the same time, MMFn can not only directly kill tumor cells in vivo, but also reshape the tumor microenvironment, stimulate innate immunity and adaptive immune responses, and inhibit tumor metastasis and recurrence.

[0030] (4) The present invention provides a safe and effective intravenous administration nanoparticle with bee venom as the main component. The nanoparticle is a biomimetic nanoparticle with ferritin as the carrier, which significantly reduces the toxicity of bee venom and changes the previous administration method to achieve intravenous administration of bee venom.

[0031] (5) The present invention further optimizes the conditions for protein expression and purification, achieving a simple, rapid, and efficient production of biomimetic ferritin nanoparticles with the characteristics of high yield and significant effect. It solves the limitation of melittin that the administration method is single due to hemolysis and can only be administered intratumorally, as well as the problems of poor specificity and rapid degradation. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of biomimetic ferritin nanoparticles; wherein, A represents the fusion expression of bee venom peptide and matrix metalloproteinase response sequence at the N-terminus of ferritin subunit; B represents the fusion expression of bee venom peptide and linker sequence at the N-terminus of ferritin subunit.

[0033] Figure 2 Figure 2 is a diagram showing the purification and biochemical characteristics of the biomimetic ferritin nanoparticle platform; A is the SDS-PAGE electrophoresis results of HFn, MMFn and MFn; B is the photographic results of biomimetic ferritin nanoparticles under a transmission electron microscope; C is a particle size characterization diagram of biomimetic ferritin nanoparticles in dynamic light scattering (DLS); D is the zeta potential diagram of biomimetic ferritin nanoparticles; E is the circular dichroism spectrum of biomimetic ferritin nanoparticles; and F is a schematic diagram showing the stability of biomimetic ferritin nanoparticles in complete culture medium DMEM.

[0034] Figure 3It is a graph of the in vitro toxicity of biomimetic ferritin nanoparticles; wherein, A is the MMP-2 / 9-dependent hemolysis induced by biomimetic iron nanoparticles in vitro; B is the MTT result graph of the toxicity of biomimetic ferritin nanoparticles to tumor cells and normal cells; C is the flow cytometry result and statistical graph of the apoptosis of tumor cells induced by biomimetic ferritin nanoparticles; D is the fluorescence graph of the apoptosis of tumor cells induced by biomimetic ferritin nanoparticles; E is the result graph of the cell clone formation experiment of biomimetic ferritin nanoparticles; F is the result graph of the cell scratch of biomimetic ferritin nanoparticles; G is the western blot result graph of the HMGB1 release promoted by biomimetic ferritin nanoparticles in tumor cells; H is the statistical graph of the ATP release promoted by biomimetic ferritin nanoparticles in tumor cells.

[0035] Figure 4 This is the fluorescent in vivo imaging of biomimetic ferritin nanoparticles.

[0036] Figure 5 These are in vivo anti-tumor research graphs of bionic ferritin nanoparticles; among them, A is a statistical graph of tumor volume and weight, tumor size, and mouse weight changes of bionic ferritin nanoparticles in the Panc02 animal model; B is a statistical graph of tumor volume and weight, tumor size, and mouse weight changes of bionic ferritin nanoparticles in the MC38 animal model; C is a statistical graph of tumor volume and weight, tumor size, and mouse weight changes of bionic ferritin nanoparticles in the 4T1 animal model.

[0037] Figure 6 This is a statistical graph of the flow cytometry results of the effects of biomimetic ferritin nanoparticles on the tumor microenvironment in three animal models: MC38, 4T1, and Panc02.

[0038] Figure 7 This is a diagram showing the safety evaluation results of biomimetic ferritin nanoparticles. DETAILED DESCRIPTION

[0039] The present invention will be described in further detail below in conjunction with the examples, but embodiments of the present invention are not limited thereto. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually based on conventional experimental conditions or the experimental conditions recommended by the manufacturer. Unless otherwise stated, the reagents and raw materials used in the present invention can be obtained commercially.

[0040] Example 1: Construction and production of a biomimetic ferritin nanoparticle platform

[0041] 1.1 Experimental Materials:

[0042] (1) Bacterial strain: BL21 (DE3) E. coli competent cells, purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0043] (2) Plasmids: pET30a(+) vector and pET30a(+)-HFn plasmid were purchased from Suzhou Jinweizhi Biotechnology Co., Ltd.

[0044] (3) Reagents: DNA agarose gel recovery kit, plasmid miniprep kit, plasmid maxiprep kit, DNA marker, Taq PCR Mastermix, and Pfu PCR Mastermix used in molecular biology experiments were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; restriction endonucleases BamHI, HindIII, and T4 ligase were purchased from New England Biolabs; LB medium, antibiotic ampicillin, and protein expression inducer IPTG used in bacterial culture were purchased from Sigma and Amresco, respectively; other reagents were of domestic analytical grade.

[0045] (4) Buffer: The buffer used for purifying the biomimetic ferritin nanoparticle platform is: 20 mM Tris-HCl, 50 mM NaCl, and the pH is adjusted to 7.

