Nano-selenium composite particle as well as preparation method and application thereof

Abstract

The invention discloses a nano-selenium composite particle as well as a preparation method and application thereof. The nano-selenium composite particle comprises an inner core of nano-selenium and a shell layer wrapping the inner core, the shell layer is made of chitosan oligosaccharide, the particle size of the nano-selenium is 15-95 nm, and the particle size of the nano-selenium composite particle is 20-100 nm. The nano-selenium composite particles prepared by the invention have the advantages of good stability and dispersibility, high free radical scavenging activity, good blood compatibility, extremely low cytotoxicity and the like, and have great application potential.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nano selenium, in particular to a nano selenium composite particle and a preparation method and application thereof. BACKGROUND

[0002] Selenium is a trace element necessary for human body, which has important effects such as scavenging free radicals, anti-tumor and immune regulation. Nano selenium is a kind of selenium element with nanoscale size (usually 1-100 nm), which has unique advantages compared with ordinary selenium element, including: (1) nanoscale particle size makes it have good dispersibility and cell penetration, which can be directly absorbed by the intestinal tract with high absorption utilization rate; (2) low toxicity and high safety; (3) high life activity, which can better play the biological function of selenium.

[0003] Based on the above unique advantages, nano selenium has application potential in the fields of biomedicine, food and feed, etc., but its shortcomings cannot be ignored, including: cytotoxicity caused by size effect; poor stability in physiological environment, easy to aggregate to form large particles or be oxidized to selenium oxide, resulting in reduced activity or increased toxicity; poor blood compatibility, which is manifested as a high hemolysis rate of more than 10% in the field of biomedicine. SUMMARY

[0004] In view of the problems in the prior art, the present application provides a nano selenium composite particle with good stability and dispersibility, high free radical scavenging activity, good blood compatibility and extremely low toxicity. A nano selenium composite particle, comprising a core of nano selenium and a shell layer wrapping the core, wherein the material of the shell layer is chitooligosaccharide, the particle size of the core is 15-95 nm, and the particle size of the nano selenium composite particle is 20-100 nm.

[0005] In the present application, the nano selenium is the active reaction site, the shell layer of chitooligosaccharide plays a protective and surface modification role for the nano selenium, which can reduce the probability of aggregation of the nano selenium in the physiological environment, thereby helping to improve the stability and dispersibility, and the blood compatibility is good and the toxicity is extremely low, which can reduce the adsorption and damage to the blood components, and therefore the application safety is high.

[0006] In addition, the surface of the shell layer has micropores, which can adsorb and capture a large number of free radicals, and the nano selenium has strong free radical scavenging ability, plus the chitooligosaccharide itself has a certain free radical scavenging ability, so that the nano selenium composite particle has excellent free radical scavenging ability.

[0007] Optionally, the particle size of the core is preferably 40-59 nm; and further preferably 50-52 nm.

[0008] Optionally, the particle size of the nano-selenium composite particle is preferably 45-64 nm; further preferably 50-60 nm.

[0009] Optionally, the thickness of the shell layer is 3-10 nm; preferably 3-6 nm.

[0010] Optionally, the number average molecular weight of the chitosan oligosaccharide is 300-2000 Da, preferably 300-1000 Da.

[0011] The application also provides a preparation method of the nano-selenium composite particle, comprising the following steps: mixing a chitosan oligosaccharide solution and a selenium source solution to obtain a premix solution, and then mixing the premix solution with a reducing agent to obtain a nano-selenium composite particle dispersion; centrifuging, filtering and freeze-drying the nano-selenium composite particle dispersion to obtain the nano-selenium composite particle.

[0012] Optionally, the concentration of the chitosan oligosaccharide solution is 2-5 wt%, preferably 3 wt%.

[0013] Optionally, the molar ratio of the effective components in the selenium source solution and the reducing agent is 1:2-25, preferably 1:4-8.

[0014] Optionally, the selenium source solution is a liquid system of an oxidizing agent (i.e. selenium source) that can provide selenium elements, such as a selenite solution.

