CN116942801A - Nanometer compound based on elastase-metal cofactor and preparation and application thereof - Google Patents
Nanometer compound based on elastase-metal cofactor and preparation and application thereof Download PDFInfo
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- CN116942801A CN116942801A CN202310906603.7A CN202310906603A CN116942801A CN 116942801 A CN116942801 A CN 116942801A CN 202310906603 A CN202310906603 A CN 202310906603A CN 116942801 A CN116942801 A CN 116942801A
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- Prior art keywords
- elastase
- solution
- nanocomposite
- cancer
- metal cofactor
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/482—Serine endopeptidases (3.4.21)
- A61K38/486—Elastase (3.4.21.36 or 3.4.21.37)
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses a nanometer compound based on elastase-metal cofactor, its preparation and application, which uses the compound of elastase and metal cofactor as inner core, and the outer layer is covered with mineralized shell formed by calcium ion and anion, to realize the enhancement of target protein cutting activity and anti-tumor effect, wherein the metal cofactor is selected from Mn 2+ 、Zn 2+ 、Ni 2+ One or more of the following. Nanocomposite of the present inventionThe substance can prevent elastase from degrading in biological environment, and can effectively convey elastase into tumor cells by inhalation route, thereby realizing organ tissue specific delivery. Compared with free elastase, the nano-composite has higher specific cutting efficiency, induces stronger cancer cell apoptosis, remarkably inhibits tumor growth, simultaneously shows good biological safety, and provides an effective research thought for the application of protein drugs in anti-tumor treatment.
Description
Technical Field
The invention relates to a nano-composite based on elastase-metal cofactor and application thereof, belonging to the field of nano-preparations.
Background
In the immune monitoring process, the organism shows a complex immune mechanism, can timely and accurately identify, kill and clear the mutated tumor cells, and plays a vital role in preventing the occurrence and development of tumors. Among them, elastase is expected to develop into a wide variety of anticancer protein drugs as an excellent representative therapeutic protein. Neutrophil elastase can release the pro-apoptotic domain by cleaving its substrate CD95 protein in cancer cells. The domain can be combined with a high-expression histone H1 subtype in cancer cells to induce apoptosis of tumor cells.
However, elastase has low therapeutic efficiency due to the non-targeting of proteins in drug delivery and their non-specific pharmacological effects. Because of the strong intratumoral pressure in tumor tissue, the protein medicine is absorbed with lower efficiency, and the effective effect is difficult to fully play. In addition, broad-spectrum protease digestion has serious side effects, for example, neutrophil elastase or its derivatives porcine pancreatic elastase can catalyze the hydrolysis of proteins such as denatured collagen and elastin. Prolonged exposure to such proteins in large amounts may lead to diseases associated with degradation of elastic fibers. In addition, protein drugs have the disadvantages of easy degradation and short half-life during in vivo administration therapy. Proteins often suffer from denaturation, inactivation, etc., for example, when neutrophil elastase is in a body fluid environment, various serine protease inhibitors in body fluids can inhibit their protein degradation activities, such as plasma α2-macroglobulin and α1-antitrypsin. These factors have severely hampered the development and use of elastase drugs.
The rapid development of nanomedicine has driven the advancement of many tumor treatment and protein delivery strategies, for example, the development of nanomesh delivery systems based on biomimetic biomineralization strategies. Biomimetic biomineralization is the process of combining inorganic ions with biological macromolecules to form mineral materials. It has the following advantages in delivering protein drugs: 1) Protecting the protein and avoiding inactivation; 2) Enhancing delivery to tumor cells; 3) Tissue specific administration to promote efficient drug accumulation. Furthermore, mineralization strategies generally have the advantage of good biocompatibility, degradability and ease of cellular uptake. These properties are important for the biosynthesis of nanomaterials using proteins as templates. Furthermore, given the interdependence between the catalytic activity of biological enzymes and their cofactors, we wish to exploit the cofactors to achieve specificity of elastase pharmacological actions. This is mainly because nearly half of the biological functions of proteins are cofactor dependent. Wherein, the cofactor exists mainly in the form of metal ions or small molecules, and plays roles of group transfer, catalysis and redox. In general, cofactors have a variety of biological functions, which can activate or inhibit the activity of proteins, depending on the enzyme and associated substrate to which they bind. For example, heme proteins require iron ions as cofactors to facilitate oxygen transport and transport. In addition, the dynamic change of cofactor and protein after combination can be accurately displayed by the current computer nanosecond molecular dynamics simulation calculation, and a convenient method is provided for the research and conversion of enzyme activity.
