Cordyceps sinensis mycelium polysaccharide-copper selenide with dual effects of inducing tumor cell apoptosis and copper death as well as preparation and application of cordyceps sinensis mycelium polysaccharide-copper selenide

Abstract

The invention belongs to the technical field of nano materials and anti-tumor treatment medicines, and discloses cordyceps sinensis mycelium polysaccharide-copper selenide with dual effects of inducing tumor cell apoptosis and copper death as well as a preparation method and application thereof. The preparation method of the cordyceps sinensis mycelium polysaccharide-copper selenide comprises the following steps: taking cordyceps sinensis mycelium polysaccharide as a template, adopting a soft template method, reducing Na2SeO3 through Vc, and adding CuSO4 to prepare the cordyceps sinensis mycelium polysaccharide-copper selenide. The cordyceps sinensis mycelium polysaccharide-copper selenide can selectively kill tumor cells and induce apoptosis and copper death of the tumor cells at the same time, and has huge application potential in the aspect of tumor prevention and treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nanomaterials and antitumor drugs, and particularly relates to a cordyceps sinensis mycelium polysaccharide-copper selenide with the dual effects of inducing tumor cell apoptosis and copper death, and a preparation method and application thereof. BACKGROUND

[0002] With the development of nanobiotechnology, the application of nanomaterials in tumor prevention and treatment has gradually become a research hotspot.

[0003] Copper selenide is an important cation-deficient transition metal selenide. Copper selenide nanomaterials have many potential applications in optoelectronic devices, medical treatment, gas sensors and catalysts, which have attracted widespread attention of researchers. Selenium is one of the essential trace elements for human body, can induce tumor cells to undergo apoptosis, and can selectively kill tumor cells. Supplementation of selenium can effectively reduce the incidence of cancer, and is known as the "king of anticancer". Copper accumulates in tumor cells when it enters the tumor microenvironment, causing oxidative stress and inducing copper death of tumor cells. Copper selenide nanomaterials have the dual functions of selenium and copper, and have great application potential in the field of antitumor. SUMMARY

[0004] In order to overcome the shortcomings and deficiencies in the prior art, the primary purpose of the present application is to provide a preparation method of cordyceps sinensis mycelium polysaccharide-copper selenide with the dual effects of inducing tumor cell apoptosis and copper death. In the method, cordyceps sinensis mycelium polysaccharide is used as a template, a nanoseelenium solution is prepared by using Vc and Na2SeO3 redox, CuSO4 is added to the nanoseelenium solution to react with the nanoseelenium solution to generate copper selenide, and the synthesis of copper selenide is assisted by the stability of cordyceps sinensis mycelium polysaccharide.

[0005] Another purpose of the present application is to provide cordyceps sinensis mycelium polysaccharide-copper selenide with the dual effects of inducing tumor cell apoptosis and copper death prepared by the above preparation method. The cordyceps sinensis mycelium polysaccharide-copper selenide can cause accumulation of selenium in cells, improve the expression of pro-apoptotic protein Bax, and further cut DNA repair enzyme PARP. The copper selenide can cause accumulation of copper in cells, consume GSH, exacerbate lipid peroxidation, cause loss of iron-sulfur cluster proteins, reduce ATP level, and induce tumor cells to undergo apoptosis and copper death, thereby providing a new strategy for tumor treatment.

[0006] Still another purpose of the present application is to provide the application of the above cordyceps sinensis mycelium polysaccharide-copper selenide with the dual effects of inducing tumor cell apoptosis and copper death.

[0007] The purposes of the present application are achieved by the following technical solutions. A method for preparing Cordyceps sinensis mycelial polysaccharide-copper selenide with dual effects of inducing tumor cell apoptosis and copper death includes the following steps: (1) Preparation of nano-selenium solution; (2) Preparation of Cordyceps sinensis mycelial polysaccharide-copper selenide.

[0008] The preparation of the nano-selenium solution in step (1) is specifically carried out according to the following steps: using Cordyceps sinensis mycelial polysaccharide as a template, Vc solution and Na2SeO3 solution are added to the Cordyceps sinensis mycelial polysaccharide solution, and the solution is allowed to stand in a water bath at 20~100℃ for 15 minutes to obtain nano-selenium solution; The preparation of copper selenide in step (2) is specifically carried out according to the following steps: CuSO4 was added to the nano-selenium solution obtained in step (1). The solution was subjected to static reaction in a water bath at 20~100℃ for 15 min, and then dialyzed for 24 h through a dialysis bag with a molecular weight cutoff of 8000 Da to obtain Cordyceps sinensis mycelial polysaccharide-copper selenide. The concentration of Na2SeO3 in the Na2SeO3 solution in the mixed solution is 2 mmol / L; the concentration of Cordyceps sinensis mycelial polysaccharide in the Cordyceps sinensis mycelial polysaccharide solution in the mixed solution is 400~2000 μg / mL; the concentration of Vc in the Vc solution in the mixed solution is 4~32 mmol / L; and the concentration of CuSO4 in the CuSO4 solution in the mixed solution is 0.5~4 mmol / L.

