Cuttlefish ink polysaccharide selenium nanoparticles as well as preparation method and application thereof

Abstract

The invention provides cuttlefish ink polysaccharide selenium nanoparticles as well as a preparation method and application thereof, and relates to the technical field of foods and biological medicines. The cuttlefish ink polysaccharide selenium nanoparticles are obtained through selenylation modification of natural marine animal extract cuttlefish ink polysaccharide. The cuttlefish ink polysaccharide selenium nanoparticles can significantly inhibit proliferation of various tumor cells, especially liver cancer cells, and can inhibit migration of the liver cancer cells at the same time. The content of selenium in the cuttlefish ink polysaccharide selenium nano-particles is 2-8%, and the cuttlefish ink polysaccharide selenium nano-particles have no obvious inhibition effect on proliferation of normal hepatocytes and have higher safety while resisting tumors.

Description

Technical Field

[0001] The invention relates to the technical field of food and biomedicine, and in particular to a kind of cuttlefish ink polysaccharide selenium nanoparticles and a preparation method thereof and application thereof in anti-tumor drugs. Background Art

[0002] Selenium (Se) is an indispensable trace element in humans and animals, and has certain benefits in preventing the formation and development of certain cancer cells. Compared with inorganic and organic selenium compounds, nanoscale zero-valent selenium has lower toxicity and better bioavailability. As a new elemental form of selenium, selenium nanoparticles (SeNPs) can inhibit tumor growth by triggering cell apoptosis and inhibiting autophagy. However, naked SeNPs are very unstable and tend to aggregate into black elemental selenium, which means that they have low biological activity. Currently, finding suitable bioactive stabilizers to improve the stability of synthetic SeNPs has received increasing attention.

[0003] Some natural biomacromolecules (such as proteins and polysaccharides) can be used as stabilizers to modify the surface of SeNPs. Many studies have also shown that polysaccharides are an effective stabilizer for SeNPs due to their low toxicity, good water solubility, high hydroxyl content, complex branched chain structure, and large specific surface area. Therefore, selenization modification of polysaccharides to further enhance their anti-tumor activity has broad prospects for medical applications.

[0004] For example, the patent document with application number 202410281870.4 discloses a Sargassum polysaccharide nano-selenium, which has a strong ability to inhibit the proliferation activity and angiogenic mimicry of non-small cell lung cancer cells. Summary of the invention

[0005] The present invention provides a cuttlefish ink polysaccharide selenium nanoparticle and a preparation method and application thereof. In the present invention, cuttlefish ink polysaccharide (SIP), a natural marine animal extract, is selenized to obtain cuttlefish ink polysaccharide selenium nanoparticles (SIP-SeNPs). The combination of the two can inhibit the migration of liver cancer cells and significantly inhibit the proliferation of various tumor cells, especially liver cancer cells.

[0006] The inventors of the present invention have found that the current selenium products have a high selenium content and have a large toxic and side effect on normal cells. For example, the Sargassum fusiformis nano-selenium product of CN202410281870.4 has a selenium content of 10%. Due to the high selenium content, the toxic and side effects on normal cells are large. The content of selenium in the present invention is 2-8%, which has no obvious inhibitory effect on the proliferation of normal liver cells.

[0007] In one aspect, the present invention provides a cuttlefish ink polysaccharide selenium nanoparticle, wherein the cuttlefish ink polysaccharide selenium nanoparticle is obtained by selenization modification of cuttlefish ink polysaccharide.

[0008] Furthermore, the selenium content in the squid ink polysaccharide selenium nanoparticles is 2% to 8%.

[0009] Preferably, the selenium content in the squid ink polysaccharide selenium nanoparticles is 3.9%.

[0010] Furthermore, the squid ink polysaccharide is prepared by the following method: (3-1) Extract squid ink from the squid ink sac, add 1 to 10 times the volume of distilled water, mix, soak overnight at 4°C, add 0.5% to 2% of the mass of squid ink enzyme for water bath enzymolysis, the enzymolysis temperature is 30 to 60°C, the enzymolysis time is 1 to 5 hours, and the enzymolysis pH is 6 to 9. Inactivate the enzyme, adjust the pH to 4 to 5, incubate at 4°C overnight, centrifuge to remove melanin, and obtain a supernatant; (3-2) Sevage reagent was added to the supernatant obtained in the above step (3-1) for protein removal, and pre-cooled ethanol was added for precipitation. The mixture was placed at 4°C overnight, and the polysaccharide precipitate was collected by centrifugation. The mixture was redissolved, dialyzed with distilled water in a dialysis bag with a molecular weight cutoff of 3500 for 65-75 hours, and lyophilized to obtain crude polysaccharide; (3-3) The crude polysaccharide obtained in step (3-2) was re-dissolved in distilled water and loaded onto a DEAE-52 anion exchange chromatography column for chromatography. Distilled water, 0.1 mol / L NaCl eluent, 0.3 mol / L NaCl eluent and 0.5 mol / L NaCl eluent were used in sequence to elute according to the ionic strength gradient from low to high. The fraction eluted with the 0.5 mol / L NaCl eluent was collected and placed in a dialysis bag with a molecular weight cutoff of 3500 Da. The fraction was dialyzed in distilled water, and the retentate was collected. After freeze-drying, squid ink polysaccharide was obtained.