Disclosure of Invention
As a novel anti-tumor protein drug, elastase has strong capability of inducing tumor cell apoptosis and killing tumor cells. For cases where elastase is less therapeutically effective in tumor treatment: the invention aims to construct a nano-composite (a cleavage activity enhanced elastase nanocomplex, CAEN) which is prepared based on elastase-metal cofactor and can enhance the elastase cleavage activity. Based on the molecular dynamics simulation theory, the research proposes that metal ions including manganese ions, zinc ions and nickel ions are used as cofactors to improve the therapeutic activity of Porcine Pancreatic Elastase (PPE), and elastase is used for realizing the specific cleavage of a substrate, namely pro-apoptotic protein CD95 protein, so that the effect of inducing apoptosis of tumor cells is enhanced. The CAEN nanocomposite can effectively protect and stabilize the activity of PPE proteins. And the CAEN nano-composite has an effective killing effect on different types of cancer cells. The nano-composite shell generated by biomineralization enhances the intracellular uptake of PPE, which is beneficial to the induction of apoptosis of tumor cells by PPE. In addition, dissolution and release of metal ions in the nanocomposite can affect homeostasis of the internal environment of tumor cells, leading to increased levels of reactive oxygen species in tumor cells and mitochondrial damage. The immune activation function of the cofactor such as manganese ion is combined, so that the growth of cancer cells can be effectively inhibited. Furthermore, biomineralization strategies play a positive role in controlling and preventing PPE-induced pulmonary cathepsin-related balance disorders. Compared with PPE protein, CAEN can effectively inhibit cell proliferation, and remarkably inhibit tumor growth in vivo by promoting tumor cell apoptosis and anti-tumor immunotherapy, and meanwhile, has good biological safety. The research provides an effective research idea for the application of protein drugs in anti-tumor treatment.
The elastase nano-Composite (CAEN) with enhanced cleavage activity is prepared by biomineralization reaction, takes the elastase-metal cofactor composite as an inner core, and takes a precipitate generated by the reaction of calcium ions and an anion salt solution as an outer shell. Wherein the model drug of elastase may be one or more of the following serine proteases: neutrophil elastase, porcine pancreatic elastase, and homologs thereof; the metal cofactor is metal ion selected from Mn 2+ 、Zn 2+ 、Ni 2+ One or more of the following.
The nanocomposite shell has positive charges, and is easy to be taken up and endocytosed by cells; in the acidic environment of the tumor cytoplasm, the nanocomposite slowly disintegrates and releases the elastase-metal cofactor. Cleavage by CAEN on elastinThe substrate CD95 (pro-apoptotic protein) of the enzyme is specific without affecting other non-target proteins, thereby significantly inducing stronger cancer cell apoptosis. In addition, released Mn 2+ Can effectively activate tumor tissues to generate I-type interferon, and further induce and promote local immune response of the tumor tissues.
Further, the shell of the nanocomposite is a mineralized shell formed from a calcium chloride solution and an anion salt solution, the anions including, but not limited to, phosphate ions, carbonate ions, oxalate ions, hydrogen phosphate ions, citrate ions, and the like, water-soluble acid ions.
The nanocomposite is preferably a nanoparticle having a particle size of 100 to 1000 nm.
The preparation method of the nanocomposite comprises the following steps:
1) Preparing elastase solution: preparing elastase powder into solution by using sterile distilled water or buffer solution such as PBS;
2) Preparing a metal cofactor solution: will contain a metal cofactor (Mn 2+ 、Ni 2+ And/or Zn 2+ ) Preparing a solution from the reagent of (a);
3) Preparing a calcium ion solution: distilled water can be used for dissolving calcium chloride to prepare a solution;
4) Preparing an anionic salt solution: dissolving an inorganic salt reagent containing anions with distilled water to prepare a solution;
5) Mixing elastase solution and metal cofactor solution according to a proper proportion, standing for 5-10 minutes, adding calcium ion solution and anion salt solution into the mixed solution, mixing uniformly, sealing, and standing for 10-24 hours at 37 ℃;
6) The precipitate is retained after high speed centrifugation and then resuspended using a medium or buffer solution to obtain the nanocomposite.
