Application of auricularia auricula-judae polysaccharide in preparation of nano-drug carrier, compound containing auricularia auricula-judae polysaccharide and application of compound

Abstract

The invention relates to the technical field of drug carriers. The invention provides a preparation method of auricularia auricula-judae polysaccharide and application of the auricularia auricula-judae polysaccharide in preparation of nano-drug carriers, and further provides a compound containing the auricularia auricula-judae polysaccharide and application of the compound in preparation of drugs related to photothermal therapy. The auricularia auricula polysaccharide is used as a nano-drug carrier, so that the problems that a photosensitizer is unstable and is easy to agglomerate and coagulate in existing photo-thermal therapy can be effectively solved, the biological compatibility is better, and the drug loading capacity is higher; meanwhile, the auricularia auricula polysaccharide serving as an anti-tumor active component also has certain benefits to patients with various tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of drug carriers, and in particular to the application of black fungus polysaccharide in the preparation of nano drug carriers, and a complex containing the black fungus polysaccharide and the application thereof. Background Art

[0002] In recent years, photothermal therapy (PTT) has been shown to be an efficient, non-invasive therapy that can eliminate various types of tumors. Applying controllable intensity external laser irradiation to the local tumor can minimize damage to surrounding healthy tissues. PTT can kill cancer cells directly or indirectly by increasing the physical killing rate, stimulating drug release, promoting oxidative stress, or affecting other biological processes. Studies have shown that PTT combined with chemodynamic therapy can increase the tumor inhibition rate by more than 20% compared with chemodynamic therapy alone, and can eliminate drug-resistant cells through physical killing and inhibition of P-glycoprotein-mediated drug pump action, thereby increasing drug treatment sensitivity.

[0003] However, many photosensitizers are currently unstable and prone to aggregation and precipitation, which limits their application in anti-tumor applications. For example, ferrous selenide (FeSe2) has the characteristics of high photothermal conversion efficiency and small molecular size. 2 It can quickly heat up to 66.8℃ within 5 minutes under light intensity, and its photothermal conversion efficiency is at the forefront of photosensitizers, but ferrous selenide is unstable and prone to agglomeration and precipitation.

[0004] Black fungus polysaccharide (BFP) is a water-soluble β-D-glucan extracted from the natural edible fungus black fungus, and is one of the main active ingredients of black fungus that exerts anti-tumor effects. Previous studies have confirmed that BFP-assisted chemotherapy can improve the patient's immune ability without increasing side effects, and is beneficial to patients with various types of tumors. Atomic force microscopy confirmed that BFP exists in aqueous solution in a rigid chain conformation and can self-assemble to form a nanofiber tubular structure. Disulfonated tetraphenylethylene and a variety of substances can be packaged in hollow nanofiber tubes through hydrophobic interactions. BFP is a natural polymer material with high stability and hydrophilicity, good biocompatibility and biodegradability. These advantages make BFP have a very broad application prospect in biomaterials. Summary of the invention

[0005] The purpose of the present invention is to provide the use of black fungus polysaccharide in the preparation of nano drug carriers and a complex containing the black fungus polysaccharide and its use. The use of black fungus polysaccharide as a nano drug carrier can effectively solve the problem that the photosensitizer is unstable and prone to agglomeration and precipitation in existing photothermal therapy, and has better biological compatibility and higher drug loading. At the same time, black fungus polysaccharide as an anti-tumor active ingredient also has certain benefits for patients with various types of tumors.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing black fungus polysaccharide, comprising the following steps:

[0008] (1) refluxing and drying the pretreated black fungus to obtain defatted black fungus powder;

[0009] (2) mixing the defatted black fungus powder with an ethanol solution and a NaCl solution in sequence, and filtering to obtain a supernatant;

[0010] (3) decolorizing the supernatant with 25-40% hydrogen peroxide, deproteinizing with a macroporous resin, dialyzing once, and then freeze-drying to obtain crude black fungus polysaccharide;

[0011] (4) The crude black fungus polysaccharide is dissolved, precipitated with alcohol, dialyzed twice and freeze-dried to obtain the black fungus polysaccharide.

[0012] Preferably, the organic solvent used for reflux in step (1) comprises ethyl acetate and / or acetone.

[0013] Preferably, the volume concentration of the ethanol solution in step (2) is 60-80%, and the solid-liquid ratio of the defatted black fungus powder to the ethanol solution is 1 g:(18-25) mL.

[0014] Preferably, step (2) includes heating and stirring during the mixing process with the NaCl solution, the temperature of the heating and stirring treatment is 80-90° C., and the time of the heating and stirring treatment is 3-5 hours.

[0015] Preferably, the solid-liquid ratio of the defatted black fungus powder to the NaCl solution is 1 g:(18-25) mL.

