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. 2026 Jul 7;16(36):38492–38504. doi: 10.1039/d6ra03287g

Construction of selenium nanoparticles for the transmembrane absorption of Polygonatum Sibiricum polysaccharides and their antioxidant activity

Kexin Zhu a,b,†, Chunyan Liu a,b,†, Haoyu Zhang a,b, Anqi Li a, Jiapeng Wang a,b, Guangdong Hu a,b, Jiangping Wu a,b, Qiuyue Lv a,b,c, Hui Che a,b,c, Guodong Wang a,b,c,✉, Dan Jin a,b,✉, Taili Shao a,b,✉
PMCID: PMC13340307  PMID: 42416705

Abstract

Using Polygonatum sibiricum rhizome polysaccharides (PSP) as a template, selenium nanoparticles (SeNPs) of approximate diameter 70–80 nm (polydispersity index = 0.117) were prepared by reducing sodium pentahydrate with ascorbic acid. Transmission electron microscopy confirmed that these nanoparticles were well-dispersed and could facilitate the transmembrane absorption of PSP. PSP-SeNPs maintained a stable particle size distribution for extended periods in physiological culture media, water, and simulated gastrointestinal fluids. In terms of activity, compared with PSP, PSP-SeNPs significantly enhanced the scavenging ability of DPPH, OH, and ABTS free radicals. Cellular experiments revealed that PSP-SeNPs were not cytotoxic at concentrations ≤400 µg mL−1, slightly promoted cell viability and reduced intracellular reactive oxygen species levels in a dose-dependent manner, effectively alleviating oxidative stress. They can simultaneously inhibit oxidative stress-induced apoptosis, maintain mitochondrial membrane potential stability, and protect mitochondrial function, with this effect being dose-dependent. These results suggest that these nanoparticles can efficiently mediate the transmembrane transport of Polygonatum Sibiricum rhizome polysaccharides and have promising applications as antioxidants in the medical field.

The prepared complex of Polygonatum sibiricum rhizome polysaccharides (PSP) and selenium nanoparticles, facilitate the transmembrane absorption of PSP with antioxidant activity.graphic file with name d6ra03287g-ga.jpg

Introduction

Selenium is an essential trace element for human health and plays a crucial role in the antioxidant defense system, immune function, and anticancer bioactivity.1,2 In recent years, selenium nanoparticles (SeNPs) have attracted widespread attention in the medical field, owing to their higher bioavailability and lower toxicity compared with those of inorganic and organic selenium compounds.3,4 However, SeNPs are unstable in the liquid phase and easily aggregate into larger selenium particles, thereby losing their bioavailability and bioactivity. Therefore, suitable stabilizers are required to maintain their stability.5

Surface modification of SeNPs can be achieved using various biomacromolecules, including proteins, polysaccharides, and polyphenols.6–8 Such modifications generally enhance the biological activity of SeNPs. Among these biomacromolecules, polysaccharides offer distinct advantages due to their abundance of hydrophilic functional groups, which confer strong stabilizing capacity to selenium nanoparticle systems, making them ideal carrier materials.9

Natural polysaccharides are considered promising therapeutic candidates owing to their low toxicity and diverse pharmacological activities including hypoglycemic, antioxidant, and immunomodulatory effects.10–15Polygonatum Sibiricum rhizomes is a plant widely recognized in traditional Chinese medicine as both a food and medicinal plant,16,17 and its main bioactive component, Polygonatum Sibiricum rhizomes polysaccharides (PSP), reportedly exhibits diverse pharmacological activities, including antioxidant, antitumor, anti-inflammatory, antibacterial, and immunomodulatory effects.18 At present, it remains unclear whether SeNPs facilitate the cellular uptake of PSP to enhance their activity, representing a novel research direction.