In the above preparation method, preferably, the concentration of the elastase solution prepared in step 1) is 1 mg/mL-10 mg/mL; the concentration of the metal cofactor solution prepared in the step 2) is 1 mmol/L-1 mol/L; the concentration of the calcium ion solution prepared in the step 3) is 1 mmol/L-1 mol/L; the concentration of the anionic salt solution prepared in the step 4) is 1 mmol/L-1 mol/L. Reagent sources of the metal cofactor include, but are not limited to, manganese chloride, zinc chloride, nickel chloride; the reagent source of the calcium ions is calcium chloride; reagent sources of the anions include, but are not limited to, hydrogen phosphate, carbonate, oxalate, and citrate. A DMEM serum-free medium (Michaelis, inc. under the trade name CM 10013) containing hydrogen phosphate ions at a concentration of 3.27mM may be used as the anionic salt solution, and in step 5), the elastase solution and the metal cofactor solution are mixed and allowed to stand, and then the calcium ion solution and the DMEM serum-free medium are added, and after being sufficiently mixed, the mixture is incubated and allowed to stand at 37 ℃.
In step 6) above, centrifugation is typically carried out at 12000rpm for 10min, and the pellet after removal of the supernatant may be resuspended in DMEM serum-free medium or PBS and then tested directly or stored for a short period of time at 4 ℃.
The above steps 1), 5) and 6) all need to be protected from light, and illumination can affect the activity of elastase.
The nano-composite based on elastase-metal cofactor is suitable for preparing the anti-tumor treatment related drugs. The inner core of the nano-composite is an elastase and metal cofactor composite, and the outer layer calcium ion precipitate has the functions of stabilizing and protecting the activity of protein, and the components of the nano-composite are biological safe and degradable substances. After the nano-composite enters cells, the ion shell of the nano-composite is slowly disintegrated due to cytoplasmic acidic environment, and the elastase-metal cofactor released further enhances the activity and the specificity of elastase for cutting substrates, so that the apoptosis of tumor cells is effectively promoted.
The cell lines of elastase-metal cofactor nanocomplex for anti-tumor therapy may be of human or murine origin, 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, U-251MG.
Cancers closely related to elastase-metal cofactor nanocomposite antitumor therapy include, but are not limited to, lung cancer, stomach 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.
The elastase-metal cofactor nano-composite provided by the invention has the following advantages:
1. the outer layer of the nanometer shell of the nanometer compound has the functions of stabilizing and protecting the activity of elastase;
2. the metal cofactor (manganese ion) in the nanometer complex can enhance the specificity of elastase for cutting substrates, and effectively promote tumor cell apoptosis
3. The biomineralization nano-composite improves the efficiency of delivering organ tissues of elastase, not only promotes the uptake of protein by tumor cells, but also effectively activates the local immune system of lung cancer tissues by utilizing metal cofactors thereof, thereby further playing a good anti-tumor synergistic effect.
In conclusion, the elastase-metal cofactor nano-composite can greatly improve the efficiency of killing tumor cells by elastase, effectively promote apoptosis of the tumor cells, has good biocompatibility, and is suitable for treating different tumor diseases.
Drawings
FIG. 1 is a schematic diagram showing the preparation and structure of a nanocomposite (CAEN) according to the present invention.
FIG. 2 is a transmission electron microscopy image of the nanocomposite (CAEN) prepared in example 1 and Porcine Pancreatic Elastase (PPE).
FIG. 3 is a graph showing the time for preparing the nanocomposite (CAEN) prepared in example 1, and the particle size.
FIG. 4. Results of infrared spectra of nanocomposites (CAEN) prepared in example 1, with Porcine Pancreatic Elastase (PPE) as control.
FIG. 5. Measurement results of X-ray electron spectroscopy analysis of the nanocomposite (CAEN) prepared in example 1.
FIG. 6. Results of cytotoxicity experiments (B16 cells in culture without fetal bovine serum) on the nanocomposites (CAEN) prepared in example 1.
FIG. 7 shows the results of cytotoxicity experiments (B16 cells in culture with 10% fetal bovine serum) on the nanocomposites (CAEN) prepared in example 1.
FIG. 8 shows the results of cytotoxicity experiments (A549 cells in culture without fetal bovine serum) performed on the nanocomposite (CAEN) prepared in example 1.
FIG. 9 shows the results of cytotoxicity test (A549 cells in culture with 10% fetal bovine serum) performed on the nanocomposite (CAEN) prepared in example 1.
FIG. 10 cell uptake experiments were performed using the fluorescent dye FITC-labeled nanocomposite (CAEN) prepared according to the method of example 1 and uptake pathways were observed by confocal microscopy.
FIG. 11. Molecular dynamics simulation of the structure of the protein metal ion complex formed by manganese ions and porcine pancreatic elastase.
