Matrix metalloproteinase-responsive multifunctional bionic ferritin nano material as well as preparation method and application thereof

Abstract

The invention discloses a matrix metalloproteinase-responsive multifunctional bionic ferritin nano material as well as a preparation method and application thereof. The method comprises the following steps: modifying melittin on the surface of a ferritin nanoparticle through a genetic engineering method, connecting by using a linker containing a matrix metalloproteinase 9 (MMP-2 / 9) restriction enzyme cutting site, expressing the obtained recombinant ferritin (SEQ ID NO: 6) in escherichia coli, and self-assembling to form the enzyme response functionalized bionic nanoparticle. According to the method, melittin can be connected to ferritin nanoparticles in series, the toxicity of melittin is remarkably reduced, and the melittin responds to matrix metalloproteinase in a tumor microenvironment so as to kill tumor cells; meanwhile, intravenous administration of melittin can be realized, and the melittin injection has a good treatment effect on 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 bionic 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 amphipathic polypeptide with a molecular weight of 2840Da, composed of 26 amino acids, with the N-terminus being mainly hydrophobic with +4 charges, while the C-terminus being mainly hydrophilic with +2 charges, resulting in a total charge of +6 at physiological pH. Melittin can directly kill tumor cells by pore-punching: Melittin usually exists as a monomer and adopts a random coil structure; however, when melittin is inserted into the lipid membrane, it aggregates into a tetramer and its structure changes to an α-helical conformation. Since melittin is positively charged, it can bind to the negatively charged membrane surface and then form pores, leading to the leakage of cell components and enhanced permeability, thereby destroying the integrity of the phospholipid bilayer and ultimately killing the cell. Melittin causes greater damage to tumor cell membranes than normal cells because of its greater membrane potential. The positive charge of melittin selectively binds to the relatively abundant negative charges in the target tumor cell membrane 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 in combination with some chemotherapy drugs.

[0003] The relevant mechanisms of bee venom peptide against tumors are as follows: ① After destroying the cell membrane, bee venom peptide directly enters the cytoplasm and interferes with various signal transductions related to the malignant growth of tumors. ② Inducing tumor cell apoptosis (cell atrophy, chromatin condensation and the formation of apoptotic bodies): bee venom peptide can increase the level of calcium ions in the cytoplasm, and can also promote the production of oxygen free radicals in cells, reduce the mitochondrial membrane potential, lead to the release of cytochrome C, and promote cell apoptosis. It can also promote the expression of death receptors (DR), thereby further inducing the activation of caspase to induce cell apoptosis. ③ Inhibit tumor invasion and migration: Rac1 is a member of the Ras superfamily of small GTP (guanosine triphosphate) binding proteins. Rac1 can promote the migration and invasion of tumor cells in various cancers. Melittin can inhibit Rac1 and thus inhibit the invasion and migration of tumor cells. Bee venom inhibits the expression and activity of MMP-9 by inhibiting the expression of NF-κB, thereby inhibiting the invasion and migration of tumors. ④ Inhibit tumor angiogenesis: Inhibit 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. Intravenous injection can cause severe toxicity and directly bind to red blood cells and release hemoglobin. It is also easily degraded rapidly in the blood. These limitations limit the application of melittin in cancer treatment. Therefore, it is necessary to select a suitable carrier to deliver melittin to the target.

[0005] Ferritin is a protein that can store iron. It plays a key role in the body's iron balance and cellular antioxidant process. The main function of ferritin is to transport iron to the tissues or cells needed by the body in a redox-inactive form. Ferritin receptors (TfR1) exist on the surface of most cells, and ferritin can enter cells by interacting with them. This biological characteristic makes ferritin have a certain targeting property. It is well known that TfR1 is highly expressed on the surface of tumor cells and has therefore long been used as a potential target for tumor diagnosis and treatment. Ferritin can spontaneously assemble into a hollow cage structure with an outer diameter of about 12nm and an inner cavity diameter of about 8nm, so it can be used as a natural drug-carrying nanomaterial. The outer shell of natural ferritin is a mixture of light and heavy chains, of which only the heavy chain can recognize tumors. The modified pure heavy chain ferritin nanoparticles can target tumor cells more efficiently, mediate entry into tumor cells by recognizing and binding to TfR1 membrane proteins, and enrich in lysosomes.