[0015] Optionally, the selenite solution is a sodium selenite or potassium selenite solution.

[0016] Optionally, the concentration of the selenite solution is 5-15 mM, preferably 10 mM.

[0017] Optionally, the reducing agent is an L-ascorbic acid solution.

[0018] Optionally, the concentration of the reducing agent is 20-250 mM, and different concentrations of the reducing agent can reduce the selenite into nano-selenium with different particle sizes.

[0019] Optionally, the temperature of the freeze-drying is -40 to -50℃, and the time is 48-96 h.

[0020] Compared with the prior art, the application has the following technical effects: (1) The nano-selenium composite particle prepared by the application has the advantages of good stability and dispersity, high free radical scavenging activity, good blood compatibility, extremely low cytotoxicity, and great application potential.

[0021] (2) The method for preparing nano selenium composite particles based on the microfluidic system can greatly reduce human operation errors and improve the convenience of operation, thereby making the particle size of the synthesized nano selenium composite particles have good repeatability. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Structure diagram of the microfluidic system for preparing nano selenium composite particles in the application; Figure 2 Mapping diagram of the medium particle size nano selenium composite particles in Example 1, wherein (a) is an element blank diagram, (b) is an N element diagram, (c) is a Se element diagram, and (d) is an N and Se element diagram; Figure 3 Spectrum diagram of the medium particle size nano selenium composite particles in Example 1, wherein (a) is an infrared spectrum diagram, (b) is an X-ray photoelectron spectrum diagram, and (c) is an X-ray diffraction diagram; Figure 4 Particle size characterization diagram of the nano selenium composite particles in Example 1, wherein (a) is an electron microscope diagram of large particle size nano selenium composite particles, (b) is a particle size distribution diagram of large particle size nano selenium composite particles, (c) is an electron microscope diagram of medium particle size nano selenium composite particles, (d) is a particle size distribution diagram of medium particle size nano selenium composite particles, (e) is an electron microscope diagram of small particle size nano selenium composite particles, and (f) is a particle size distribution diagram of small particle size nano selenium composite particles; Figure 5 Effect diagram of the nano selenium composite particles on ABTS + radical scavenging effect in Application Example 1; Figure 6 Effect diagram of the nano selenium composite particles on DPPH radical scavenging effect in Application Example; Figure 7 Hemolysis result diagram of the nano selenium composite particles in Application Example 2; Figure 8 Stability result diagram of nano selenium composite particles of different particle sizes in simulated gastrointestinal fluid in Application Example 3, wherein (a) is a stability result diagram of small particle size nano selenium composite particles in simulated intestinal fluid, (b) is a stability result diagram of small particle size nano selenium composite particles in simulated gastric fluid, (c) is a stability result diagram of medium particle size nano selenium composite particles in simulated intestinal fluid, (d) is a stability result diagram of medium particle size nano selenium composite particles in simulated gastric fluid, (e) is a stability result diagram of large particle size nano selenium composite particles in simulated intestinal fluid, and (f) is a stability result diagram of large particle size nano selenium composite particles in simulated intestinal fluid; Figure 9 Test result diagram of the toxicity of nano selenium composite particles of different particle sizes on fibroblast L929. DETAILED DESCRIPTION

[0023] The technical solutions described in the present application will be further described in conjunction with the specific embodiments. However, the present application is not limited to this.

[0024] Referring to Figure 1 The present application provides a microfluidic system, comprising a microfluidic device, a mixing chip, a reaction chip and a collection container. The microfluidic device comprises a syringe pump controller and a plurality of syringe pumps, each of which is used to hold different raw materials; the mixing chip comprises two first input ends, a first output end and an annular mixing channel, and the two first input ends are arranged in parallel; the reaction chip comprises two second input ends, an annular reaction channel and a second output end, and the two second input ends are arranged at an angle of 79°. The syringe pumps, the mixing chip, the reaction chip and the collection container are connected by hoses.