[0011] Furthermore, the enzyme in the above step (3-1) includes papain, trypsin or alkaline protease.

[0012] Furthermore, the squid ink polysaccharide selenium nanoparticles are prepared by the following method: (4-1) mixing the squid ink polysaccharide solution and the selenium-containing compound solution to obtain a mixed solution; (4-2) adding Tween-80 solution and ascorbic acid solution to the mixed solution obtained in step (4-1), stirring at room temperature to react, and obtaining a squid ink polysaccharide selenium nanoparticle solution; (4-3) The cuttlefish ink polysaccharide selenium nanoparticle solution obtained in step (4-2) is dialyzed and freeze-dried to obtain cuttlefish ink polysaccharide selenium nanoparticles.

[0013] Furthermore, the concentration of the squid ink polysaccharide solution described in step (4-1) of the above preparation method is 1~20 mg / mL, the concentration of the selenium-containing compound solution is 5~100 mmol / L, the selenium-containing compound includes sodium selenite (Na2SeO3), selenious acid (H2SeO3), and selenium oxide (SeO2), and the volume ratio of the squid ink polysaccharide solution to the selenium-containing compound solution is 0.25~10:1.

[0014] Furthermore, in step (4-1) of the above preparation method, the volume ratio of the squid ink polysaccharide solution to the selenium-containing compound solution is 0.4-4:1.

[0015] Furthermore, the concentration of the Tween-80 solution in step (4-2) of the above preparation method is 5-40 mg / mL, the concentration of the ascorbic acid solution is 20-400 mmol / L, the volume ratio of the polysaccharide solution to the Tween-80 solution in the mixed solution is 1-8:1; the volume ratio of the selenium-containing compound solution to the ascorbic acid solution in the mixed solution is 1:1-5; and the time of the room temperature stirring reaction is 6-12 hours.

[0016] Furthermore, the dialysis described in step (4-3) of the above preparation method is performed using a dialysis bag with a molecular weight cutoff of 3500 in distilled water for 65 to 75 hours.

[0017] Furthermore, the squid ink polysaccharide selenium nanoparticles prepared according to the above method have a particle size of 40-100 nm and a polymer dispersibility index PDI of 0.002-0.2.

[0018] In another aspect, the present invention provides a method for preparing squid ink polysaccharide selenium nanoparticles, comprising the following steps: (6-1) mixing the squid ink polysaccharide solution and the selenium-containing compound solution to obtain a mixed solution; (6-2) Add Tween-80 solution and ascorbic acid solution to the mixed solution obtained in step (6-1), and stir the mixture at room temperature to obtain a squid ink polysaccharide selenium nanoparticle solution.

[0019] (6-3) The cuttlefish ink polysaccharide selenium nanoparticle solution obtained in step (6-2) is dialyzed and freeze-dried to obtain cuttlefish ink polysaccharide selenium nanoparticles.

[0020] Furthermore, in the above step (6-1), the concentration of the squid ink polysaccharide solution is 1~20 mg / mL, the concentration of the selenium-containing compound solution is 5~100 mmol / L, the selenium-containing compound includes sodium selenite (Na2SeO3), selenious acid (H2SeO3), selenium oxide (SeO2), etc., and the volume ratio of the squid ink polysaccharide solution to the selenium-containing compound solution is 0.4~4:1.

[0021] Furthermore, in the above step (6-1), the volume ratio of the squid ink polysaccharide solution to the selenium-containing compound solution is 0.5-2:1.

[0022] Furthermore, in the above step (6-1), the squid ink polysaccharide solution and the selenium-containing compound solution are mixed at room temperature for 1 to 5 hours.

[0023] Furthermore, the concentration of the Tween-80 solution in the above step (6-2) is 5-40 mg / mL, and the concentration of the ascorbic acid solution is 20-400 mmol / L; The volume ratio of the polysaccharide solution to the Tween-80 solution in the mixed solution is 1-8:1; the volume ratio of the selenium compound solution to the ascorbic acid solution in the mixed solution is 1:1-5; The reaction time of stirring at room temperature is 6 to 12 hours.

[0024] Furthermore, the dialysis in the above step (6-3) is performed using a dialysis bag with a molecular weight cut-off of 3500 and distilled water for 72 hours.

[0025] On the other hand, the present invention provides a use of squid ink polysaccharide selenium nanoparticles in the preparation of anti-tumor drugs.

[0026] Furthermore, the above-mentioned anti-tumor drugs are drugs against liver cancer, breast cancer, and colorectal cancer.

[0027] Preferably, the anti-tumor drug is a drug for inhibiting liver cancer proliferation and migration.

[0028] On the other hand, the present invention provides a use of squid ink polysaccharide selenium nanoparticles in preparing a selenium supplement.

[0029] Preferably, the selenium supplement is a health food or a medicine.