[0006] At present, most studies are limited to using the role of ferritin nanocarriers to encapsulate chemotherapy drugs to reach the lysosomes of tumor cells and release drugs to kill tumors, but they are still helpless against drug-resistant tumors. In recent years, peptide anti-tumor drugs have attracted more and more attention due to their advantages such as low molecular weight, simple structure, high anti-cancer activity, high selectivity, fewer side effects, multiple administration methods, and low risk of causing multi-drug resistance. Among them, melittin is the main active ingredient of bee venom, and its functions are diverse, such as anti-inflammatory, analgesic, antibacterial, anti-HIV and anti-tumor pharmacological activities. It has been reported to have a broad spectrum of anti-tumor activity, including human hepatocellular carcinoma, leukemia, breast cancer, etc., and can form ion pores that penetrate the plasma membrane during the aggregation process, destroying membrane structures such as cell membranes and lysosomes. Due to its extremely strong non-specific membrane-breaking ability, it is easy to cause thrombosis and other toxic side effects during aggregation, which seriously restricts its application.

[0007] Matrix metalloproteinases (MMPs) regulate extracellular matrix degradation, cell proliferation, adhesion and migration, thus affecting cell activity and function. MMPs are expressed at low levels in normal tissues, but at high levels in tumor tissues. Matrix metalloproteinase 2 / 9 (MMP-2 / 9) promotes the growth of tumor cells by promoting the formation of new blood vessels, and promotes the infiltration and invasion of tumor cells to the basement membrane by degrading and destroying the basement membrane. Many studies have shown that MMP2 / 9 is significantly increased in the tissues of malignant tumors such as colon cancer, gastric cancer, lung cancer and cervical cancer, and has become a target for anti-tumor drugs. However, so far, there have been no reports on the use of matrix metalloproteinase-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 bionic ferritin nanomaterial responsive to matrix metalloproteinases.

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

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

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

[0012] A multifunctional bionic ferritin nanomaterial responsive to matrix metalloproteinases, wherein the active component 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 metalloproteinase comprises the following steps:

[0015] (1) connecting the gene sequence of recombinant ferritin (MMFn protein) as shown in SEQ ID NO: 5 to 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 bionic 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 bee venom peptide at the N-terminus of ferritin by PCR, and then the sticky end is 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-thiogalactoside (IPTG) is 1 mmol / L, the induction temperature is 18° C., and the induction time is 4 hours.

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

[0022] The multifunctional bionic ferritin nanomaterial responsive to matrix metalloproteinase is used in the preparation of anti-tumor drugs and / or tumor vaccines.

[0023] The multifunctional bionic ferritin nanomaterial responsive to matrix metalloproteinases can target tumor cells, induce tumor cell apoptosis, inhibit tumor cell proliferation, and achieve 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] Compared with the prior art, the present invention has the following advantages and effects:

[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 non-specific membrane-permeating peptide. In the present invention, melittin is modified on the surface of ferritin nanoparticles by a genetic engineering method, 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 bionic nanoparticles MMFn. The bionic nanoparticles MMFn specifically target the tumor site through active targeting mediated by enhanced permeability and retention effect (EPR) and transferrin receptor 1 (TfR1). MMP-2 / 9 highly expressed in tumor cells cuts it to release melittin, which combines with the negatively charged cell membrane to form pores in the cell membrane, destroying the integrity of the phospholipid bilayer and causing the cell contents to flow out. After breaking the membrane, melittin enters the cytoplasm and induces apoptosis by inducing the activation of caspase, thereby further killing tumor cells.

[0028] (2) Based on ferritin nanoparticles, the present invention uses genetic engineering to perform genetic modification on the pET30a(+)-HFn plasmid that induces the expression of ferritin, constructs a plasmid that can express ferritin, enzyme response elements and bee venom peptide fusion protein in series, and sequentially adds MMP-2 / 9 specific enzyme cleavage sites and melittin with membrane lysis function after aggregation at the N-terminus to construct enzyme-responsive multifunctional biomimetic ferritin nanomaterial MMFn; in addition, the enzyme response site is replaced by a linker to modify bee venom peptide 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 uses this system to connect bee venom peptide 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 bee venom peptide 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 a matrix metalloproteinase cleavage site (enzyme response site), and constructs an enzyme-responsive multifunctional bionic 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 response, and inhibit tumor metastasis and recurrence.

