Nanocomplex based on elastase-metal cofactor and its preparation and application

Abstract

The present invention discloses a nanocomposite based on elastase-metal cofactor and its preparation and application, wherein the complex of elastase and metal cofactor is used as the core, and the outer layer is covered with a mineralized shell formed by calcium ions and anions, so as to enhance the target protein cleavage activity and anti-tumor effect, wherein the metal cofactor is selected from Mn 2+ 、Zn 2+ 、Ni 2+ One or more of the above. The nanocomplex of the present invention can prevent elastase from being degraded in a biological environment, effectively deliver it to tumor cells through an inhalation pathway, and achieve organ and tissue specific delivery. Compared with free elastase, the nanocomplex has a higher specific cleavage efficiency, induces stronger cancer cell apoptosis, significantly inhibits tumor growth, and shows good biosafety, providing an effective research idea for the application of protein drugs in anti-tumor therapy.

Description

Technical Field

[0001] The invention relates to an elastase-metal cofactor-based nanocomposite and application thereof, belonging to the field of nano preparations. Background Art

[0002] During the immune surveillance process, the body exhibits a complex immune mechanism that can timely and accurately identify, kill and eliminate mutated tumor cells, which plays a vital role in preventing the occurrence and development of tumors. Among them, elastase, as an excellent representative therapeutic protein, is expected to develop into a broad anti-cancer protein drug. Neutrophil elastase can release the pro-apoptotic domain by cleaving its substrate CD95 protein in cancer cells. This domain can bind to the highly expressed histone H1 subtype in cancer cells and induce tumor cell apoptosis.

[0003] However, due to the non-targeting of proteins in drug delivery and their non-specific pharmacological effects, the therapeutic efficiency of elastase is low. Due to the strong intratumoral pressure in tumor tissue, protein drugs are absorbed inefficiently and it is difficult to fully exert their effective effects. In addition, broad-spectrum protease digestion has serious side effects. For example, neutrophil elastase or its derivative porcine pancreatic elastase can catalyze the hydrolysis of proteins, such as denatured collagen and elastin. Long-term and large-scale exposure to such proteins may cause diseases related to elastic fiber degradation. In addition, during in vivo drug administration, protein drugs have the defects of easy degradation and short half-life. Proteins often have problems such as denaturation and inactivation. For example, when neutrophil elastase is in a body fluid environment, a variety of serine protease inhibitors in the body fluid can inhibit its protein degradation activity, such as plasma α2-macroglobulin and α1-antitrypsin. These factors have seriously hindered the development and application of elastase drugs.

[0004] The rapid development of nanomedicine has promoted the progress of many tumor treatments and protein delivery strategies, such as the development of nanodelivery systems based on biomimetic biomineralization strategies. Biomimetic biomineralization is the process by which inorganic ions combine with biomacromolecules to form mineral materials. It has the following advantages in delivering protein drugs: 1) protecting proteins and avoiding inactivation; 2) enhancing delivery to tumor cells; 3) tissue-specific administration to promote effective drug accumulation. In addition, mineralization strategies generally have the advantages of good biocompatibility, degradability and easy cellular uptake. These properties are very important for the biosynthesis of nanomaterials using proteins as templates. In addition, inspired by the interdependence between the catalytic activity of biological enzymes and their cofactors, we hope to use cofactors to achieve the specificity of the pharmacological effects of elastase. This is mainly because nearly half of the biological functions of proteins are cofactor-dependent. Among them, cofactors mainly exist in the form of metal ions or small molecules, playing the role of group transfer, catalysis and redox. In general, cofactors have various biological functions and can activate or inhibit the activity of proteins, depending on the enzymes and related substrates they bind to. For example, heme proteins require iron ions as cofactors to promote the transmission and transport of oxygen. Moreover, computer nanosecond molecular dynamics simulation calculations can now accurately display the kinetic changes after the binding of cofactors to proteins, providing a convenient method for the study and transformation of enzyme activity. Summary of the invention

