Synthesis And Biological Activity Of Grateloupia Livida Polysaccharides-Functionalized Selenium Nanoparticles

Selenium(Se)is an essential trace element for human.Selenium nanoparticles(SeNPs)has gradually become a research hotspot in the field of nanomedicine due to its unique physical and chemical properties,low toxicity,and high biological activity.However,its stability is poor and easily transform into thermally stable biologically inert selenium.In order to improve the stability and biological activity of selenium nanoparticles,the functional selenium nanoparticles(GLP-SeNPs)were prepared with Grateloupia Livida polysaccharide(GLP)as dispersant and stabilizer.A series of characterization methods were used to analyze the morphology and stability of GLP-SeNPs.The antioxidant activity,antitumor activity,safety and drug delivery performance were also studied,so as to provide a theoretical basis for the preparation and application of green,high-efficiency and low-toxic selenium dietary supplements and anti-tumor drugs.The research results are as follows:(1)Single factor test and response surface analysis were used to optimize the extraction process of GLP,and the optimum extraction separating technics parameter the best process conditions were obtained as follows:ultrasonic temperature 80℃,ultrasonic time 60 min,liquid-to-material ratio 90 mL/g,ultrasonic power 360 W,under these conditions,the extraction rate of GLP was 50.74±0.12%.After deproteinization,alcohol precipitation,reconstitution,dialysis and freeze-drying of GLP,the polysaccharide content measured by the phenol-sulfuric acid method was 89.56%,and the uronic acid content measured by the sulfuric acid-carbazole method was 3.28%.The results of gel chromatography-differential-multi-angle laser light scattering technology(GPC-RI-MALLS)demonstrated that number-average molar weight(Mn)and average molecular weight(Mw)of GLP were 178.9 kDa and 323.1 kDa,respectively.The results of HPLC showed that GLP is composed of fucose(0.21%),galactose(89.13%),glucose(4.03%),xylose(1.08%),mannose(0.71%),fructose(1.81%),guluronic acid(0.38%)and glucuronic acid(2.65%).(2)GLP-SeNPs was prepared by in-situ reduction of ascorbic acid(Vc)by using GLP as dispersant and stabilizer and sodium selenite(Na2SeO3)as selenium source,and the optimal synthesis process was discussed through single factor and orthogonal experiments.Under the conditions of reaction temperature of 45℃,reaction time of 3 h,polysaccharide concentration of 1.0 mg/mL and Vc concentration of 0.04 M,the prepared GLP-SeNPs have a particle size of 115.54±2.7 nm.The morphology of GLP-SeNPs was analyzed by transmission electron microscope(TEM)and scanning electron microscope(SEM),and the results showed that GLP-SeNPs are monodisperse,uniform spherical particles.The results of X-ray photoelectron spectroscopy(XPS),X-ray diffraction(XRD)and energy dispersive X-ray spectroscopy(EDX)analysis show that Se in GLP-SeNPs is amorphous elemental selenium,with a content of 36.49%.In addition,the stability of GLP-SeNPs was evaluated by observing changes in sample color,particle size distribution and Zeta potential.The results showed that the stability of GLP-SeNPs has been significantly improved compared with SeNPs without GLP modification.(3)The antioxidant activity of GLP-SeNPs was investigated by free radical scavenging experiment.The results showed that GLP-SeNPs possess stronger scavenging ability to DPPH,ABTS,hydroxyl radical and superoxide anion radical compared with GLP and Na2SeO3.Within the experimental concentration range,the scavenging ability increased with the increase of concentration.The IC50 of scavenging DPPH,ABTS,hydroxyl radical and superoxide anion radical were 79.57±5.23 μg/mL,27.13±3.25 μg/mL,258.48±6.15μg/mL and 314.54±7.61 μg/mL,respectively,indicating that GLP-SeNPs possess good antioxidant activity and has the potential to be developed into antioxidants.(4)The CCK-8 method was used to study the inhibitory effect of GLP-SeNPs on the proliferation of human lung cancer cell A549,human breast cancer cell MCF-7,human liver cancer cell HepG2 and human renal tubular epithelial cell HK-2.The results showed that GLP-SeNPs can effectively inhibit the proliferation of a variety of tumor cells,among them,the inhibitory effect on A549 cells was the strongest(IC50=5.73±2.33 μM),and the effect on normal cells HK-2 was small(IC50=270.36±5.59 μM).In addition,flow cytometry,annexin V-FITC/PI double staining,TUNEL and DAPI double staining and JC-1 staining showed that GLP senps could inhibit the proliferation of A549 cells by inducing S-phase arrest and apoptosis.The results of hemolysis experiments showed that the hemolysis rate of GLP-SeNPs was much lower than that of Na2SeO3 at the same concentration of Se,indicating that GLP-SeNPs possess good blood compatibility.The Bliss method was used to study the acute oral toxicity of GLP-SeNPs to SPF-grade KM mice,and the median lethal dose(LD50)was calculated to be 104.72 mg Se/kg(with 95%confidence limits of 80.53-135.97 mg Se/kg),indicating that GLP-SeNPs are less toxic and have broad application prospects in the field of in food industry and medicine.(5)The drug loading and release properties of GLP-SeNPs were studied using 5-fluorouracil(5-Fu)was used as a model drug.The results of infrared spectroscopy(FT-IR),XRD and DLS proved that 5-Fu was successfully loaded into the carrier.The average particle size of GLP-SeNPs@5-Fu is 154.8 nm,and the drug loading is 21.36%.The in vitro release test results show that the release of 5-Fu has a certain pH selectivity,which provides a reference for subsequent research on its targeted anticancer effect.