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

[0031] (5) The present invention further optimizes the conditions for protein expression and purification, thereby achieving the simple, rapid and efficient production of biomimetic ferritin nanoparticles, which have the characteristics of high yield and significant effect, and 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; and B represents the fusion expression of bee venom peptide and linker sequence at the N-terminus of ferritin subunit.

[0033] Figure 2 : It is a purification and biochemical characterization diagram of the biomimetic ferritin nanoparticle platform; wherein, A is the SDS-PAGE electrophoresis result diagram of HFn, MMFn and MFn; B is the photographic result diagram 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; F is a schematic diagram of the characterization of the stability of biomimetic ferritin nanoparticles in complete culture medium DMEM.

[0034] Figure 3It is a graph of in vitro toxicity of bionic ferritin nanoparticles; wherein, A is a graph showing MMP-2 / 9-dependent hemolysis induced by bionic iron nanoparticles in vitro; B is a graph showing the MTT results of the toxicity of bionic ferritin nanoparticles to tumor cells and normal cells; C is a flow cytometry result and statistical graph showing that bionic ferritin nanoparticles induce apoptosis of tumor cells; D is a fluorescence graph showing that bionic ferritin nanoparticles induce apoptosis of tumor cells; E is a graph showing the results of a cell clone formation experiment of bionic ferritin nanoparticles; F is a graph showing the results of cell scratching of bionic ferritin nanoparticles; G is a graph showing the western blot results of bionic ferritin nanoparticles promoting the release of HMGB1 from tumor cells; and H is a statistical graph showing that bionic ferritin nanoparticles promote the release of ATP from 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; wherein, 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 flow cytometry statistical graph 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 the embodiments of the present invention are not limited thereto. Unless otherwise specified, 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 carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0040] Example 1: Construction and production of 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 Genewise 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, with the pH 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 ferritin expression vector The nucleic acid sequence of human heavy-chain ferritin (HFn) was optimized for Escherichia coli host codons according to its protein sequence. The optimized ferritin gene sequence (HFn, SEQ ID NO: 4) was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. According to the amino acid sequence of melittin (SEQ ID NO: 1), the amino acid sequence of melittin and the enzyme response site (SEQ ID NO: 2), and the amino acid sequence of melittin and the linker (SEQ ID NO: 3), primers with restriction sites were designed (primer sequences are shown below) to perform PCR amplification on the optimized HFn ferritin fragment to obtain an amplified fragment (MMFn, SEQ ID NO: 5); then the amplified MMFn fragment and the pET30a (+) plasmid were double-digested with BamHⅠ and HindⅢ enzymes, respectively, and DNA agarose gel was recovered, and then the pET30a (+) vector after restriction digestion: ferritin fragment (MMFn) was connected at a molar ratio of 1:5 under the action of T4 ligase at 16°C overnight. For specific steps, refer to the instructions of the Takara-dna ligation kit. The ligation product was transformed into BL21 / DE3 E.coli competent cells to screen the correctly connected clones to obtain the nanoparticle carrier Melittin-MMP-HFn. At the same time, the MMP-2 / 9 enzyme response sequence was replaced with an equal length flexible linker (GGSGGS) to construct a mutant MFn as a control, and the MFn fragment (SEQ ID NO: 7) was obtained by PCR amplification and then connected to 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 out. Among them, the optimal induction conditions for MFn and MMFn were OD600 of 0.8, isopropyl-β-D-thiogalactoside (IPTG) 1mmol / L, and induction at 18℃ for 4 hours. The expression levels of MFn and MMFn biomimetic ferritin nanoparticles were both about 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 about 31 mg per liter of bacterial liquid.

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

[0075] 2.1 Experimental materials: CM-agarose gel CM-Sepharose was purchased from GE HealthCare. 50 kDa ultrafiltration tube was 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 configuration 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 placed in a 70°C water bath and heated for 15 min to denature and precipitate most of the impurities that are not resistant to high temperatures. The denatured protein precipitate was removed by centrifugation at 12,000 rpm for 15 min, and then the fine precipitate was removed by a 0.45 μm filter to obtain a crude ferritin solution. The crude ferritin solution was concentrated in a 50 kDa cut-off concentration tube and filtered through a 0.22 μm filter to remove the fine precipitate.

