Water-soluble monodisperse selenium nanoparticles as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nanoparticles, and particularly relates to water-soluble monodisperse selenium nanoparticles as well as a preparation method and application thereof. The water-soluble monodisperse selenium nano-particles have spherical structures and comprise selenium nano-particles and polysaccharide coated on the surfaces of the selenium nano-particles; the polysaccharide is one of oxidized xyloglucan and xyloglucan. Xyloglucan is extracted from tamarind fruits, oxidized xyloglucan is obtained by oxidizing xyloglucan, the selenium nanoparticles are coated with oxidized xyloglucan or a xyloglucan solution as a soft template, a selenium source and a reducing agent are used as raw materials, a one-pot oxidation-reduction reaction is carried out, and the selenium nanoparticles are obtained through the processes of centrifugation, washing, freeze-drying and the like. The prepared oxidized xyloglucan / xyloglucan selenium nanoparticles have the advantages of small particle size, dispersion stability, uniform morphology and the like, and have great potential in the fields of nano-selenium supplements, wound repair, immunoregulation, anti-tumor treatment and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanoparticles, and in particular relates to water-soluble monodisperse selenium nanoparticles and a preparation method and application thereof. Background Art

[0002] Selenium is an essential trace element for the human body, and selenium deficiency has been reported to be associated with various diseases. Selenium has antibacterial, antiviral, antitumor, and immunomodulatory effects. Selenium is primarily formed in the human body into certain enzymes, most of which have antioxidant properties, such as glutathione peroxidase (GSH-Px). Glutathione peroxidase participates in cell protection by scavenging intracellular free radicals. However, its availability is significantly limited by the narrow gap between its biologically effective dose and toxic dose. Consequently, considerable effort has been devoted to developing a low-toxic, highly effective source of selenium for use in dietary supplements.

[0003] Selenium nanoparticles (SeNPs) are increasingly favored due to their high bioactivity and low toxicity, and exhibit unique functions due to their nanosize. However, SeNPs are generally unstable and easily aggregate into gray and black elemental selenium, resulting in low bioactivity and bioavailability, which limits their application. Since the concept of "green chemistry" was proposed, chemical reduction has been a common environmentally friendly method for synthesizing SeNPs. In particular, chemical reduction methods in the presence of dispersants or stabilizers provide a good method for preparing SeNPs. During the preparation process, soft template regulators are usually used to control the agglomeration or aggregation of selenium nanoparticles to obtain SeNPs with ideal particle size and morphology. However, their efficacy is based on the stabilizing effect of the template. That is, SeNPs without the addition of a template are extremely prone to aggregation and passivation and cannot exert their activity, which has become a bottleneck restricting the application of SeNPs. Summary of the Invention

[0004] In the prior art, selenium nanoparticles are unstable and prone to aggregation, resulting in low bioactivity and bioavailability. The present invention aims to provide water-soluble, monodisperse selenium nanoparticles, their preparation method, and applications, thereby overcoming the shortcomings of the prior art. By introducing oxidized xyloglucan / xyloglucan in a one-pot process, the stability and dispersibility of nano-selenium are improved. The resulting oxidized xyloglucan / xyloglucan selenium nanoparticles have significant potential in the fields of nano-selenium supplementation, wound repair, immunomodulation, and anti-tumor therapy.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] In a first aspect, the present invention provides a water-soluble monodisperse selenium nanoparticle having a spherical structure, comprising selenium nanoparticles and a polysaccharide coated on their surface;

[0007] The polysaccharide is one of oxidized xyloglucan and xyloglucan.

[0008] In some other embodiments, the mass ratio of selenium nanoparticles to polysaccharide in the water-soluble monodisperse selenium nanoparticles is (30-35): (6.0-105);

[0009] The average molecular weight of the unit sugar ring of the oxidized xyloglucan is 90-100 g / mol, the degree of oxidation is 34-38%, and the aldehyde content is 4.0-5.0 mmol / g;

[0010] The average molecular weight of the unit sugar ring of the xyloglucan is 145-150 g / mol, and the hydroxyl content is 13.0-14 mmol / g;

[0011] Preferably, the mass ratio of selenium nanoparticles to polysaccharide in the water-soluble monodisperse selenium nanoparticles is 34.6:(6.4-102.4);

[0012] The average molecular weight of the unit sugar ring of the oxidized xyloglucan is 95.8 g / mol, the degree of oxidation is 35.78±1.5%, and the aldehyde content is 4.8 mmol / g;

[0013] The average molecular weight of the unit sugar ring of the xyloglucan is 149.1 g / mol, and the hydroxyl content is 13.4 mmol / g.

[0014] In some other embodiments, the average particle size of the water-soluble monodisperse selenium nanoparticles is 30-120 nm.

[0015] In a second aspect, the present invention provides a method for preparing the water-soluble monodisperse selenium nanoparticles according to the first aspect, comprising the following steps:

[0016] (1) mixing a polysaccharide solution with a selenium source solution to obtain a mixed solution;

[0017] (2) adding the reducing agent solution dropwise into the mixed solution to react, thereby preparing an aqueous solution of polysaccharide selenium nanoparticles;

[0018] (3) The polysaccharide selenium nanoparticles aqueous solution is centrifuged, washed, and freeze-dried to obtain polysaccharide selenium nanoparticles.