FIG. 12 is a graph comparing in vitro cleavage effects of Porcine Pancreatic Elastase (PPE) and CAEN on CD95, wherein A shows the molecular sieve results of purified MBP-CD95 protein and the molecular size determined by Coomassie Brilliant blue staining, and B shows the case of CAEN cleaving MBP-CD95, wherein PBS is phosphate buffered solution control, NP is metal ion precipitation control, and PPE is PPE protein alone control.
FIG. 13 shows a graph comparing mitochondrial membrane potential changes in tumor cells after treatment of murine melanoma cells B16 with Porcine Pancreatic Elastase (PPE) and CAEN.
FIG. 14 shows a graph comparing changes in apoptosis marker Caspase3 protein and its cleaved forms after treatment of murine melanoma cells B16 with Porcine Pancreatic Elastase (PPE) and CAEN.
FIG. 15 shows a comparative graph of changes in cell membrane apoptosis marker AnnexinV after treatment of murine melanoma cells B16 with Porcine Pancreatic Elastase (PPE) and CAEN.
FIG. 16 data analysis results of flow assays of cell membrane apoptosis marker Annexin V after treatment of murine melanoma cells B16 with Porcine Pancreatic Elastase (PPE) and CAEN.
FIG. 17. Schedule of tumor inoculation and tracheal administration for mice in example 14.
FIG. 18 shows lung tumor pictures of mice after tracheal administration treatment (control PBS, porcine pancreatic elastase PPE, and nanocomposite CAED) in example 14.
FIG. 19 survival curves of mice following tracheal administration treatment in example 14 (control PBS, porcine pancreatic elastase PPE and nanocomposite CAED).
FIG. 20 HE staining of the lungs of mice following tracheal administration treatment in example 14 (control PBS, porcine pancreatic elastase PPE and nanocomposite CAED). FIG. 21 HE staining of tissues and organs of mice after tracheal administration treatment (control PBS, porcine pancreatic elastase PPE and nanocomposite CAED) in example 14, scale 200 μm.
FIG. 22 TUNEL staining of the lungs of mice following tracheal administration treatment in example 14 (control PBS, porcine pancreatic elastase PPE and nanocomposite CAED) on a scale of 100. Mu.m.
FIG. 23 CD8 positive staining of mice lungs after tracheal administration treatment in example 14 (control group PBS, porcine pancreatic elastase group PPE and nanocomposite group CAED), scale 50 μm.
FIG. 24 shows the structure of a protein metal ion complex formed by zinc ions and porcine pancreatic elastase in a molecular dynamics simulation.
FIG. 25 shows the structure of a protein metal ion complex formed by nickel ions and porcine pancreatic elastase in a molecular dynamics simulation.
Detailed Description
The invention mainly provides a strategy based on metal cofactor for enhancing target protein cleavage activity and anti-tumor effect based on a nano-composite of elastase and cofactor thereof, which takes porcine pancreatic elastase as a model drug (neutrophil elastase derivative). Briefly, an elastase nanocomposite (a cleavage activity enhanced elastase nanocomplex, CAEN) with enhanced cleavage activity was prepared and optimized in vitro to prevent degradation of elastase in a biological environment. Subsequent efficient delivery into lung tumor cells by the inhaled route, the elastase nanocomposite significantly induces stronger cancer cell apoptosis after specific cleavage of transmembrane protein CD95 with higher efficiency than free elastase. Importantly, the cleavage of the nanocomposite is specific for CD95 without affecting other non-target protein species. In addition, released metal cofactors such as divalent manganese ions can be effective in activating type I interferons in macrophages, further promoting and enhancing the local immune response of lung tumor tissue. In a word, the invention provides a new idea for greatly improving the specificity in the aspects of protein drug delivery and pharmacological actions.
The invention is further illustrated and explained below by means of examples, without being limiting thereto.
Example 1, in Mn 2+ Elastase-metal cofactor nanocomplex prepared for cofactors
1) The elastase (PPE) solution was prepared using distilled water at a concentration of 10mg/mL;
2) Dissolving manganese chloride by using distilled water to prepare a metal cofactor solution, wherein the concentration is 1mol/L; dissolving calcium chloride by using distilled water to prepare a calcium ion solution, wherein the concentration is 1mol/L;
3) 1mL of DMEM was pipetted as anionic solution containing NaH 2 PO 4 The concentration is 125mg/L;
4) 100 mu L of elastase solution and 10 mu L of manganese ion solution are respectively sucked up, uniformly mixed and stood for 5 minutes;
5) Adding 10 mu L of calcium ion solution and 1mL of anion salt solution into the mixed solution, fully and uniformly mixing and sealing;
6) Standing in a incubator at 37 ℃ for 30 minutes to 24 hours;
7) Centrifuging at a high speed for 5-15 minutes at 12000rpm, and keeping the sediment at the bottom;
8) The elastase-metal cofactor nanocomposite can be obtained by resuspending the nanoprecipitates with PBS buffer.