[0079] (2) Anion exchange agarose gel (CM-Sepharose) is soaked in 200mM Tris-HCl (pH7.8) for column equilibration. A small amount of sample buffer is added to the column; the outlet of the column is opened and a small amount of loading buffer is introduced to drive out the air at the bottom of the column; the resin is shaken and introduced into the column with a glass rod without introducing bubbles; the outlet of the column is opened and more sample buffer is added when the resin accumulates; the resin is allowed to reach exchange equilibrium. After loading the column, the resin is finally rinsed with sample buffer so that its pH value and ionic strength finally meet the experimental requirements and the resin is washed 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. The outlet at the bottom of the column is opened and the loading buffer is introduced into the column until the liquid level reaches the surface of the resin. The outlet at the bottom of the column is closed; the protein solution is slowly added to the resin surface with a pipette.

[0080] (3) Open the outlet of the chromatography column to 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 drip a small amount of loading buffer onto 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 A: After the crude protein extract was purified by simple heating, more than 80% of the impurities were removed by heat denaturation. After elution by anion exchange chromatography column, SDS-PAGE electrophoresis results showed that the position of the purified protein band was basically consistent with the predicted protein size, and the molecular weight of the purified HFn was about 21.1kDa. The protein purity was above 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), respectively, and centrifuged at 4500 rpm and 4°C for 20 min. After removing the supernatant, the mixture was resuspended in a buffer solution (20 mM Tris, 500 mM NaCl, pH = 7), and after ultrasonic disruption, the mixture 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 tiny precipitate to obtain a crude pure solution of MMFn or MFn ferritin.

[0084] (2) Take the purified Ni column, clean the column, add 3-4 ml of nickel agarose gel, wash the column with ddH2O 5 times, and then balance the column with equilibrium solution 2 times. Combine the column and supernatant thoroughly, place it on a four-dimensional rotating mixer and incubate it for 1-2 hours before taking it out for purification. Equilibrium buffer: 20 mM Tris buffer at pH 8.0 (containing 500 mM NaCl). Elution buffer: 20 mMTris buffer at pH 8.0 (containing 500 mM NaCl and 50 mM imidazole), 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 equilibrium buffer to wash away the unadsorbed sample, with a flow rate of 1-2 ml / min. The column was eluted in stages with 50mM, 100mM, 200mM, and 300mM imidazole concentrations, with elution of about 5 to 10 column volumes (if more protein is adsorbed, more elution solution is used), the flow rate was 1 to 2ml / min, and 5ml / tube was collected. The collected proteins eluted from each concentration gradient were sampled and analyzed by SDS-PAGE electrophoresis. According to the SDS-PAGE electrophoresis diagram, if there is protein, it was collected and concentrated with a 50kDa ultrafiltration tube.

[0085] (3) Figure 2 As shown in A: After MFn and MMFn were purified by HIS tag affinity, more than 80% of the impurities were removed. The SDS-PAGE electrophoresis results showed that the position of the purified protein band was basically consistent with the predicted protein size (molecular weight was about 24.9 kDa). The protein purity was above 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 the copper mesh with the support film, let it sit for a few minutes, then use filter paper to absorb the excess liquid from the edge of the copper mesh, drop the dye solution on it and dye it for 1 to 2 minutes, then use filter paper to absorb the dye solution, and observe it with a transmission electron microscope after it is dried. 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 20nm.

[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 with a lid, and measure the particle size of the solution. Set three replicates. The dynamic light scattering results are as follows: Figure 2 As shown in C: The particle sizes of MFn and MMFn are slightly larger than those of HFn, indicating that the modification of melittin increases the particle size. The particle size of HFn is about 10nm, and the particle sizes of MFn and MMFn are about 20nm.

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

[0092] Add the solution to be tested into the potential detection cell, be careful not to have bubbles, cover the lid, and perform potential detection on the solution. Set three repetitions. The potential detection results are as follows: Figure 2 As shown in D: MFn and MMFn have larger potentials than HFn. This is because bee venom peptide carries a positive charge, which neutralizes the negative charge carried by ferritin.

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

[0094] HFn, MFn and MMFn were quantified to 100ug / ml, added to the sample pool, and scanned at full wavelength. Three replicates were set. The circular dichroism results of biomimetic ferritin nanoparticles are shown in 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 bee venom peptide 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, placed at 4°C, and 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 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 red blood cells were separated and purified and diluted to 10 7 / mL for hemolysis test. Melittin, MFn, MFn+MMP-2 / 9, MMFn, MMFn+MMP-2 / 9 of different concentrations were incubated with mouse erythrocytes at 37°C for 3 hours, centrifuged at 3000rpm for 3 minutes, and the absorbance of the supernatant was measured at 540nm; the final concentrations of each drug added were 0, 2, 4, 6, 8, 10, 20μM; matrix metalloproteinase MMP2 / 9 was purchased from abcam, item number ab81550, and mouse erythrocytes treated with TritonX-100 at a final concentration of 1% (v / v) were used as positive controls, and the hemoglobin release rate of this group was set to 100%. The hemolytic effect of nanoparticles on erythrocytes with or without matrix metalloproteinases was studied.