[0030] Compared with the prior art, the technical solution of the present invention has the following advantages: (1) The SIP-SeNPs prepared by the present invention have a particle size of 40-100 nm, a polymer dispersibility index (pdi) of 0.002-0.2, a uniform particle size distribution, good stability and targeting, and can significantly inhibit the proliferation of cancer cells (liver cancer cell HepG2, liver cancer cell PLC-PRF-5, breast cancer cell MDA-MB-231, colorectal cancer cell HCT-8). Figure 6 It can be seen that the combination of SIP and SeNPs has an obvious synergistic effect. Compared with the use of SIP or SeNPs alone, the inhibitory effect of SIP-SeNPs on various cancer cells is significantly improved.

[0031] (2) The SIP-SeNPs prepared by the present invention exhibit the strongest anti-proliferation activity against liver cancer cells, that is, SIP-SeNPs have liver cancer targeting properties. Figure 6 As shown, the IC50 values ​​of SIP-SeNPs for HepG2 and PLC-PRF-5 were 13.85µg / mL and 19.4µg / mL, respectively, which were significantly lower than the IC50 values ​​of SIP-SeNPs for MDA-MB-231 and HCT-8.

[0032] (3) The SIP-SeNPs prepared by the present invention have a selenium content of 2% to 8%. While having a significant inhibitory effect on cancer cells, they have no significant inhibitory effect on the proliferation of normal liver cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the elemental composition analysis diagram of SIP-SeNPs.

[0034] Figure 2 Fourier infrared spectra of SIP and SIP-SeNPs.

[0035] Figure 3 It is the particle size distribution histogram of SeNPs and SIP-SeNPs.

[0036] Figure 4 Scanning electron microscopy images of SIP and SIP-SeNPs.

[0037] Figure 5 This is the result of SIP-SeNPs-1, SIP, and SeNPs inhibiting the migration of liver cancer cells.

[0038] Figure 6 The graph shows the results of SIP-SeNPs-1, SIP, and SeNPs inhibiting the proliferation of tumor cells (HepG2, PLC-PRF-5, MDA-MB-231, and HCT-8).

[0039] Figure 7 The graph shows the results of SIP-SeNPs-1, 2, 3, 4, and 5 inhibiting the proliferation of tumor cells HepG2, where ** indicates that there are significant differences between SIP-SeNPs-1, SIP-SeNPs-2, SIP-SeNPs-3 and SIP-SeNPs-4, P < 0.01; ## indicates that there are significant differences between SIP-SeNPs-1, SIP-SeNPs-2, SIP-SeNPs-3 and SIP-SeNPs-5, P < 0.01.

[0040] Figure 8The figure shows the effects of SIP-SeNPs-1, 3, 5, SIP, and SeNPs on the proliferation of normal hepatocytes THLE-2, where ** indicates that there are significant differences between SIP-SeNPs-5, SeNPs and SIP-SeNPs-1, P < 0.01; ## indicates that there are significant differences between SIP-SeNPs-5, SeNPs and SIP-SeNPs-3, P < 0.01. DETAILED DESCRIPTION

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0042] An embodiment of the present invention provides a cuttlefish ink polysaccharide selenium nanoparticle, which is obtained by selenization modification of cuttlefish ink polysaccharide.

[0043] As a new elemental form of selenium, selenium nanoparticles (SeNPs) can inhibit tumor growth by triggering cell apoptosis, blocking the cell cycle, inhibiting angiogenesis, etc. Marine polysaccharides have special sulfate group modifications and more N-acetylgalactosamine. These structures make marine polysaccharides have significant inhibitory effects and targeting on cancer cell invasion and migration and angiogenesis. The present invention provides a squid ink polysaccharide selenium nanoparticle, which synergistically enhances the anti-tumor activity of marine polysaccharides and selenium nanoparticles, and the selenium content in the present invention does not need to be too high, less than or equal to 8%, and has good selectivity, high liver cancer targeting, and will not significantly inhibit the proliferation of normal cells.

[0044] In one embodiment of the present invention, the selenium content in the squid ink polysaccharide selenium nanoparticles is 2% to 8%, for example, 2% to 7%, 2% to 6%, 3% to 5%, 3% to 4%, 2% to 4%, 2% to 5%, 3% to 6%, 4% to 5%.

[0045] In another embodiment of the present invention, squid ink polysaccharide is prepared by the following method: (1) Extracting squid ink from the squid ink sac, adding distilled water, soaking at 4°C overnight, adding enzyme for enzymatic hydrolysis, inactivating the enzyme in a boiling water bath for 15 min, adjusting the pH to 4-5, placing at 4°C overnight, and high-speed centrifugation to remove melanin to obtain a supernatant; (2) Sevage reagent is added to the supernatant obtained in step (1) above to remove protein, and pre-cooled ethanol is added for precipitation, and the mixture is placed at 4°C overnight, and the polysaccharide precipitate is collected by centrifugation, and distilled water is added for re-dissolution, dialyzed, and freeze-dried to obtain crude polysaccharide; (3) Add distilled water to the crude polysaccharide obtained in the above step (2) for redissolution and chromatography, collect the target polysaccharide by gradient elution, dialyze and freeze-dry to obtain squid ink polysaccharide.