Example 2 with Zn 2+ Elastase-metal cofactor nanocomplex prepared for cofactors
1) The elastase (PPE) solution was prepared using distilled water at a concentration of 10mg/mL;
2) Dissolving zinc chloride by using distilled water to prepare a metal cofactor solution, wherein the concentration is 1mol/L; dissolving calcium chloride by using distilled water to prepare a calcium ion solution, wherein the concentration is 1mol/L;
3) 1mL of DMEM was taken as the anionic salt solution containing NaH 2 PO 4 The concentration is 125mg/L;
4) 100 mu L of elastase solution and 10 mu L of zinc ion solution are respectively sucked up, uniformly mixed and stood for 5 minutes;
5) Adding 10 mu L of calcium ion solution and 1mL of anion salt solution into the mixed solution, fully and uniformly mixing and sealing;
6) Standing in a incubator at 37 ℃ for 30 minutes to 24 hours;
7) Centrifuging at high speed at 10000 rpm for 5 min, and keeping the bottom sediment;
8) The elastase-metal cofactor nanocomposite can be obtained by resuspending the nanoprecipitates with PBS buffer.
Example 3 with Ni 2+ Elastase-metal cofactor nanocomplex prepared for cofactors
1) The elastase (PPE) solution was prepared using distilled water at a concentration of 10mg/mL;
2) Dissolving nickel chloride by using distilled water to prepare a metal cofactor solution, wherein the concentration is 1mol/L; dissolving calcium chloride by using distilled water to prepare a calcium ion solution, wherein the concentration is 1mol/L;
3) 1mL of DMEM was taken as the anionic salt solution containing NaH 2 PO 4 The concentration is 125mg/L;
4) 100. Mu.L of elastase solution and 10. Mu.L of nickel ion solution were respectively aspirated, mixed well and left to stand for 5 minutes;
5) Adding 10 mu L of calcium ion solution and 1mL of anion salt solution into the mixed solution, fully and uniformly mixing and sealing;
6) Standing in a incubator at 37 ℃ for 30 minutes to 24 hours;
7) Centrifuging at high speed at 10000 rpm for 5 min, and keeping the bottom sediment;
8) The elastase-metal cofactor nanocomposite can be obtained by resuspending the nanoprecipitates with PBS buffer.
EXAMPLE 4 elastase-Metal cofactor nanocomposites prepared with carbonate anions
1) The elastase (PPE) solution was prepared using distilled water at a concentration of 10mg/mL;
2) Dissolving zinc chloride by using distilled water to prepare a metal cofactor solution, wherein the concentration is 1mol/L; dissolving calcium chloride by using distilled water to prepare a calcium ion solution, wherein the concentration is 1mol/L;
3) Dissolving sodium carbonate by using distilled water to prepare an anion salt solution, wherein the concentration is 1 mmol/L-1 mol/L;
4) 100 mu L of elastase solution and 10 mu L of zinc ion solution are respectively sucked up, uniformly mixed and stood for 5 minutes;
5) Adding 10 mu L of calcium ion solution and 1mL of anion salt solution into the mixed solution, fully and uniformly mixing and sealing;
6) Standing in a incubator at 37 ℃ for 30 minutes to 24 hours;
7) Centrifuging at high speed at 10000 rpm for 5 min, and keeping the bottom sediment;
8) The elastase-metal cofactor nanocomposite can be obtained by resuspending the nanoprecipitates with PBS buffer.