[0016] The invention provides application of black fungus polysaccharide prepared by the preparation method in preparing nano drug carriers.

[0017] The present invention also provides a complex comprising the black fungus polysaccharide prepared by the preparation method.

[0018] The present invention also provides a method for preparing the above-mentioned composite, comprising the following steps:

[0019] The black fungus polysaccharide is prepared into a black fungus polysaccharide solution and then heated. Then, ferrous selenide powder is added, ultrasonicated, stirred and centrifuged to obtain the solution.

[0020] Preferably, the concentration of the black fungus polysaccharide solution is 0.8-1.3 mg / mL.

[0021] Preferably, the heating temperature is 130-150° C., and the heating time is 0.3-0.8 h.

[0022] Preferably, the centrifugal speed is 3000-4500 rpm, and the centrifugal time is 5-10 min.

[0023] The present invention also provides the use of the complex in preparing drugs related to photothermal therapy.

[0024] The present invention also provides a photosensitizer that can be used for anti-tumor treatment, comprising the above-mentioned complex.

[0025] By adopting the above technical solution, the present invention has the following beneficial effects:

[0026] 1. The technical solution of the present invention utilizes black fungus polysaccharide as a nano drug carrier, which can effectively solve the problem that the photosensitizer in the existing photothermal therapy is unstable and prone to agglomeration and precipitation; and has better biosafety and biocompatibility; at the same time, black fungus polysaccharide as an anti-tumor active ingredient also has certain benefits for patients with various types of tumors.

[0027] 2. The present invention uses black fungus polysaccharide as a nano drug carrier, and utilizes the nanofiber tubes formed by self-assembly in an aqueous solution to load ferrous selenide, thereby solving the problem that ferrous selenide is unstable and prone to agglomeration and flocculation. The embodiments also show that the BFP-compound is non-toxic and has good biocompatibility, and also show that the BFP-FeSe2 complex is easily taken up by tumor cells and can effectively induce immunogenic death of tumor cells.

[0028] 3. The technical solution of the present invention utilizes black fungus polysaccharide as a nano drug carrier, the preparation technology operation process is simple, the source of selectable materials is wide, and the drug loading capacity is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the Fourier infrared spectrum measurement result of BFP;

[0030] Figure 2 The morphology of BFP observed by scanning electron microscope;

[0031] Figure 3 The morphology of BFP observed by transmission electron microscope;

[0032] Figure 4This is the morphology of the BFP molecular chain observed by atomic force microscopy;

[0033] Figure 5 is the diffraction spectrum of FeSe2 powder;

[0034] Figure 6 This is the morphology of FeSe2 observed by transmission electron microscopy;

[0035] Figure 7 This is the morphology of BFP-FeSe2 observed by scanning electron microscope;

[0036] Figure 8 The element distribution of BFP-FeSe2 detected by the energy spectrometer attached to the field emission scanning electron microscope;

[0037] Fig. 9 The effect of different concentrations of BFP-FeSe2 on HUVEC cell activity;

[0038] Fig.10 The results of the effects of BFP-FeSe2 and saline on mouse blood biochemical indicators and tissue section staining ( Fig.10 A in the figure indicates the results of tissue section staining, and B indicates the results of blood biochemical index detection);

[0039] Fig.11 The blood compatibility results of different concentrations of BFP-FeSe2;

[0040] Fig.12 This is the laser confocal image of the distribution of BFP-FeSe2 in HCC-LM3 cells;

[0041] Fig.13 The effects of different treatment groups on the activity of HCC-LM3 cells;

[0042] Fig.14 The effects of different treatment groups on the expression levels of CRT and HMGB1 in H22 cells. DETAILED DESCRIPTION

[0043] The present invention provides a method for preparing black fungus polysaccharide, comprising the following steps:

[0044] (1) refluxing and drying the pretreated black fungus to obtain defatted black fungus powder;

[0045] (2) mixing the defatted black fungus powder with an ethanol solution and a NaCl solution in sequence, and filtering to obtain a supernatant;

[0046] (3) decolorizing the supernatant with 25-40% hydrogen peroxide, deproteinizing with a macroporous resin, dialyzing once, and then freeze-drying to obtain crude black fungus polysaccharide;

[0047] (4) The crude black fungus polysaccharide is dissolved, precipitated with alcohol, dialyzed twice and freeze-dried to obtain the black fungus polysaccharide.

[0048] In the present invention, the black fungus is dried and crushed, and then passed through a sieve to collect the sieve. The drying time of the present invention is preferably 10-15 hours, more preferably 11-14 hours, and more preferably 12 hours; the drying temperature is preferably 35-45°C, more preferably 38-42°C, and more preferably 40°C.