[0009] The Cordyceps sinensis mycelium polysaccharide is a homogeneous polysaccharide PHP-1 prepared from the aqueous extract of Cordyceps sinensis mycelium through dialysis, alcohol precipitation, protein removal, anion exchange column chromatography, and gel column chromatography. PHP-1 has a weight-average molecular weight of 24.5 kDa and is characterized by →4)-α-Glc p -(1→ residues form the main chain structure.)

[0010] The concentration of Cordyceps sinensis mycelial polysaccharide in the mixed solution is preferably 1600 μg / mL; The concentration of Vc in the Vc solution in the mixed solution is preferably 16 mmol / L; The concentration of CuSO4 in the CuSO4 solution in the mixed solution is preferably 2 mmol / L; The preferred temperature for the reaction is 40°C.

[0011] The average particle size of the Cordyceps sinensis mycelial polysaccharide-copper selenide is 116.9 nm, the Zeta potential is -34.4 mV, and its morphology is spherical or elliptical.

[0012] The cordyceps mycelium polysaccharide-copper selenide is composed of the following elements in percentage by mass: Se 34.8%, Cu 21.6%, O 1.8%, and C 41.8%.

[0013] The cordyceps mycelium polysaccharide-copper selenide is composed of copper selenide crystals and cordyceps mycelium polysaccharides that interact with the copper selenide through hydrogen bonds between -OH groups.

[0014] A cordyceps mycelium polysaccharide-copper selenide with dual effects of inducing tumor cell apoptosis and copper death, prepared by the above preparation method.

[0015] The above cordyceps mycelium polysaccharide-copper selenide with dual effects of inducing tumor cell apoptosis and copper death in the preparation of a tumor cell apoptosis and copper death inducer.

[0016] The tumor cells are human colon cancer cells HT-29, human cervical cancer cells Hela, or human lung cancer cells A549, preferably human lung cancer cells A549.

[0017] The toxicity of the cordyceps mycelium polysaccharide-copper selenide prepared by the above preparation method on different tumor cells and normal cells is detected by the CCK-8 method to evaluate the anti-tumor activity and selectivity of the cordyceps mycelium polysaccharide-copper selenide. The normal cell models include human embryonic kidney cells 293 T and mouse macrophage cells RAW264.7, and the toxicity of the cordyceps mycelium polysaccharide-copper selenide on normal cells is significantly lower than that on tumor cells.

[0018] The tumor cell models include human colon cancer cells HT-29, human cervical cancer cells Hela, and human lung cancer cells A549, preferably A549 cells.

[0019] The present application has the following advantages and beneficial effects relative to the prior art: (1) The present application explores the influence of different template addition amount, reaction temperature, molar ratio of Vc and Na2SeO3, molar ratio of Na2SeO3 and CuSO4 on the particle size of cordyceps sinensis mycelium polysaccharide-copper selenide; on the basis of determining the optimal single factor, the prepared cordyceps sinensis mycelium polysaccharide-copper selenide is characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FT-IR) and other technical means. The present application uses cordyceps sinensis mycelium polysaccharide as a template, and Vc, Na2SeO3 and CuSO4 as raw materials to synthesize cordyceps sinensis mycelium polysaccharide-copper selenide by soft template method, which has the advantages of simple and fast preparation process, green, small particle size of copper selenide, uniform distribution, good stability, and the cordyceps sinensis mycelium polysaccharide itself has immunomodulatory activity, so that the prepared copper selenide composite material has the potential of synergistic effect.

[0020] (2) The cordyceps sinensis mycelium polysaccharide-copper selenide prepared by the present application can selectively kill tumor cells. Cordyceps sinensis mycelium polysaccharide-copper selenide can cause intracellular selenium accumulation, improve Bax expression and further cut DNA repair enzyme PARP; cordyceps sinensis mycelium polysaccharide-copper selenide can lead to intracellular copper accumulation, consume GSH, exacerbate lipid peroxidation, and cause iron-sulfur cluster protein loss, reduce ATP levels, and further induce tumor cell apoptosis and copper death, which can provide a new strategy for tumor prevention and treatment.