EXAMPLE 5 elastase-Metal cofactor nanocomposites prepared with Hydrogen phosphate as anion
1) The elastase (PPE) solution was prepared using distilled water at a concentration of 10mg/mL;
2) Dissolving zinc chloride by using distilled water to prepare a metal cofactor solution, wherein the concentration is 1mol/L; dissolving calcium chloride by using distilled water to prepare a calcium ion solution, wherein the concentration is 1mol/L;
3) Dissolving sodium dihydrogen phosphate with distilled water to prepare an anion salt solution with the concentration of 1 mmol/L-1 mol/L;
4) 100 mu L of elastase solution and 10 mu L of zinc ion solution are respectively sucked up, uniformly mixed and stood for 5 minutes;
5) Adding 10 mu L of calcium ion solution and 1mL of anion salt solution into the mixed solution, fully and uniformly mixing and sealing;
6) Standing in a incubator at 37 ℃ for 30 minutes to 24 hours;
7) Centrifuging at high speed at 10000 rpm for 5 min, and keeping the bottom sediment;
8) The elastase-metal cofactor nanocomposite can be obtained by resuspending the nanoprecipitates with PBS buffer.
EXAMPLE 6 elastase-Metal cofactor nanocomposites prepared with citrate as anion
1) The elastase (PPE) solution was prepared using distilled water at a concentration of 10mg/mL;
2) Dissolving zinc chloride by using distilled water to prepare a metal cofactor solution, wherein the concentration is 1mol/L; dissolving calcium chloride by using distilled water to prepare a calcium ion solution, wherein the concentration is 1mol/L;
3) Dissolving sodium citrate by using distilled water to prepare an anion salt solution, wherein the concentration is 1 mmol/L-1 mol/L;
4) 100 mu L of elastase solution and 10 mu L of zinc ion solution are respectively sucked up, uniformly mixed and stood for 5 minutes;
5) Adding 10 mu L of calcium ion solution and 1mL of anion salt solution into the mixed solution, fully and uniformly mixing and sealing;
6) Standing in a incubator at 37 ℃ for 30 minutes to 24 hours;
7) Centrifuging at high speed at 10000 rpm for 5 min, and keeping the bottom sediment;
8) The elastase-metal cofactor nanocomposite can be obtained by resuspending the nanoprecipitates with PBS buffer.
EXAMPLE 7 Transmission electron microscopy of nanoparticle Complexes (CAEN) (FIGS. 2, 3)
The mineralized nanocomposite prepared in example 1 was diluted 5-fold with deionized water, and its structure was observed by transmission electron microscopy (JEM-200X, JEOL, japan). And (3) after keeping the diluted sample at the constant temperature for 30min at room temperature, dripping the diluted sample on a copper mesh of a common carbon support film, directly loading the diluted sample into a JEM-200X transmission electron microscope after the water is volatilized, and observing the particle size and the morphology, wherein the acceleration voltage is 80 kV. As a result of measurement, the CAEN was spherical and had a particle diameter of about 148.79.+ -. 6.00nm, as shown in FIG. 2. The longer the incubation time at 37 ℃ the larger the nanoparticle diameter (figure 3).
Example 8 Infrared spectroscopic analysis of nanocomposite (CAEN)
The composition of the nanoparticles was measured by an infrared spectrum analyzer after drying the nanocomposite prepared in example 1. The infrared spectrum is an infrared absorption spectrum of a substance obtained by detecting the condition that infrared rays are absorbed by a molecule which can selectively absorb infrared rays with certain wavelengths and cause transition of vibration energy level and rotation energy level in the molecule. The results of the assay are shown in FIG. 4, where the shell of the nanocomposite (CAEN) consists essentially of phosphate groups, while the core consists of amino acids.
EXAMPLE 9X-ray Spectrometry analysis of nanocomposite (CAEN) (FIG. 5)
The CAEN prepared in example 1 was dried and then subjected to elemental composition measurement by an X-ray photoelectron spectrometer (Thermo escalab 250 Xi). As shown in FIG. 5, CAEN mainly contains C, N, O, ca, P elements.
Example 10 cytotoxicity experiments of nanocomposites (CAEN) (FIGS. 6-9)
To examine the toxic effect of nanocomposite CAEN on tumor cells, 100. Mu.L of B16 cell suspension was seeded in 96-well plates (cell number: 5X 10) at night the day before administration 3 Well) in a cell incubator for 12h (37 ℃,5% co 2 ). Then adding PPE and CAEN with different concentrations, incubating for 6 hours, adding 10 mu L of CCK-8 reagent into the cell culture solution of the 96-well plate, mixing uniformly, continuing incubating for 1.5 hours in an incubator, reading by an enzyme-labeling instrument, and obtaining the cell activity, wherein the detection wavelength is 450nm. The results are shown in FIGS. 6-9, where both PPE and CAEN have killing effects on tumor cells in serum-free medium, as PPE activity is unaffected. After serum is added, the activity of free PPE is inhibited, the free PPE has no killing effect on tumor cells, but CAEN has still killing effect, which indicates that a metal ion microprecipitate shell formed on the surface of PPE in the nano-composite has the effect of protecting core protein in extracellular fluid.