According to relevant reports, selenium nanoparticles stabilized by natural polysaccharides with a size of 50–100 nm are mainly internalized by cells through clathrin-mediated endocytosis and macropinocytosis, which provides a theoretical basis for the transmembrane transport mechanism of PSP-SeNPs.19–21 In recent years, great advances have been achieved in the intracellular delivery mechanism, colloidal stability, and structure–activity relationship of polysaccharide-stabilized selenium nanoparticles, providing important theoretical guidance for the design of highly stable and bioactive polysaccharide-selenium nanosystems.22,23 This study used the neutral polysaccharides isolated from Polygonatum Sibiricum rhizomes as a template to prepare stable and low-toxicity monodisperse SeNPs. The core objective was to explore the transmembrane transport effect of SeNPs on PSP. The synthesized nanoparticles were structurally characterized, their stability under various biologically relevant conditions was evaluated, and studies were conducted to determine whether they could permeate cells to exert their effects, as well as to investigate their antioxidant activity in vitro and at the cellular level. This study provides a basis for the development and application of PSP-SeNPs as natural antioxidants, particularly by facilitating the endocytic uptake of PSP with the aid of SeNPs, thereby enhancing their pharmacological effects and laying the foundation for medical applications.

Materials and methods

Materials

The dried Polygonatum Sibiricum rhizomes (7–8 years old) was purchased from Chanyue Food Co., Ltd, Qingyang County, Chizhou, Anhui Province, identified by Liu Chunyan, an associate researcher at Wannan Medical University. The authenticated specimen was deposited in the Herbarium of Wannan Medical University (Wuhu, China) under voucher number WN-20240327. RAW264.7 cells were purchased from iCell Bioscience Inc. Sodium selenite pentahydrate and l-ascorbic acid were purchased from Shanghai Yien Chemical Technology Co., Ltd (Shanghai, China); DEAE-52 cellulose, Sephadex G-75 dextran gel, 10% fetal bovine serum, 1% penicillin–streptomycin, and DMEM were all purchased from Beijing Soleibao Technology Co., Ltd (Beijing, China) 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH), hydroxyl radicals (˙OH), and 2,2′-azot-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging kits were purchased from Nanjing Jiancheng Institute of Bioengineering (Nanjing, China). The CCK8 assay kit was purchased from Xi'an Aorui Jingchuang Biotechnology Co., Ltd (Xi'an, China). The reactive oxygen species detection kit, mitochondrial membrane potential detection kit, and Annexin V-FITC/PI dual-staining apoptosis kit were all purchased from Beijing Lanjie Ke Technology Co., Ltd (Beijing, China).The remaining reagents and solvents were all of analytical grade.

Extraction and purification of PSP

Crude Polygonatum Sibiricum rhizomes polysaccharides (PSP) were obtained through water extraction and alcohol precipitation, followed by deproteinization using Sevage reagent (chloroform:n-butanol = 4 : 1, v/v) repeatedly 4–5 times until no protein precipitate appeared at the interface, and subsequent decolorized using D101 resin. Next, 200 mg of freeze-dried crude PSP powder was dissolved in 40 mL of deionized water. A DEAE-52 anion exchange column was then used for sequential elution with deionized water followed by NaCl solutions (0.1–0.5 M) to remove the acidic sugar polysaccharide fractions, thereby yielding the neutral polysaccharide components of PSP. Thereafter, a Sephadex G-75 gel chromatography column (eluted with deionized water) was used to obtain purified PSP. Analysis was performed using an LC-10avp high-performance gel permeation chromatography (HPGPC) system (Shimadzu, Japan) equipped with an RID-10A differential refractive index detector. Elution was conducted using a TSKgel G3000PWXL column (7.8 × 300 mm, Tosoh Corporation, Japan). Samples were prepared at 2 mg mL−1 in deionized water and analyzed at a flow rate of 0.7 mL min−1. Fractions corresponding to peaks with the same retention time and shape were pooled, frozen and freeze-dried to obtain purified PSP.

Synthesis of PSP-SeNPs

10 mL of PSP solution (3 mg mL−1) were mixed with 1.14 mL of sodium selenite pentahydrate (2.63 mg mL−1) in a 50 mL three-necked flask and stirred magnetically at room temperature in the dark. Thereafter, 1.14 mL of ascorbic acid solution (7.04 mg mL−1) was added dropwise. The mixture was continuously stirred at room temperature for 4 h, during which the solution gradually changed from colorless to light yellow, eventually turning a stable orange-red, indicating SeNPs formation. The solution was purified using dialysis in a dialysis bag (1000 Da) against distilled water at 4 °C for 48 h, with the water replaced every 12 h to remove excess ascorbic acid.