[0099] The results of biomimetic iron nanoparticles inducing MMP-2 / 9-dependent cell hemolysis in vitro are as follows Figure 3 As shown in A: Only when MMFn is added with matrix metalloproteinases, will hemolysis of erythrocytes occur. Single MMFn or MFn, MFn+MMP-2 / 9 and HFn will not cause hemolysis of erythrocytes. This indicates that MMFn releases bee venom peptide under the action of matrix metalloproteinases, and then forms tetramers, which rupture the erythrocyte membrane and release hemoglobin.

[0100] 2.2.5 Cytotoxicity assay

[0101] The day before, pancreatic cancer cells Panc02 and MIA PaCa-2 (referred to 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, and then the cultured cells in the logarithmic phase were digested, centrifuged and counted. 100 μL containing 3000 to 7000 cells / well was inoculated on a 96-well plate and cultured for 24 hours at 37°C, 5% CO2, and 90% humidity. Then, melittin, HFn, MFn and MMFn (final concentrations were all 2 μM) were added respectively, and PBS buffer was added as a control. The drug treatment was performed for 12 hours. Before the experiment, MTT solvent needs to be added to MTT powder to prepare 5 mg / mL MTT solvent (unused can be stored in a -20°C refrigerator). 10 μL of MTT solution was added to each well to a final concentration of 0.5 mg / mL. Then incubate for 4 hours at 37°C, 5% CO2, and 90% humidity. Carefully aspirate all the supernatant in the wells to prevent the monolayer from rupturing. Add 200 μL of dimethyl sulfoxide (DMSO) to each well, place the plate on an oscillator at 300 rpm / min for 10 minutes, and measure the absorbance at 570 nm using a microplate reader. The experiment was repeated three times.

[0102] The results of cytotoxicity experiments were as follows Figure 3 As shown in B: The killing effect of biomimetic ferritin nanoparticles MMFn was observed in pancreatic cancer cells Panc02, MIA PaCa-2, breast cancer cells MDA-MB-231, 4T1 and colon cancer cells MC38, while no killing of tumor cells was observed in the control group that did not express matrix metalloproteinases. It was also verified that MMFn had almost no killing effect in normal human umbilical vein epithelial cells HUVEC, which may be due to the fact that normal cells do not express matrix metalloproteinases, so bee venom peptide cannot fall off the ferritin nanoparticles and kill the cells.

[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, and 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, 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 1000g for 5 minutes, the supernatant was discarded, the cells were collected, and the cells were gently resuspended with PBS and counted. Take 50,000 to 100,000 resuspended cells, centrifuge at 1000g for 5 minutes, discard the supernatant, add 195μl Annexin V-FITC binding solution to gently resuspend the cells, then add 5μl Annexin V-FITC and 10μl propidium iodide staining solution, and mix gently. Incubate at room temperature (20-25℃) in the dark for 10-20 minutes, and then perform flow cytometry detection. The experiment was repeated three times.

[0105] Biomimetic ferritin nanoparticles induce tumor cell apoptosis 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 ) were cultured in a 24-well plate, and melittin, HFn, MFn and MMFn (final concentration of 2 μM) were added respectively, with PBS buffer added as the control. After drug treatment for 4 hours, the multi-well plate was centrifuged at 1000g for 5 minutes. The cell culture medium was removed and PBS was added to wash once. 195 μl Annexin V-FITC binding solution was added, and then 5 μl Annexin V-FITC and 10 μl propidium iodide staining solution were added, and gently mixed. Incubate at room temperature (20-25°C) in the dark for 10-20 minutes. Then 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 Experiment on clone formation of biomimetic ferritin nanoparticles to inhibit tumor cell proliferation