Example 11 cell uptake pathway experiments were performed using fluorescent dye FITC-labeled nanocomposites (CAEN) prepared according to the method of example 1.
The PPE protein marked by FITC is prepared into a nano compound. First, 8X 10 was added to a confocal cuvette 5 After 12 hours of incubation, 1mL of the nanocomposite solution (final concentration 40. Mu.g/mL) was added to each well; 37 ℃ 5% CO 2 After incubation for 0,2,4,8 hours, respectively, observations were made under a confocal laser microscope. As shown in fig. 10, fluorescence in the cytoplasm increases with prolonged incubation time, and cytoplasmic dispersion distribution indicates that the nanocomposite can be efficiently taken up by tumor cells and release elastase in the cytoplasm.
Example 12 Effect of binding mode of manganese ion to PPE and specific cleavage function thereof
The molecular dynamics simulation results (FIG. 11) indicate that CaHPO 4 And MnHPO 4 The surface of PPE is covered mainly by arginine residues on the surface of PPE, where manganese ions will bind to PPE preventing its non-specific cleavage of the substrate. To verify the difference in the ability of free PPE and nanocomposite CAEN to cleave CD95, we purified the MBP-CD95 protein (see a in fig. 12) and performed cleavage experiments, as shown in fig. 12. MBP-CD95 (200-335) has a theoretical molecular weight of 58.3kD. First, we incubated the different preparation groups with MBP-CD95 protein for 30min, and the results are shown in FIG. 12B. Both the CAEN group and the PPE protein group effectively cut MBP-CD95, but the difference between the two groups is that the PPE group completely cut MBP-CD95, whereas the CAEN group selectively cut only CD95 protein. Thus, the biomineralization complex of elastase and manganese ions can effect specific cleavage of CD95 protein.
EXAMPLE 13 nanoparticle Complex (CAEN) induces apoptosis in tumor cells (FIGS. 13-16)
As shown in fig. 13, intracellular Reactive Oxygen Species (ROS) levels in the CAEN group increased significantly after 6 hours incubation with tumor cells compared to PBS and PPE groups. ROS is one of the major inducers of apoptosis. Excessive ROS affect mitochondrial function, lowering its membrane potential, and thus promoting apoptosis of tumor cells. We tested the mitochondrial membrane potential of cells treated with different formulations, where the CAEN group mitochondrial membrane potential was significantly reduced, and the intracellular mitochondrial function was impaired, suggesting increased apoptosis.
We further examined the changes in the apoptosis indicator protein Caspase3 in B16 cells treated with the different groups (fig. 14). The apoptosis index was changed for PPE and CAEN groups compared to PBS groups. With the occurrence of tumor apoptosis, the expression level of cysteine protease 3 (Caspase 3) is down-regulated, and the corresponding sheared form of cysteine protease 3 (clean Caspase 3) is up-regulated. These results indicate that CAEN has a strong ability to induce apoptosis. To further elucidate the mechanism by which CAEN induces cancer cell death, we examined the change in cell membrane expression of annexin v as an indicator of apoptosis of B16 after drug treatment, as the results in fig. 15-16 show that the late apoptosis of CAEN is most pronounced with high levels of apoptosis.
Example 14 in vivo validation of nanoparticle Complex CAEN on treatment of Lung tumors in mice
To verify the antitumor effect of CAEN, C57BL/6 mice were injected with B16-F10 cells via the tail vein to form a model of lung cancer for local antitumor therapy (fig. 17). On days 5, 13 and 21, mice were dissected and their lung tissue was collected. Subsequently, we photographed B16 cell metastases in mouse lung tissue. As shown in fig. 18, the CAEN group maintained good therapeutic effect during anti-tumor treatment without significant metastasis. Free PPE has no obvious cancer inhibiting effect in vivo because it is easy to be inhibited by various factors in extracellular fluid in vivo due to the lack of nano-shell protection. However, the nano shell of CAEN can improve the cutting efficiency of PPE, reduce toxicity, promote the effective uptake of nano particles by tumor cells, and exert better inhibition effect on the tumor cells. Survival analysis showed that the CAEN group mice survived more than the other group after 21 days post-withdrawal (fig. 19). The staining results of the major organs of mice in fig. 20, such as heart, liver, spleen, lung and kidney, indicate that CAEN does not cause significant pathological changes in these organs. According to HE staining of tumor tissue in fig. 21, fewer cancer lesions were found in tissue sections of CAEN-dosed mice, whereas more cancer lesions were found in control PBS and PPE groups. We further confirmed that CAEN was effective in inducing apoptosis by TUNEL staining (figure 22). And as shown in fig. 23, in all treatment groups, the proportion of the CD8 positive T cells in the CAEN group is highest, which indicates that the nano-composite not only promotes PPE to better exert the effect of inducing tumor cell apoptosis, but also can enhance the local immune response of the administration part, promote the infiltration of the CD8 positive killer T cells in tumor focus and synergistically exert the inhibition effect on tumors.