[0046] In step (1), the amount of distilled water added is 1 to 10 times that of the squid ink, for example, 1 to 10 times, 2 to 8 times, 3 to 7 times, or 4 to 6 times.

[0047] In step (1), the type of enzyme can be papain, trypsin, or alkaline protease; The amount of enzyme added is 0.5% to 2% of the mass of the squid ink, for example, 0.5% to 1%, 1% to 1.5%, 1.5% to 2%, 0.5% to 1.5%, 1% to 2%.

[0048] The temperature of the enzymatic hydrolysis treatment in step (1) is 30-60°C, for example, 30-40°C, 40-50°C, or 50-60°C; The enzymatic hydrolysis time is 1 to 5 hours, for example, 1 to 2 hours, 2 to 3 hours, 3 to 4 hours, or 4 to 5 hours; The enzymatic hydrolysis pH is 6-9, for example, 6-7, 7-8, or 8-9.

[0049] The amount of distilled water added in step (2) is the same as the volume of the supernatant.

[0050] In step (2), the dialysis bag with a molecular weight cut-off of 3500 is used for dialysis, and the dialysis time is 65-75 hours, for example, 65-70 hours or 70-75 hours.

[0051] The chromatography treatment in step (3) uses a DEAE-52 anion exchange chromatography column. The amount of distilled water added in step (3) is 0.1 ml / mg crude polysaccharide.

[0052] In step (3), the dialysis bag with a molecular weight cutoff of 3500 is used for dialysis, and the dialysis time is 65-75 hours, for example, 65-70 hours or 70-75 hours.

[0053] Another embodiment of the present invention provides a method for preparing squid ink polysaccharide selenium nanoparticles, which specifically comprises the following steps: (1) mixing a squid ink polysaccharide solution and a selenium-containing compound solution to obtain a mixed solution; (2) adding Tween-80 solution and ascorbic acid solution to the mixed solution obtained in step (1), stirring at room temperature to react, and obtaining a squid ink polysaccharide selenium nanoparticle solution; (3) The squid ink polysaccharide selenium nanoparticle solution obtained in step (2) is dialyzed against distilled water in a dialysis bag with a molecular weight cutoff of 3500, and freeze-dried to obtain squid ink polysaccharide selenium nanoparticles.

[0054] In step (1), the volume ratio of the squid ink polysaccharide solution to the selenium-containing compound solution is 0.4-4:1. For example, it can be 0.4-1:1, 1-2:1, 2-3:1, or 3-4:1.

[0055] The concentration of the applicable squid ink polysaccharide solution can be 1-20 mg / mL.

[0056] The concentration of the applicable selenium compound solution may be 5-100 mmol / L.

[0057] Suitable selenium-containing compounds may be sodium selenite (Na2SeO3), selenious acid (H2SeO3), selenium oxide (SeO2) and other selenium-containing compounds.

[0058] In step (2), the volume ratio of the polysaccharide solution to the Tween-80 solution in the mixed solution is 1-8:1, for example, 1:1, 2:1, 4:1, 6:1, 8:1; the volume ratio of the selenium compound solution to the ascorbic acid solution in the mixed solution is 1:1-5, for example, 1:1, 1:2, 1:3, 1:4, 1:5; The applicable Tween-80 solution concentration can be 5~40mg / mL.

[0059] The applicable ascorbic acid solution may be 20-400 mmol / L.

[0060] The dialysis time of step (3) is 65-75 h, for example, 65-70 h, 75-75 h.

[0061] The following is a detailed description of the squid ink polysaccharide selenium nanoparticles and their preparation method and application described in the embodiments of the present invention in conjunction with specific examples.

[0062] Example 1 A. Cut open the squid ink sac, take out the ink, add 3 times the volume of distilled water, and soak overnight at 4℃. Add 1% of the ink weight of trypsin for water bath enzymolysis, the enzymolysis temperature is 37℃, the enzymolysis time is 4h, and the enzymolysis pH is 7. After enzymolysis, inactivate the enzyme in boiling water for 15min, then adjust the pH to 4.5 with 4mol / L hydrochloric acid, place at 4℃ overnight, remove melanin by high-speed refrigerated centrifugation, and take the supernatant.

[0063] B. Add Sevage reagent to the extract obtained in step A to remove residual protein, then add pre-cooled ethanol to precipitate, leave at 4°C overnight, collect the precipitate by centrifugation, add distilled water of the same volume as the original extract to reconstitute, dialyze with distilled water in a dialysis bag with a molecular weight cutoff of 3500 for 72 hours, and then lyophilize to obtain light yellow squid ink crude polysaccharide.

[0064] C. Weigh 150 mg of the crude polysaccharide obtained in step B, add 5 ml of distilled water to re-dissolve it, and then pass it through a DEAE-52 anion exchange chromatography column (2.6×50 mm). Elute it with distilled water, 0.1 mol / L NaCl, 0.3 mol / L NaCl and 0.5 mol / L NaCl eluents according to the ionic strength gradient from low to high, respectively. Collect the components eluted with 0.5 mol / L NaCl eluent, dialyze it with distilled water in a dialysis bag with a molecular weight of 3500 for 72 hours, collect the retentate, and then lyophilize it to obtain white squid ink polysaccharide SIP.