[0046] (5) Primer fragments: The primer sequences were synthesized by Shanghai Biotechnology Co., Ltd.

[0047] 1.2 Experimental methods:

[0048] Construction of the ferritin expression vector The nucleic acid sequence of human heavy-chain ferritin (HFn) was codon-optimized for Escherichia coli based on its protein sequence. The optimized ferritin gene sequence (HFn, SEQ ID NO: 4) was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. Based on the amino acid sequence of melittin (SEQ ID NO: 1), the amino acid sequence of melittin and the enzyme-responsive site (SEQ ID NO: 2), and the amino acid sequence of melittin and the linker (SEQ ID NO: 3), primers with restriction enzyme cleavage sites were designed (primer sequences are shown below) to amplify the optimized HFn ferritin fragment by PCR, yielding the amplified fragment (MMFn, SEQ ID NO: 5). The amplified MMFn fragment and the pET30a(+) plasmid were then double-digested with BamHI and HindIII, respectively, and the DNA was recovered from an agarose gel. The digested pET30a(+) vector and the ferritin fragment (MMFn) were then ligated overnight at 16°C using T4 ligase at a molar ratio of 1:5. For specific steps, refer to the Takara DNA Ligation Kit manual. The ligation product was transformed into BL21 / DE3 E. coli competent cells, and clones with correct ligation were screened to obtain the nanoparticle vector Melittin-MMP-HFn. At the same time, the MMP-2 / 9 enzyme response sequence was replaced with a flexible linker of equal length (GGSGGS) to construct a mutant MFn as a control. The MFn fragment (SEQ ID NO: 7) was amplified by PCR and ligated into the pET30a(+) vector. The remaining steps were the same as above. The sequences involved are as follows:

[0049] Amino acid sequence of melittin (SEQ ID NO: 1):

[0050] GIGAVLKVLTTGLPALISWIKRKRQQ;

[0051] Amino acid sequence of melittin and enzyme response site (SEQ ID NO: 2):

[0052] GIGAVLKVLTTGLPALISWIKRKRQQGPLGVR;

[0053] Amino acid sequence of melittin and linker (SEQ ID NO: 3)

[0054] GIGAVLKVLTTGLPALISWIKRKRQQGGSGGS;

[0055] The primers for amplification of the MMFn ferritin fragment are:

[0056] MMFn-1: 5'-TCTTGGATCAAACGCAAACGTCAGCAGGGTCCACTGGGTGTACGTGGTGGCGGTAGCACC-3';

[0057] MMFn-2: 5'-GTGCTGACCACTGGTCTGCCGGCACTGATTTCTTGGATCAAACGCAAA CGTCAGCAG-3';

[0058] MMFn-3: 5'-CGgGATCCATGGGTATTGGTGCTGTTCTGAAAGTGCTGACCACTGGTCT GCCG-3';

[0059] MMFn-4: 5'-CCCAAGCTTTTAAGATTCGTTGTCAGAATCGCCC-3';

[0060] The amplification primers for the MFn ferritin fragment are:

[0061] MFn-1: 5'-TCTTGGATCAAACGCAAACGTCAGCAGGGTGGCGGTAGCGGCGGTAGCGGCGGTAGCACC-3';

[0062] MFn-2: 5'-GTGCTGACCACTGGTCTGCCGGCACTGATTTCTTGGATCAAACGCAAAC GTCAGCAG-3';

[0063] MFn-3: 5'-CGgGATCCATGGGTATTGGTGCTGTTCTGAAAGTGCTGACCACTGGTCTG CCG-3';

[0064] MFn-4: 5'-CCCAAGCTTTTAAGATTCGTTGTCAGAATCGCCC-3'.

[0065] The nucleic acid sequence of HFn (SEQ ID NO:4):

[0066] ACCACCGCGTCCACCTCCCAGGTTCGTCAGAACTACCACCAAGATTCCGAAGCAGCTATCAACCGTCAGATCAACCTGGAACTGTACGCGTCTTACGTGTACCTGTCTATGAGCTATTACTTCGACCGTGATGACGTTGCACTGAAAAACTTCGCTAAGTACTTCCTGCACCAGTCCCACGAGGAACGCGAGCACGCTGAGAAACTGATGAAACTGCAGAACCAGCGTGGCGGTCGTATTTTCCTGCAGGACATCAAAAAACCGGATTGTGACGATTGGGAAAGCGGTCTGAACGCTATGGAATGTGCGCTGCACCTGGAGAAAAATGTTAACCAGTCCCTGCTGGAACTGCACAAACTGGCGACCGACAAAAACGATCCGCACCTGTGTGACTTCATTGAAACTCACTACCTGAATGAACAGGTGAAAGCAATCAAGGAGCTGGGCGACCACGTCACTAACCTGCGTAAAATGGGTGCACCGGAAAGCGGCCTGGCTGAGTACCTGTTCGATAAGCACACTCTGGGCGATTCTGACAACGAATCT。