[0019] In some other embodiments, in step (1), the polysaccharide solution is one of an oxidized xyloglucan solution and a xyloglucan solution;

[0020] The concentration of the oxidized xyloglucan solution is 0.64-5.12 mg / mL;

[0021] The concentration of the xyloglucan solution is 0.64-2.56 mg / mL;

[0022] The concentration of the selenium source solution is 10-20 mM;

[0023] The volume ratio of the polysaccharide solution to the selenium source solution is (0.5-1.5):1;

[0024] The selenium source is one or more of sodium selenite, selenous acid, selenium dioxide and sodium selenite pentahydrate.

[0025] In some other embodiments, in step (1), the method for preparing oxidized xyloglucan comprises the following steps: dissolving xyloglucan in water to obtain a xyloglucan solution, adding sodium periodate to carry out a light-proof reaction, adding ethylene glycol to terminate the reaction and keeping the solution in the dark overnight, dialyzing, filtering, pre-freezing, and freeze-drying to obtain the oxidized xyloglucan.

[0026] In some other embodiments, the mass ratio of xyloglucan to sodium periodate is 1:(0.05-0.15);

[0027] The mixing ratio of xyloglucan to water is 1 g: (90-110) mL;

[0028] The light-proof reaction is carried out at room temperature for 3-5 hours;

[0029] The dialysis is performed in deionized water for 20-25 hours using a dialysis bag with a molecular weight cut-off of 8-14 kDa;

[0030] In some other embodiments, the xyloglucan is prepared by mixing tamarind gum with water, stirring and centrifuging, and then freeze-drying the supernatant to obtain xyloglucan.

[0031] Preferably, the mixing ratio of tamarind gum and water is 10g:(0.9-1.1)L;

[0032] The stirring is performed at 35-45° C. for 20-30 hours;

[0033] The centrifugation is performed at 7500-8000 rpm for 3-6 minutes.

[0034] In some other embodiments, in step (2), the concentration of the reducing agent solution is 40-80 mM;

[0035] The volume ratio of the reducing agent solution to the mixed solution is 1:(1-1.5);

[0036] The reaction is stirred at room temperature in the dark for 4-12 hours;

[0037] The reducing agent is one of L-ascorbic acid solution and hydrazine monohydrate.

[0038] In some other embodiments, in step (3), the centrifugal force of the centrifugation is 16000-18000 g, the centrifugation time is 15-45 min, and the centrifugation temperature is 20-25° C.;

[0039] The freeze-drying temperature is -20°C to -80°C, and the time is 2-8 hours.

[0040] In a third aspect, the present invention provides the use of the water-soluble monodisperse selenium nanoparticles described in the first aspect in the preparation of injectable hydrogels and selenium supplement products.

[0041] Beneficial effects of the present invention:

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1. The present invention provides an oxidized xyloglucan / xyloglucan selenium nanoparticle. The present invention stabilizes selenium nanoparticles with oxidized xyloglucan or xyloglucan to obtain uniformly dispersed selenium nanoparticles, thereby improving the utilization rate of nano-selenium and further enhancing the application potential of nano-selenium in the fields of nano-selenium supplements, wound repair, immunomodulation and anti-tumor treatment.

[0044] 2. The present invention provides a method for preparing oxidized xyloglucan-selenium nanoparticles that is simple, easy to operate, and has a high success rate. Furthermore, the oxidized xyloglucan coating of the selenium nanoparticles can be used to form a special injectable hydrogel, further expanding the application of nano-selenium. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0046] Figure 1 Transmission electron microscopy image and particle size statistics of oxidized xyloglucan selenium nanoparticles prepared in Example 1 of the present invention;

[0047] Figure 2 The scanning electron microscope image and particle size statistics of oxidized xyloglucan selenium nanoparticles prepared in Example 1 of the present invention;

[0048] Figure 3 These are the ultraviolet and infrared spectra of sodium selenite (Na2SeO3), L-ascorbic acid (Vc), selenium nanoparticles (Se), and oxidized xyloglucan selenium nanoparticles (OXG@SeNPs) in Example 1 of the present invention, where (a) is the ultraviolet spectrum; (b) is the infrared spectrum;

[0049] Figure 4 IR spectra of selenium nanoparticles (Se), oxidized xyloglucan (OXG), and oxidized xyloglucan selenium nanoparticles (OXG@SeNPs) in Example 1 of the present invention;

[0050] Figure 5 1 is an X-ray diffraction analysis diagram of selenium nanoparticles (Se), oxidized xyloglucan (OXG), and oxidized xyloglucan selenium nanoparticles (OXG@SeNPs) in Example 1 of the present invention;

[0051] Figure 6 The structural formula of xyloglucan extracted from Example 1 of the present invention and the schematic diagram of the reaction to generate oxidized xyloglucan are shown in Figure 1. (a) is the chemical structure of xyloglucan extracted from tamarind gum and (b) is a schematic diagram of the periodate oxidation of XG.