[0005] As a new type of anti-tumor protein drug, elastase has a strong ability to induce tumor cell apoptosis and kill tumor cells. In view of the low therapeutic efficiency of elastase in tumor treatment: difficult protein delivery, easy degradation or easy inhibition of activity, the present invention intends to construct a nanocomplex (a cleavage activity enhanced elastasenanocomplex, CAEN) prepared based on elastase-metal cofactors to enhance the cleavage activity of elastase. Based on molecular dynamics simulation theory, this study proposes to use metal ions, including manganese ions, zinc ions, and nickel ions, as cofactors to improve the therapeutic activity of porcine pancreatic elastase (PPE), so that elastase can achieve specific cleavage of its substrate, the pro-apoptotic protein CD95 protein, thereby enhancing the effect of inducing tumor cell apoptosis. CAEN nanocomplexes can effectively protect and stabilize the activity of PPE protein. Moreover, CAEN nanocomplexes have an effective killing effect on different types of cancer cells. The nanocomplex shell produced by biomineralization enhances the intracellular uptake of PPE, which is beneficial for PPE to induce tumor cell apoptosis. In addition, the dissolution and release of metal ions in the nanocomplexes can affect the homeostasis of the internal environment of tumor cells, leading to increased levels of reactive oxygen species and mitochondrial damage in tumor cells. Combined with the immune activation function of cofactors such as manganese ions, the growth of cancer cells can be effectively inhibited. In addition, the biomineralization strategy has played a positive role in controlling and preventing PPE-induced protease-related balance disorders in lung tissues. Compared with PPE protein, CAEN can effectively inhibit cell proliferation and significantly inhibit tumor growth in vivo by promoting tumor cell apoptosis and anti-tumor immunotherapy, while showing that CAEN has good biosafety. This study provides an effective research idea for the application of protein drugs in anti-tumor therapy.

[0006] The elastase nanocomplex with enhanced cleavage activity (CAEN) provided by the present invention is prepared by biomineralization reaction, with the elastase-metal cofactor complex as the core and the precipitate produced by the reaction of calcium ions and anionic salt solution as the shell. The model drug of elastase can be one or more of the following serine proteases: neutrophil elastase, porcine pancreatic elastase, and their homologues; the metal cofactor is a metal ion selected from Mn 2+ 、Zn 2+ 、Ni 2+ One or more of .

[0007] The nanocomplex shell is positively charged and is easily taken up and internalized by cells; in the acidic environment of tumor cytoplasm, the nanocomplex slowly disintegrates and releases elastase-metal cofactors. The cleavage of CAEN is specific to CD95 (pro-apoptotic protein), the substrate of elastase, without affecting other non-target proteins, thereby significantly inducing stronger cancer cell apoptosis. In addition, the released Mn 2+ It can effectively activate tumor tissue to produce type I interferon, further inducing and promoting local immune response in tumor tissue.

[0008] Furthermore, the shell of the nanocomposite is a mineralized shell formed by a calcium chloride solution and an anion salt solution, wherein the anions include but are not limited to water-soluble acid ions such as phosphate ions, carbonate ions, oxalate ions, hydrogen phosphate ions, and citrate ions.

[0009] The nanocomposite is preferably nanoparticles with a particle size of 100 to 1000 nm.

[0010] The method for preparing the nanocomposite of the present invention comprises the following steps:

[0011] 1) Preparation of elastase solution: prepare elastase powder into a solution with sterile distilled water or a buffer solution such as PBS;

[0012] 2) Prepare metal cofactor solution: 2+ 、Ni 2+ and / or Zn 2+ ) is prepared into a solution;

[0013] 3) Preparation of calcium ion solution: calcium chloride can be dissolved in distilled water to prepare a solution;

[0014] 4) preparing anion salt solution: dissolving an inorganic salt reagent containing the anion in distilled water to prepare a solution;

[0015] 5) Mix the elastase solution and the metal cofactor solution in appropriate proportions and let stand for 5 to 10 minutes, then add the calcium ion solution and the anion salt solution to the mixed solution, mix well, seal and let stand at 37° C. for 10 minutes to 24 hours;

[0016] 6) After high-speed centrifugation, the precipitate is retained and then resuspended in culture medium or buffer solution to obtain a nanocomplex.

[0017] In the above preparation method, preferably, the concentration of the elastase solution prepared in step 1) is 1 mg / mL to 10 mg / mL; the concentration of the metal cofactor solution prepared in step 2) is 1 mmol / L to 1 mol / L; the concentration of the calcium ion solution prepared in step 3) is 1 mmol / L to 1 mol / L; the concentration of the anion salt solution prepared in step 4) is 1 mmol / L to 1 mol / L. The reagent source of the metal cofactor includes but is not limited to manganese chloride, zinc chloride, and nickel chloride; the reagent source of the calcium ion is calcium chloride; the reagent source of the anion includes but is not limited to hydrogen phosphate, phosphate, carbonate, oxalate, and citrate. DMEM serum-free medium (Zhongke Maichen, catalog number CM10013) can also be used as an anion salt solution, which contains a concentration of 3.27 mM hydrogen phosphate ions. In step 5), after the elastase solution and the metal cofactor solution are mixed and allowed to stand, the calcium ion solution and the DMEM serum-free medium are added, and the mixture is fully mixed and incubated at 37°C.