[0025] Specifically, the syringe pumps comprise a first syringe pump for holding chitosan oligosaccharide solution, a second syringe pump for holding sodium selenite solution and a third syringe pump for holding L-ascorbic acid solution, wherein the first syringe pump and the second syringe pump are respectively connected with the two first input ends, the first output end is connected with one of the second input ends, the third syringe pump is connected with the other second input end, and the second output end is connected with the collection container. The mixing chip is a PDMS chip; the reaction chip is a PDMS chip. The injection flow rate of each syringe pump is 0.5-1.5 mL / h.

[0026] Example 1 1.1 Synthesis of nano selenium composite particles (1) Prepare chitosan oligosaccharide solution with a concentration of 3wt% (chitosan oligosaccharide purchased from Shanghai Aladdin Bio-Technology Co., Ltd., product number C302935), 10mM sodium selenite solution and L-ascorbic acid solution with concentrations of 20mM, 40mM and 250mM respectively.

[0027] (2) Use three groups of syringe pumps to suck 1mL of chitosan oligosaccharide solution, sodium selenite solution and air respectively, install the three groups of syringe pumps on the syringe pump controller, debug the microfluidic device, set the flow rate to 1mL / h and the time to 60min, start the microfluidic device, and let the chitosan oligosaccharide solution and the sodium selenite solution mix and fill the two chips.

[0028] (3) Remove the three groups of syringe pumps of step (1), install three groups of syringe pumps for sucking 1mL of L-ascorbic acid solution (40mM), chitosan oligosaccharide solution and sodium selenite solution respectively on the microfluidic device which has been debugged, start the device, set the flow rate to 1mL / h and the time to 60min, and collect the nano selenium dispersion.

[0029] (4) Repeat steps (2) and (3), and only change the concentration of L-ascorbic acid solution in step (3), and collect three groups of nano selenium dispersion.

[0030] (5) The three groups of nano selenium dispersion liquid were centrifuged, filtered and freeze-dried, the temperature of freeze-drying was -48℃, and the time was 72h; after freeze-drying, three kinds of nano selenium composite particles with different particle sizes were obtained, and they were all red solid powders.

[0031] 1.2 Characterization Referring to Figure 2 (a) The medium particle size nano selenium composite particles were characterized by element distribution mapping, Se element was from nano selenium, N element was from chitooligosaccharide, N element at nano selenium was obviously more concentrated, so chitooligosaccharide was wrapped in nano selenium, that is, the nano selenium composite particles were core-shell structure.

[0032] Referring to Figure 3 (a), the medium particle size nano selenium composite particles were characterized by infrared spectrum (FITC), the -OH group absorption band of COS was red shifted from 3406.87 to 3385.4, which indicated that there was hydrogen bond interaction between COS and SeNPs, the spectral absorption peak of C-O-H bond in COS-SeNPs was 1064.48, which was lower than 1069.80 of COS, it could be inferred that the C-O-H bond of COS was destroyed and combined with Se to form C-O-Se bond.

[0033] Referring to Figure 3 (b), the medium particle size nano selenium composite particles were characterized by X-ray photoelectron spectroscopy (XPS), the XPS spectrum of chitooligosaccharide nano selenium and chitooligosaccharide was compared, the Se peak was added in the spectrum of chitooligosaccharide nano selenium, which indicated that selenium element was introduced into chitooligosaccharide.

[0034] Referring to Figure 3 (c), the medium particle size nano selenium composite particles were characterized by X-ray diffraction (XRD), chitooligosaccharide and chitooligosaccharide nano selenium both had only one peak, and no typical diffraction peak attributed to the typical crystal structure of SeNP was detected, which was because chitooligosaccharide covered nano selenium, that is, the nano selenium composite particles were core-shell structure.

[0035] Similarly, the large particle size and small particle size nano selenium composite particles were characterized, both of which were core-shell structure of chitooligosaccharide and nano selenium.