[0067] Nucleic acid sequence of MMFn (SEQ ID NO:5):

[0068] GGTATTGGTGCTGTTCTGAAAGTGCTGACCACTGGTCTGCCGGCACTGATTTCTTGGATCAAACGCAAACGTCAGCAGGGTCCACTGGGTGTACGTGGTGGCGGTAGCACCACCGCGTCCACCTCCCAGGTTCGTCAGAACTACCACCAAGATTCCGAAGCAGCTATCAACCGTCAGATCAACCTGGAACTGTACGCGTCTTACGTGTACCTGTCTATGAGCTATTACTTCGACCGTGATGACGTTGCACTGAAAAACTTCGCTAAGTACTTCCTGCACCAGTCCCACGAGGAACGCGAGCACGCTGAGAAACTGATGAAACTGCAGAACCAGCGTGGCGGTCGTATTTTCCTGCAGGACATCAAAAAACCGGATTGTGACGATTGGGAAAGCGGTCTGAACGCTATGGAATGTGCGCTGCACCTGGAGAAAAATGTTAACCAGTCCCTGCTGGAACTGCACAAACTGGCGACCGACAAAAACGATCCGCACCTGTGTGACTTCATTGAAACTCACTACCTGAATGAACAGGTGAAAGCAATCAAGGAGCTGGGCGACCACGTCACTAACCTGCGTAAAATGGGTGCACCGGAAAGCGGCCTGGCTGAGTACCTGTTCGATAAGCACACTCTGGGCGATTCTGACAACGAATCT;

[0069] MMFn amino acid sequence (SEQ ID NO:6):

[0070] GIGAVLKVLTTGLPALISWIKRKRQQGPLGVRGGGSTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES;

[0071] Nucleic acid sequence of MFn (SEQ ID NO:7):

[0072] .

[0073] After cloning, construction and screening, MFn and MMFn biomimetic ferritins with correct sequences were obtained. After trial expression, the optimal induction conditions and expression strains were screened. The optimal induction conditions for MFn and MMFn were OD600 of 0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) 1mmol / L, and induction at 18℃ for 4 hours. The expression levels of MFn and MMFn biomimetic ferritin nanoparticles were both approximately 22 mg per liter of bacterial liquid. The optimal induction conditions for HFn were OD600 of 0.8, IPTG 1mmol / L, and induction at 37℃ for 4 hours. The expression levels of HFn protein nanoparticles were both approximately 31 mg per liter of bacterial liquid.

[0074] Example 2: Purification and characterization of biomimetic ferritin nanoparticle platform

[0075] 2.1 Experimental Materials: CM-Sepharose was purchased from GE Healthcare. 50 kDa ultrafiltration tubes were purchased from Merck Millipore. 0.22 μm and 0.45 μm filters were purchased from Pall Corporation. Transmission electron microscope, Tecnai Spirit (120 kV), was manufactured by FEI (USA). Common chemical reagents (SDS-PAGE gel preparation and electrophoresis buffer, glycine, Tris, Coomassie Brilliant Blue, etc.) were purchased from Shanghai Biotechnology Co., Ltd.

[0076] 2.2 Experimental methods:

[0077] 2.2.1 Isolation and purification of HFn

[0078] (1) Purification of crude protein: The HFn ferritin crude extract was heated in a 70°C water bath for 15 min to denature and precipitate most of the heat-sensitive impurities. The denatured protein precipitate was removed by centrifugation at 12,000 rpm for 15 min. The crude ferritin solution was then filtered through a 0.45 μm filter to remove the fine precipitate to obtain a crude ferritin solution. The crude ferritin solution was concentrated in a 50 kDa cut-off concentrator and filtered through a 0.22 μm filter to remove the fine precipitate.

[0079] (2) Soak the anion exchange agarose gel (CM-Sepharose) in 200mM Tris-HCl (pH7.8) for column equilibration. Add a small amount of sample buffer to the column; open the column outlet and drain a small amount of loading buffer to expel the air at the bottom of the column; shake the resin and guide it into the column with a glass rod without introducing bubbles; open the column outlet and add more sample buffer when the resin accumulates; allow the resin to reach exchange equilibrium. After loading the column, rinse the resin with sample buffer until its pH and ionic strength finally meet the experimental requirements and wash the resin to the baseline level. The sample buffer used in this step is generally not more than 10 times the volume of the resin. The sample solution must first be clarified by centrifugation or filtration with 0.45μm pore size filter paper. Open the bottom outlet of the column and introduce the loading buffer into the column until the liquid level reaches the resin surface. Close the bottom outlet of the column; use a pipette to slowly add the protein solution to the resin surface.

[0080] (3) Open the outlet of the chromatography column and allow the protein solution to enter the resin. When the surface of the solution overlaps with the surface of the resin, close the outlet of the chromatography column. Slowly add a small amount of loading buffer to the surface of the resin. Then, perform protein chromatography and elution according to the gradient concentration. The biochemical characteristics of the protein are analyzed by SDS-PAGE electrophoresis.