Ultraviolet-visible spectrum

Samples were measured in the 200–800 nm range using a NanoDrop One ultramicro spectrophotometer (Thermo, USA) with distilled water as the blank to confirm PSP-SeNPs formation.

Dynamic light scattering

The hydrodynamic size and polydispersity index of PSP-SeNPs were measured by dynamic light scattering using a Litesizer 500 (Anton Paar, Austria). All measurements were replicated thrice at 25 °C. (Samples were appropriately diluted with distilled water).

Transmission electron microscopy

The morphology and size distribution of PSP-SeNPs were observed using a transmission electron microscope (Hitachi HT7800, Japan). The nanoparticle suspension was dropped onto a carbon-coated copper grid and observed after air drying at room temperature.

Energy dispersive X-ray spectroscopy

The elemental composition of PSP-SeNPs was determined using energy-dispersive X-ray spectroscopy combined with scanning electron microscopy. Freeze-dried samples were fixed on an aluminum sample stage with conductive carbon tape, and the atomic percentages of C, O, and Se were quantitatively measured.

X-ray photoelectron spectroscopy

Use the Thermo Scientific K-Alpha spectrometer. The analysis parameters are as follows: chamber pressure, <2.0 × 10−7 mbar; spot size, 400 µm; voltage, 12 kV; filament current, 6 mA. The pass energy for the full spectrum scan is set to 150 eV (step size 0.1 eV). Peak fitting is performed using Avantage software.

Stability study

PSP-SeNPs were diluted in different media [deionized water, simulated gastric fluid (pH 1.45), simulated intestinal fluid (pH 7.8), DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin solution]. To evaluate the colloidal stability of PSP-SeNPs. The mixtures were incubated at 37 °C for 4 h (water, gastrointestinal fluids) or 4 days (physiological medium). Equal portions of the samples were obtained and particle size was determined using DLS.

In vitro antioxidant activity test

The in vitro antioxidant capacity of PSP-SeNPs was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radicals (˙OH), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging kits.

Cell culture

RAW264.7 cells were cultured in DMEM medium containing 10% FBS and 1% penicillin–streptomycin in a cell culture incubator at 37 °C with 5% CO2.

Cell viability assay

The CCK8 method was used to evaluate the effects of PSP and PSP-SeNPs on the proliferation of RAW264.7 cells. Briefly RAW264.7 cells were seeded into 96-well plates and incubated for 24 h. Thereafter the cells were incubated with different concentrations (0–400 µg mL−1) of PSP and PSP-SeNPs in serum-free medium (DMEM) for 24 h. Next, CCK8 reagent was added, and the absorbance at 450 nm was measured using a microplate reader to quantify cell viability. Cell viability (%) = (As − Ab)/(Ac − Ab) × 100%, where As is the absorbance of cells treated with PSP or PSP-SeNPs, Ab is the absorbance of wells without cells, and Ac is the absorbance of untreated control cells. All experiments were repeated thrice, with six replicates per concentration.

Cell uptake study

After the cultured cells were treated, they were incubated for 24 h. The culture medium was then discarded and electron microscopy fixative was added immediately. Cells were gently scraped in a single direction using a cell scraper and transferred into a centrifuge tube. The suspension was centrifuged until a visible cell pellet approximately the size of a mung bean was obtained. After removing the fixative, fresh electron microscopy fixative was added, and the cells were fixed at room temperature for 2 h. The fixed cells were washed with phosphate-buffered saline and refixed. Subsequently, samples were dehydrated through a graded ethanol series, placed into embedding molds, infiltrated with embedding medium, and sectioned using an ultramicrotome (Leica, EM UC7, Leica Microsystems, Wetzlar, Germany). The sections were allowed to dry at room temperature overnight and then observed under a transmission electron microscope.

Intracellular reactive oxygen species (ROS) assay

Intracellular ROS was assessed using a DCFH-DA probe. Briefly, RAW264.7 cells in the logarithmic growth phase (2 × 105 cells per well, six-well plate, treated with the target compound for 24 h) were collected, centrifuged (1000×g, 5 minutes), and resuspended in 1 mL of diluted DCFH-DA (incubated at 37 °C in the dark for 20 minutes, inverted every 3–4 minutes). The cells were then centrifuged, washed thrice with serum-free basal medium, filtered, and analyzed for ROS using flow cytometry.