[0036] The nano selenium composite particle powder was redissolved in water, and the particle size of each nano selenium composite particle was determined by transmission electron microscope (TEM), and the results were shown in Figure 4 Figure 4 (a) and Figure 4 ​(b) Nanometer selenium composite particles of large particle size (number average particle size D is 79.11004 nm) obtained by reducing sodium selenite solution with 250 mM L-ascorbic acid solution, wherein the particle size of the nanometer selenium core is 75 nm, and the thickness of the shell layer is 4.1104 nm; Figure 4 (c) and Figure 4 (b) Nanometer selenium composite particles of medium particle size (number average particle size D is 55.03827 nm) obtained by reducing sodium selenite solution with 40 mM L-ascorbic acid solution, wherein the particle size of the nanometer selenium core is 51 nm, and the thickness of the shell layer is 4.03827 nm; Figure 4 (e) and Figure 4 (f) Nanometer selenium composite particles of small particle size (number average particle size D is 28.44061 nm) obtained by reducing sodium selenite solution with 20 mM L-ascorbic acid solution, wherein the particle size of the nanometer selenium core is 25 nm, and the thickness of the shell layer is 3.44061 nm.

[0037] Example 1: Ability to scavenge in vitro oxidative substances 1.1 Scavenging activity on ABTS + radical (1) An ABTS radical solution was prepared by mixing a 7 mM 2,2'-azino-di (3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt solution (ABTS solution) and a 2.5 mM potassium persulfate solution; the ABTS radical stock solution was allowed to stand in the dark for 12 h, and then diluted with a phosphate buffer solution at pH 7.4 to obtain an ABTS radical solution having an absorbance of 1.0 ± 0.05 Abs at 734 nm. + radical stock solution was allowed to stand in the dark for 12 h, and then diluted with a phosphate buffer solution at pH 7.4 to obtain an ABTS radical solution having an absorbance of 1.0 ± 0.05 Abs at 734 nm. + radical stock solution was allowed to stand in the dark for 12 h, and then diluted with a phosphate buffer solution at pH 7.4 to obtain an ABTS radical solution having an absorbance of 1.0 ± 0.05 Abs at 734 nm. + radical solution.

[0038] (2) Each kind of nanometer selenium composite particle powder was respectively prepared into a nanometer selenium composite particle solution having a concentration of 0.6 mM, 0.5 mM, 0.4 mM, 0.3 mM and 0.2 mM by using water; 1 mL of the ABTS radical stock solution and 1 mL of the nanometer selenium composite particle solution were mixed, and the absorbance of the reaction solution at 734 nm was measured after mixing and reaction for 6 min in the dark, and a L-ascorbic acid solution having a concentration of 0.6 mM, 0.5 mM, 0.4 mM, 0.3 mM and 0.2 mM was used as a positive control. + radical stock solution and 1 mL of the nanometer selenium composite particle solution were mixed, and the absorbance of the reaction solution at 734 nm was measured after mixing and reaction for 6 min in the dark, and a L-ascorbic acid solution having a concentration of 0.6 mM, 0.5 mM, 0.4 mM, 0.3 mM and 0.2 mM was used as a positive control.

[0039] (3) The scavenging activity of each nanometer selenium composite particle solution on ABTS + radical (%) was calculated by using the following formula: ABTS + radical scavenging rate = 100% A a Absorbance of sample mixture, A b Absorbance of sample solution without ABTS, A c Absorbance of ABTS solution without nanose- lenium composite particles + Absorbance of free radical solution.

[0040] See Figure 5 There are differences in the scavenging effects of different particles, and the scavenging rate of small-particle-size nanose- lenium composite particles on ABTS + is higher than that of large-particle-size nanose- lenium composite particles.

[0041] 1.2 Scavenging activity on DPPH free radicals (1) Three kinds of nanose- lenium composite particles were each prepared into a nanose- lenium composite particle solution with a concentration of 0.4 mM, 0.3 mM, 0.2 mM, 0.1 mM and 0.05 mM using water; and a 50 mg / L DPPH ethanol solution was freshly prepared.