[0081] (4) The results are as follows Figure 2As shown in Figure A: After a simple heat purification of the crude protein extract, over 80% of the impurities were removed by heat denaturation. Elution via an anion exchange chromatography column and SDS-PAGE electrophoresis revealed that the position of the purified protein bands was consistent with the predicted size. The molecular weight of the purified HFn was approximately 21.1 kDa. The protein purity was over 95%.

[0082] 2.2.2 Affinity chromatography purification of MMFn and MFn

[0083] (1) The MFn and MMFn biomimetic ferritins prepared in Example 1 were resuspended in a buffer solution (20 mM Tris, 500 mM NaCl, pH = 7), centrifuged at 4500 rpm and 4°C for 20 min, and the supernatant was removed and resuspended in a buffer solution (20 mM Tris, 500 mM NaCl, pH = 7). After ultrasonic disruption, the supernatant was centrifuged at 15000 rpm and 4°C for 20 min. The supernatant was collected and placed on ice, and then filtered through a 0.45 μm filter to remove the fine precipitate to obtain a crude MMFn or MFn ferritin solution.

[0084] (2) Take the purified Ni column, clean the column, add 3-4 ml nickel agarose gel, wash the column with ddH2O 5 times, and then equilibrate the column with equilibration solution 2 times. Combine the column and supernatant thoroughly, place it on a four-dimensional rotating mixer and incubate for 1-2 hours before taking it out for purification. Equilibration buffer: 20 mM Tris buffer (containing 500 mM NaCl) at pH 8.0. Elution buffer: 20 mMTris buffer (containing 500 mM NaCl and 50 mM imidazole) at pH 8.0, add imidazole according to the concentration gradient. Pass through the column, flow through the liquid, and sample it. Use 20 times the column volume of equilibration buffer to wash away the unadsorbed sample, with a flow rate of 1-2 ml / min. The column was eluted in stages using 50mM, 100mM, 200mM, and 300mM imidazole concentrations for approximately 5-10 column volumes (if more protein is adsorbed, use more eluent) at a flow rate of 1-2ml / min, collecting 5ml / tube. The collected protein from each concentration gradient was sampled and analyzed by SDS-PAGE electrophoresis. Based on the SDS-PAGE electrophoretogram, if protein was present, it was collected and concentrated using a 50kDa ultrafiltration tube.

[0085] (3) Figure 2 As shown in A: After HIS tag affinity purification of MFn and MMFn, over 80% of impurities were removed. SDS-PAGE electrophoresis results showed that the positions of the purified protein bands were consistent with the predicted protein size (molecular weight approximately 24.9 kDa). The protein purity was over 95%.

[0086] 2.2.3 Characterization of HFn, MMFn, and MFn

[0087] (1) Transmission electron microscopy observation of nanoparticle morphology

[0088] Drop the purified HFn, MFn and MMFn onto a copper mesh with a support film. After a few minutes, use filter paper to absorb excess liquid from the edge of the copper mesh. Then drop the dye solution on it and stain it for 1 to 2 minutes. Then use filter paper to absorb the dye solution. After drying, it can be observed with a transmission electron microscope. The transmission electron microscope results are as follows: Figure 2 As shown in B: After purification, HFn, MFn and MMFn all have uniform size, stable structure and morphology, and a diameter of about 20 nm.

[0089] (2) Characterization of particle size of biomimetic ferritin nanoparticles in dynamic light scattering (DLS)

[0090] Add the solution to be tested into the cuvette, cover it, and perform particle size measurement on the solution. Set up three replicates. The dynamic light scattering results are as follows: Figure 2 As shown in Figure C, the particle sizes of MFn and MMFn are slightly larger than those of HFn, indicating that the modification with melittin increases the particle size. The particle size of HFn is approximately 10 nm, while the particle sizes of MFn and MMFn are approximately 20 nm.

[0091] (3) Characterization of the potential size of biomimetic ferritin nanoparticles

[0092] Add the solution to be tested into the potential detection cell, making sure there are no bubbles, close the lid, and perform potential detection on the solution. Set up three repetitions. The potential detection results are as follows: Figure 2 As shown in D: MFn, MMFn have a larger potential than HFn. This is because bee venom peptide carries a positive charge, which neutralizes the negative charge of ferritin.

[0093] (4) Circular dichroism spectra of biomimetic ferritin nanoparticles

[0094] HFn, MFn and MMFn were quantified to 100ug / ml and added to the sample pool for full wavelength scanning. Three replicates were set. The circular dichroism results of biomimetic ferritin nanoparticles are shown in Figure 2. Figure 2 As shown in E: The circular dichroism results of biomimetic ferritin nanoparticles show that there is not much difference between MFn, MMFn and HFn, indicating that the modification of melittin does not change the secondary structure of ferritin nanoparticles.