Example 15, molecular dynamics simulation zinc ion, nickel ion and porcine pancreatic elastase formed protein metal ion complex structures respectively.
Molecular dynamics simulation was used to analyze the binding between metal ions and porcine pancreatic elastase. All molecular dynamics simulations used a combination of Gromacs 5.1 package, CHARMM-GUI website and UCSF chip software to process and analyze protein structure. First, the CHARMM-GUI website is used to process protein files and add negative charge to render the system uncharged. The energy of the system is then optimized to eliminate unreasonable collisions between water molecules and proteins. The equilibrium kinetics and constant temperature and pressure kinetics were then adjusted and a 400ns molecular dynamics simulation was performed at a constant temperature of 300K. In this system we added 500mol/L zinc chloride or nickel chloride to verify the binding of ions to PPE proteins. Finally, UCSF chip was used to examine and analyze protein and ion binding. As shown in fig. 24 and 25, the molecular dynamics simulation results show that both zinc ions and nickel ions can bind to porcine pancreatic elastase, which is beneficial to improving the specificity of porcine pancreatic elastase binding to a substrate, preventing the binding to serine protease inhibitors, and thus improving the activity of elastase.
Claims (10)
1. An elastase nanocomposite with enhanced cleavage activity, characterized in that the nanocomposite has a complex of elastase and a metal cofactor as an inner core and an outer layer covered with a complex of calcium ions and anionsMineralized crust, wherein the metal cofactor is a metal ion selected from Mn 2+ 、Zn 2+ 、Ni 2+ One or more of the following.
2. The nanocomposite of claim 1, wherein the anion is selected from one or more of the following acid ions: phosphate ion, hydrogen phosphate ion, carbonate ion, oxalate ion, citrate ion.
3. The nanocomposite of claim 1, wherein the elastase is selected from one or more of the following serine proteases: neutrophil elastase, porcine pancreatic elastase, and homologs thereof.
4. The nanocomposite of claim 1, wherein the nanocomposite is a nanoparticle having a particle size of 100 to 1000 nm.
5. The method for preparing the cleavage activity-enhanced elastase nanocomposite according to any one of claims 1 to 4, comprising the steps of:
1) Preparing elastase solution;
2) Preparing a metal cofactor solution;
3) Preparing a calcium ion solution;
4) Preparing an anion salt solution;
5) Mixing elastase solution and metal cofactor solution in proportion, standing for 5-10 min, adding calcium ion solution and anion salt solution, mixing, sealing, standing at 37 deg.C for 10 min-24 hr;
6) The precipitate remained after high speed centrifugation and then was resuspended using solution to obtain the nanocomposite.
6. The method according to claim 5, wherein the concentration of the elastase solution prepared in step 1) is 1mg/mL to 10mg/mL; the concentration of the metal cofactor solution prepared in the step 2) is 1 mmol/L-1 mol/L; the concentration of the calcium ion solution prepared in the step 3) is 1 mmol/L-1 mol/L; the concentration of the anionic salt solution prepared in the step 4) is 1 mmol/L-1 mol/L.
7. The method of claim 5, wherein step 2) employs manganese chloride, zinc chloride and/or nickel chloride to formulate a metal cofactor solution; and 3) preparing the calcium ion solution by adopting calcium chloride.
8. The method according to claim 5, wherein step 4) uses DMEM serum-free medium as the anionic salt solution.
9. Use of the elastase nanocomposite with enhanced cleavage activity according to any one of claims 1 to 4 for the preparation of an antitumor drug.
10. The use of claim 9, wherein the neoplasm comprises lung cancer, gastric cancer, liver cancer, pancreatic cancer, uterine cancer, melanoma, skin cancer, ovarian cancer, head and neck cancer, breast cancer, cholangiocarcinoma, renal cancer, colorectal cancer, and cervical cancer.
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