[0042] (2) 1 mL of the nanose- lenium composite particle solution with different concentrations and 1 mL of the DPPH ethanol solution were mixed, and the absorbance was measured at 517 nm after 30 min of light shielding, with ascorbic acid as a positive control. The following formula was used to calculate the DPPH free radical activity (%): DPPH scavenging activity = 100% A a Absorbance of sample mixture, A b Absorbance of sample solution without DPPH, A c Absorbance of DPPH solution without nanose- lenium composite particles.

[0043] See Figure 6 The scavenging rate of small-particle-size nanose- lenium composite particles on DPPH is higher than that of large-particle-size nanose- lenium composite particles.

[0044] Application Example 2 Hemolysis rate Fresh rabbit blood 1 mL was centrifuged at 3000 rpm for 10 min. The upper plasma was removed to separate the red blood cells and plasma, and PBS was added to the red blood cells for washing, followed by centrifugation. When the upper solution was colorless, PBS was finally used for expansion.

[0045] Positive control group: 900 μL of pure water was added to 100 μL of blood, and incubated in a 37° water bath for 1 h. The supernatant 100 μL was detected in the enzyme-labeled plate for the absorbance at 540 nm.

[0046] Negative control group: 900 μL of PBS was added to 100 μL of blood, which was incubated in a 37° water bath for 1 h, centrifuged using a centrifuge, and 100 μL of supernatant was taken to detect the absorbance at 540 nm in an enzyme-labeled plate.

[0047] Sample group: The nano-selenium composite particles were dissolved in PBS buffer as a nano-selenium composite particle solution (concentration of 25 mg / L, 50 mg / L, 100 mg / L), 900 μL of the nano-selenium composite particle solution was added to 100 μL of blood, which was incubated in a 37° water bath for 1 h, centrifuged using a centrifuge, and 100 μL of supernatant was taken to detect the absorbance at 540 nm in an enzyme-labeled plate. The hemolysis rate was calculated using the following formula: Hemolysis rate = (A540 nm of sample - A540 nm of negative control) / (A540 nm of positive control - A540 nm of negative control) × 100%. Referring to Figure 7 , the hemolysis rates of the three nano-selenium composite particles were all less than 1%, indicating that they had good blood compatibility.

[0048] Example 3 Stability in simulated gastrointestinal fluid Simulated gastric fluid: 0.1% w / v pepsin was added to 45 mL of pure water, the pH of the solution was adjusted to 1.2 using HCL, and the solution was mixed. The prepared nano-selenium composite particle solution (1 mg / mL, 5 mL) was added to the artificial gastric fluid and divided into centrifuge tubes, 5 mL in each tube, and incubated in a 37°C constant temperature shaker at 160 r / min. Every 1 h, one tube was taken out for centrifugation (15000 rpm, 15 min), the precipitate was removed, and the supernatant was collected. The supernatant was boiled and filtered for ICP-MS testing.

[0049] Simulated intestinal fluid: 1% w / v trypsin was added to 45 mL of pure water, the pH of the solution was adjusted to 7 using NaOH, and the solution was mixed. The prepared nano-selenium composite particle solution (1 mg / mL, 5 mL) was added to the artificial intestinal fluid and divided into centrifuge tubes, 5 mL in each tube, and incubated in a 37°C constant temperature shaker at 160 r / min. Every 1 h, one tube was taken out for centrifugation (15000 rpm, 15 min), the precipitate was removed, and the supernatant was collected. The supernatant was boiled and filtered for ICP-MS testing.

[0050] The release rate of the nano-selenium composite particles in the simulated gastrointestinal fluid was calculated using the following formula: Release rate = (A540 nm of sample - A540 nm of negative control) / (A540 nm of positive control - A540 nm of negative control) × 100%.

[0051] Referring to Figure 8 , the release rates of the three nano-selenium composite particles increased with time in the simulated intestinal fluid and simulated gastric fluid.