[0095] (5) Stability testing of biomimetic ferritin nanoparticles

[0096] HFn, MFn and MMFn were diluted with DMEM complete medium and placed at 4°C. The particle size was tested at the same time every day. After the test, they were placed back at 4°C and tested for one week to test the stability of biomimetic ferritin nanoparticles. In order to prevent protein contamination, an appropriate amount of double antibody (penicillin-streptomycin antibiotics, 100U / ml) can be added. The experiment was repeated three times. The stability test results of biomimetic ferritin nanoparticles are shown in Figure 2. Figure 2 As shown in Figure F: The results show that the particle size of HFn, MFn and MMFn did not change significantly within one week, indicating that they have good stability.

[0097] 2.2.4 Biomimetic iron nanoparticles induce MMP-2 / 9-dependent cell hemolysis in vitro

[0098] Blood was collected from 8-week-old female Bal b / c mice (weight 20 g / mouse, purchased from Hunan Slake Company) in EDTA 32K anticoagulant tubes. Mouse erythrocytes were isolated and purified and diluted to 10 7 Hemolysis assays were performed at 100 μM / mL. Different concentrations of melittin, MFn, MFn + MMP-2 / 9, MMFn, and MMFn + MMP-2 / 9 were incubated with mouse erythrocytes at 37°C for 3 hours, centrifuged at 3000 rpm for 3 minutes, and the absorbance of the supernatant was measured at 540 nm. The final concentrations of each drug were 0, 2, 4, 6, 8, 10, and 20 μM. Matrix metalloproteinase (MMP2 / 9) was purchased from abcam, catalog number ab81550. Mouse erythrocytes treated with a final concentration of 1% (v / v) TritonX-100 served as a positive control, and the hemoglobin release rate of this group was set as 100%. The hemolytic effect of the nanoparticles on erythrocytes was investigated in the presence or absence of matrix metalloproteinases.

[0099] The results of biomimetic iron nanoparticles inducing MMP-2 / 9-dependent cell hemolysis in vitro are as follows Figure 3 As shown in Figure A: Only when MMFn is added in conjunction with the addition of matrix metalloproteinases does erythrocyte hemolysis occur. MMFn, MFn alone, MFn + MMP-2 / 9, and HFn do not cause erythrocyte hemolysis. This suggests that MMFn, under the action of matrix metalloproteinases, releases melittin, which then forms tetramers, rupturing the erythrocyte membrane and releasing hemoglobin.

[0100] 2.2.5 Cytotoxicity assay

[0101] The day before, pancreatic cancer cells Panc02 and MIA PaCa-2 (abbreviated as MiaPaCa), breast cancer cells MDA-MB-231 and 4T1, colon cancer cells MC38, and human umbilical vein epithelial cells HUVEC (all purchased from ATCC) were cultured to the logarithmic growth phase. The cells were then digested, centrifuged, and counted. 100 μL of culture medium containing 3,000 to 7,000 cells per well was seeded onto a 96-well plate and incubated at 37°C, 5% CO2, and 90% humidity for 24 hours. Melittin, HFn, MFn, and MMFn were then added (all at a final concentration of 2 μM), with PBS buffer serving as a control. Treatment was continued for 12 hours. Prior to the experiment, MTT solution was added to MTT powder to make a 5 mg / mL MTT solution (any unused portion can be stored at -20°C). 10 μL of MTT solution was added to each well to a final concentration of 0.5 mg / mL. Incubate the cells at 37°C, 5% CO₂, and 90% humidity for 4 hours. Carefully aspirate all supernatant from the wells to prevent disruption of the cell monolayer. Add 200 μL of dimethyl sulfoxide (DMSO) to each well. Shake the plate at 300 rpm / min for 10 minutes. Measure absorbance at 570 nm using a microplate reader. Perform the experiment in triplicate.

[0102] The results of cytotoxicity experiments were as follows Figure 3 Figure B: The biomimetic ferritin nanoparticles (MMFn) demonstrated a cytotoxic effect on pancreatic cancer cells Panc02 and MIA PaCa-2, breast cancer cells MDA-MB-231 and 4T1, and colon cancer cells MC38, whereas no cytotoxic effect was observed in the control group, which lacks matrix metalloproteinase expression. MMFn also demonstrated little cytotoxicity against normal human umbilical vein epithelial cells (HUVECs). This may be because normal cells lack matrix metalloproteinase expression, preventing melittin from being shed from the ferritin nanoparticles and causing cell cytotoxicity.

[0103] 2.2.6 Biomimetic ferritin nanoparticles induce tumor cell apoptosis

[0104] MIA Paca-2 cells (5×10 5After the cells were plated, melittin, HFn, MFn, and MMFn (final concentration of 2 μM) were added respectively. PBS buffer was added as a control and the drugs were treated for 4 hours. The cell culture medium was aspirated into a suitable centrifuge tube, the adherent cells were washed once with PBS, and an appropriate amount of trypsin was added to digest the cells and the adherent cells were collected. The culture medium and the collected adherent cells were centrifuged at 1000 g for 5 minutes, the supernatant was discarded, the cells were collected, and the cells were gently resuspended in PBS and counted. 50,000 to 100,000 resuspended cells were taken, centrifuged at 1000 g for 5 minutes, the supernatant was discarded, 195 μl of Annexin V-FITC conjugate solution was added to gently resuspend the cells, and then 5 μl of Annexin V-FITC and 10 μl of propidium iodide staining solution were added and gently mixed. Incubate at room temperature (20-25°C) in the dark for 10-20 minutes, and then perform flow cytometry. The experiment was repeated three times.