Measurement of mitochondrial membrane potential (MMP)

ΔΨm was measured using a JC-1 fluorescent probe. Logarithmic-phase RAW264.7 cells were seeded at a density of 5 × 105 cells per mL in a six-well plate and incubated overnight until cell adhesion was achieved. Except for the control group, all groups were treated with 1 µg mL−1 LPS for 24 hours to establish an inflammatory model. Subsequently, cells were treated with specified concentrations of PSP or PSP-SeNPs for 24 hours, with controls (normal culture) and model groups (only LPS stimulation) established. Cells were harvested (1000×g, 5 min), washed two to three times with PBS, resuspended in 500 µL of a JC-1 working solution, and incubated at 37 °C for 20 min in the dark. After two washes with a JC-1 staining buffer, cells were resuspended in assay buffer for flow cytometry, with data analyzed using FlowJo software.

Apoptosis rate detection

The apoptotic rate was determined using a commercial apoptosis detection kit. RAW264.7 cells were plated in six-well plates at 5 × 105 cells per well and cultured for 24 hours until cell adhesion was achieved. With control and model included. Except for the control group, all groups were treated with 1 µg mL−1 LPS for 24 hours to establish an inflammatory model. Subsequently, cells were treated with specified concentrations of PSP or PSP-SeNPs for 24 hours, with controls (normal culture) and model groups (only LPS stimulation) established. Apoptosis was assessed via Annexin V-FITC/PI double staining. Post-treatment, cells were washed with PBS, harvested with ethylenediaminetetraacetic acid-free trypsin, centrifuged at 1000×g for 5 min, and resuspended in 500 µL of 1×Binding Buffer. Next, 100 µL of cell suspension (1 × 105 cells) was incubated with 5 µL of Annexin V-FITC for 15 min at room temperature in the dark, supplemented with 5 µL of propidium iodide 5 min pre-detection, filtered through a 300-mesh sieve, and analyzed via flow cytometry.

Statistical analysis

Data analyses were performed using GraphPad Prism (v9, Windows, GraphPad). Data were presented as mean ± standard deviation of the mean. Statistical comparisons between groups of different parameters were performed using a one-way analysis of variance. The significance levels were set as follows: *P < 0.05 and **P < 0.01.

Results and discussions

Extraction and characterization of PSP

As shown in the flowchart (Fig. 1A), the neutral polysaccharide components were successfully extracted from Polygonatum Sibiricum rhizomes using the hot water extraction-alcohol precipitation method and DEAE-cellulose column chromatography.24 The crude polysaccharides were then purified using a Sephadex G-75 gel chromatography column (Fig. 1B), and analyzed using high-performance liquid chromatography (Fig. 1C), indicating that the PSP fractions were obtained.

Fig. 1. (A) Polygonatum Sibiricum rhizomes polysaccharide extraction flowchart. (B) DEAE-cellulose and G-75 elution curves. (C) High-performance gel permeation chromatography chart.

Fig. 1

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Preparation and structural characterization of PSP-SeNPs

The neutral PSP fraction was used as a stabilizer and dispersant for the subsequent synthesis of SeNPs. Ascorbic acid was used to reduce sodium selenite pentahydrate, successfully synthesizing PSP-SeNPs. During the reaction, the solution eventually turned a characteristic orange-red color,25 visually indicating the formation of SeNPs, whereas without PSP, precipitation occurred (Fig. 2A). Ultraviolet-visible spectroscopy showed that PSP-SeNPs exhibited an absorption peak at approximately 265 nm, confirming the successful synthesis of SeNPs (Fig. 2B).

Fig. 2. (A) PSP-SeNPs preparation flowchart. (B) UV-visible spectra of PSP and PSP-SeNPs. (C) Particle size diagram of PSP-SeNPs. (D) Transmission electron microscopy images of PSP-SeNPs at different magnifications.