[0052] Figure 7 The infrared spectrum and hydrogen nuclear magnetic resonance spectrum of the oxidized xyloglucan produced in Example 1 of the present invention are shown, wherein (a) is the infrared spectrum; (b) is the hydrogen nuclear magnetic resonance spectrum;

[0053] Figure 8 The titration curve and its first-order derivative of the oxidized xyloglucan produced in Example 1 of the present invention;

[0054] Figure 9 Transmission electron micrographs of selenium nanoparticles prepared using deionized water (H2O) and oxidized xyloglucan (OXG 2.56 mg / mL) in Example 1 of the present invention;

[0055] Figure 10 The scanning electron micrographs of selenium nanoparticles prepared using deionized water (H2O) and oxidized xyloglucan (OXG 2.56 mg / mL) in Example 1 of the present invention are shown;

[0056] Figure 11 Gaussian particle size distribution and average particle size statistical histogram of selenium nanoparticles prepared with deionized water (H2O) and oxidized xyloglucan (OXG2.56 mg / mL) in Example 1 of the present invention, wherein (a) Gaussian particle size distribution and (b) average particle size statistical histogram;

[0057] Figure 12 This is a graph showing the scavenging capacity of Na2SeO3, Se and SeNPs@OXG for DPPH free radicals in Example 1 of the present invention;

[0058] Figure 13 This is a diagram showing macroscopic observation and Tyndall effect observation of selenium nanoparticles prepared from oxidized xyloglucan solutions of different concentrations in Example 2 of the present invention;

[0059] Figure 14Transmission electron microscopy observations of selenium nanoparticles prepared from oxidized xyloglucan solutions of different concentrations in Example 2 of the present invention;

[0060] Figure 15 Gaussian particle size distribution diagram and average particle size statistical histogram of selenium nanoparticles prepared from oxidized xyloglucan solutions of different concentrations in Example 2 of the present invention, wherein (a) Gaussian particle size distribution diagram and (b) average particle size statistical histogram;

[0061] Figure 16 Transmission electron microscopy and macroscopic observation images of selenium nanoparticles prepared from xyloglucan solutions with different concentrations in Example 3 of the present invention. DETAILED DESCRIPTION

[0062] Those skilled in the art will appreciate that the following examples are intended to illustrate the present invention only and should not be construed as limiting the scope of the invention. Unless otherwise specified, the examples were performed under conventional conditions or manufacturer's recommended conditions. Components used without manufacturer's indication are commercially available conventional products.

[0063] Polymeric nanoparticles based on biocompatible and biodegradable polymers have attracted considerable interest in recent years. Among these bioinspired nanocomposites, polysaccharides are considered the most promising source for nanoparticle production due to their relative stability and cost-effectiveness. Therefore, polysaccharides can serve as ideal templates for synthesizing nanoparticles using modern nanotechnology.

[0064] Xyloglucan (XG) is derived from the primary cell wall of monocot seeds. It is a high molecular weight neutral branched polysaccharide with a cellulose backbone containing galactose-substituted side chains. It is characterized by the fact that approximately 75% of the glucose units on the cellulose backbone are replaced by (1-6)-α-xylose branches, and some xylose residues are further replaced by (1-2)-β-galactose. The unique branched structure of XG not only makes it highly viscous in water, but also plays a role in promoting cell aggregation and tissue regeneration during wound healing. In particular, XG contains a large number of hydroxyl groups, which react with the selective oxidant periodate (NaIO4) to specifically oxidize and cleave the vicinal diols in the XG polysaccharide chain into dialdehyde structures to generate oxidized xyloglucan (OXG).

[0065] The present invention provides oxidized xyloglucan / xyloglucan-selenium nanoparticles. The oxidized xyloglucan / xyloglucan-selenium nanoparticles are prepared using oxidized xyloglucan or xyloglucan, a selenium source, and a reducing agent as raw materials through an oxidation-reduction reaction. The present invention employs a one-pot preparation process for the oxidation-reduction reaction, comprising the following steps:

[0066] The oxidized xyloglucan solution is mixed with a sodium selenite solution to obtain a uniform clear mixed solution; an appropriate amount of ascorbic acid solution is added dropwise into the stirred mixed solution, and the mixture is reacted in a light-proof environment to finally obtain an aqueous solution of oxidized xyloglucan selenium nanoparticles; the aqueous solution of oxidized xyloglucan selenium nanoparticles is centrifuged and washed multiple times, and the final precipitate is freeze-dried to obtain oxidized xyloglucan selenium nanoparticles.

[0067] The materials and reagents used in the following examples are as follows:

[0068] (1) Main reagents

[0069] Xyloglucan (XG) was extracted from tamarind seed powder using a water-immersion method: 10 g of tamarind seed powder was added to 1 L of deionized water and stirred at 40°C for 24 hours. The resulting suspension was centrifuged at 8000 rpm for 5 minutes to remove insoluble matter, and the supernatant was freeze-dried to obtain the xyloglucan raw material.

[0070] The average molecular weight of the sugar ring of the obtained xyloglucan is 149.1 g / mol, and the hydroxyl content is 13.4 mmol / g.

[0071] The temperature during the centrifugation process must be controlled at 4°C;

[0072] Among them, it must be pre-frozen overnight at -20℃ before freeze-drying.

[0073] The source of tamarind powder is Tokyo Chemical Industry Co., Ltd.; the specific reagent name is tamarind gum.