[0105] The results of biomimetic ferritin nanoparticles inducing tumor cell apoptosis are as follows Figure 3 As shown in C: MMFn and melittin can significantly induce apoptosis of tumor cells, while HFn and MFn have no effect on tumor cells.

[0106] 2.2.7 In situ fluorescence detection of tumor cell apoptosis induced by biomimetic ferritin nanoparticles

[0107] MIAPaca-2 cells (5×10 5 Cells were cultured in 24-well plates, and melittin, HFn, MFn, and MMFn (final concentration of 2 μM) were added respectively. PBS buffer was added as a control. After 4 hours of drug treatment, the multiwell plates were centrifuged at 1000 g for 5 minutes. The cell culture medium was aspirated and washed once with PBS. 195 μl of Annexin V-FITC binding solution was added, followed by 5 μl of Annexin V-FITC and 10 μl of propidium iodide staining solution, and the mixture was gently mixed. Incubate at room temperature (20-25°C) in the dark for 10-20 minutes. Observe under a fluorescence microscope. Annexin V-FITC is green fluorescence, and propidium iodide (PI) is red fluorescence. The experiment was repeated three times.

[0108] In situ fluorescence detection results of biomimetic ferritin nanoparticles inducing tumor cell apoptosis Figure 3 As shown in D: The results showed that MMFn and melittin could significantly induce apoptosis of tumor cells, while HFn and MFn had no effect on tumor cells.

[0109] 2.2.8 Colony formation experiment of biomimetic ferritin nanoparticles to inhibit tumor cell proliferation

[0110] MIAPaca-2 cells in the logarithmic growth phase were trypsinized, resuspended in complete DMEM medium, and counted. 500–1000 cells / well were seeded in each experimental group in a 6-well culture plate. Melittin, HFn, MFn, and MMFn (all at a final concentration of 2 μM) were added, respectively. PBS buffer was used as a control. The cells were treated for 4 hours and cultured until the majority of individual clones contained >50 cells. The medium was changed every 3 days, and the cells were observed. After cloning, the cells were photographed under a microscope, washed once with PBS, fixed with 1 mL of 4% paraformaldehyde per well for 30–60 minutes, washed once with PBS, and then stained with 1 mL of crystal violet solution per well for 10–20 minutes. Finally, the cells were washed several times with PBS, air-dried, and photographed. The experiment was repeated three times.

[0111] The results of the clone formation experiment of biomimetic ferritin nanoparticles inhibiting tumor cell proliferation are as follows Figure 3 As shown in E: MMFn and melittin can significantly inhibit the proliferation of tumor cells, while MFn and HFn have no significant effect on the proliferation of tumor cells.

[0112] 2.2.9 Cell scratch assay to inhibit tumor cell migration using biomimetic ferritin nanoparticles

[0113] MIAPaca-2 cells in the logarithmic growth phase were seeded in 6-well plates. When the cells adhered and reached 100% fusion, the plate was scratched with a 10-μl pipette tip, either parallel or perpendicular to the plate. The tip should be vertical and not tilted. The cells were rinsed three times with PBS, the scratched cells removed, and serum-free culture medium was added. The plates were first photographed under a 4x microscope. The cells were then treated with melittin, HFn, MFn, and MMFn (all at a final concentration of 2 μM). PBS buffer was used as a control. The plates were photographed again 24 hours later. The experiment was repeated three times.

[0114] The results of cell scratch experiments on the inhibition of tumor cell migration by biomimetic ferritin nanoparticles are as follows Figure 3 As shown in F: MMFn and melittin can significantly inhibit tumor cell migration, while HFn and MFn have no effect on tumor cells.

[0115] 2.2.10 Biomimetic ferritin nanoparticles induce cell release of HMGB1 and ATP

[0116] MIA Paca-2 cells in the logarithmic growth phase were seeded into 24-well plates. When the cells adhered to the wall and the fusion rate reached 80%, melittin, HFn, MFn and MMFn (final concentration of 2 μM) were added respectively. PBS buffer was added as a control and the drugs were treated for 12 hours. The cell supernatant was collected and the amount of ATP released by the cells was detected using an ATP detection kit. Divide another plate and repeat the above operation. The collected supernatant was centrifuged at 10,000 rpm and 4°C for 10 minutes. Collect the supernatant, add 500 ul methanol, 200 ul chloroform, and centrifuge at 10,000 rpm for ten minutes at room temperature. Remove the supernatant, add 500 ul methanol, centrifuge at 10,000 rpm for ten minutes at room temperature, and remove the supernatant. Add 50 ul sample buffer. Boil the sample and run the gel. The experiment was repeated three times. The results are as follows Figure 3 G and 3H.