[0049] In the present invention, the above-mentioned sieve underflow is refluxed and dried to obtain defatted black fungus powder. The organic solvent used for the reflux in the present invention preferably includes ethyl acetate and / or acetone, and is further preferably ethyl acetate and acetone, that is, the present invention refluxes the sieve underflow using ethyl acetate and acetone in sequence. The reflux time of the present invention is preferably 7-9h, further preferably 7.5-8.5h, and further preferably 8h; the drying temperature is preferably 50-70°C, further preferably 55-65°C, and further preferably 60°C.

[0050] In the present invention, the obtained defatted black fungus powder is mixed with an ethanol solution, stirred evenly, filtered, and the filter residue is collected. The volume concentration of the ethanol solution of the present invention is preferably 60-80%, further preferably 65-75%, and further preferably 70%; the solid-liquid ratio of the defatted black fungus powder to the ethanol solution is preferably 1g:(18-25)mL, further preferably 1g:(19-22)mL, and further preferably 1g:20mL; the stirring time is preferably 20-26h, further preferably 22-25h, and further preferably 24h.

[0051] In the present invention, the collected filter residue is mixed with a NaCl solution, then heated and stirred, and then stirred at room temperature overnight, and the supernatant is collected after filtration. The mass concentration of the NaCl solution of the present invention is preferably 0.7-1.2%, further preferably 0.8-1.1%, and further preferably 0.9%; the solid-liquid ratio of the defatted black fungus powder to the NaCl solution is preferably 1g:(18-25)mL, further preferably 1g:(19-22)mL, and further preferably 1g:20mL. The temperature of the heating and stirring treatment of the present invention is preferably 80-90°C, further preferably 82-88°C, and further preferably 85°C; the time of the heating and stirring treatment is preferably 3-5h, further preferably 3.5-4.5h, and further preferably 4h.

[0052] In the present invention, the supernatant is decolorized by hydrogen peroxide, added dropwise and stirred until the color turns light yellow, the dropwise addition is stopped and the stirring is continued until the color fades, and the mass concentration of the hydrogen peroxide is preferably 25-40%, further preferably 28-35%, and further preferably 30%. Then, a macroporous resin is used for deproteinization, and the macroporous resin is preferably a 717 anion exchange resin. The deproteinized solution is dialyzed once with ultrapure water, and then freeze-dried to obtain black fungus crude polysaccharide. The freeze-drying temperature in the present invention is preferably -18 to -25°C, further preferably -19 to -22°C, and further preferably -20°C.

[0053] In the present invention, the black fungus crude polysaccharide is dissolved in water, the obtained black fungus crude polysaccharide aqueous solution is reprecipitated with ethanol, the precipitate is collected and redissolved in water, and then dialyzed twice with ultrapure water, and freeze-dried to obtain the black fungus polysaccharide. The freeze-drying temperature in the present invention is preferably -18 to -25°C, more preferably -19 to -22°C, and more preferably -20°C. The black fungus polysaccharide in the present invention is white cotton-like.

[0054] The invention provides application of black fungus polysaccharide prepared by the preparation method in preparing nano drug carriers.

[0055] The invention provides a complex comprising black fungus polysaccharide prepared by the preparation method.

[0056] The present invention also provides a method for preparing the composite, comprising the following steps:

[0057] The black fungus polysaccharide is prepared into a black fungus polysaccharide solution and then heated. Then, ferrous selenide powder is added, ultrasonicated, stirred and centrifuged to obtain the solution.

[0058] In the present invention, black fungus polysaccharide is configured into a black fungus polysaccharide solution, and the concentration of the black fungus polysaccharide solution is preferably 0.8-1.3 mg / mL, further preferably 0.9-1.1 mg / mL, and further preferably 1 mg / mL. The black fungus polysaccharide solution is heated, and the heating temperature is preferably 130-150°C, further preferably 135-145°C, and further preferably 140°C; the heating time is preferably 0.3-0.8h, further preferably 0.4-0.6h, and further preferably 0.5h. The heated black fungus polysaccharide solution is quenched to room temperature, and then an excess of ferrous selenide powder is added for ultrasonic treatment, and the ultrasonic time is preferably 8-12min, further preferably 9-11min, and further preferably 10min. The ultrasonic treatment of the present invention is to better disperse the ferrous selenide powder. In the present invention, it is preferred to ensure that the ferrous selenide powder is added in excess. When the excess ferrous selenide gathers and sinks to the bottom, it indicates that the ferrous selenide powder is added in excess.