[0018] In the above step 6), centrifugation is generally performed at 12000 rpm for 10 min, and the precipitate after removing the supernatant can be resuspended in DMEM serum-free medium or PBS, and then directly tested or stored at 4°C for a short period of time.

[0019] The above steps 1), 5) and 6) all need to be protected from light, as light can affect the activity of elastase.

[0020] The elastase-metal cofactor-based nanocomplex of the present invention is suitable for preparing anti-tumor treatment-related drugs. The inner core of the nanocomplex is an elastase-metal cofactor complex, and the outer layer of calcium ion precipitation has the function of stabilizing and protecting protein activity, and its components are biosafe and degradable substances. After the nanocomplex enters the cell, its ion shell slowly disintegrates due to the acidic environment of the cytoplasm, and the further released elastase-metal cofactor enhances the activity and specificity of elastase cleavage substrates, effectively promoting tumor cell apoptosis.

[0021] The cell lines for anti-tumor therapy using elastase-metal cofactor nanocomplexes can be human or mouse cell lines, including but not limited to the following tumor cell lines: A549, A-431, B16-F10, HL-60, HBEC3-KT, Hela, HSkMC, HAP1, hTCEpi, HSC-T6, HepG2, HDLM-2, HT29, HBF TERT88, Jurkat, Karpas-707, Lovo, MOLT-4, NCI-H1299, NB-4, OE19, PC-12, SuSa, SiHa, SW620, SK-BR-3, THP-1, U266 / 84, U-138MG, and U-251MG.

[0022] Cancers closely related to the anti-tumor therapy of elastase-metal cofactor nanocomplexes include, but are not limited to, lung cancer, gastric cancer, liver cancer, pancreatic cancer, uterine cancer, melanoma, skin cancer, ovarian cancer, head and neck cancer, breast cancer, bile duct cancer, kidney cancer, colorectal cancer and cervical cancer.

[0023] The elastase-metal cofactor nanocomplex provided by the present invention has the following advantages:

[0024] 1. The outer layer of the nanoshell of the nanocomplex has the function of stabilizing and protecting the activity of elastase;

[0025] 2. The metal cofactor (manganese ion) in the nanocomplex can enhance the specificity of elastase cleavage substrates and effectively promote tumor cell apoptosis

[0026] 3. The form of biomineralized nanocomplex improves the efficiency of organ tissue delivery of elastase, which not only promotes the uptake of protein by tumor cells, but also utilizes its metal cofactors to effectively activate the local immune system of lung cancer tissue, further exerting a good anti-tumor synergistic effect.

[0027] In summary, the elastase-metal cofactor nanocomplex of the present invention can greatly improve the efficiency of elastase in killing tumor cells, effectively promote the apoptosis of tumor cells, and also has good biocompatibility, and is suitable for the treatment of different tumor diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 . Schematic diagram of the preparation and structure of the nanocomposite (CAEN) in the present invention.

[0029] Figure 2 . Transmission electron microscopy image of the nanocomposite (CAEN) and porcine pancreatic elastase (PPE) prepared in Example 1.

[0030] Figure 3 .A graph showing the change in preparation time and particle size of the nanocomposite (CAEN) prepared in Example 1.

[0031] Figure 4 .The infrared spectrum measurement results of the nanocomposite (CAEN) prepared in Example 1 were taken as a control with porcine pancreatic elastase (PPE).

[0032] Figure 5 .Measurement results of X-ray electron spectroscopy analysis of the nanocomposite (CAEN) prepared in Example 1.

[0033] Figure 6Results of cytotoxicity test (B16 cells cultured without fetal bovine serum) on the nanocomplex (CAEN) prepared in Example 1.

[0034] Figure 7 The results of the cytotoxicity test (B16 cells cultured in the presence of 10% fetal bovine serum) on the nanocomplex (CAEN) prepared in Example 1.

[0035] Figure 8 Results of cytotoxicity test (A549 cells cultured without fetal bovine serum) on the nanocomplex (CAEN) prepared in Example 1.

[0036] Fig. 9 Results of cytotoxicity test (A549 cells cultured in the presence of 10% fetal bovine serum) on the nanocomplex (CAEN) prepared in Example 1.

[0037] Fig.10 . The nanocomplex (CAEN) labeled with fluorescent dye FITC prepared according to the method of Example 1 was subjected to a cell uptake experiment and the uptake pathway was observed by confocal microscopy.