[0021] (3) The present inventors have previously conducted research on the induction of pyroptosis in tumor cells using a copper selenide nanoscale enzyme prepared using black tea extract as a template, and have filed a related patent (Chinese patent application No. 202510461554X). However, the present invention is a further study on the induction of apoptosis in tumor cells. Pyroptosis is mainly mediated by inflammasomes (such as NLRP3) and the Gasdermin protein family. Inflammasomes activate caspase-1, which cleaves the Gasdermin-D protein, releasing its N-terminal portion, which inserts into the cell membrane to form pores, causing cell swelling and leading to cell membrane rupture. This process is accompanied by the release of pro-inflammatory cytokines such as IL-1β and IL-18, which induce a strong inflammatory response. Apoptosis, on the other hand, is a process mediated by caspase family enzymes and mainly includes the extrinsic and intrinsic pathways. The intrinsic pathway is regulated by mitochondria and the Bcl-2 protein family. Anti-apoptotic proteins (such as Bcl-2) in the Bcl-2 family interact with pro-apoptotic proteins (such as Bax) to regulate changes in mitochondrial membrane potential, leading to the activation of caspase-9 and caspase-3, which cleave DNA repair enzyme PARP, ultimately leading to programmed cell death. Morphologically, the cell membrane remains intact, the cell shrinks, and there is no inflammatory response, but apoptotic bodies are gradually formed. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a graph showing the effect of different PHP-1 addition amounts on the particle size of Ophiocordyceps sinensis mycelium polysaccharide-copper selenide (PHP-1-Cu 2-x Se).

[0023] Figure 2 is a graph showing the effect of different reaction temperatures on the particle size of Ophiocordyceps sinensis mycelium polysaccharide-copper selenide (PHP-1-Cu 2-x Se).

[0024] Figure 3 is a graph showing the effect of different Vc and Na2SeO3 molar ratios on the particle size of Ophiocordyceps sinensis mycelium polysaccharide-copper selenide (PHP-1-Cu 2-x Se).

[0025] Figure 4 is a graph showing the effect of different Na2SeO3 and CuSO4 molar ratios on the particle size of Ophiocordyceps sinensis mycelium polysaccharide-copper selenide (PHP-1-Cu 2-x Se).

[0026] Figure 5 is a graph showing the particle size and Zeta potential distribution of PHP-1-Cu 2-x Se (A: particle size; B: Zeta potential).

[0027] Figure 6 PHP-1-Cu 2-x TEM images of Se (A: 100 nm; B: 20 nm).

[0028] Figure 7 PHP-1-Cu 2-x EDX analysis images of Se (A: original state; B: C element; C: O oxygen element; D: Se element; E: Cu element; F: O, Se, Cu element fusion).

[0029] Figure 8 PHP-1-Cu 2-x XRD spectrum of Se.

[0030] Figure 9 PHP-1 and PHP-1-Cu 2-x FT-IR spectrum of Se.

[0031] Figure 10 PHP-1 vs PHP-1-Cu 2-x Effect of Se stabilization (left: template group; right: blank control group).

[0032] Figure 11 PHP-1 vs PHP-1-Cu 2-x Scanning electron microscope images of the effect of Se stabilization (A: template group; B: blank control group).

[0033] Figure 12 PHP-1-Cu 2-x Inhibitory effect of Se on the proliferation of different tumor cells.

[0034] Figure 13 PHP-1-Cu 2-x Inhibitory effect of Se on the proliferation of normal cells.

[0035] Figure 14 Inhibitory effect of PHP-1 on the proliferation of A549 cells.

[0036] Figure 15 PHP-1-Cu 2-x Effect of Se treatment on the Cu and Se content in A549 cells (A: Cu; B: Se).

[0037] Figure 16 PHP-1-Cu 2-x Effect of Se treatment on the GSH content in A549 cells.

[0038] Figure 17 PHP-1-Cu 2-x Effect of Se treatment on the MDA content in A549 cells.

[0039] Figure 18 PHP-1-Cu is PHP-1-Cu 2-x Effect of Se treatment on ATP content of A549 cells.

[0040] Figure 19 PHP-1-Cu is PHP-1-Cu 2-x Effect of Se treatment on apoptosis and copper death-related protein expression of A549 cells (A: apoptosis protein; B: copper death protein). DETAILED DESCRIPTION

[0041] The application will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the application are not limited thereto.