[0065] D. Weigh an appropriate amount of squid ink polysaccharide and add distilled water to make a 2mg / ml polysaccharide solution, mix 50ml of the polysaccharide solution with 50ml of 10mmol / L sodium selenite solution, and stir thoroughly for 3h at room temperature. Then add 10ml of 20mg / ml Tween-80 solution and 50ml of 40mmol / L ascorbic acid solution and stir for 10h at room temperature in the dark to allow the redox reaction to fully proceed.

[0066] E. The solution after the reaction was placed in a dialysis bag (molecular weight cutoff of 3500Da) and dialyzed in distilled water for 72 hours, and then freeze-dried to obtain squid ink polysaccharide selenium nanoparticles SIP-SeNPs-1.

[0067] The elemental analysis results of SIP-SeNPs-1 were measured using energy dispersive X-ray spectrometer (EDS), such as Figure 1 As shown, the content of selenium is 3.9%, and the contents of C and O are 84.3% and 11.7% respectively.

[0068] Example 2 A. Cut open the squid ink sac, take out the ink, add 1 volume of distilled water, and soak overnight at 4℃. Add 0.5% papain of the ink weight for water bath enzymolysis, the enzymolysis temperature is 55℃, the enzymolysis time is 3h, and the enzymolysis pH is 6.5. After enzymolysis, inactivate the enzyme in boiling water for 15min, then adjust the pH to 4 with 4mol / L hydrochloric acid, place at 4℃ overnight, remove melanin by high-speed refrigerated centrifugation (9000rpm, 20min), and take the supernatant.

[0069] B. Add Sevage reagent to the extract obtained in step A to remove residual protein, then add pre-cooled ethanol to precipitate, leave at 4°C overnight, collect the precipitate by centrifugation, add distilled water of the same volume as the original extract to reconstitute, dialyze with distilled water in a dialysis bag with a molecular weight cutoff of 3500 for 72 hours, and then lyophilize (-80°C, p<20Pa) to obtain light yellow squid ink crude polysaccharide.

[0070] C. Weigh 150 mg of the crude polysaccharide obtained in step B, add 5 ml of distilled water to re-dissolve it, and then pass it through a DEAE-52 anion exchange chromatography column (2.6×50 mm). Use distilled water, 0.1 mol / L NaCl, 0.3 mol / L NaCl and 0.5 mol / L NaCl eluents to elute according to the ionic strength gradient from low to high, respectively. Collect the components eluted with 0.5 mol / L NaCl eluent, dialyze with distilled water in a dialysis bag with a molecular weight cutoff of 3500 for 72 hours, collect the retentate, and then lyophilize (-80℃, p<20Pa) to obtain white squid ink polysaccharide SIP.

[0071] D. Weigh an appropriate amount of squid ink polysaccharide and add distilled water to prepare a 5 mg / ml polysaccharide solution. Mix 20 ml of the polysaccharide solution with 10 ml of 25 mmol / L sodium selenite solution and stir thoroughly at room temperature for 1 hour. Then, 2 ml of 40 mg / ml Tween-80 solution and 20 ml of 50 mmol / L ascorbic acid solution were added, and the mixture was stirred at room temperature in the dark for 6 h to allow the redox reaction to proceed fully.

[0072] E. After the reaction, the solution was placed in a dialysis bag (molecular weight cutoff: 3500Da) and dialyzed in distilled water for 72 hours, and freeze-dried to obtain squid ink polysaccharide selenium nanoparticles SIP-SeNPs-2.

[0073] The elemental analysis results of SIP-SeNPs-2 measured by EDS showed that the content of selenium was 2%.

[0074] Example 3 A. Cut open the squid ink sac, take out the ink, add 5 times the volume of distilled water, and soak overnight at 4℃. Add 1.5% of the ink weight of alkaline protease for water bath enzymolysis, the enzymolysis temperature is 45℃, the enzymolysis time is 2h, and the enzymolysis pH is 9. After enzymolysis, inactivate the enzyme in boiling water for 15min, then adjust the pH to 4 with 4mol / L hydrochloric acid, place at 4℃ overnight, remove melanin by high-speed refrigerated centrifugation, and take the supernatant.

[0075] B. Add Sevage reagent to the extract obtained in step A to remove residual protein, then add pre-cooled ethanol to precipitate, leave at 4°C overnight, collect the precipitate by centrifugation, add distilled water of the same volume as the original extract to reconstitute, dialyze with distilled water in a dialysis bag with a molecular weight cutoff of 3500 for 72 hours, and then lyophilize to obtain light yellow squid ink crude polysaccharide.

[0076] C. Weigh 150 mg of the crude polysaccharide obtained in step B, add 5 ml of distilled water to re-dissolve it, and pass it through a DEAE-52 anion exchange chromatography column (2.6×50 mm). Elute it with distilled water, 0.1 mol / L NaCl, 0.3 mol / L NaCl and 0.5 mol / L NaCl eluents according to the ionic strength gradient from low to high, respectively. Collect the components eluted with 0.5 mol / L NaCl eluent, dialyze it with distilled water in a dialysis bag with a molecular weight cutoff of 3500 for 72 hours, collect the retentate, and then lyophilize it to obtain white squid ink polysaccharide SIP.