[0038] Fig.11 .The structure of the protein-metal ion complex formed by manganese ion and porcine pancreatic elastase simulated by molecular dynamics.

[0039] Fig.12 .Comparison of the cleavage effects of porcine pancreatic elastase (PPE) and CAEN on CD95 in vitro, wherein A shows the molecular screening results of the purified MBP-CD95 protein and the molecular size determined by Coomassie Brilliant Blue staining, and B shows the cleavage of MBP-CD95 by CAEN, wherein PBS is a phosphate buffer solution control, NP is a metal ion precipitation control, and PPE is a single PPE protein control.

[0040] Fig.13 .Comparison of changes in mitochondrial membrane potential of tumor cells after mouse melanoma cells B16 were treated with porcine pancreatic elastase (PPE) and CAEN.

[0041] Fig.14 .Comparison of the changes in the apoptosis marker Caspase3 protein and its splice form after mouse melanoma cells B16 were treated with porcine pancreatic elastase (PPE) and CAEN.

[0042] Fig.15 .Comparison of the changes in Annexin V, a cell membrane apoptosis marker, after mouse melanoma cells B16 were treated with porcine pancreatic elastase (PPE) and CAEN by flow cytometry.

[0043] Fig.16 .Data analysis results of flow cytometry detection of Annexin V, a cell membrane apoptosis marker, in mouse melanoma cells B16 after treatment with porcine pancreatic elastase (PPE) and CAEN.

[0044] Fig.17 .The schedule of tumor inoculation and tracheal administration in mice in Example 14.

[0045] Fig.18 .Pictures of mouse lung tumors after tracheal administration in Example 14 (control group PBS, porcine pancreatic elastase group PPE and nanocomplex group CAED).

[0046] Fig.19 .Survival curve of mice after tracheal administration treatment in Example 14 (control group PBS, porcine pancreatic elastase group PPE and nanocomplex group CAED).

[0047] Fig. 20 HE staining of the mouse lungs after tracheal administration in Example 14 (control group PBS, porcine pancreatic elastase group PPE and nanocomplex group CAED). Fig.21 HE staining of various tissues and organs of mice after tracheal administration in Example 14 (control group PBS, porcine pancreatic elastase group PPE and nanocomplex group CAED), scale bar 200 μm.

[0048] Fig. 22 .TUNEL staining of the lungs of mice after tracheal administration in Example 14 (control group PBS, porcine pancreatic elastase group PPE and nanocomplex group CAED), scale bar 100 μm.

[0049] Fig.23 CD8 positive staining in the lungs of mice after tracheal administration in Example 14 (control group PBS, porcine pancreatic elastase group PPE and nanocomplex group CAED), scale bar 50 μm.

[0050] Fig.24 .The structure of the protein-metal ion complex formed by zinc ion and porcine pancreatic elastase simulated by molecular dynamics.

[0051] Fig.25 .The structure of the protein-metal ion complex formed by nickel ion and porcine pancreatic elastase simulated by molecular dynamics. DETAILED DESCRIPTION

[0052] The present invention is mainly based on the nanocomplex of elastase and its cofactors, and takes porcine pancreatic elastase as a model drug (neutrophil elastase derivative), and proposes a strategy based on metal cofactors to enhance the target protein cleavage activity and anti-tumor effect. In short, a cleavage activity enhanced elastase nanocomplex (CAEN) was prepared and optimized in vitro to prevent the degradation of elastase in the biological environment. Subsequently, it was effectively delivered to lung tumor cells through the inhalation route. After specifically cleaving the transmembrane protein CD95 with a higher efficiency than free elastase, the elastase nanocomplex significantly induced stronger cancer cell apoptosis. Importantly, the cleavage effect of the nanocomplex is specific to CD95 without affecting other non-target protein substances. In addition, the released metal cofactors such as divalent manganese ions can effectively activate type I interferon in macrophages, further promoting and enhancing the local immune response of lung tumor tissues. In summary, the present invention provides a new idea for greatly improving the specificity of protein drug delivery and pharmacological effects.

[0053] The present invention is further described and explained by the following examples, but is not intended to limit the present invention.