[0042] The cordyceps mycelium polysaccharide used in the following examples is prepared according to the following method: The cordyceps mycelium water extract is dialyzed in an 8 kDa dialysis bag for 72 h, and the solution in the dialysis bag is collected. Anhydrous ethanol is added to the dialysate to make the final concentration of ethanol 80 vol%, and the solution is allowed to stand at 4°C for 16 h for alcohol precipitation. The precipitate is collected by centrifugation at 6000 r / min for 10 min, and the precipitate is dissolved in ultrapure water to obtain a crude polysaccharide solution; Sevage reagent is added to the crude polysaccharide solution in an equal volume, and the Sevage reagent is a chloroform-n-butanol mixture with a volume ratio of 4:1. The solution is stirred with a magnetic stirrer for 40 min, and centrifuged at 6000 r / min for 10 min at room temperature to remove the protein. The supernatant is collected, and the protein removal is repeated until there is no white flocculent material at the interface between the two phases. The organic solvent is removed by rotary evaporation, and then the protein-removed polysaccharide solution is freeze-dried to obtain a crude polysaccharide freeze-dried powder; The crude polysaccharide freeze-dried powder is prepared into a solution with a concentration of 35 mg / mL, and separated by a DEAE-Sepharose fast flow ion exchange column (2.6 cm x 30 cm). The column is eluted with ultrapure water at a flow rate of 2.5 mL / min and an elution time of 4 min / tube. The first 40 tubes of elution component solution are received by a full-automatic partial collector. The polysaccharide content of each tube is detected by the phenol-sulfuric acid method, and an elution curve is drawn. The main component peaks are collected according to the elution curve, concentrated, dialyzed in an 8 kDa dialysis bag for 24 h, and freeze-dried to obtain the PHP-1 component; PHP-1 component was assigned to prepare a solution with a concentration of 10 mg / mL, purified by Sephacryl S-200 HR gel column (2.6 cm x 60 cm), eluted with phosphate buffer containing 0.15 M NaCl at pH 7.4, the elution flow rate was 1 mL / min, the elution time was 8 min / tube, the first 60 tubes of elution solution were collected using the automatic partial collector, the polysaccharide content was determined by the phenol-sulfuric acid method and the elution curve was drawn, and the main component peak was collected according to the elution curve, then concentrated, dialyzed in an 8 kDa dialysis bag for 72 h, and freeze-dried to obtain the cordyceps mycelium polysaccharide, named PHP-1.

[0043] The weight average molecular weight of cordyceps mycelium polysaccharide PHP-1 was determined by high-performance gel permeation chromatography. An appropriate amount of polysaccharide was dissolved in the mobile phase to prepare a solution with a concentration of 10 mg / mL, and then filtered through a 0.22 μm microporous filter for testing. A Shimadzu SIL-20A chromatograph was used, equipped with a differential detector (RID-20A), and the gel chromatography column was a TSK-GEL G5000 PWXL (7.8 mm x 300 mm) and a TSK-GEL 3000 PWXL (7.8 mm x 300 mm) in series. The weight average molecular weight of PHP-1 was determined to be 24.5 kDa by calculating the dextran standard curve.

[0044] The glycosidic bond type and proportion of cordyceps mycelium polysaccharide PHP-1 were determined by methylation kit combined with GC-MS method. An appropriate amount of polysaccharide was weighed and operated according to the methylation kit instructions, and the acetylated product obtained was determined and analyzed by Agilent GCMS 6890-5973 gas chromatography-mass spectrometry. The chromatography column was RXI-5 SIL MS (30 m x 0.25 mm x 0.25 μm). By database cross comparison, it was found that the polysaccharide PHP-1 contained Glc p -(1→,→4)-Glc p -(1→,→3,4)-Glc p -(1→ and →4,6)-Glc p -(1→ four glycosidic bonds, the molar percentage was 8.3%, 84.3%, 1.8% and 5.6% respectively. The configuration of cordyceps mycelium polysaccharide PHP-1 was determined by nuclear magnetic resonance technology, and the four glycosidic bonds were all in α configuration, i.e. α-Glc p -(1→,→4)-α-Glc p -(1→,→3,4)-α-Glc p -(1→ and →4,6)-α-Glc p -(1→,→4)-α-Glc p- (1→ Residues are predominantly.

[0045] Example 1: Effect of different PHP-1 addition amount on the particle size of PHP-1-Cu 2-x Se (1) Dissolve PHP-1 in ultrapure water to obtain a PHP-1 solution with a concentration of 5 mg / mL; take 2 mL, 4 mL, 6 mL, 8 mL, and 10 mL of the above solution (so that the final concentration of PHP-1 in the mixed solution in subsequent step (2) is 400 μg / mL, 800 μg / mL, 1200 μg / mL, 1600 μg / mL, and 2000 μg / mL, respectively), add ultrapure water to make up to 23.2 mL, and magnetically stir until uniform; add 0.8 mL of a Vc solution with a concentration of 500 mM, and magnetically stir for 5 min; after standing for 5 min, add 0.5 mL of a Na2SeO3 solution with a concentration of 100 mmol / L, and magnetically stir for 5 min; place in a 40°C water bath and stand for 15 min to obtain a nano selenium solution; (2) Then add 0.5 mL of a CuSO4 solution with a concentration of 100 mmol / L, and magnetically stir for 5 min; place the obtained mixed solution in a 40°C water bath and stand for 15 min; dialyze in a dialysis bag with a molecular weight cutoff of 8000 Da in ultrapure water for 24 h, and change the ultrapure water every 3 h; after dialysis, a series of PHP-1-Cu 2-x Se solutions are obtained.