Fig. 2

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Dynamic light scattering (DLS) analysis showed that the prepared PSP-SeNPs were uniformly dispersed with an average particle size of 125 nm and a polydispersity index (PDI) of 0.117 (Fig. 2C). The low PDI value (≤0.2) indicates the excellent monodispersity of these nanoparticles,18 which is attributed to the steric hindrance and electrostatic stabilization effects generated by PSP molecules adsorbed on the surface of SeNPs, effectively preventing particle aggregation.26 Transmission electron microscopy (TEM) observation further confirmed the successful preparation and good dispersion of PSP-SeNPs (Fig. 2D). The TEM images showed that the SeNPs were spherical, with no obvious aggregation observed, consistent with the DLS test results.

Energy-dispersive X-ray spectroscopy (EDX) was used to determine the elemental composition and distribution, and a characteristic selenium absorption peak was detected at approximately 1.37 keV, confirming the presence of SeNPs (Fig. 3A).

Fig. 3. (A) PSP-SeNPs energy-dispersive X-ray spectroscopy (EDX) spectrum. (B) XPS full spectrum and XPS high-resolution spectra of PSP-SeNPs.

Fig. 3

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X-ray photoelectron spectroscopy (XPS) analysis was used to characterize the surface chemical state of PSP-SeNPs (Fig. 3B). In the XPS survey spectrum, characteristic Se 3d, C 1s, and O 1s peaks were detected, with the presence of Se 3d confirming selenium as the core component of SeNPs. In the high-resolution XPS spectra, the C 1s, O 1s, and Se 3d regions were analyzed separately. Se 3d3/2 (56.28 eV) and Se 3d5/2 (55.38 eV) confirmed that selenium was present in the zero-valent state;27 and the absence of a binding-energy peak at 59.1 eV indicated that all Se4+ in the reaction system had been fully reduced to Se0.

Stability of PSP-SeNPs

The stability of nanoparticles is a key factor affecting their biological applications. PSP-SeNPs stability was evaluated by monitoring particle size changes under different simulated physiological conditions, including deionized water and simulated gastrointestinal fluids. The results showed that the PSP-SeNPs solution remained stable in water and simulated gastric and intestinal fluids for 4 hours, with almost no change in particle size (Fig. 4A), and remained uniformly dispersed without aggregation (Fig. 4B). The results indicated that after incubation in physiological culture media for 4 days, the UV-visible spectrum showed almost no change (Fig. 4C), and the particle size also remained stable (Fig. 4D). The protective effect of the PSP-derived shell reduces the interaction between the nanoparticles and ions, enzymes, or other environmental factors, thereby preventing particle aggregation or degradation and ensuring the applicability of PSP-SeNPs in a complex physiological environment. Studies have shown that SeNPs modified with chitosan, goji polysaccharides, and other coatings also exhibit good stability in physiological environments due to the protective effect of the polysaccharide shell.28

Fig. 4. (A) Changes in the particle size of a PSP-SeNPs solution in simulated gastric fluid, simulated intestinal fluid, and water within 4 h. (B) State changes in PSP-SeNPs solution in simulated gastric fluid, simulated intestinal fluid, and water within 4 h. (C) UV-visible spectra of PSP-SeNPs cultured in physiological medium for 0–4 days. (D) Particle size chart of PSP-SeNPs cultured in physiological medium for 0–4 days.

Fig. 4

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In vitro antioxidant activity

Using purified PSP as the control and vitamin C (VC) as the positive control, the antioxidant activity of PSP-SeNPs was systematically evaluated by measuring their scavenging capacities for diphenylpicrylhydrazyl (DPPH), hydroxyl (˙OH), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals. The results showed that both PSP and PSP-SeNPs exhibited dose-dependent scavenging effects on DPPH, ˙OH, and ABTS radicals; however, PSP-SeNPs demonstrated significantly higher antioxidant activity than that of pure PSP at the same concentration (Fig. 5). The enhanced antioxidant activity of PSP-SeNPs may originate from the synergistic interaction between PSP and SeNPs: PSP can exert intrinsic antioxidant effects through hydrogen donation, while SeNPs can modulate the redox balance of biological systems and scavenge free radicals through selenium-containing functional groups. When combined, these complementary mechanisms produce a more pronounced antioxidant effect. This synergistic mechanism has been confirmed in studies on selenium-modified corn silk polysaccharides and Ganoderma lucidum polysaccharides, showing that the introduction of selenium can significantly improve the free radical scavenging ability of the original polysaccharides.28 This effect arises from the fact that the bond dissociation energy of selenium-containing groups is lower than that of O–H bonds, enabling more efficient hydrogen atom donation to free radicals, thereby enhancing antioxidant efficacy.26

Fig. 5. PSP-SeNPs scavenging rates of DPPH, hydroxyl, and ABTS radicals. VC was used as the positive control. Data are presented as mean ± SD (n = 3).