[0074] Oxidized xyloglucan (OXG) is prepared by oxidizing xyloglucan with sodium periodate. The selective oxidant, periodate (NaIO4), specifically oxidizes and cleaves the vicinal diols in the XG polysaccharide chains into dialdehyde structures, thereby generating OXG. The specific steps are as follows: 1 g of xyloglucan is completely dissolved in 100 mL of deionized water. In the dark, 0.1 g of sodium periodate is slowly added while stirring to dissolve. After 4 hours of reaction, 1 mL of ethylene glycol is slowly added dropwise and stirred for 2 hours to terminate the reaction. The reaction solution is then transferred to a dialysis bag and dialyzed against deionized water for 24 hours. After dialysis, the OXG is freeze-dried to obtain the resulting solution.

[0075] The dialysis was performed using a dialysis bag with a molecular weight cutoff of 8-14 kDa. The obtained oxidized xyloglucan had an average molecular weight per sugar ring of 95.8 g / mol, an oxidation degree of 35.78±1.5%, and an aldehyde content of 4.8 mmol / g.

[0076] Sodium selenite was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0077] L-Ascorbic acid was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0078] (2) Main instruments

[0079] LCA-MSA-HD magnetic stirrer: Shanghai Lichen Technology Co., Ltd., China; H2050R benchtop high-speed refrigerated centrifuge: Hunan Xiangyi Co., Ltd., China; FTIR-850 Fourier transform infrared spectrometer: Tianjin Gangdong Technology Co., Ltd., China; Malvern Zetasizer Nano ZSE nanoparticle size analyzer: Malvern, UK; Hitachi transmission electron microscope HT7700 Exalens: Hitachi, Japan; UV spectrophotometer TU-1900: Beijing Puxi General Instrument Co., Ltd., China; X-ray diffractometer DMAX-2500PC: Rigaku Corporation, Japan; AVANCE NEO 400 nuclear magnetic resonance spectrometer: Bruker Scientific Instruments, Germany.

[0080] Example 1

[0081] A method for preparing oxidized xyloglucan-coated selenium nanoparticles comprises the following steps:

[0082] 51.2 mg of oxidized xyloglucan (OXG) prepared as described above was weighed and dissolved in 20 mL of deionized water to prepare a 2.56 mg / mL oxidized xyloglucan solution. 10 mL of a 20 mM sodium selenite solution was added to the oxidized xyloglucan solution, and a uniform mixture was formed under magnetic stirring. Then, 10 mL of a freshly prepared 80 mM L-ascorbic acid solution was added dropwise to the mixture under vigorous stirring. The mixture was stirred in the dark at 37°C for 12 hours, and the solution gradually changed from colorless and transparent to orange-red and eventually to reddish-brown. Oxidized xyloglucan selenium nanoparticles were generated in the solution and labeled as OXG@SeNPs.

[0083] In this embodiment, an excess of L-ascorbic acid is used to completely react sodium selenite, so the oxidized xyloglucan selenium nanoparticles are subsequently purified and washed by centrifugation and repeated washing.

[0084] The total solution was divided into 30 mL centrifuge tubes. 10 mL of the reacted mixed solution was added to each tube. The centrifugation parameters were set as follows: centrifugal force 18,000 g, centrifugation time 30 min, and centrifugation temperature 20°C. After centrifugation, the upper clear solution was discarded, retaining the reddish-brown precipitate. 10 mL of deionized water was then added to each tube, and the centrifugation operation was repeated three times to ensure cleanliness. The remaining precipitate was pre-frozen at -20°C for 2 hours and freeze-dried for 24 hours to obtain oxidized xyloglucan selenium nanoparticles.

[0085] The oxidized xyloglucan-selenium nanoparticles prepared in this example were dissolved in deionized water, added dropwise to a 200-mesh copper grid and allowed to dry. The particle size of the oxidized xyloglucan-selenium nanoparticles was observed by transmission electron microscopy. Figure 1 Transmission electron microscopy images and particle size statistics of oxidized xyloglucan selenium nanoparticles prepared for this example.

[0086] like Figure 1 As shown, OXG@SeNPs are uniform in size, water-soluble, and well-dispersed. This indicates that they are uniformly dispersed in the solution as spheres. Compared to existing selenium nanoparticles, the particle size is relatively small, with a particle size distribution of 31.67 ± 8.55 nm, as shown in the particle size statistics.

[0087] The OXG@SeNPs prepared in this example were evenly pasted onto the surface of the conductive adhesive and then treated with gold spraying for 15 seconds to take a scanning electron microscope photo. Figure 2 Scanning electron microscope images and particle size statistics of oxidized xyloglucan selenium nanoparticles prepared for this example.

[0088] like Figure 2 As shown, the freeze-dried powder of OXG@SeNPs prepared in this example (the sample surface is coated with more oxidized xyloglucan dissolved in the solution) appears as uniform spheres of relatively consistent size and a relatively complete spherical structure, with an average particle size of 75.52±17.13nm. Due to the surface coating of OXG, the larger average size observed in transmission electron microscopy indicates the successful formation of selenium nanoparticles and the maintenance of a complete and uniform spherical structure.

[0089] To further illustrate the improvement of the present invention's oxidized xyloglucan on existing problems in the preparation of selenium nanoparticles, in this example, selenium nanoparticles without the addition of oxidized xyloglucan were prepared using the same steps and parameters as above. The only difference was that 20 mL of the 2.56 mg / mL OXG solution was replaced with 20 mL of deionized water.