[0046] The average particle size of the PHP-1-Cu 2-x Se solution is determined by DLS technology; by comparing the particle size of PHP-1-Cu Figure 1 Se at different PHP-1 addition amounts, the optimal PHP-1 addition amount is determined, as shown in Table 1. When the PHP-1 addition amount is 1600 μg / mL, the average particle size of PHP-1-Cu Se is the smallest, so the PHP-1 addition amount for preparing PHP-1-Cu 2-x Se is determined to be 1600 μg / mL. The other steps are the same as in Example 1, the fixed PHP-1 addition amount is 1600 μg / mL, the water bath reaction temperature is changed, the water bath standing reaction temperature is set to 20℃, 40℃, 60℃, 80℃, 100℃ (the water bath standing reaction temperature of steps (1) and (2) is the same), the average particle size of the Cordyceps sinensis mycelium polysaccharide-copper selenite is determined by DLS technology, and the optimal reaction temperature is determined by comparing the particle size of the Cordyceps sinensis mycelium polysaccharide-copper selenite at different reaction temperatures, as shown in Figure 2 When the reaction temperature is 40℃, the average particle size of the Cordyceps sinensis mycelium polysaccharide-copper selenite is the smallest, so the reaction temperature for preparing the Cordyceps sinensis mycelium polysaccharide-copper selenite is determined to be 40℃.

[0047] Example 3: Effect of different Vc and Na2SeO3 molar ratios on the particle size of Cordyceps sinensis mycelium polysaccharide-copper selenite (PHP-1-Cu 2-x Se) The other steps are the same as in Example 1, the fixed PHP-1 addition amount is 1600 μg / mL, the molar ratio of Vc:Na2SeO3 is set to 4:2, 8:2, 16:2, 24:2, 32:2; because the molar concentration of Na2SeO3 in the mixed solution is 2 mmol / L, the Vc concentration is changed to be 4 mmol / L, 8 mmol / L, 16 mmol / L, 24 mmol / L, 32 mmol / L in the mixed solution, the average particle size of the Cordyceps sinensis mycelium polysaccharide-copper selenite is determined by DLS technology, and the optimal Vc and Na2SeO3 molar ratio is determined by comparing the particle size of the Cordyceps sinensis mycelium polysaccharide-copper selenite at different Vc and Na2SeO3 molar ratios, as shown in Figure 3 When the molar ratio of Vc to Na2SeO3 is 16:2, the average particle size of the Cordyceps sinensis mycelium polysaccharide-copper selenite is the smallest, so the molar ratio of Vc to Na2SeO3 for preparing the Cordyceps sinensis mycelium polysaccharide-copper selenite is determined to be 16:2. Example 4: Effect of different Na2SeO3 and CuSO4 molar ratios on the particle size of Cordyceps sinensis mycelium polysaccharide-copper selenite (PHP-1-Cu 2- x Se) The other steps are the same as in Example 1, the fixed PHP-1 addition amount is 1600 μg / mL, and the molar ratio of Na2SeO3:CuSO4 is set to be 2:0.5, 2:1, 2:2, 2:3, and 2:4; because the molar concentration of Na2SeO3 in the mixed solution is 2 mmol / L, the concentration of CuSO4 is 0.5 mmol / L, 1 mmol / L, 2 mmol / L, 3 mmol / L, and 4 mmol / L. The average particle size of the cordyceps mycelium polysaccharide-copper selenide is determined by using the DLS technology, and the optimal molar ratio of Na2SeO3 to CuSO4 is determined by comparing the particle size of the cordyceps mycelium polysaccharide-copper selenide at different molar ratios of Na2SeO3 to CuSO4, as shown in Figure 4 When the molar ratio of Na2SeO3 to CuSO4 is 2:2, the average particle size of the cordyceps mycelium polysaccharide-copper selenide is the smallest, and therefore the molar ratio of Na2SeO3 to CuSO4 for preparing the cordyceps mycelium polysaccharide-copper selenide is determined to be 2:2.

[0048] Example 5: Structure characterization of cordyceps mycelium polysaccharide-copper selenide (PHP-1-Cu 2-x Se) (1) Particle size and Zeta potential analysis of PHP-1-Cu 2-x Se On the basis of the single-factor experiment, the optimal conditions are selected to prepare PHP-1-Cu 2-x Se: PHP-1 is used as a template, Na2SeO3 solution and Vc solution are added to the PHP-1 solution to undergo a reduction reaction, and then CuSO4 solution is added to obtain a mixed solution (in the mixed solution, the concentration of PHP-1 is 1600 μg / mL, the concentration of Na2SeO3 is 2 mmol / L, the concentration of Vc is 16 mmol / L, and the concentration of CuSO4 is 2 mmol / L), and PHP-1-Cu 2-x Se solution is prepared by reaction; the reaction time is 15 min, and the reaction temperature is 40℃.