[0059] In the present invention, after ultrasonic treatment, it is allowed to stand to room temperature and then stirred, and the stirring time is preferably 45-52h, further preferably 47-50h, and further preferably 48h. Then centrifugation is performed to remove suspended matter, and the supernatant is collected to obtain a complex containing black fungus polysaccharide. The centrifugal speed of the present invention is preferably 3000-4500rpm, further preferably 3500-4200rpm, and further preferably 4000rpm; the centrifugal time is preferably 5-10min, further preferably 6-9min, and further preferably 8min. In the present invention, the collected supernatant is freeze-dried to obtain a brown flocculent product, which is a complex powder (BFP-FeSe2 complex) containing black fungus polysaccharide.

[0060] In the present invention, the preparation method of the ferrous selenide powder comprises the following steps:

[0061] (1) oleylamine and 1-octadecene are mixed in a volume ratio of (2-4):(1-3), heated, and then FeCl2·4H2O powder is added to obtain a mixed solution A;

[0062] (2) Selenium powder and oleylamine are mixed at a ratio of (0.5-1.5) g: (20-30) mL and added to mixed solution A. After heating, the mixture is centrifuged, washed, and freeze-dried to obtain ferrous selenide powder.

[0063] In the present invention, oleylamine and 1-octadecene are mixed and then heated, and the volume ratio of the oleylamine and 1-octadecene is preferably (2-4):(1-3), more preferably (2.4-3.5):(1.2-2.5), and more preferably 3:2. In the present invention, the temperature is preferably heated to 100-130°C, more preferably 110-125°C, and more preferably 120°C; the heating temperature is preferably maintained for 25-35 minutes, more preferably 28-32 minutes, and more preferably 30 minutes.

[0064] In the present invention, after the heating treatment is completed, FeCl2·4H2O powder is added and stirred to obtain a mixed solution A. The volume mass ratio of the mixed solution of oleylamine+1-octadecene to FeCl2·4H2O powder of the present invention is preferably (80-120) mL:(0.5-1) g, further preferably (90-110) mL:(0.7-0.9) g, and further preferably 100 mL:0.8 g; the stirring time is preferably 25-35 min, further preferably 28-32 min, and further preferably 30 min.

[0065] In the present invention, selenium powder is mixed with oleylamine, and then added to the mixed solution A and stirred evenly. The ratio of the selenium powder to oleylamine is preferably (0.5-1.5) g: (20-30) mL, further preferably (0.8-1.3) g: (22-28) mL, and further preferably 1 g: 25 mL; the stirring time is preferably 7-15 min, further preferably 8-12 min, and further preferably 10 min.

[0066] In the present invention, the mixture is heated after being stirred evenly, preferably to 140-160°C, more preferably to 145-155°C, and even more preferably to 150°C; the temperature after heating is preferably maintained for 25-35min, more preferably for 28-32min, and even more preferably for 30min.

[0067] In the present invention, after the heating is completed, the mixture is cooled to room temperature, and then centrifuged and washed. The centrifugal speed of the present invention is preferably 4000-6000rpm, further preferably 4500-5500rpm, and further preferably 5000rpm; the centrifugal time is preferably 5-10min, further preferably 7-9min, and further preferably 8min; and acetone is preferably used for the washing. After the washing in the present invention is completed, secondary centrifugation and secondary washing are preferably performed, and the secondary centrifugal speed is preferably 4000-6000rpm, further preferably 4500-5500rpm, and further preferably 5000rpm; the secondary centrifugal time is preferably 5-10min, further preferably 7-9min, and further preferably 8min; and anhydrous ethanol solution is preferably used for the secondary washing. In the present invention, repeated washing can be performed as appropriate, and the repeated washing operation process is the same as the secondary washing operation process.

[0068] In the present invention, after washing, freeze-drying is performed to obtain ferrous selenide powder.

[0069] The present invention also provides the use of the complex in preparing drugs related to photothermal therapy.

[0070] The present invention also provides a photosensitizer that can be used for anti-tumor treatment, comprising the above-mentioned complex.

[0071] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0072] Example 1

[0073] A method for preparing black fungus polysaccharide, comprising the following steps:

[0074] (1) drying black fungus at 40° C. for 12 h, crushing the mixture, passing it through a sieve, collecting the sieve contents, and refluxing the mixture with ethyl acetate and acetone in sequence for 8 h each, and then drying the mixture at 60° C. to obtain defatted black fungus powder;

[0075] (2) Mix the defatted black fungus powder with 70% ethanol solution at a ratio of 1 g: 20 mL, stir for 24 h, and then filter to collect the residue;

[0076] (3) The filter residue was mixed with 0.9% NaCl solution at a ratio of 1 g:20 mL, stirred at 85° C. for 4 h, then stirred at room temperature overnight, and then filtered to collect the supernatant;

[0077] (4) decolorizing the supernatant with 30% hydrogen peroxide, adding it dropwise and stirring until the color turns light yellow, stopping the addition and continuing stirring until the color fades; using 717 anion exchange resin for adsorption and deproteinization; placing the solution in a dialysis bag and dialyzing it with ultrapure water for 7 days; freezing the solution in a -20°C refrigerator and then placing it in a freeze dryer for freeze drying to obtain black fungus crude polysaccharide;

[0078] (5) The crude black fungus polysaccharide is dissolved in water, reprecipitated with ethanol, the precipitate is collected and redissolved in water, and then dialyzed twice with ultrapure water, and then frozen in a -20°C refrigerator and freeze-dried in a freeze dryer to obtain the black fungus polysaccharide.