[0090] The UV absorbance in the range of 210-400 nm was measured using a UV-visible spectrophotometer. The Fourier transform infrared (FT-IR) spectrum was obtained in the wavenumber range of 4000-600 cm -1 The wavelength range was recorded by ATR accessory. The UV and IR spectra of the raw materials were measured respectively. The results are shown in Figure 3 .

[0091] The results are as follows Figure 3As shown in the figure, the ultraviolet and infrared spectra of sodium selenite (Na2SeO3), L-ascorbic acid (Vc), selenium nanoparticles (Se) and oxidized xyloglucan selenium nanoparticles (OXG@SeNPs), where (a) is ultraviolet and (b) is infrared spectrum.

[0092] observe Figure 3 In (a), the absorption peak of selenium nanoparticles was observed at 269 nm, consistent with relevant reports. The successful cleaning of the surface preparation materials and the successful formation of selenium nanoparticles are consistent with relevant reports. The absorption peak of OXG@SeNPs shifted after the introduction of oxidized xyloglucan, indicating that the successful coating of OXG on the surface of selenium nanoparticles caused the shift in the absorption peak.

[0093] To further illustrate the successful cleaning of the raw materials and further illustrate the chemical bonds contained within the selenium nanoparticles, the Figure 3 The Fourier transform infrared spectrum (FTIR) in (b) shows that the selenium nanoparticles (Se) lack obvious chemical bonding, possibly indicating an amorphous state. Oxidized xyloglucan selenium nanoparticles (OXG@SeNPs) exhibit characteristic peaks of oxidized xyloglucan. Neither of these exhibits characteristic peaks of Na₂SeO₃ nor Vc, indicating that the cleaning method used in this example meets cleaning standards.

[0094] To further demonstrate that oxidized xyloglucan was successfully coated on the surface of selenium nanoparticles, selenium nanoparticles (Se), oxidized xyloglucan (OXG), and oxidized xyloglucan selenium nanoparticles (OXG@SeNPs) were placed on the ATR accessory of a Fourier transform infrared (FTIR) spectrometer, and the wavenumbers were recorded at 4000-600 cm -1 Infrared spectra in the wavenumber range, such as Figure 4 shown.

[0095] observe Figure 4 , it was found that Se itself does not contain relevant groups, but after adding OXG solution during the preparation process, OXG@SeNPs were prepared, and the characteristic groups of OXG continued on its surface, such as Figure 4 Medium 1100cm -1 Ether peaks (COC) on the left and right, 3400 cm -1 The hydroxyl peaks (OH) on the left and right and 1730 cm -1 The aldehyde peaks (C=O) on the left and right were well continued, indicating that OXG was successfully coated on the surface of SeNPs.

[0096] The diffraction intensity of selenium nanoparticles (Se), oxidized xyloglucan (OXG) and oxidized xyloglucan selenium nanoparticles (OXG@SeNPs) at 5-90° was observed by X-ray diffractometer to analyze their phase structures, such as Figure 5 shown.

[0097] observe Figure 5 It was found that the X-ray diffraction peaks of selenium nanoparticles (Se), oxidized xyloglucan (OXG) and oxidized xyloglucan selenium nanoparticles (OXG@SeNPs) were all large broad peaks, indicating that all three were amorphous, and OXG was coated on the surface of SeNPs as a soft template, forming a uniform spherical morphology.

[0098] The present invention is directed to the structural formula of xyloglucan extracted from tamarind gum fruit and the basic structure of oxidized xyloglucan generated by the reaction, such as Figure 6 As shown in the figure, (a) is the chemical structure of xyloglucan extracted from tamarind gum and (b) is a schematic diagram of the periodate oxidation of XG.

[0099] like Figure 6 As shown, periodate is a highly specific and selective oxidant that can specifically oxidize and cleave vicinal diols in polysaccharide chains into dialdehyde structures. Periodate oxidation has been widely used in the biopharmaceutical field for the oxidative modification of polysaccharides. The present invention utilizes this reaction to obtain aldehyde-modified oxidized xyloglucan.

[0100] In order to further illustrate the successful synthesis of oxidized xyloglucan and further prove its structural characteristics, infrared spectroscopy and nuclear magnetic resonance spectroscopy were used to verify the successful modification of xyloglucan, such as Figure 7 As shown, (a) is the infrared spectrum and (b) is the nuclear magnetic resonance hydrogen spectrum.

[0101] observe Figure 7 (a) Infrared spectrum of OXG, compared with XG, the 1730cm -1 The new peak at 3370 cm-1 can be attributed to the symmetrical vibration of -C=O in the aldehyde group, but this peak is not obvious, which may be due to the formation of hemiacetal between the free aldehyde group and the hydroxyl group, resulting in a decrease in the peak intensity. In addition, the generation of -C=O promotes the strengthening of hydrogen bonds, resulting in a peak at 3370 cm-1. -1 The hydroxyl stretching vibration peak at 3370 cm -1 Move to 3363cm -1 ).observe Figure 7 (b) H NMR spectrum: The new peaks at 5.51 and 5.66 ppm are the result of the shift of the methylene peaks around the aldehyde groups. Both IR and H NMR spectra confirm the successful modification of xyloglucan.