[0054] Example 1: Mn 2+ Elastase-metal cofactor nanocomplex prepared as cofactor

[0055] 1) Prepare an elastase (from porcine pancreas, abbreviated as PPE) solution using distilled water at a concentration of 10 mg / mL;

[0056] 2) dissolving manganese chloride in distilled water to prepare a metal cofactor solution with a concentration of 1 mol / L; dissolving calcium chloride in distilled water to prepare a calcium ion solution with a concentration of 1 mol / L;

[0057] 3) Pipette 1 mL of DMEM as anion solution, containing 125 mg / L NaH2PO4;

[0058] 4) Pipette 100 μL of elastase solution and 10 μL of manganese ion solution respectively, mix them and let stand for 5 minutes;

[0059] 5) Add 10 μL of calcium ion solution and 1 mL of anion salt solution to the above mixed solution, mix thoroughly and seal;

[0060] 6) Incubate at 37°C for 30 minutes to 24 hours;

[0061] 7) High-speed centrifugation, 12000 rpm, for 5 to 15 minutes, retaining the bottom precipitate;

[0062] 8) Resuspending the nanoprecipitate in PBS buffer can obtain the elastase-metal cofactor nanocomplex.

[0063] Example 2: Using Zn 2+ Elastase-metal cofactor nanocomplex prepared as cofactor

[0064] 1) Prepare an elastase (from porcine pancreas, abbreviated as PPE) solution using distilled water at a concentration of 10 mg / mL;

[0065] 2) dissolving zinc chloride in distilled water to prepare a metal cofactor solution with a concentration of 1 mol / L; dissolving calcium chloride in distilled water to prepare a calcium ion solution with a concentration of 1 mol / L;

[0066] 3) Pipette 1 mL of DMEM as anionic salt solution, containing 125 mg / L NaH2PO4;

[0067] 4) Pipette 100 μL of elastase solution and 10 μL of zinc ion solution respectively, mix them and let stand for 5 minutes;

[0068] 5) Add 10 μL of calcium ion solution and 1 mL of anion salt solution to the above mixed solution, mix thoroughly and seal;

[0069] 6) Incubate at 37°C for 30 minutes to 24 hours;

[0070] 7) High-speed centrifugation, 10,000 rpm, centrifugation time 5 min, retaining the bottom precipitate;

[0071] 8) Resuspend the nanoprecipitate in PBS buffer to obtain the elastase-metal cofactor nanocomplex.

[0072] Example 3: Ni 2+ Elastase-metal cofactor nanocomplex prepared as cofactor

[0073] 1) Prepare an elastase (from porcine pancreas, abbreviated as PPE) solution using distilled water at a concentration of 10 mg / mL;

[0074] 2) using distilled water to dissolve nickel chloride to prepare a metal cofactor solution with a concentration of 1 mol / L; using distilled water to dissolve calcium chloride to prepare a calcium ion solution with a concentration of 1 mol / L;

[0075] 3) Pipette 1 mL of DMEM as anionic salt solution, containing 125 mg / L NaH2PO4;

[0076] 4) Pipette 100 μL of elastase solution and 10 μL of nickel ion solution respectively, mix them and let stand for 5 minutes;

[0077] 5) Add 10 μL of calcium ion solution and 1 mL of anion salt solution to the above mixed solution, mix thoroughly and seal;

[0078] 6) Incubate at 37°C for 30 minutes to 24 hours;

[0079] 7) High-speed centrifugation, 10,000 rpm, centrifugation time 5 min, retaining the bottom precipitate;

[0080] 8) Resuspend the nanoprecipitate in PBS buffer to obtain the elastase-metal cofactor nanocomplex.

[0081] Example 4: Elastase-metal cofactor nanocomplex prepared with carbonate as anion

[0082] 1) Prepare an elastase (from porcine pancreas, abbreviated as PPE) solution using distilled water at a concentration of 10 mg / mL;

[0083] 2) dissolving zinc chloride in distilled water to prepare a metal cofactor solution with a concentration of 1 mol / L; dissolving calcium chloride in distilled water to prepare a calcium ion solution with a concentration of 1 mol / L;

[0084] 3) using distilled water to dissolve sodium carbonate to prepare an anion salt solution with a concentration of 1 mmol / L to 1 mol / L;

[0085] 4) Pipette 100 μL of elastase solution and 10 μL of zinc ion solution respectively, mix them and let stand for 5 minutes;

[0086] 5) Add 10 μL of calcium ion solution and 1 mL of anion salt solution to the above mixed solution, mix thoroughly and seal;

[0087] 6) Incubate at 37°C for 30 minutes to 24 hours;

[0088] 7) High-speed centrifugation, 10,000 rpm, centrifugation time 5 min, retaining the bottom precipitate;

[0089] 8) Resuspend the nanoprecipitate in PBS buffer to obtain the elastase-metal cofactor nanocomplex.