[0079] Example 2

[0080] A method for preparing black fungus polysaccharide, comprising the following steps:

[0081] (1) Drying black fungus at 35° C. for 14 h, crushing the powder, passing it through a sieve, collecting the sieve, and refluxing the sieve with ethyl acetate and acetone in turn, each for 7 h, and then drying at 70° C. to obtain defatted black fungus powder;

[0082] (2) Mix the defatted black fungus powder and 60% ethanol solution at a ratio of 1 g: 25 mL, stir for 20 h, and then filter to collect the residue;

[0083] (3) The filter residue was mixed with 0.9% NaCl solution at a ratio of 1 g:18 mL, stirred at 80° C. for 5 h, then stirred at room temperature overnight, and then filtered to collect the supernatant;

[0084] (4) decolorizing the supernatant with 30% hydrogen peroxide, adding it dropwise and stirring until the color turns light yellow, stopping the addition and continuing stirring until the color fades; using 717 anion exchange resin for adsorption and deproteinization; placing the solution in a dialysis bag and dialyzing it with ultrapure water for 7 days; freezing the solution in a -20°C refrigerator and then placing it in a freeze dryer for freeze drying to obtain black fungus crude polysaccharide;

[0085] (5) The crude black fungus polysaccharide is dissolved in water, reprecipitated with ethanol, the precipitate is collected and redissolved in water, and then dialyzed twice with ultrapure water, and then frozen in a -20°C refrigerator and freeze-dried in a freeze dryer to obtain the black fungus polysaccharide.

[0086] Example 3

[0087] A method for preparing black fungus polysaccharide, comprising the following steps:

[0088] (1) Drying black fungus at 45° C. for 10 h, crushing the mixture, passing it through a sieve, collecting the sieve contents, and refluxing the mixture with ethyl acetate and acetone in sequence for 9 h, respectively, and then drying the mixture at 50° C. to obtain defatted black fungus powder;

[0089] (2) Mix the defatted black fungus powder and 80% ethanol solution at a ratio of 1 g:18 mL, stir for 26 h, and then filter to collect the residue;

[0090] (3) The filter residue was mixed with 0.9% NaCl solution at a ratio of 1 g:25 mL, stirred at 90° C. for 3 h, then stirred at room temperature overnight, and then filtered to collect the supernatant;

[0091] (4) decolorizing the supernatant with 30% hydrogen peroxide, adding it dropwise and stirring until the color turns light yellow, stopping the addition and continuing stirring until the color fades; using 717 anion exchange resin for adsorption and deproteinization; placing the solution in a dialysis bag and dialyzing it with ultrapure water for 7 days; freezing the solution in a -20°C refrigerator and then placing it in a freeze dryer for freeze drying to obtain black fungus crude polysaccharide;

[0092] (5) The crude black fungus polysaccharide is dissolved in water, reprecipitated with ethanol, the precipitate is collected and redissolved in water, and then dialyzed twice with ultrapure water, and then frozen in a -20°C refrigerator and freeze-dried in a freeze dryer to obtain the black fungus polysaccharide.

[0093] Example 4

[0094] A BFP-FeSe2 composite, the preparation method is as follows:

[0095] (1) Preparation of ferrous selenide powder:

[0096] a. Mix oleylamine and 1-octadecene in a volume ratio of 3:2, heat to 120°C, and maintain for 30 min. Then, mix the mixed solution of oleylamine + 1-octadecene with FeCl2·4H2O powder in a ratio of 100 mL:0.8 g, and stir for 30 min to obtain a mixed solution A.

[0097] b. Selenium powder and oleylamine were mixed at a ratio of 1 g: 25 mL and added to the mixture A, and stirred for 10 min; then the temperature was raised to 150°C, and the reaction was stopped after 30 min; then the mixture was cooled to room temperature, centrifuged at 5000 rpm for 8 min, and washed with acetone; centrifuged at 5000 rpm for 8 min again, and washed with anhydrous ethanol; and ferrous selenide powder was obtained after freeze-drying;

[0098] (2) The black fungus polysaccharide was prepared into a 1 mg / mL black fungus polysaccharide solution, then heated at 140°C for 0.5 h, and then quenched to room temperature; an excess of the above-mentioned ferrous selenide powder was added, ultrasonically dispersed for 10 min, and then stirred at room temperature for 48 h. The suspended matter was removed by centrifugation at 4000 rpm for 8 min, and the supernatant was freeze-dried to obtain a brown flocculent product, which was the BFP-FeSe2 complex.