[0077] D. Weigh an appropriate amount of squid ink polysaccharide and add distilled water to make a 4 mg / ml polysaccharide solution. Mix 10 ml of the polysaccharide solution with 20 ml of 20 mmol / L sodium selenite solution and stir thoroughly at room temperature for 5 hours. Then add 8 ml of 5 mg / ml Tween-80 solution and 20 ml of 80 mmol / L ascorbic acid solution and stir for 12 hours at room temperature in the dark to allow the redox reaction to fully proceed.

[0078] E. The solution after the reaction was placed in a dialysis bag (molecular weight cutoff of 3500Da) and dialyzed in distilled water for 72 hours, and then freeze-dried to obtain squid ink polysaccharide selenium nanoparticles SIP-SeNPs-3.

[0079] The elemental analysis results of SIP-SeNPs-3 measured by EDS showed that the content of selenium was 8%.

[0080] Comparative Example 1 Preparation of SeNPs 10 mL of 10 mmol / L sodium selenite solution was mixed evenly with 10 mL of 40 mmol / L ascorbic acid aqueous solution, and stirred in the dark at room temperature for 12 h to allow the redox reaction to proceed fully. After the reaction, the solution was put into a dialysis bag (MW: 3500, molecular weight cutoff 3500 Da) and dialyzed in double distilled water for 48 h. The retained liquid in the dialysis bag was taken and freeze-dried to obtain powdered nano-selenium.

[0081] Comparative Example 2 The preparation method is the same as that of Example 1, except for the following steps.

[0082] D. Weigh an appropriate amount of squid ink polysaccharide and add distilled water to make a 20 mg / ml polysaccharide solution. Mix 20 ml of the polysaccharide solution with 4 ml of 100 mmol / L sodium selenite solution and stir thoroughly at room temperature for 5 hours. Then add 4 ml of 20 mg / ml Tween-80 solution and 4 ml of 400 mmol / L ascorbic acid solution and stir for 10 hours at room temperature in the dark to allow the redox reaction to proceed fully.

[0083] Squid ink polysaccharide selenium nanoparticles SIP-SeNPs-4 were prepared, and the elemental analysis results of SIP-SeNPs-4 measured by EDS showed that the content of selenium was 0.8%.

[0084] Comparative Example 3 The preparation method is the same as that of Example 1, except for the following steps.

[0085] D. Weigh an appropriate amount of squid ink polysaccharide and add distilled water to make a 10mg / ml polysaccharide solution. Mix 40ml of the polysaccharide solution with 120ml of 50mmol / L sodium selenite solution and stir thoroughly for 1h at room temperature. Then add 8ml of 20mg / ml Tween-80 solution and 30ml of 200mmol / L ascorbic acid solution and stir for 12h at room temperature in the dark to allow the redox reaction to fully proceed.

[0086] Squid ink polysaccharide selenium nanoparticles SIP-SeNPs-5 were prepared, and the elemental analysis results of SIP-SeNPs-5 measured by EDS showed that the selenium content was 12%.

[0087] Test Example 1: Structural Characterization and Analysis of Cuttlefish Ink Polysaccharide Selenium Nanoparticles (1) Fourier transform infrared spectroscopy The freeze-dried powdered sample of SIP-SeNPs in Example 1 was used, and 1.0 mg of the sample was mixed with potassium bromide (KBr) powder at a ratio of 1:150. After grinding, the mixture was pressed into a transparent sheet and scanned at a wavelength of 4000-400 cm -1 The conditions were tested and the results were as follows Figure 2 As shown: 1129.1cm -1 The absorption peak at 3419.7 cm indicates that SIP is an α-configuration pyranose. Compared with SIP, there is no new absorption peak in SIP-SeNPs, but a significant shift is observed, indicating that the reaction between SIP and SeNPs does not produce any new covalent bonds. -1 To 3401.2cm -1 The blue shift indicated that there was an interaction between -OH in the polysaccharide and SeNPs, and SIP-SeNPs were formed.

[0088] (2) Particle size analysis The freeze-dried powdered sample of SIP-SeNPs in Example 1 was re-dissolved in distilled water to prepare a 1 mg / mL squid ink polysaccharide selenium nanoparticle solution. The average particle size of the squid ink polysaccharide selenium nanoparticles was measured using a Malvern nanoparticle size and Zeta potential analyzer. The results are as follows: Figure 3 As shown: The particle size of the squid ink polysaccharide selenium nanoparticle powder obtained in the above example is 48.68 nm, and dynamic light scattering analysis shows that it has a good particle size distribution. The polymer dispersity index (PDI) of the obtained squid ink polysaccharide selenium nanoparticle complex is 0.002.

[0089] (3) Scanning electron microscopy (SEM) The freeze-dried powdered sample of SIP-SeNPs in Example 1 was evenly dispersed on a conductive double-sided tape for fixation, and then placed in an ion sputtering coating instrument. The surface of the sample was sprayed with gold in a vacuum environment. The gold plating conditions were an operating voltage of 15 keV and an operating current of 15 mA, and then the sample morphology was observed.