[0049] The PHP-1-Cu 2-x Se solution obtained in the above embodiment is determined for particle size and Zeta potential by using DLS, as shown in Figure 5 According to A in Figure 5 , the particle size distribution of PHP-1-Cu 2-x Se is between 59 nm and 295 nm, and the average particle size is 116.9 nm. According to B in Figure 5 , the Zeta potential of PHP-1-Cu 2-xThe Zeta average potential of Se is -34.4 mV, indicating that the PHP-1 prepared cordyceps mycelium polysaccharide-copper selenide prepared by the application has strong stability.

[0050] (2) PHP-1-Cu 2-x Se morphology observation To the PHP-1-Cu obtained in the above embodiment 2-x Se was observed under a transmission electron microscope, and Figure 6 It can be seen that the PHP-1-Cu observed under different magnifications 2-x Se is spherical or elliptical.

[0051] (3) PHP-1-Cu 2-x Se surface element composition analysis To determine the PHP-1-Cu obtained in the above embodiment 2-x Se element composition, TEM-EDX was used for element composition analysis. From Figure 7 It can be seen that the PHP-1-Cu 2-x Se surface contains Se, Cu, O, C elements, and the mass percentage of elements Se, Cu, O, C is 34.8%, 21.6%, 1.8%, 41.8% respectively; from Figure 7 F observation can be known, PHP-1 is successfully combined on the surface of copper selenide particles, and PHP-1 is likely to play a stabilizing role on copper selenide.

[0052] (4) PHP-1-Cu 2-x Se crystal form analysis From Figure 8 It can be seen that the PHP-1-Cu obtained in the above embodiment 2-x Se has diffraction peaks at 2θ angles of 27°, 31°, 45°, 53°, 65°, 72°, 83°, 89°, which correspond to the diffraction peaks of the standard card PDF # 06-0680 of copper selenide, and conforms to the standard spectrum of copper selenide crystal.

[0053] (5) PHP-1 and copper selenide interaction analysis According to Figure 9 It can be seen that the PHP-1-Cu obtained in the above embodiment 2-x Se has similar characteristic peaks with PHP-1, indicating that the PHP-1-Cu 2-x Se has PHP-1. Through FT-IR analysis, it can be seen that the stretching vibration of -OH of PHP-1 is shifted from 3392 cm -1 to 3415 cm -1 , and other absorption peaks do not change significantly, indicating that there is a hydrogen bond interaction between the -OH group of PHP-1 and copper selenide, and indicating that PHP-1 can stabilize copper selenide.

[0054] Example 6: Effect of PHP-1 on the stability of copper selenide The other steps are the same as in Example 5, and a blank control group and a template group are set up. The template group is added with PHP-1 at a concentration of 1600 μg / mL, and the blank control group is replaced with ultrapure water instead of PHP-1, so as to explore the effect of PHP-1 on the stability of copper selenide. From Figure 10 It can be seen that when PHP-1 is not added, the product appears obvious aggregation, while when 1600 μg / mL PHP-1 is added, the product is a uniform colloidal solution. Further observation by scanning electron microscopy shows that Figure 11 It can be seen that the product prepared without the addition of PHP-1 has a large particle size and irregular shape, while the product prepared by adding 1600 μg / mL PHP-1 has a small particle size and is spherical or elliptical, which is consistent with the result of transmission electron microscopy observation in Example 5, indicating that the addition of PHP-1 is indispensable for the preparation of stable copper selenide.

[0055] Example 7: PHP-1-Cu 2-x Effect of Se on the proliferation of different tumor cells Human colon cancer cells HT-29, human cervical cancer cells Hela and human lung cancer cells A549 were inoculated in 96-well plates at a concentration of 5×10 4 cells / well, and cultured for 24 h, then co-incubated with 0, 50, 100, 200, 400, 600 μg / mL PHP-1-Cu 2-x Se prepared in Example 5 for 48 h. The CCK-8 method was used to detect the inhibitory effect of PHP-1-Cu 2-x Se on the proliferation of different tumor cells, and the absorbance at 450 nm was read by a microplate reader, and the results are shown in Figure 12 .

[0056] As shown in Figure 12 , with the increase of the concentration of PHP-1-Cu 2-x Se, the survival rates of the three different tumor cells gradually decreased, indicating that PHP-1-Cu 2-x Se had a proliferation inhibitory effect on the three different tumor cells, and the inhibitory effect on A549 cells was the strongest. Example 8: Effect of PHP-1-Cu 2-x Se on the proliferation of normal cells Human embryonic kidney cells 293 T and mouse macrophage cells RAW264.7 were inoculated in 96-well plates at a concentration of 5×10 4 cells / well, and cultured for 24 h, then co-incubated with 0, 50, 100, 200 μg / mL PHP-1-Cu 2-xSe co-incubated for 48 h. The CCK-8 method was used to detect the inhibition of PHP-1-Cu 2-x Se on normal cell proliferation, and the absorbance at 450 nm was read by a microplate reader. The results are shown in Figure 13 .