Fig. 5

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Cellular antioxidant activity and biocompatibility

The CCK8 method was used to evaluate the effect of PSP-SeNPs on the cell viability of mouse mononuclear macrophage leukemia cells (RAW264.7), and the DCFH-DA fluorescent probe was used to measure intracellular reactive oxygen species levels to assess intracellular antioxidant activity.

Cell viability

After treatment with PSP-SeNPs at concentrations of 0–400 µg mL−1 for 24 hours, no cytotoxicity was observed; instead, both polysaccharides increased RAW264.7 cell viability in a dose-dependent manner (p < 0.05, p < 0.01), with statistical significance determined by Among them, the proliferative effect of PSP-SeNPs was overall better than that of PSP, with the greatest increase in activity at 400 µg mL−1. This indicates that PSP (especially after selenium modification) can safely and effectively improve RAW264.7 cell viability and metabolic activity under the experimental conditions (Fig. 6A).29,30

Fig. 6. (A) Cell viability of RAW264.7 cells treated with PSP-SeNPs and PSP. Data are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01. (B) Transmission electron microscopy images of PSP-SeNPs entering RAW264.7 cells. (C) Measurement of intracellular reactive oxygen species (ROS) levels using DCFH-DA staining. Data are presented as mean ± SD (n = 3). #P < 0.05, ##P < 0.01 compared to control group. *P < 0.05, **P < 0.01 compared to model group.

Fig. 6

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Cellular uptake

After administering PSP-SeNPs to cells, the embedded sections were examined using TEM to investigate their transmembrane uptake. TEM images (Fig. 6B) confirmed that intact spherical PSP-SeNPs were internalized into cells as nanocomposites without extracellular dissociation, which was further supported by the excellent colloidal stability in culture medium. Nano-sized SeNPs have a larger specific surface area and higher surface energy, which facilitates interaction with the cell membrane and endocytic cell entry. Additionally, the PSP coating contains abundant hydroxyl and carboxyl groups, which may mediate nanoparticle internalization by interacting with receptors or transport proteins on the cell membrane. These results confirm that PSP-SeNPs can not only be effectively taken up by cells combined with the particle size of 70–80 nm and hydrophilic polysaccharide coating, PSP-SeNPs were inferred to enter RAW264.7 cells mainly via clathrin-mediated endocytosis, which is consistent with the endocytic pathways of polysaccharide-based selenium nanoparticles documented in previous literature., as nanocarriers, may also promote the transmembrane transport of PSP, providing a basis for the observed intracellular ROS scavenging activity.

Intracellular ROS clearance ability

PSP-SeNPs effectively inhibited lipopolysaccharide (LPS)-induced ROS generation in RAW264.7 macrophages, exhibiting significant antioxidative stress effects in a dose-dependent manner. Compared with the control group, treatment with LPS alone significantly increased intracellular ROS fluorescence intensity, confirming the successful establishment of the oxidative stress model (Fig. 6C). PSP-SeNP treatment dose-dependently reduced the elevated ROS levels. This decrease in intracellular ROS indicates that PSP-SeNPs can effectively enter cells and exert their antioxidant function.

Mitochondrial membrane potential

PSP-SeNPs can reversibly and concentration-dependently reverse the abnormal elevation of mitochondrial membrane potential induced by LPS in RAW cells, thereby maintaining mitochondrial functional homeostasis. Compared with the blank control group, the fluorescence intensity of JC-1 aggregates in LPS-treated cells was significantly enhanced, indicating a marked increase in mitochondrial membrane potential (Fig. 7). However, after pretreatment with PSP-SeNPs at different concentrations, the fluorescence intensity of JC-1 aggregates gradually decreased with increasing drug concentration, demonstrating a dose-dependent recovery of mitochondrial membrane potential toward normal homeostasis rather than mitochondrial depolarization. These findings suggest that PSP-SeNPs effectively antagonize the abnormal regulation of mitochondrial membrane potential induced by LPS in RAW cells, protecting mitochondrial structural and functional integrity. These results confirm the mitochondrial protective effect of PSP-SeNPs against LPS-induced oxidative damage.