[0090] Example 5: Elastase-metal cofactor nanocomplex prepared with hydrogen phosphate as anion

[0091] 1) Prepare an elastase (from porcine pancreas, abbreviated as PPE) solution using distilled water at a concentration of 10 mg / mL;

[0092] 2) dissolving zinc chloride in distilled water to prepare a metal cofactor solution with a concentration of 1 mol / L; dissolving calcium chloride in distilled water to prepare a calcium ion solution with a concentration of 1 mol / L;

[0093] 3) Dissolve sodium dihydrogen phosphate in distilled water to prepare an anion salt solution with a concentration of 1 mmol / L to 1 mol / L;

[0094] 4) Pipette 100 μL of elastase solution and 10 μL of zinc ion solution respectively, mix them and let stand for 5 minutes;

[0095] 5) Add 10 μL of calcium ion solution and 1 mL of anion salt solution to the above mixed solution, mix thoroughly and seal;

[0096] 6) Incubate at 37°C for 30 minutes to 24 hours;

[0097] 7) High-speed centrifugation, 10,000 rpm, centrifugation time 5 min, retaining the bottom precipitate;

[0098] 8) Resuspending the nanoprecipitate in PBS buffer can obtain the elastase-metal cofactor nanocomplex.

[0099] Example 6: Elastase-metal cofactor nanocomplex prepared with citrate as anion

[0100] 1) Prepare an elastase (from porcine pancreas, abbreviated as PPE) solution using distilled water at a concentration of 10 mg / mL;

[0101] 2) dissolving zinc chloride in distilled water to prepare a metal cofactor solution with a concentration of 1 mol / L; dissolving calcium chloride in distilled water to prepare a calcium ion solution with a concentration of 1 mol / L;

[0102] 3) Dissolve sodium citrate in distilled water to prepare an anion salt solution with a concentration of 1 mmol / L to 1 mol / L;

[0103] 4) Pipette 100 μL of elastase solution and 10 μL of zinc ion solution respectively, mix them and let stand for 5 minutes;

[0104] 5) Add 10 μL of calcium ion solution and 1 mL of anion salt solution to the above mixed solution, mix thoroughly and seal;

[0105] 6) Incubate at 37°C for 30 minutes to 24 hours;

[0106] 7) High-speed centrifugation, 10,000 rpm, centrifugation time 5 min, retaining the bottom precipitate;

[0107] 8) Resuspending the nanoprecipitate in PBS buffer can obtain the elastase-metal cofactor nanocomplex.

[0108] Example 7, transmission electron microscopy image of nanoparticle complex (CAEN) ( Figure 2 , Figure 3 )

[0109] The mineralized nanocomposite prepared in Example 1 was diluted 5 times with deionized water and its structure was observed by transmission electron microscopy (JEM-200X, JEOL, Japan). The diluted sample was kept at room temperature for 30 minutes and then dripped onto a copper mesh of a common carbon support film. After the water evaporated, it was directly loaded into a JEM-200X transmission electron microscope with an accelerating voltage of 80 kV to observe the particle size and morphology. The measurement results are shown in Figure 2. Figure 2 As shown in the figure, the prepared CAEN is spherical with a particle size of about 148.79±6.00nm. The longer the incubation time at 37℃, the larger the diameter of the nanoparticles ( Figure 3 ).

[0110] Example 8: Infrared spectroscopy analysis of nanocomposite (CAEN)

[0111] The nanocomposite prepared in Example 1 was dried and then the composition of the nanoparticles was detected by infrared spectrometer. Infrared spectrum is the ability of molecules to selectively absorb infrared rays of certain wavelengths, which causes the transition of vibrational energy levels and rotational energy levels in molecules. The infrared absorption spectrum of the substance can be obtained by detecting the absorption of infrared rays. The measurement results are as follows: Figure 4 As shown, the outer shell of the nanocomplex (CAEN) is mainly composed of phosphate groups, while the core is composed of amino acids.

[0112] Example 9, X-ray energy spectrum analysis of nanocomposite (CAEN) Figure 5 )

[0113] The CAEN prepared in Example 1 was dried and then tested for elemental composition using an X-ray photoelectron spectrometer (Thermo escalab 250Xi). Figure 5 As shown, CAEN mainly contains C, N, O, Ca, and P elements.