[0102] In order to further determine the oxidation degree of the oxidized xyloglucan prepared in the present invention, the oxidation degree of OXG was determined by hydroxylamine hydrochloride titration. One molecule of aldehyde group reacts with hydroxylamine hydrochloride to generate one molecule of oxime and hydrochloric acid. Therefore, the content of aldehyde group can be converted by titrating the hydrochloric acid in the reaction solution with sodium hydroxide, as shown in FIG. Figure 8 shown.

[0103] observe Figure 8 A titration curve was plotted with the volume of NaOH consumed as the abscissa and the corresponding pH value as the ordinate. The highest point after the first differentiation of the titration curve was the titration endpoint. The NaOH consumption corresponding to the titration endpoint was used to calculate the OXG oxidation degree to be 35.78±1.5%, and the aldehyde content to be 4.8mmol / g.

[0104] To further illustrate the improvement of the present invention on the problem that SeNPs are easy to agglomerate and thus have limited applications, the selenium nanoparticles prepared with deionized water (H2O) and the selenium nanoparticles prepared with the concentration of OXG 2.56 mg / mL in this example were observed by transmission electron microscopy. Figure 9 shown.

[0105] observe Figure 9 It was found that selenium nanoparticles prepared by deionized water were severely agglomerated and could not be evenly distributed in the solution, which limited their application. However, the samples prepared according to this example showed a uniform spherical shape.

[0106] The selenium nanoparticles prepared with deionized water (H2O) and the concentration of OXG (2.56 mg / mL) in this example were observed by scanning electron microscopy. Figure 10 shown.

[0107] observe Figure 10 It was found that selenium nanoparticles prepared by deionized water were severely agglomerated and could not be evenly distributed in the solution, which limited their application. However, the samples prepared according to this example showed a uniform spherical shape.

[0108] To further illustrate the optimization effect of oxidized xyloglucan on the average particle size of selenium nanoparticles in this example, the hydrated particle size was tested using a Malvern particle size analyzer. (a) is a Gaussian distribution diagram, and (b) is an average particle size histogram. Figure 11 shown.

[0109] according to Figure 11 (a) Gaussian distribution diagram of particle size analyzer and Figure 11 From the average particle size histogram in (b), it can be clearly observed that the hydrated particle size of oxidized xyloglucan selenium nanoparticles is much smaller than the particle size of directly prepared selenium nanoparticles, which proves the obvious optimization of particle size.

[0110] To demonstrate the improvement of the antioxidant effect of selenium nanoparticles in the present invention, a DPPH free radical scavenging experiment was used to determine its antioxidant activity. 2 mL of sample solutions of different concentrations (0.2-1.2 mg / mL) were mixed with an equal volume of DPPH ethanol solution (2 mL, 0.1 mmol / L). Shake well and incubate at room temperature in the dark for 30 minutes. Then, a UV-visible spectrophotometer was used to measure the absorbance at a wavelength of 517 nm for calculating the clearance rate. The calculation results are shown in FIG. Figure 12 shown.

[0111] Figure 12 The DPPH free radical scavenging abilities of Na2SeO3 (an inorganic selenium source), Se (template-free selenium nanoparticles), and SeNPs@OXG (oxidized xyloglucan-coated selenium nanoparticles), with Vc as a positive control, were demonstrated. Within the 0.2-1.2 mg / mL concentration range, the Na2SeO3 selenium source had limited free radical scavenging activity. Se's free radical scavenging ability slowed with increasing concentration, likely due to aggregation. The DPPH scavenging efficiency of SeNPs@OXG increased linearly with concentration, reaching 95.87% at 1.2 mg / mL, approaching the 96.44% efficiency of the positive control Vc.

[0112] This example provides oxidized xyloglucan selenium nanoparticles. The experimental results demonstrate that the oxidized xyloglucan selenium nanoparticles provided by the present invention can be better dispersed in solution, exhibiting a uniform and complete spherical structure. Coating the selenium nanoparticles with oxidized xyloglucan improves the utilization of nano-selenium and further enhances its potential applications in areas such as nano-selenium supplementation, wound repair, immunomodulation, and anti-tumor therapy.

[0113] Example 2

[0114] To determine the excellent performance of oxidized xyloglucan on selenium nanoparticles, the concentration of OXG solution was screened. The specific steps are as follows:

[0115] 6.4, 12.8, 25.6, 51.2, and 102.4 mg of OXG were weighed and dissolved in 20 mL of deionized water to prepare OXG solutions at concentrations of 0.32, 0.64, 1.28, 2.56, and 5.12 mg / mL, respectively. 10 mL of a 20 mM sodium selenite solution was added to each of these solutions, and a uniform mixture was formed under magnetic stirring. Then, 10 mL of a freshly prepared 80 mM L-ascorbic acid solution was added dropwise to each mixture under vigorous stirring. The mixture was stirred in the dark at 37°C for 12 hours, and the solution gradually changed from colorless and transparent to orange-red and eventually to reddish-brown, indicating the formation of oxidized xyloglucan selenium nanoparticles in the solution.