[0090] like Figure 4 As shown in Figure 2, the SEM image of SIP shows that its surface is in the form of thin flakes with smooth edges. The SEM image of SIP-SeNPs shows that selenium nanoparticles (SeNPs) are uniformly distributed on the surface of SIP in the form of spherical particles.

[0091] Test Example 2: Antitumor Activity of Cuttlefish Ink Polysaccharide Selenium Nanoparticles (1) HepG2 cell migration assay HepG2 hepatoma cells were prepared into a cell suspension using serum-containing cell culture medium and inoculated into a 24-well plate with an inoculation volume of 500 μl and a cell number of 1×10 per well. 5 , and cultured in a 5% CO2 cell culture incubator. When the monolayer cell confluence reached 70%-80%, a new 200μl pipette tip was used to gently scratch the monolayer culture cells. After washing with PBS, serum-free medium containing equal concentrations of SIP-SeNPs-1, SIP, and SeNPs was added to continue culturing. The control group was cultured in normal serum-free medium. The cells were observed at fixed time points (24h, 48h) and the migration rate was calculated.

[0092] HepG2 cell migration results Figure 5 As shown, compared with SIP and SeNPs, SIP-SeNPs-1 can significantly inhibit the migration and healing of HepG2. When cultured for 48 hours, SIP-SeNPs-1 can reduce the migration area of ​​liver cancer cells HepG2 by about 45% compared with the control group.

[0093] (2) Cell proliferation inhibition test of different tumor cells Tumor cells (HepG2, PLC-PRF-5, MDA-MB-231, HCT-8) were made into cell suspension using serum-containing cell culture medium and inoculated into 96-well plates with an inoculation volume of 150 μL and 5,000 cells per well. The plates were cultured in a 5% CO2 cell culture incubator.

[0094] After the cells adhered to the wall, the culture medium in the well plate was aspirated, and SIP-SeNPs-1, SIP, and SeNPs were prepared into solutions of different concentrations using fresh cell culture medium, added to the above 96-well plate, and cultured for 48 hours.

[0095] CCK-8 at 10% of the volume of the culture medium was added to each well, and after culturing for 30 minutes, the absorbance was measured at a wavelength of 450 nm, and the effects of SIP-SeNPs-1, SIP, and SeNPs on tumor cell growth were calculated based on the absorbance.

[0096] CCK-8 results Figure 6 The results showed that SIP and SeNPs had a weak inhibitory effect on the proliferation of various tumor cells, while SIP-SeNPs-1 could significantly inhibit the proliferation of various tumor cells, and the effect was significantly better than that of SIP and SeNPs, especially for liver cancer cells. This may be because the ASGR1 receptor (asialoglycoprotein receptor 1) highly expressed in liver cancer cells can recognize a variety of ligands containing terminal galactose (Gal) or N-acetylgalactosamine (GalNAc) residues, mainly recognizing N-acetylgalactosamine. The ASGR1 receptor has been confirmed as a highly selective receptor on liver cancer cells, and the expression of this receptor is low in other tumor cells. Cuttlefish ink polysaccharide contains about 25.6% acetylgalactosamine. Cuttlefish ink selenium polysaccharide can combine polysaccharide and selenium nanoparticles to exert a synergistic effect. Through the targeting of cuttlefish ink polysaccharide, selenium nanoparticles are delivered to liver cancer cells, thereby exerting a significant anti-tumor effect. (3) Experimental study on the inhibitory effect of squid ink polysaccharide selenium nanoparticles with different selenium contents on the proliferation of HepG2 cells and human normal liver cells THLE-2 Using the squid ink polysaccharide selenium nanoparticles with different selenium contents obtained in the examples and comparative examples, as well as SIP and SeNPs as different treatment groups, comparative experiments were carried out on HepG2 cells and human normal liver cells THLE-2 to detect their proliferation inhibitory effects on HepG2 cells and human normal liver cells THLE-2, respectively.

[0097] like Figure 7As shown in the figure, the inhibitory effects of SIP-SeNPs-1 (IC50=13.85μg / ml), SIP-SeNPs-2 (IC50=26.27μg / ml), and SIP-SeNPs-3 (IC50=21.1μg / ml) on the proliferation of HepG2 cells were significantly better than those of SIP-SeNPs-4 (IC50=142.9μg / ml) and SIP-SeNPs-5 (IC50=134.7μg / ml). When the selenium content was 3.9%, the inhibitory effect on the proliferation of HepG2 cells was the strongest. When the selenium content was greater than 8%, the inhibitory effect on the proliferation of HepG2 cells tended to gradually weaken with the increase of selenium content. It may be that when the selenium content was high, selenium nanoparticles were prone to aggregation and precipitation, thereby affecting the anti-tumor activity of SIP-SeNPs. When the selenium content is less than 2%, the inhibitory effect on HepG2 cell proliferation tends to weaken gradually with the decrease of selenium content, and gradually approaches the inhibitory effect of SIP.