[0099] Example 5

[0100] A BFP-FeSe2 composite, the preparation method is as follows:

[0101] (1) Preparation of ferrous selenide powder:

[0102] a. Mix oleylamine and 1-octadecene in a volume ratio of 4:2, heat to 100°C, and maintain for 35 min. Then, mix the mixed solution of oleylamine + 1-octadecene with FeCl2·4H2O powder in a ratio of 110 mL:0.5 g, stir for 25 min, and obtain a mixed solution A.

[0103] b. Selenium powder and oleylamine were mixed in a ratio of 1.5 g:30 mL and added to the mixture A, and stirred for 15 min; then the temperature was raised to 150 ° C, and the reaction was stopped after 30 min; then the mixture was cooled to room temperature, centrifuged at 4000 rpm for 10 min, and washed with acetone; centrifuged at 4000 rpm for 10 min again, and washed with anhydrous ethanol; and ferrous selenide powder was obtained after freeze-drying;

[0104] (2) The black fungus polysaccharide was prepared into a 1.3 mg / mL black fungus polysaccharide solution, then heated at 130°C for 0.8 h, and then quenched to room temperature; an excess of the above-mentioned ferrous selenide powder was added, ultrasonically dispersed for 12 min, and then stirred at room temperature for 45 h. The suspended matter was removed by centrifugation at 4200 rpm for 5 min, and the supernatant was freeze-dried to obtain a brown flocculent product, which was the BFP-FeSe2 complex.

[0105] Example 6

[0106] A BFP-FeSe2 composite, the preparation method is as follows:

[0107] (1) Preparation of ferrous selenide powder:

[0108] a. Mix oleylamine and 1-octadecene in a volume ratio of 3.5:1.2, heat to 130°C, and maintain for 25 min. Then, mix the mixed solution of oleylamine + 1-octadecene with FeCl2·4H2O powder in a ratio of 120 mL:1 g, and stir for 35 min to obtain a mixed solution A.

[0109] b. Selenium powder and oleylamine were mixed at a ratio of 0.8 g:20 mL and added to the mixture A, and stirred for 7 min; then the temperature was raised to 150°C, and the reaction was stopped after 35 min; then the mixture was cooled to room temperature, centrifuged at 6000 rpm for 5 min, and washed with acetone; centrifuged at 6000 rpm for 5 min again, and washed with anhydrous ethanol; and ferrous selenide powder was obtained after freeze-drying;

[0110] (2) The black fungus polysaccharide was prepared into a 0.8 mg / mL black fungus polysaccharide solution, then heated at 150°C for 0.3 h, and then quenched to room temperature; an excess of the above-mentioned ferrous selenide powder was added, ultrasonically dispersed for 8 min, and then stirred at room temperature for 50 h. The suspended matter was removed by centrifugation at 3500 rpm for 10 min, and the supernatant was freeze-dried to obtain a brown flocculent product, which was the BFP-FeSe2 complex.

[0111] Test Example 1

[0112] (I) Structure and morphology of BFP

[0113] Fourier transform infrared spectroscopy was used to determine the glycosidic bond type of BFP. After an appropriate amount of BFP was fully dried in a vacuum drying oven, potassium bromide tableting method (samples were sent to Wuhan University Testing Center for testing) was used. The glycosidic bond type of BFP was determined by Fourier transform infrared spectrometer at 4000-400 cm -1 The infrared absorption of BFP was measured in the wave number range (e.g. Figure 1 ).

[0114] The morphology of BFP was observed using scanning electron microscopy and transmission electron microscopy. The fully dried BFP was prepared into a 0.1 mg / mL solution with deionized water, and then quickly frozen with liquid nitrogen after ultrasonic treatment to maintain its morphology in aqueous solution. The quick-frozen polysaccharide was evenly spread on the conductive glue after freeze-drying. The polysaccharide was sprayed with platinum and then the morphology of the polysaccharide was observed using a field emission scanning electron microscope. 2.5 μL of the fully dispersed solution was dropped on a 230-mesh carbon support film and dried naturally. The morphology of the polysaccharide (such as Figure 2 and Figure 3 ).

[0115] The molecular chain morphology of BFP was observed using an atomic force microscope. BFP was prepared into a 1 mg / mL aqueous solution using deionized water and filtered through a 0.22 μm microporous membrane. The solution was gradually diluted to 1×10 -6 g / mL and ultrasonically treated, 5 μL of the solution was dropped onto a fresh mica sheet and allowed to dry naturally. The image was taken by atomic force microscopy in tapping mode (e.g. Figure 4 ).