Fig. 7. (A) JC-1 staining was used to detect mitochondrial membrane potential. (B) Statistical analysis of JC-1 detection by flow cytometry. Data are presented as mean ± SD (n = 3). #P < 0.05, ##P < 0.01 compared to control group. *P < 0.05, **P < 0.01 compared to model group.

Fig. 7

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Apoptosis

Flow cytometry results demonstrated that the apoptosis rate in the control group was only 3.52%; in the LPS model group, the apoptosis rate significantly increased to 11.94%, indicating that oxidative stress can induce massive apoptosis of macrophages. In the PSP treatment group, the apoptosis rate was 10.59% (Fig. 8). The apoptosis rates in PSP-SeNPs groups at various doses showed dose-dependent reduction. PSP-SeNPs effectively inhibited oxidative stress-induced macrophage apoptosis, exerting a protective effect on cells.

Fig. 8. (A) Detection of apoptosis using Annexin V-FITC/PI double staining method. (B) Statistical analysis of apoptosis detection by flow cytometry. Data are presented as mean ± SD (n = 3). #P < 0.05, ##P < 0.01 compared to control group. *P < 0.05, **P < 0.01 compared to model group.

Fig. 8

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Conclusions

In this study, neutral polysaccharides extracted from Polygonatum sibiricum rhizomes were used as stabilizers to synthesize exhibited good short-term colloidal stability under simulated physiological and gastrointestinal conditions SeNPs, with EDX and XPS analyses confirming the elemental composition and chemical state. These nanoparticles exhibited good stability in water, simulated gastrointestinal fluids, and physiological culture media, overcoming the limitations of conventional nanoparticles. In terms of activity, compared with natural PSP, PSP-SeNPs exhibited significantly enhanced scavenging abilities against DPPH, ˙OH, and ABTS free radicals. Cell experiments indicated that PSP-SeNPs exhibited no cytotoxicity at concentrations up to 400 µg mL−1. TEM observation showed that PSP-SeNPs effectively entered RAW264.7 cells, mainly through clathrin-mediated endocytosis and macropinocytosis based on particle size and surface property analysis, and dose-dependently reduced intracellular ROS levels in the LPS-induced oxidative stress model. PSP-SeNPs significantly inhibited oxidative stress-induced macrophage apoptosis, maintained mitochondrial membrane potential homeostasis, and protected mitochondrial function in a dose-dependent manner. These results suggest that PSP-SeNPs can efficiently mediate the transmembrane transport of PSP and exert strong intracellular antioxidant activity. This study provides a feasible strategy for constructing highly stable polysaccharide-functionalized selenium nanoparticles and clarifies the possible endocytic pathways for their cellular uptake, thereby improving the mechanistic understanding of their enhanced bioactivity. It shows promising prospects for the application of PSP-SeNPs as safe and efficient antioxidants in the biomedical field.

Author contributions

Kexin Zhu: project administration, writing – original draft, methodology. Chunyan Liu: writing – original draft, investigation. Haoyu Zhang: validation. Anqi Li: formal analysis. Jiapeng Wang: visualization. Guangdong Hu: visualization. Jiangping Wu: formal analysis. Qiuyue Lv: data curation. Hiu Che: data curation. Guodong Wang: conceptualization, resources. Dan Jin: funding acquisition, supervision. Taili Shao: conceptualization, writing – reviewing & editing, supervision.

Conflicts of interest

There are no conflicts to declare.

Acknowledgments

This work was supported by the Project of Natural Science Foundation of the Department of Education of Anhui Province (2024AH051923, 2024AH051934), Anhui Provincial Quality Project for Education in the New Era (Postgraduate Education) (2024cxcysj181, 2024cxcysj182, 2025zyxwjxalk369).

Data availability

The data supporting the findings of this study are available within the article.

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Data Availability Statement

The data supporting the findings of this study are available within the article.

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