[0052] ​​In simulated intestinal fluid for 5 hours, Figure 8 (a) the selenium release rate of the small-particle-size nano-selenium composite particles is less than 0.10%; Figure 8 (c) the selenium release rate of the particle-size nano-selenium composite particles is less than 1.3%; Figure 8 (e) the selenium release rate of the large-particle-size nano-selenium composite particles is less than 3.9%.

[0053] In simulated gastric fluid for 5 hours, Figure 8 (b) the selenium release rate of the small-particle-size nano-selenium composite particles is less than 0.015%. Figure 8 (d) the selenium release rate of the particle-size nano-selenium composite particles is less than 0.4%. Figure 8 (f) the selenium release rate of the large-particle-size nano-selenium composite particles is less than 0.95%. The results show that the nano-selenium composite particles have good stability in simulated intestinal fluid and simulated gastric fluid.

[0054] Application Example 4 Cytotoxicity The cytotoxicity of the three groups of nano-selenium composite particles on fibroblast L929 cells was determined by the MTT method. 1×10 4 L929 cells were inoculated in a 96-well plate, and the plate was incubated at 37°C in 5% CO2 for 24 hours. After the cells adhered, the old culture medium was removed. Different concentrations of nano-selenium composite particle solutions (0 mg / L, 10 mg / L, 20 mg / L, 40 mg / L, 80 mg / L, and 160 mg / L, respectively) were added to the L929 complete culture medium, and the medium was added to the 96-well plate as the new culture medium. After 24 hours in the incubator, 10 μL of MTT solution was added to each well, and the plate was incubated for 4 hours. The supernatant was removed, 100 μL of DMSO was added, and the 96-well plate was placed in a constant temperature shaker for 10 minutes. The absorbance at 570 nm was measured using a microplate reader. The cell viability was calculated using the following formula: Cell survival rate (%) = 100%.

[0055] Referring to Figure 9 , the nano-selenium composite particles of the present application have low cytotoxicity to L929 cells. When the concentration of the nano-selenium composite particle solution is as high as 160 mg / L, the cell survival rate is more than 40%. Since the particle sizes of the three nano-selenium composite particles are not significantly different, the toxicity difference is not significant.

[0056] The above-described embodiments are merely illustrative of several embodiments of the present application, and the description is relatively specific and detailed, but should not be understood as a limitation on the scope of the patent. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the scope of the present application. Therefore, the scope of protection of the present application should be subject to the appended claims.

Claims

1. Nanoselenium composite particles, characterized in that, The nano selenium composite particle comprises a core of nano selenium and a shell layer wrapping the core, the material of the shell layer is chitooligosaccharide, the particle size of the core is 15-95 nm, and the particle size of the nano selenium composite particle is 20-100 nm.

2. The nano-selenium composite particle according to claim 1, characterized by, The particle size of the nano selenium composite particle is 50-60 nm.

3. The nano-selenium composite particle according to claim 1, characterized by, The thickness of the shell layer is 3-10 nm.

4. The nano-selenium composite particle according to claim 1, characterized by, The number average molecular weight of the chitooligosaccharide is 300-2000 Da.

5. The method of producing nano-selenium composite particles according to any one of claims 1 to 4, characterized by, The method comprises the following steps: Mixing a chitooligosaccharide solution and a selenium source solution to obtain a premix solution, and then mixing the premix solution with a reducing agent to obtain a nano selenium composite particle dispersion liquid; Centrifuging, filtering and freeze-drying the nano selenium composite particle dispersion liquid to obtain the nano selenium composite particle.

6. The production method according to claim 5, wherein The molar ratio of effective components in the selenium source solution and the reducing agent is 1:2-25.

7. The preparation method according to claim 5, characterized in that, The concentration of the chitooligosaccharide solution is 2-5 wt%.

8. The preparation method according to claim 5, characterized in that, The selenium source solution is a selenite solution with a concentration of 5-15 mM.

9. The preparation method according to claim 5, characterized in that, The temperature of the freeze-drying is-40--50℃, and the time is 48-96 h.

10. The nano-selenium composite particles according to any one of claims 1 to 4 for use in scavenging ABTS + radicals and DPPH radicals.

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