[0057] As shown in Figure 13 , PHP-1-Cu 2-x Se had no significant inhibitory effect on the proliferation of A549 cells in the concentration range of 50-600 μg / mL, indicating that the PHP-1-Cu 2-x Se prepared in the application had anti-tumor activity. Example 9: Effect of PHP-1 on the proliferation of A549 cells A549 cells were inoculated in a 96-well plate at a concentration of 5×10 4 cells / well, and after 24 h of culture, they were co-incubated with 0, 50, 100, 200, 400, 600 μg / mL PHP-1 for 48 h. The CCK-8 method was used to detect the inhibition of PHP-1 on the proliferation of A549 cells, and the absorbance at 450 nm was read by a microplate reader. The results are shown in Figure 14 .

[0058] As shown in Figure 14 , PHP-1 had no significant inhibitory effect on the proliferation of A549 cells in the concentration range of 50-600 μg / mL, indicating that the PHP-1-Cu 2-x Se prepared in the application had anti-tumor activity. Example 10: Effect of PHP-1-Cu 2-x Se treatment on the content of Cu and Se in A549 cells A549 cells were inoculated in a 70 mm culture dish at a concentration of 1.0×10 5 / mL, and after 24 h of culture, they were co-incubated with 0, 50, 100, 200 μg / mL PHP-1-Cu 2-x Se prepared in Example 5 for 6 h. The cells were collected, and the content of Cu and Se in A549 cells was detected by inductively coupled plasma mass spectrometry. The results are shown in Figure 15 .

[0059] As can be seen from Figure 15 , with the increase of the concentration of PHP-1-Cu 2-x Se, the content of Cu and Se in A549 cells increased significantly. It is indicated that PHP-1-Cu 2-x Se can cause excessive accumulation of Cu and Se in A549 cells. Example 11: PHP-1-Cu 2-x Effect of Se treatment on GSH content in A549 cells A549 cells were seeded in 70 mm culture dishes at a concentration of 1.0 x 10 5 / mL and incubated for 24 h, then co-incubated with 0, 50, 100, 200 μg / mL PHP-1-Cu 2-x Se prepared in Example 5 for 48 h. The cells were collected and the GSH content in A549 cells was detected according to the GSH kit method. The results are shown in Figure 16 .

[0060] As shown in Figure 16 , with the increase of the concentration of PHP-1-Cu 2-x Se, the GSH content in A549 cells decreased significantly. After PHP-1-Cu 2-x Se entered the cells, the Cu 2+ contained in PHP-1-Cu 2-x Se could react with GSH to consume GSH. It is indicated that PHP-1-Cu 2-x Se can consume GSH in A549 cells, destroy the redox balance in cells, and further cause cell damage. Example 12: Effect of PHP-1-Cu 2-x Se treatment on MDA content in A549 cells Cell culture and PHP-1-Cu 2-x Se treatment were the same as in Example 11. After the cells were collected, the MDA content in A549 cells was detected according to the MDA kit method. The results are shown in Figure 17 .

[0061] As shown in Figure 17 , with the increase of the concentration of PHP-1-Cu 2-x Se, the MDA content in A549 cells increased significantly. It is indicated that PHP-1-Cu 2-x Se entered A549 cells and caused lipid peroxidation. Example 13: Effect of PHP-1-Cu 2-x Se treatment on ATP content in A549 cells Cell culture and PHP-1-Cu 2-x Se treatment were the same as in Example 11. After the cells were collected, the ATP content in A549 cells was detected according to the ATP kit method. The results are shown in Figure 18 .

[0062] As shown in Figure 18 , with the increase of the concentration of PHP-1-Cu 2-x Se, the ATP content in A549 cells decreased significantly. It is indicated that PHP-1-Cu2-x Se enters A549 cells, causing oxidative stress, causing mitochondrial damage, and hindering ATP synthesis. Example 14: PHP-1-Cu 2-x Effects of Se treatment on apoptosis of A549 cells and expression of copper death-related proteins Cell culture and PHP-1-Cu 2-x Se treatment was the same as in Example 11. After the cells were collected, total proteins of each treatment group were extracted, electrophoresed on 10% Bis-Tris precast gels at 4°C, and the protein was electrophoresed onto a PVDF membrane in an ice bath. The PVDF membrane strip was cut according to the position of the Marker, blocked with 5% skim milk powder blocking solution for 1 h, and the strip was placed in PARP, Bax, LIAS, FDX1, and β-actin primary antibody diluent, respectively, and incubated overnight on a shaking table at 4°C. After the strip was washed thoroughly with TBST, it was placed in secondary antibody at room temperature for 1 h. The strip was washed again, the protein was developed using a hypersensitive ECL chemiluminescence reagent, and the gray value was analyzed using FluorChem5500 software. The results are shown in Figure 19

[0063] As can be seen from Figure 19 A, as the concentration of PHP-1-Cu 2-x Se increases, the expression of cleaved PARP and pro-apoptotic protein Bax increases, indicating that the PHP-1-Cu 2-x Se prepared by the present application indeed has the effect of inducing cell apoptosis.