[0116] The five groups of sample solutions were divided into five 30 mL centrifuge tubes, and 10 mL of the reacted mixed solution was added to each centrifuge tube. The centrifugation parameters were set as follows: centrifugal force 18000 g, centrifugation time 30 min, and centrifugation temperature 20 ° C. After centrifugation, the upper clear solution was discarded and the reddish-brown precipitate was retained. Then 10 mL of deionized water was added to each centrifuge tube and the centrifugation operation was repeated 3 times to ensure cleanliness. The remaining precipitate was pre-frozen at -20 ° C for 2 hours and freeze-dried for 24 hours to obtain OXG-coated oxidized xyloglucan selenium nanoparticles with different solution concentrations.

[0117] OXG@SeNPs prepared with different solution concentrations of OXG were prepared. 10 mg of selenium nanoparticle powder was dispersed in 10 mL of deionized water by ultrasound, added to a transparent sample bottle, and the solution was irradiated with a 655 nm laser to observe its Tyndall effect, such as Figure 13 shown.

[0118] observe Figure 13 It was found that except for the sample prepared with 0.32 mg / mL OXG concentration, the laser formed a straight path in the solution for all other samples, indicating that the samples prepared with 0.64-5.12 mg / mL OXG concentration formed a colloidal solution containing nanoparticles.

[0119] The particle sizes of the oxidized xyloglucan selenium nanoparticles prepared by coating with OXG at different solution concentrations were observed by transmission electron microscopy. Figure 14 shown.

[0120] observe Figure 14 The researchers found that, with the exception of the sample prepared at 0.32 mg / mL OXG concentration, which contained less nano-selenium and had larger particle size, the nano-selenium prepared at concentrations of 0.64-5.12 mg / mL exhibited uniform dispersion and spherical shapes. Furthermore, as the OXG solution concentration increased, the nano-selenium particle size initially decreased and then slightly increased, with the OXG@SeNPs prepared at 2.56 mg / mL having the smallest particle size.

[0121] In order to further prove the trend of particle size change, the hydrated particle size of the above samples was tested using a Malvern laser particle size analyzer. Figure 15 shown.

[0122] observe Figure 15 It was found that the trend of change in the hydrated particle size was consistent with the transmission electron microscopy observation, indicating that the increase in OXG concentration would gradually increase the coating force of the soft template and thus limit its particle size, while too high an OXG concentration would lead to excessive viscosity, resulting in uneven reaction and increased particle size.

[0123] In summary, this example provides a method for preparing selenium nanoparticles coated with oxidized xyloglucan at varying concentrations. The experimental results demonstrate that by varying the concentration of the oxidized xyloglucan soft template during the preparation process, selenium nanoparticles with a uniform particle size of 30-120 nm can be produced, stably present in solution. The minimum-sized selenium nanoparticles produced were produced at a xyloglucan concentration of 2.56 mg / mL, resulting in a particle size of approximately 30 nm. This further broadens the practical application of selenium nanoparticles.

[0124] Example 3

[0125] Similarly, the oxidized xyloglucan solution can be replaced with a xyloglucan solution, that is, xyloglucan selenium nanoparticles can be prepared by referring to the above process, and the concentration of the xyloglucan solution is: 0.64-5.12 mg / mL.

[0126] The specific steps for preparing xyloglucan nanoparticles (XG@SeNPs) with different concentrations of xyloglucan (XG) are as follows:

[0127] 6.4, 12.8, 25.6, 51.2, and 102.4 mg of XG were weighed and dissolved in 20 mL of deionized water to prepare XG solutions with concentrations of 0.32, 0.64, 1.28, 2.56, and 5.12 mg / mL, respectively. 10 mL of 20 mM sodium selenite solution was added to each of these solutions, and a uniform mixture was formed under magnetic stirring. Then, 10 mL of freshly prepared 80 mM L-ascorbic acid solution was added dropwise to each mixture under vigorous stirring. The mixture was stirred in the dark at 37°C for 12 hours, and the solution gradually changed from colorless and transparent to orange-red and eventually to reddish-brown, indicating the formation of xyloglucan selenium nanoparticles in the solution.

[0128] The five groups of sample solutions were divided into five 30 mL centrifuge tubes, and 10 mL of the reacted mixed solution was added to each centrifuge tube. The centrifugation parameters were set as follows: centrifugal force 18000 g, centrifugation time 30 min, and centrifugation temperature 20 ° C. After centrifugation, the upper clear solution was discarded and the reddish-brown precipitate was retained. Then 10 mL of deionized water was added to each centrifuge tube and the centrifugation operation was repeated 3 times to ensure cleanliness. The remaining precipitate was pre-frozen in a -20 ° C environment for 2 hours and freeze-dried for 24 hours to obtain xyloglucan selenium nanoparticles coated with XG at different solution concentrations.

[0129] The samples prepared above were compared with transmission electron microscopy and macroscopic observation, such as Figure 16 shown.

[0130] observe Figure 16It can be seen from transmission electron microscopy observation that the nano-selenium prepared with XG concentrations of 0.64-2.56 mg / mL is evenly dispersed and spherical in uniform size, indicating that the raw material xyloglucan for the preparation of oxidized xyloglucan also has excellent soft template coating ability, solving the existing problems in the preparation of selenium nanoparticles.

[0131] Therefore, the present invention solves the problems of instability, easy aggregation and uneven particle size of selenium nanoparticles in the prior art.