[0098] like Figure 8 As shown in the figure, the CCK-8 experimental results of human normal liver cells THLE-2 showed that SeNPs in inorganic form and SIP-SeNPs-5 with high selenium content significantly inhibited the growth of normal liver cells, while SIP-SeNPs-1 and SIP-SeNPs-3 had no obvious inhibitory effect on their proliferation, indicating that after the squid ink polysaccharide SIP was combined with SeNPs, when the selenium content was 2-8%, normal liver cells had no obvious side effects, and when the selenium content was higher than 8%, it began to have a significant inhibitory effect on normal liver cells, and with the increase of selenium content, the inhibitory effect on normal liver cells will gradually increase. When the selenium content is less than 8%, the lower the selenium content, the smaller the inhibitory effect on normal liver cells.

Claims

1. A squid ink polysaccharide selenium nanoparticle, characterized in that: It is obtained by selenization modification of squid ink polysaccharide.

2. The squid ink polysaccharide selenium nanoparticles according to claim 1, characterized in that: The selenium content of the squid ink polysaccharide selenium nanoparticles is 2%-8%, and the particle size is 40-100nm.

3. The squid ink polysaccharide selenium nanoparticles according to claim 1, characterized in that: The squid ink polysaccharide is prepared by the following method: (3-1) extracting squid ink from a squid ink sac, adding distilled water to soak overnight, adding enzymes to perform enzymatic hydrolysis, and centrifuging to remove melanin to obtain a supernatant; (3-2) removing protein from the supernatant obtained in step (3-1), then adding pre-cooled ethanol for alcohol precipitation, leaving overnight, collecting the polysaccharide precipitate, adding distilled water for re-dissolution, dialyzing the retained product in distilled water using a dialysis bag with a molecular weight cut-off of 3500, and lyophilizing to obtain crude polysaccharide; (3-3) The crude polysaccharide obtained in step (3-2) was re-dissolved in distilled water and loaded onto a DEAE-52 anion exchange chromatography column for chromatography. Distilled water, 0.1 mol / L NaCl eluent, 0.3 mol / L NaCl eluent and 0.5 mol / L NaCl eluent were used in sequence to elute according to an ionic strength gradient from low to high. The fraction eluted with the 0.5 mol / L NaCl eluent was collected and placed in a dialysis bag with a molecular weight cutoff of 3500 Da. The fraction was dialyzed in distilled water, and the retentate was collected and freeze-dried to obtain cuttlefish ink polysaccharide.

4. The squid ink polysaccharide selenium nanoparticles according to claim 1, characterized in that: The squid ink polysaccharide selenium nanoparticles are prepared by the following method: (4-1) mixing the squid ink polysaccharide solution and the selenium-containing compound solution to obtain a mixed solution; (4-2) adding Tween-80 solution and ascorbic acid solution to the mixed solution obtained in step (4-1), stirring at room temperature to react, and obtaining a squid ink polysaccharide selenium nanoparticle solution; (4-3) The cuttlefish ink polysaccharide selenium nanoparticle solution obtained in step (4-2) is dialyzed and freeze-dried to obtain cuttlefish ink polysaccharide selenium nanoparticles.

5. The squid ink polysaccharide selenium nanoparticles according to claim 4, characterized in that: In step (4-1), the concentration of the squid ink polysaccharide solution is 1-20 mg / mL, the concentration of the selenium-containing compound solution is 5-100 mmol / L, the selenium-containing compound includes sodium selenite, selenious acid, and selenium oxide, and the volume ratio of the squid ink polysaccharide solution to the selenium-containing compound solution is 0.4-4:1; The concentration of the Tween-80 solution in step (4-2) is 5-40 mg / mL, and the concentration of the ascorbic acid solution is 20-400 mmol / L; the volume ratio of the polysaccharide solution to the Tween-80 solution in the mixed solution is 1-8:1; the volume ratio of the selenium compound solution to the ascorbic acid solution in the mixed solution is 1:1-5; The dialysis described in step (4-3) is performed in distilled water using a dialysis bag with a molecular weight cut-off of 3500.

6. A method for preparing squid ink polysaccharide selenium nanoparticles according to any one of claims 1 to 5, characterized in that: The method comprises the following preparation steps: (6-1) mixing the squid ink polysaccharide solution and the selenium-containing compound solution to obtain a mixed solution; (6-2) adding Tween-80 solution and ascorbic acid solution to the mixed solution obtained in step (6-1), stirring at room temperature to react, and obtaining a squid ink polysaccharide selenium nanoparticle solution; (6-3) The cuttlefish ink polysaccharide selenium nanoparticle solution obtained in step (6-2) is dialyzed and freeze-dried to obtain cuttlefish ink polysaccharide selenium nanoparticles.

7. A use of the squid ink polysaccharide selenium nanoparticles according to any one of claims 1 to 5 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug is a drug for liver cancer, breast cancer and colorectal cancer.

8. A use of the squid ink polysaccharide selenium nanoparticles according to any one of claims 1 to 5 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug is an anti-liver cancer drug.

9. Use of the squid ink polysaccharide selenium nanoparticles according to any one of claims 1 to 5 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug is a drug for resisting liver cancer proliferation and migration.

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