[0064] As can be seen from Figure 19 B, as the concentration of PHP-1-Cu 2-x Se increases, the expression of iron-sulfur cluster proteins LIAS and FDX1 decreases, indicating that the PHP-1-Cu 2-x Se prepared by the present application indeed has the effect of inducing cell apoptosis. The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.​

Claims

1. A method for preparing a cordyceps sinensis mycelium polysaccharide-copper selenide with dual efficacy of inducing tumor cell apoptosis and copper death, comprising the following steps: (1) preparation of a nano-selenium solution; (2) preparation of copper selenide; The preparation of the nano-selenium solution in step (1) is specifically performed according to the following steps: adding a Vc solution and a Na2SeO3 solution to a cordyceps sinensis mycelium polysaccharide solution as a template, and then placing the mixture in a water bath at 20-100 ℃ for 15 min to obtain a nano-selenium solution; The preparation of the copper selenide in step (2) is specifically performed according to the following steps: adding a CuSO4 solution to the nano-selenium solution obtained in step (1), placing the obtained mixture in a water bath at 20-100 ℃ for 15 min, and then dialyzing the mixture through a dialysis bag with a molecular weight cut-off of 8000 Da for 24 h to obtain a cordyceps sinensis mycelium polysaccharide-copper selenide; The concentration of Na2SeO3 in the Na2SeO3 solution in the mixture is 2 mmol / L; the concentration of cordyceps sinensis mycelium polysaccharide in the cordyceps sinensis mycelium polysaccharide solution in the mixture is 400-2000 μg / mL; the concentration of Vc in the Vc solution in the mixture is 4-32 mmol / L; and the concentration of CuSO4 in the CuSO4 solution in the mixture is 0.5-4 mmol / L.

2. The preparation method of the Cordyceps sinensis mycelium polysaccharide-copper selenide with the dual effects of inducing tumor cell apoptosis and copper death according to claim 1, characterized in that: The Cordyceps sinensis mycelium polysaccharide is a uniform polysaccharide PHP-1 prepared from Cordyceps sinensis mycelium water extract as raw material through dialysis, alcohol precipitation, protein removal, anion exchange column chromatography and gel column chromatography separation and purification, the weight average molecular weight of PHP-1 is 24.5 kDa, and the →4)-α-Glc p The main chain structure is mainly composed of (1→ residues.

3. The preparation method of the Cordyceps sinensis mycelium polysaccharide-copper selenide with the dual effects of inducing tumor cell apoptosis and copper death according to claim 1, characterized in that: The concentration of cordyceps sinensis mycelium polysaccharide in the cordyceps sinensis mycelium polysaccharide solution in the mixture is 1600 μg / mL; the concentration of Vc in the Vc solution in the mixture is 16 mmol / L; the concentration of CuSO4 in the CuSO4 solution in the mixture is 2 mmol / L; and the temperature of the reaction is 40 ℃.

4. The preparation method of the Cordyceps sinensis mycelium polysaccharide-copper selenide with the dual effects of inducing tumor cell apoptosis and copper death according to claim 1, characterized in that: The average particle size of the cordyceps sinensis mycelium polysaccharide-copper selenide is 116.9 nm, the Zeta potential is -34.4 mV, and the morphology is spherical or elliptical.

5. The method for preparing the Cordyceps sinensis mycelium polysaccharide-copper selenide with the dual efficacy of inducing tumor cell apoptosis and copper death according to claim 1, characterized in that: The cordyceps sinensis mycelium polysaccharide-copper selenide is composed of the following elements in terms of mass percentage: Se 34.8%, Cu 21.6%, O 1.8%, and C 41.8%.

6. The method for preparing the Cordyceps sinensis mycelium polysaccharide-copper selenide with the dual efficacy of inducing tumor cell apoptosis and copper death according to claim 1, characterized in that: The cordyceps sinensis mycelium polysaccharide-copper selenide is composed of copper selenide crystals and cordyceps sinensis mycelium polysaccharide interacting with copper selenide through hydrogen bonds between -OH groups. 7.A cordyceps sinensis mycelium polysaccharide-copper selenide with dual efficacy of inducing tumor cell apoptosis and copper death, prepared by the method of any one of claims 1-6. 8.The use of the cordyceps sinensis mycelium polysaccharide-copper selenide with dual efficacy of inducing tumor cell apoptosis and copper death according to claim 7 in the preparation of a tumor cell apoptosis and copper death inducer.

9. Use according to claim 8, characterized in that: The tumor cells are human colon cancer cells HT-29, human cervical cancer cells Hela, or human lung cancer cells A549.

10. Use according to claim 8, characterized in that: The tumor cells are human lung cancer cells A549.

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