[0116] According to the measurement results, BFP has typical absorption peaks of β-glycosyl bonds, polysaccharide CO stretching vibration, and CH2 vibration; BFP is dispersed in the form of rigid chains in its dilute solution; BFP can self-assemble into nanofiber tubular morphology at a certain concentration.

[0117] (II) Structure and morphology of FeSe2

[0118] The diffraction spectrum of the FeSe2 sample powder was measured using an X-ray diffractometer, with the scanning rate of the X-ray diffractometer set to 2° / min, the voltage to 40 kV, the current to 15 mA, and the scanning from 5° to 80°.

[0119] The morphology of FeSe2 was observed using a transmission electron microscope. An appropriate amount of FeSe2 powder was fully dispersed in ethanol. 2.5 μL of the dispersion was dropped on a 230-mesh carbon support film and dried naturally. The morphology was observed using a transmission electron microscope, and the particle size distribution was calculated.

[0120] The results of the test are as follows Figure 5 and Figure 6 As shown, the XRD spectrum of the FeSe2 sample powder matches the standard card PDF#21-0432 of FeSe2, and the FeSe2 particle size is mainly between 10-20 nm.

[0121] (III) Structure and morphology of BFP-FeSe2 composite

[0122] The morphology of BFP-FeSe2 was observed using a scanning electron microscope. 2.5 μL of 0.1 mg / mL BFP-FeSe2 solution was dropped on a 230-mesh carbon support film and dried naturally. The morphology was observed using a transmission electron microscope.

[0123] The element distribution of BFP-FeSe2 was detected using an energy spectrometer attached to a field emission scanning electron microscope, and the mapping scan of Fe and Se elements was performed on the BFP-FeSe2 dry sample using the energy spectrometer.

[0124] The results of the test are as follows Figure 7 and Figure 8 As shown, FeSe2 particles are distributed in the nanofiber tubes formed by BFP; Fe elements and Se elements are distributed in the nanofiber tubes formed by BFP.

[0125] Test Example 2 Evaluation of biocompatibility of BFP-FeSe2 composite (one)

[0127] HUVEC (purchased from the Typical Culture Collection Room of Shanghai Cell Bank, Chinese Academy of Sciences) was used as a model cell to investigate the cell compatibility of BFP-FeSe2 complex. 3 Cells were seeded into 96-well plates, and different concentrations (0, 1, 10, 100, 250, 500 and 750 μg / mL) of BFP-FeSe2 dissolved in PBS were added for intervention 8 hours later. The absorbance at 450 nm after treatment with CCK-8 cell proliferation-toxicity detection kit was measured by microplate reader, and the cell viability was calculated by the absorbance ratio of the treatment group and the control group (i.e., 0 μg / mL group, containing only PBS), and the results were expressed as percentages.

[0128] The results are as follows Fig. 9 As shown, BFP-FeSe2 had no effect on cell proliferation. (two)

[0130] The systemic acute toxicity experiment was carried out on Balb / c mice (6-8 weeks, 18-22g, purchased from Wuhan Wanqian Jiaxing Biotechnology Co., Ltd., and raised in the ABSL-3 laboratory of the Animal Experiment Center of Wuhan University). 0.25mg / mL BFP-FeSe2 solution and normal saline were intravenously injected into healthy mice at a dose of 5mg / kg. After 21 days, orbital blood was taken to measure blood biochemical indicators (including ALT, AST, ALB, TBIL, UREA and CREA), and the mice were dissected alive, and the heart, liver, spleen, lung and kidney were taken to prepare tissue sections, and HE staining was used to observe the tissue compatibility of BFP-FeSe2 with various organs of mice.

[0131] The systemic acute toxicity experiment in mice showed that BFP-FeSe2 had no effect on the normal life of mice. Fig.10 The results of visceral pathological sections showed that BFP-FeSe2 had no obvious toxicity to mouse organs. (three)

[0133] New Zealand rabbit blood (purchased from Wuhan Wanqian Jiaxing Biotechnology Co., Ltd.) was collected in an anticoagulant tube containing EDTA, shaken evenly and centrifuged at 3000 rpm for 15 min to obtain blood cells. 1 mL of BFP-FeSe2 solution dissolved in PBS with different concentrations (100, 250, 500 μg / mL), ultrapure water (ddH2O) and PBS solution were mixed with 20 μL of blood cells and incubated at 37°C for 6 h to investigate the blood compatibility of the BFP-FeSe2 complex.

[0134] Hemolysis test further showed that BFP-FeSe2 has good blood compatibility. Fig.11 shown.