[0110] After trypsin digestion of MIAPaca-2 cells in the logarithmic growth phase, resuspend them in DMEM complete medium to form a cell suspension and count them; 500-1000 cells / well were inoculated in each experimental group in a 6-well culture plate, and melittin, HFn, MFn and MMFn (final concentration of 2 μM) were added respectively, with PBS buffer added as a control, and the drug treatment was continued for 4 hours, and the number of cells in most single clones was greater than 50. The medium was changed every 3 days and the cell state was observed. After the cloning was completed, the cells were photographed under a microscope, then washed once with PBS, 1 mL of 4% paraformaldehyde was added to each well for fixation for 30-60 minutes, washed once with PBS, and then 1 ml of crystal violet stain was added to each well, and the cells were stained for 10-20 minutes. Finally, the cells were washed several times with PBS, 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 test of biomimetic ferritin nanoparticles to inhibit tumor cell migration

[0113] MIAPaca-2 cells in the logarithmic growth phase were inoculated into 6-well plates. When the cells adhered to the wall and the fusion rate reached 100%, a 10-μl pipette tip was used to scratch the culture plate parallel or perpendicular to the plate. The tip should be vertical and not tilted. The cells were rinsed with PBS three times, the scratched cells were removed, serum-free culture medium was added, and the cells were first placed under a 4x microscope for photography. Then, the cells were treated with drugs, and melittin, HFn, MFn and MMFn (final concentrations were all 2 μM) were added, respectively. PBS buffer was added as a control, and the photos were taken again after 24 hours. 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 shown in Figure 2. 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 in 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, and PBS buffer was added as a control. 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 subcutaneous pancreatic cancer tumor model of Panc02 mice was constructed (male C57, 6 weeks old, 20 g, purchased from Hunan Slake, with 1 million Panc02 cells inoculated per mouse). 3 The biodistribution of biomimetic ferritin nanoparticles in mice was investigated.

[0119] HFn and MMFn biomimetic ferritin nanoparticles were connected with fluorescent tags with cy5.5, connected overnight at 4°C, and then dialyzed to remove unconnected cy5.5 to obtain Cy5.5-HFn and Cy5.5-MMFn. Then, the mouse model was injected with PBS, Cy5.5-HFn and Cy5.5-MMFn (all at 2μM concentration and 100μl injection volume) through the tail vein, and the mice were subjected to fluorescent in vivo imaging at 4, 8, 12 and 24 hours after injection.

[0120] The in vivo distribution results of biomimetic ferritin nanoparticles are shown in Figure 4As shown: No fluorescence signal was observed in the PBS control group at 4 time points, but a strong fluorescence signal could be observed in the tumor site 4 hours after the injection of Cy5.5-MMFn, indicating that it was enriched in the tumor in large quantities, and this enrichment lasted until 24 hours. In contrast, 4 hours after the injection of Cy5.5-MMFn, although the fluorescence signal could be observed in the tumor site, the fluorescence was weaker than that in the lungs. After 24 hours, the fluorescence signal was mainly distributed in the tumor site and the lungs. After 24 hours, the mice were dissected, the tumors and major internal organs were separated and in vitro imaging was performed. It was found that the fluorescence signal in the tumor of the Cy5.5-MMFn group was the strongest, and Cy5.5-MMFn also showed a strong fluorescence signal in the lungs. The above results show that MMFn can be specifically enriched in the tumor site of mice after tail vein injection, and has a certain tumor targeting ability.

[0121] 2.2.12 Antitumor effect of biomimetic ferritin nanoparticles in vivo

[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 100-150mm 3 The mice were given melittin (MEL), HFn, MFn and MMFn (5 mg / kg) by tail vein administration. Each group of 5 mice was injected with an equal volume of PBS buffer as a control. The mice were given the drug three times a week. The mice were weighed every other day, and the tumor volume was measured and recorded with a vernier caliper. When the tumor volume of the PBS group grew to 1500mm 3 The mice were killed at 1:10 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 4 000 s, which was recorded as the survival endpoint.

[0123] In vivo antitumor effect of biomimetic ferritin nanoparticles Figure 5As shown: Compared with the PBS group, the injection of HFn had no significant effect on tumor growth. The tumors in these three groups grew rapidly after the start of treatment. After receiving MMFn treatment, the change trend of tumor volume was gentle, indicating that tumor growth was inhibited. MMFn has a better inhibitory effect on tumors than free bee venom peptide. After the treatment, the mice were killed and the tumors were removed for weighing and photographing. There was no significant difference in tumor weight and size between the HFn group and the PBS group. Compared with the HFn group and the bee venom peptide group, the tumor weight and size of the MMFn group were significantly reduced. During the treatment, the body weight of the bee venom peptide group decreased slightly compared with the PBS group. There was no significant difference in the body weight of mice in the other groups, and there was no drastic change in body weight, indicating that compared with bee venom peptide alone, MMFn biomimetic ferritin nanoparticles significantly improved biosafety.