[0117] 2.2.11 Distribution of biomimetic ferritin nanoparticles in vivo

[0118] First, a Panc02 mouse pancreatic cancer subcutaneous tumor model was established (male C57, 6 weeks old, 20 g, purchased from Hunan Slake, with a Panc02 cell inoculation volume of 1 million per mouse). 3 The biodistribution of biomimetic ferritin nanoparticles in mice was investigated.

[0119] HFn and MMFn biomimetic ferritin nanoparticles were attached with a fluorescent label containing Cy5.5 at 4°C overnight. Unattached Cy5.5 was then dialyzed to remove the resulting Cy5.5, yielding Cy5.5-HFn and Cy5.5-MMFn. Mice were then injected via the tail vein with PBS, Cy5.5-HFn, and Cy5.5-MMFn (all at 2 μM concentration, 100 μl injection volume), respectively. In vivo fluorescence imaging was performed on the mice at 4, 8, 12, and 24 hours after injection.

[0120] The in vivo distribution results of biomimetic ferritin nanoparticles are as follows Figure 4As shown, no fluorescence signal was observed in the PBS control group at all four time points. However, strong fluorescence signal was observed in the tumor site 4 hours after Cy5.5-MMFn injection, indicating substantial tumor accumulation, which persisted until 24 hours later. In contrast, although fluorescence signal was observed in the tumor site 4 hours after Cy5.5-MMFn injection, it was weaker than in the lungs. After 24 hours, the fluorescence signal was primarily concentrated in the tumor site and lungs. Twenty-four hours later, mice were dissected, and tumors and major internal organs were isolated and imaged ex vivo. The strongest fluorescence signal was observed in the tumors of the Cy5.5-MMFn group, while Cy5.5-MMFn also showed strong fluorescence signal in the lungs. These results demonstrate that MMFn can specifically accumulate in mouse tumors after tail vein injection, demonstrating a certain tumor-targeting capability.

[0121] 2.2.12 In vivo antitumor effect of biomimetic ferritin nanoparticles

[0122] Mice (purchased from Hunan Slake, 6-week-old C57 or Bal b / c) were intraperitoneally injected with 8% (w / v) chloral hydrate according to different body weights. After anesthesia, tumor cells (Panc02, MC38, and 4T1) resuspended in PBS were injected subcutaneously into the mice. When the tumor volume grew to 100-150 mm 3 Melittin (MEL), HFn, MFn and MMFn (5 mg / kg) were administered by tail vein, with 5 mice in each group receiving an equal volume of PBS buffer as control. The mice were administered 3 times a week, and the mice were weighed every other day. The volume of the tumor was measured and recorded using a vernier caliper. When the tumor volume of the PBS group grew to 1500 mm 3 The mice were killed at 4 pm, and the tumors were collected and weighed. The same grouping and administration method were used to treat the breast cancer mouse model. The administration was stopped after two weeks of treatment. The tumor volume grew to 1800 mm. 3 The mice were killed at 4 pm, which was recorded as the survival endpoint.

[0123] In vivo antitumor effect of biomimetic ferritin nanoparticles Figure 5As shown, compared with the PBS group, HFn injection had no significant effect on tumor growth. Tumors in all three groups grew rapidly after the start of treatment. However, after MMFn treatment, the change in tumor volume was gentle, indicating that tumor growth was inhibited. MMFn had a superior inhibitory effect on tumors than free melittin. After treatment, mice were sacrificed, and tumors were removed, weighed, and photographed. No significant differences in tumor weight and size were found between the HFn and PBS groups. Compared with the HFn and melittin groups, tumor weight and size were significantly reduced in the MMFn group. During treatment, body weight decreased slightly in the melittin group compared with the PBS group. There were no significant differences in body weight between the other groups, and no dramatic changes in body weight were observed. This suggests that MMFn biomimetic ferritin nanoparticles significantly improve biosafety compared with melittin alone.

[0124] 2.2.13 Effects of biomimetic ferritin nanoparticles on the tumor microenvironment

[0125] After all the above tumors (Panc02, MC38 and 4T1) were peeled off, the tumors were transferred to 5mL EP tubes. 1ml collagenase IV (purchased from Bio sharp) was added to each EP tube. The tumors were fully minced with hand-held curved scissors, and then the collagenase was added to 4ml. Digestion and incubation were carried out at 37℃ and 180rpm on a shaker for 1-2 hours. After digestion, it was diluted with PBS, filtered through a 200-mesh filter into a 15ml centrifuge tube, and centrifuged at 450g×5min to discard the supernatant. The precipitate was resuspended with 6ml 40% (v / v) Percoll solution, centrifuged, and the supernatant was discarded. 2ml red blood cell lysis buffer (purchased from Biyuntian) was added to the precipitate, and the red blood cells were lysed at 4℃ for 10min. Then PBS was filled up and the cells were collected by centrifugation at 800g×5min. Finally, the cells were resuspended with an appropriate amount of PBS, filtered through a 200-mesh filter into a new 15ml centrifuge tube, and counted with trypan blue staining. After counting, the cells were divided into 1×10 6 Resuspend the cells in 30 μl of CD16 / 32 antibody (purchased from Thermo Fisher Scientific) diluted in buffer and block at 4°C for 20 minutes. Then, add 30 μl of the antibody mixture (diluted in PBS) and incubate at 4°C in the dark for at least 30 minutes. Top up with PBS and centrifuge at 400-600g for 5 minutes at room temperature. Discard the supernatant. Finally, resuspend the cells in 300 μl of PBS, filter through a 200-mesh filter, and transfer to a flow cytometer for analysis.