[0135] Experimental Example 3 Evaluation of the anti-tumor effect of BFP-FeSe2 complex in vitro (one)

[0137] Fluorescence confocal observation was used to observe the intracellular distribution of drug-loaded nanoparticles BFP-FeSe2 (0.25 mg / mL) during the co-culture of HCC-LM3 cells (purchased from the Typical Culture Collection Room of Shanghai Cell Bank, Chinese Academy of Sciences) for 12 hours.

[0138] The results showed that BFP-FeSe2 was easily taken up by cells, such as Fig.12 The laser confocal image shown shows the distribution of drug-loaded nanoparticles in cells, which are basically completely taken up within 8 hours. (two)

[0140] HCC-LM3 cells were used as model cells and 8×10 3 Cells / well The cells were seeded into 96-well plates. After 8 hours, different concentrations (0, 250, 500 μg / mL) of BFP-FeSe2 dissolved in PBS were added for intervention. After 8 hours, different concentrations of BFP-FeSe2 were aspirated and serum-free medium was added. After 24 hours, 808 nm near-infrared light (NIR) was given according to the grouping, 5 min per well. The phototoxicity and dark toxicity of BFP-FeSe2 were determined by CCK-8 method.

[0141] The results are as follows Fig.13 , BFP-FeSe2 has a certain dark toxicity to HCC-LM3 in vitro, but its phototoxicity is stronger, and it can also effectively induce immunogenic death of tumor cells. (three)

[0143] Confocal laser scanning microscopy was used to observe the CRT exposure of BFP-FeSe2 on the surface of H22 cells (purchased from the Typical Culture Collection Room of Shanghai Cell Bank, Chinese Academy of Sciences) and the expression of HMGB1 in the cell nucleus.

[0144] The results are as follows Fig.14 After H22 was pretreated with BFP-FeSe2 and irradiated with near-infrared light, the expression of CRT on the cell membrane surface was significantly increased, and the expression level of HMGB1 in the cell nucleus was significantly decreased compared with the untreated control group.

[0145] In summary, it can be seen that the present invention uses black fungus polysaccharide as a nano drug carrier, and uses the nanofiber tubes formed by self-assembly in an aqueous solution to load ferrous selenide, thereby solving the problem that ferrous selenide is unstable and prone to agglomeration and flocculation. Moreover, the BFP-FeSe2 complex has good biosafety and biocompatibility, is easily taken up by tumor cells, and can effectively induce immunogenic death of tumor cells.

[0146] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing black fungus polysaccharide, characterized in that: The steps include: (1) refluxing and drying the pretreated black fungus to obtain defatted black fungus powder; (2) mixing the defatted black fungus powder with an ethanol solution and a NaCl solution in sequence, and filtering to obtain a supernatant; (3) decolorizing the supernatant with 25-40% hydrogen peroxide, deproteinizing with a macroporous resin, dialyzing once, and then freeze-drying to obtain crude black fungus polysaccharide; (4) dissolving the crude black fungus polysaccharide, precipitating with alcohol, dialyzing twice and freeze-drying to obtain the black fungus polysaccharide; The organic solvent used for reflux in step (1) includes ethyl acetate and / or acetone.

2. The preparation method according to claim 1, characterized in that: The volume concentration of the ethanol solution in step (2) is 60-80%, and the solid-liquid ratio of the defatted black fungus powder to the ethanol solution is 1g:(18-25)mL.

3. The preparation method according to claim 1, characterized in that: The mixing process of step (2) with the NaCl solution includes heating and stirring treatment, the temperature of the heating and stirring treatment is 80-90° C., and the time of the heating and stirring treatment is 3-5 hours; The solid-liquid ratio of the defatted black fungus powder to the NaCl solution is 1 g: (18-25) mL.

4. Use of the black fungus polysaccharide prepared by the preparation method according to any one of claims 1 to 3 in the preparation of nano drug carriers.

5. A complex comprising black fungus polysaccharide prepared by the preparation method according to any one of claims 1 to 3.

6. The method for preparing the composite according to claim 5, characterized in that: The steps include: The black fungus polysaccharide is prepared into a black fungus polysaccharide solution and then heated. Then, ferrous selenide powder is added, ultrasonicated, stirred and centrifuged to obtain the solution.

7. The preparation method according to claim 6, characterized in that: The concentration of the black fungus polysaccharide solution is 0.8-1.3 mg / mL.

8. The preparation method according to claim 6, characterized in that: The heating temperature is 130-150° C., and the heating time is 0.3-0.8 h; The centrifugal speed is 3000-4500 rpm, and the centrifugal time is 5-10 min.

9. Use of the complex according to claim 5 or the complex prepared by the preparation method according to any one of claims 6 to 8 in the preparation of drugs related to photothermal therapy.

10. A photosensitizer that can be used for anti-tumor treatment, characterized in that: The invention comprises the complex according to claim 5 or the complex prepared by the preparation method according to any one of claims 6 to 8.

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