[0114] Example 10, Cytotoxicity Test of Nanocomplex (CAEN) Figure 6-Figure 9 )

[0115] In order to detect the toxic effect of nanocomplex CAEN on tumor cells, 100 μL of B16 cell suspension was inoculated in a 96-well plate (cell number: 5 × 10 3 / well), and incubated in a cell incubator for 12 hours (37°C, 5% CO2). Then, different concentrations of PPE and CAEN were added. After incubation for 6 hours, 10 μL of CCK-8 reagent was added to the cell culture medium of the 96-well plate, mixed, and continued to incubate in the incubator for 1.5 hours. The cell activity was obtained by reading the microplate reader at a detection wavelength of 450 nm. The results are shown in Figure 6-Figure 9 The results showed that in serum-free culture medium, since the activity of PPE was not affected, both PPE and CAEN had a killing effect on tumor cells. After adding serum, the activity of free PPE was inhibited and it had no killing effect on tumor cells, while CAEN still had a killing effect, suggesting that the metal ion micro-precipitation shell formed on the surface of PPE in the nanocomplex has the function of protecting the core protein in the extracellular fluid.

[0116] Example 11: A cellular uptake pathway experiment was conducted using the nanocomplex (CAEN) prepared according to the method of Example 1 and labeled with the fluorescent dye FITC.

[0117] The nanocomplex was prepared by using FITC-labeled PPE protein. First, 8×10 5 After culturing B16 cells for 12 hours, 1 mL of nanocomplex solution (final concentration 40 μg / mL) was added to each well; the cells were incubated at 37°C and 5% CO2 for 0, 2, 4, and 8 hours, respectively, and then observed under a laser confocal microscope. Fig.10 As shown, with the extension of incubation time, the fluorescence in the cytoplasm increased and the cytoplasm was diffusely distributed, indicating that the nanocomplex can be effectively taken up by tumor cells and release elastase in the cytoplasm.

[0118] Example 12: Binding mode of manganese ions to PPE and its effect on its specific cleavage function

[0119] Molecular dynamics simulation results ( Fig.11 ) showed that CaHPO4 and MnHPO4 mainly covered the PPE surface through the arginine residues on the PPE surface, where manganese ions would bind to PPE and prevent its nonspecific cleavage of the substrate. In order to verify the difference in the ability of free PPE and nanocomplex CAEN to cut CD95, Fig.12 As shown, we purified the MBP-CD95 protein (see Fig.12 A) and a cleavage experiment was performed. The theoretical molecular weight of MBP-CD95 (200-335) is 58.3 kD. First, we incubated different preparation groups with MBP-CD95 protein for 30 minutes. Fig.12As shown in B. Both the CAEN group and the PPE protein group effectively cleaved MBP-CD95, but the difference between the two groups was that the PPE group completely cleaved MBP-CD95, while the CAEN group only selectively cleaved CD95 protein. Therefore, the biomineralization complex of elastase and manganese ions can achieve specific cleavage of CD95 protein.

[0120] Example 13: Nanoparticle complex (CAEN) induces tumor cell apoptosis ( Figure 13-Figure 16 )

[0121] like Fig.13 As shown, after 6 hours of incubation with tumor cells, the intracellular reactive oxygen species (ROS) level in the CAEN group increased significantly compared with the PBS and PPE groups. ROS is one of the main inducers of apoptosis. Excessive ROS affects the function of mitochondria and reduces their membrane potential, thereby promoting apoptosis of tumor cells. We tested the mitochondrial membrane potential of cells treated with different preparations, among which the mitochondrial membrane potential of the CAEN group was significantly reduced, and the intracellular mitochondrial function was impaired, indicating an increase in apoptosis.

[0122] We further determined the changes in the apoptosis indicator protein Caspase3 in B16 cells treated with different groups ( Fig.14 ). Compared with the PBS group, the cell apoptosis indicators in the PPE group and CAEN group changed. With the occurrence of tumor cell apoptosis, the expression of cysteine ​​protease 3 (Caspase3) was downregulated, and the corresponding cleaved form of cysteine ​​protease 3 (CleavedCaspase3) was upregulated. These results indicate that CAEN has a strong ability to induce cell apoptosis. In order to further clarify the mechanism of CAEN-induced cancer cell death, we detected the expression changes of the apoptosis indicator AnnexinV on the cell membrane of B16 after drug treatment, such as Figure 15-16 The results showed that CAEN had the most significant late-stage apoptosis, with a high level of apoptosis.