[0126] The results of the effects of biomimetic ferritin nanoparticles on the tumor microenvironment are as follows Figure 6As shown: In all three animal models (Panc02, MC38, and 4T1), flow cytometry data showed that the percentage of CD11c+MHCⅡ+ cells was higher in the MMFn-treated group than in the other groups. The proportions of CD4+ and CD8+ T cells also increased significantly. This result suggests that MMFn exposes tumor antigens for presentation by dendritic cells, thereby stimulating the adaptive immune response. Effective CD4+ T cell anti-tumor responses promote robust and sustained CD8+ T cell responses, which also facilitates dendritic cell maturation. MMFn also activates innate immune responses, with the percentage of NK cells in the MMFn-treated group significantly higher than in the other groups. Significant increases in NKT cells were also observed in the MC38 and 4T1 animal models, but not in Panc02. Furthermore, we also measured levels of the cytokine IFN-γ in the 4T1 tumor model and found that IFN-γ levels were significantly elevated in the MMFn-treated group, also demonstrating that MMFn triggers an adaptive immune response. Together, these results suggest that MMFn initiates both innate and adaptive immune responses, thereby reshaping the tumor microenvironment.

[0127] 2.2.14 Biosafety of Biomimetic Ferritin Nanoparticles

[0128] Healthy female BALB / c mice (purchased from Hunan Slake) were randomly divided into 5 groups, with 5 mice in each group. Orthotopic tumors were injected with MC38 tumor cells (1 million / mouse) to establish animal models. When the tumor volume grew to 150-200 mm, the mice were injected with MC38 tumor cells (1 million / mouse) to establish animal models. 3 At 4 hr, patients were injected with PBS, HFn, MFn, Melittin, and MMFn (5 mg / kg), respectively. Blood was collected after treatment. Serum was collected by centrifugation and assayed for levels of aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), creatinine creatine oxidase (CREA), urea nitrogen (UREA), creatine kinase (CK), and lactate dehydrogenase (LDH).

[0129] The biosafety results of biomimetic ferritin nanoparticles are as follows Figure 7 As shown in the figure, there was no difference in ALT, AST, UREA, CREA, LDH, and CK between HFn and PBS, indicating that the HFn vector has good biosafety. Melittin showed significant toxicity compared with the PBS group, while there was no significant difference between the MMFn and PBS groups, indicating that modifying melittin onto ferritin not only retains the killing effect of melittin but also reduces its toxicity.

[0130] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A multifunctional biomimetic ferritin nanomaterial responsive to matrix metalloproteinases, characterized by: Its active ingredient is recombinant ferritin, the amino acid sequence of which is shown in SEQ ID NO:6; The multifunctional biomimetic ferritin nanomaterial responsive to matrix metalloproteinases is prepared by the following method: (1) ligating the recombinant ferritin gene sequence shown in SEQ ID NO:5 into the pET30a(+) vector to obtain a ferritin expression vector; (2) The ferritin expression vector is transformed into Escherichia coli competent cells, and the matrix metalloproteinase-responsive multifunctional biomimetic ferritin nanomaterial is obtained through induced expression and purification.

2. The multifunctional biomimetic ferritin nanomaterial responsive to matrix metalloproteinases according to claim 1, characterized in that: The E. coli competent cells described in step (2) are BL21 / DE3 E. coli competent cells.

3. The multifunctional biomimetic ferritin nanomaterial responsive to matrix metalloproteinases according to claim 1, characterized in that: The induction conditions described in step (2) are: E. coli cell OD600 is 0.8, the induction concentration of isopropyl-β-D-thiogalactopyranoside is 1 mmol / L, the induction temperature is 18°C, and the induction time is 4 hours.

4. Use of the matrix metalloproteinase-responsive multifunctional biomimetic ferritin nanomaterial according to claim 1 in the preparation of anti-tumor drugs.

5. The use according to claim 4, characterized in that: The tumor is a malignant tumor.

6. The use according to claim 5, characterized in that: The tumor is at least one of pancreatic cancer, breast cancer and colon cancer.

7. The use according to claim 4, characterized in that: The anti-tumor drug is administered intravenously.

8. The use according to claim 7, characterized in that: The anti-tumor drug is administered by intravenous injection.

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