[0132] The present invention stabilizes selenium nanoparticles with oxidized xyloglucan or xyloglucan to obtain evenly dispersed selenium nanoparticles, thereby improving the utilization rate of nano-selenium and further enhancing the application potential of nano-selenium in the fields of nano-selenium supplements, wound repair, immunomodulation and anti-tumor treatment.

[0133] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes the preferred embodiment 1.

[0134] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0135] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A water-soluble monodisperse selenium nanoparticle, characterized in that: The water-soluble monodisperse selenium nanoparticles have a spherical structure and include selenium nanoparticles and polysaccharides coated on their surfaces; The polysaccharide is one of oxidized xyloglucan and xyloglucan.

2. The water-soluble monodisperse selenium nanoparticles according to claim 1, characterized in that The mass ratio of selenium nanoparticles to polysaccharide in the water-soluble monodisperse selenium nanoparticles is (30-35): (6.0-105); The average molecular weight of the unit sugar ring of the oxidized xyloglucan is 90-100 g / mol, the degree of oxidation is 34-38%, and the aldehyde content is 4.0-5.0 mmol / g; The average molecular weight of the unit sugar ring of the xyloglucan is 145-150 g / mol, and the hydroxyl content is 13.0-14 mmol / g; Preferably, the mass ratio of selenium nanoparticles to polysaccharide in the water-soluble monodisperse selenium nanoparticles is 34.6:(6.4-102.4); The average molecular weight of the unit sugar ring of the oxidized xyloglucan is 95.8 g / mol, the degree of oxidation is 35.78±1.5%, and the aldehyde content is 4.8 mmol / g; The average molecular weight of the unit sugar ring of the xyloglucan is 149.1 g / mol, and the hydroxyl content is 13.4 mmol / g.

3. A method for preparing water-soluble monodisperse selenium nanoparticles according to claim 1 or 2, characterized in that: The following steps are involved: (1) mixing a polysaccharide solution with a selenium source solution to obtain a mixed solution; (2) adding the reducing agent solution dropwise into the mixed solution to react, thereby preparing an aqueous solution of polysaccharide selenium nanoparticles; (3) The polysaccharide selenium nanoparticles aqueous solution is centrifuged, washed, and freeze-dried to obtain polysaccharide selenium nanoparticles.

4. The method for preparing water-soluble monodisperse selenium nanoparticles according to claim 3, characterized in that: In step (1), the polysaccharide solution is one of an oxidized xyloglucan solution and a xyloglucan solution; The concentration of the oxidized xyloglucan solution is 0.64-5.12 mg / mL; The concentration of the xyloglucan solution is 0.64-2.56 mg / mL; The concentration of the selenium source solution is 10-20 mM; The volume ratio of the polysaccharide solution to the selenium source solution is (0.5-1.5):1; The selenium source is one or more of sodium selenite, selenous acid, selenium dioxide and sodium selenite pentahydrate.

5. The method for preparing water-soluble monodisperse selenium nanoparticles according to claim 4, characterized in that: In step (1), the preparation method of the oxidized xyloglucan comprises the following steps: dissolving xyloglucan in water to obtain a xyloglucan solution, adding sodium periodate to carry out a light-proof reaction, adding ethylene glycol to terminate the reaction and keeping the solution in the dark overnight, dialyzing, filtering, pre-freezing, and freeze-drying to obtain the oxidized xyloglucan.

6. The method for preparing water-soluble monodisperse selenium nanoparticles according to claim 5, characterized in that: The mass ratio of xyloglucan to sodium periodate is 1:(0.05-0.15); The mixing ratio of xyloglucan to water is 1 g: (90-110) mL; The light-proof reaction is carried out at room temperature for 3-5 hours; The dialysis is performed in deionized water for 20-25 hours using a dialysis bag with a molecular weight cut-off of 8-14 kDa.

7. The method for preparing water-soluble monodisperse selenium nanoparticles according to claim 6, characterized in that: The preparation method of xyloglucan comprises the following steps: mixing tamarind gum with water, stirring and centrifuging, and then taking the supernatant and freeze-drying the mixture to obtain xyloglucan; Preferably, the mixing ratio of tamarind gum and water is 10g:(0.9-1.1)L; The stirring is performed at 35-45° C. for 20-30 hours; The centrifugation is performed at 7500-8000 rpm for 3-6 minutes.

8. The method for preparing water-soluble monodisperse selenium nanoparticles according to claim 3, characterized in that: In step (2), the concentration of the reducing agent solution is 40-80 mM; The volume ratio of the reducing agent solution to the mixed solution is 1:(1-1.5); The reaction is stirred at room temperature in the dark for 4-12 hours; The reducing agent is one of L-ascorbic acid solution and hydrazine monohydrate.

9. The method for preparing water-soluble monodisperse selenium nanoparticles according to claim 3, characterized in that: In step (3), the centrifugal force is 16000-18000g, the centrifugal time is 15-45min, and the centrifugal temperature is 20-25°C; The freeze-drying temperature is -20°C to -80°C, and the time is 2-8 hours.

10. Use of the water-soluble monodisperse selenium nanoparticles according to claim 1 or 2 in the preparation of injectable hydrogels and selenium supplement products.

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