[0123] Example 14: Verification of the therapeutic effect of the nanoparticle complex CAEN on lung tumors in mice

[0124] To verify the antitumor effect of CAEN, B16-F10 cells were injected into C57BL / 6 mice via the tail vein to form a lung cancer model for local antitumor therapy ( Fig.17 ). On the 5th, 13th and 21st days, the mice were dissected and their lung tissues were collected. Subsequently, we took pictures of the B16 cell metastasis in the lung tissues of the mice. Fig.18As shown in the figure, the CAEN group maintained a good therapeutic effect during the anti-tumor treatment without significant metastasis. Due to the lack of nanoshell protection, free PPE is easily inhibited by various factors in the extracellular fluid in the body, so it has no obvious anti-cancer effect in the body. However, the nanoshell of CAEN can improve the cutting efficiency of PPE, reduce toxicity, promote the effective uptake of nanoparticles by tumor cells, and exert a better inhibitory effect on tumor cells. The results of survival analysis showed that after stopping the drug after 21 days, the survival rate of mice in the CAEN group was higher than that of mice in other groups ( Fig.19 ). Fig. 20 The staining results of the heart, liver, spleen, lung, and kidney of mice showed that CAEN did not cause significant pathological changes in these organs. Fig.21 HE staining of tumor tissues showed that fewer cancer lesions were found in tissue sections of mice in the CAEN-treated group, while more cancer lesions were found in the control PBS and PPE groups. We further confirmed that CAEN effectively induced cell apoptosis by TUNEL staining ( Fig. 22 ). And if Fig.23 As shown, among all treatment groups, the proportion of CD8-positive T cells in the CAEN group was the highest, indicating that the nanocomplex not only promotes PPE to better play its role in inducing tumor cell apoptosis, but also can enhance the local immune response at the administration site, increase the infiltration of CD8-positive cytotoxic T cells in tumor lesions, and synergistically exert an inhibitory effect on tumors.

[0125] Example 15: Molecular dynamics simulation of the protein metal ion complex structures formed by zinc ions, nickel ions and porcine pancreatic elastase.

[0126] Molecular dynamics simulations were used to analyze the binding between metal ions and porcine pancreatic elastase. All molecular dynamics simulations used the Gromacs 5.1 software package, the CHARMM-GUI website, and the UCSF Chimera software in combination to process and analyze the protein structure. First, the CHARMM-GUI website was used to process the protein file and add negative charges to make the system uncharged. The energy of the system was then optimized to eliminate unreasonable collisions between water molecules and proteins. Next, the equilibrium dynamics and constant temperature and pressure dynamics were adjusted, and molecular dynamics simulations were performed for 400ns at a constant temperature of 300K. In this system, we added 500 mol / L zinc chloride or nickel chloride to verify the binding of ions to PPE protein. Finally, UCSF Chimera was used to examine and analyze the binding of proteins and ions. As Fig.24 and Fig.25As shown, the molecular dynamics simulation results show that both zinc ions and nickel ions can bind to porcine pancreatic elastase, which is beneficial to improve the specificity of porcine pancreatic elastase binding to substrates, prevent it from binding to serine protease inhibitors, and thus increase the activity of elastase.

Claims

1. An elastase nanocomplex with enhanced cleavage activity, characterized in that: The nanocomplex has a complex of elastase and a metal cofactor as the core, and an outer layer is covered with a mineralized shell formed by calcium ions and anions, wherein the metal cofactor is Mn 2+ , the elastase is porcine pancreatic elastase, and the anion is a hydrogen phosphate ion.

2. The nanocomposite according to claim 1, characterized in that The nanocomposite is nanoparticles with a particle size of 100-1000 nm.

3. The method for preparing the elastase nanocomplex with enhanced cleavage activity according to claim 1 or 2, comprising the following steps: 1) Prepare elastase solution; 2) Prepare metal cofactor solution; 3) Prepare calcium ion solution; 4) Prepare anionic salt solution; 5) Mix the elastase solution and the metal cofactor solution in proportion and let it stand for 5 to 10 minutes, then add the calcium ion solution and the anion salt solution, mix well, seal and let it stand at 37°C for 10 minutes to 24 hours; 6) After high-speed centrifugation, the precipitate is retained, and then resuspended with a solution to obtain the nanocomplex.

4. The preparation method according to claim 3, characterized in that: The concentration of the prepared elastase solution in step 1) is 1 mg / mL ~ 10 mg / mL; the concentration of the prepared metal cofactor solution in step 2) is 1 mmol / L ~ 1 mol / L; the concentration of the prepared calcium ion solution in step 3) is 1 mmol / L ~ 1 mol / L; the concentration of the prepared anion salt solution in step 4) is 1 mmol / L ~ 1 mol / L.

5. The preparation method according to claim 3, characterized in that: Step 2) using manganese chloride to prepare a metal cofactor solution; Step 3) using calcium chloride to prepare the calcium ion solution.

6. The preparation method according to claim 3, characterized in that: Step 4) DMEM serum-free medium is used as the anionic salt solution.

7. Use of the elastase nanocomplex with enhanced cleavage activity according to claim 1 or 2 in the preparation of anti-tumor drugs, wherein the tumor is lung cancer or melanoma.

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