[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 cut into pieces with hand-held curved scissors, and then the collagenase was added to 4ml. Digestion and incubation were carried out at 37℃ and 180rpm shaking for 1-2h. 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, it was 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 / tube), resuspend the cells with 30ul CD16 / 32 antibody (purchased from Thermo Fisher Scientific) diluent, and block at 4℃ for 20min. Then add 30ul antibody mixture (diluted with PBS), incubate at 4℃ in the dark for more than 30min. Fill with PBS, centrifuge at 400-600g for 5min at room temperature, discard the supernatant; finally, resuspend the cells with 300ul PBS, filter through a 200-mesh filter into a flow tube, and then test on the machine.

[0126] The results of the effects of biomimetic ferritin nanoparticles on the tumor microenvironment are as follows Figure 6As shown: In the three animal models (Panc02, MC38 and 4T1), flow cytometry data showed that the percentage of CD11c+MHCⅡ+ in the MMFn treatment group was higher than that in other groups. The proportion of CD4+T cells and CD8+T cells also increased significantly. This result shows that MMFn exposes tumor antigens and is presented by dendritic cells, thereby stimulating the occurrence of adaptive immune responses. The effective CD4+T cell anti-tumor response promotes a strong and lasting CD8+T cell response, which also contributes to the maturation of dendritic cells. At the same time, MMFn also activated the innate immune response. Among them, the percentage of NK cells in the MMFn treatment group was also much higher than that in other groups. NKT cells were also significantly increased in the MC38 and 4T1 animal models, but not in Panc02. At the same time, we also detected the level of cytokine IFN-γ in the 4T1 tumor model and found that the level of IFN-γ in the MMFn treatment group was significantly increased. This result also shows that MMFn initiated 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, 5 mice in each group. Orthotopic tumor-bearing mice were injected with MC38 tumor cells (1 million / mouse) to establish animal models. When the tumor volume grew to 150-200 mm 3 PBS, HFn, MFn, Melittin and MMFn (5 mg / kg) were injected at 4 pm, respectively. Blood was collected after treatment. The blood was centrifuged to collect serum, and the levels of aspartate aminotransferase AST, serum alanine aminotransferase ALT, creatinine creatine oxidase CREA, urea nitrogen UREA, creatine kinase CK and lactate dehydrogenase LDH were detected.

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

[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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A multifunctional bionic ferritin nanomaterial responsive to matrix metalloproteinases, characterized in that: The active ingredient is recombinant ferritin, and the amino acid sequence is shown in SEQ ID NO:

6.

2. The gene encoding the recombinant ferritin according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO:

5.

3. The method for preparing the multifunctional biomimetic ferritin nanomaterial responsive to matrix metalloproteinase according to claim 1, characterized in that: The steps include: (1) connecting the gene sequence of recombinant ferritin as shown in SEQ ID NO:5 to the pET30a(+) vector to obtain a ferritin expression vector; (2) The ferritin expression vector is transformed into Escherichia coli competent cells, and the multifunctional bionic ferritin nanomaterial responsive to matrix metalloproteinase is obtained through induction expression and purification.

4. The method according to claim 3, characterized in that: The E. coli competent cells described in step (2) are BL21 / DE3 E. coli competent cells.

5. The method according to claim 3, characterized in that: The induction conditions described in step (2) are: the OD600 of the Escherichia coli cells is 0.8, the induction concentration of isopropyl-β-D-thiogalactoside is 1 mmol / L, the induction temperature is 18° C., and the induction time is 4 hours.

6. Use of the matrix metalloproteinase-responsive multifunctional bionic ferritin nanomaterial according to claim 1 in the preparation of anti-tumor drugs and / or tumor vaccines.

7. The use according to claim 6, characterized in that: The tumor is a malignant tumor.

8. The use according to claim 7, characterized in that: The tumor is at least one of pancreatic cancer, breast cancer and colon cancer.

9. The use according to claim 6, characterized in that: The anti-tumor drug is administered intravenously.

10. The use according to claim 9, characterized in that: The anti-tumor drug is administered by intravenous injection.

Images