Selenium nanoparticles combined with Calycosin treated sepsis through synergistic anti-inflammatory and antioxidant effects

doi:10.21203/rs.3.rs-5148313/v1

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Selenium nanoparticles combined with Calycosin treated sepsis through synergistic anti-inflammatory and antioxidant effects

https://doi.org/10.21203/rs.3.rs-5148313/v1

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Sepsis is a heterogeneous disease with high morbidity and mortality due to the limited therapeutic. Calycosin (CA), one of the main active ingredients of Astragalus, can potentially treat sepsis, but its therapeutic effect is limited by low blood circulation concentration and poor bioavailability. To address this challenge, we have successfully prepared BSA@Se-CA nanocomposite system (BSC) through self-assembly loading calycosin (CA) onto BSA@Se nanoparticles (BS). Compared to CA, BSC enhances the scavenging of ROS more effectively than CA alone by enhancing the activity of glutathione peroxidase (GPX). Notably, BSC reducing the expression level of inflammatory factors (NO, IL-6, IL-1β, and TNF-α) in inflammatory macrophages by synergistically inhibit the NF-κB signaling pathway. Moreover, the in vitro experiments demonstrated that BSC can also effectively alleviate RAW264.7 cells and HUVEC cells damage caused by oxidative stress, which can maintain the normal cells physiological function. In vivo, BSC exhibit significantly improve the therapeutic effect of sepsis by intraperitoneal injection, such as increase the survival rate of sepsis mice, and alleviate normal organ damage. Thus, this study provides a new strategy for improving the utilization efficiency of natural products with poor treatment effect and provides a reference for improving the therapeutic effect of sepsis.

Biological sciences/Drug discovery/Drug delivery

Biological sciences/Drug discovery/Pharmaceutics

Physical sciences/Nanoscience and technology

selenium nanoparticles

antioxidant

sepsis

calycosin

anti-inflammatory

Sepsis is a prevalent and potentially diverse complication associated with a significant mortality rate(22,5%)^{[1, 2]}. Pathogen-induced hyperinflammation and excessive reactive oxygen species (ROS) are the dominant features responsible for the high modality of sepsis^[3–6], which cannot be adequately addressed by current therapeutic strategies such as antibiotics, fluid resuscitation, or organ support therapy^{[7, 8]}. Hence, there is an avid need to develop a new therapeutic agent that can clear excess inflammatory factors and ROS^{[9, 10]}.

According to previous research, many natural products with high biocompatibility and diverse pharmacological activities have potential therapeutic effects on sepsis. Calycosin(CA), one of the primary active components found in Astragalus^[11], belongs to a class of natural flavonoid phytoestrogens that possess various pharmacological activities including anti-inflammatory^[12], anti-tumor^[13], antioxidant^[14], and anti-apoptotic effects^[15] et al. Previous studies have shown that CA can inhibit the expression of NF-κB signaling pathway related proteins, and thus inhibit the production of LPS-induced pro-inflammatory cytokines NO, TNF-α, IL-1β and IL-6 in macrophages^{[16, 17]}. However, despite their favorable pharmacological effects, natural products are often constrained by low blood circulation concentration and poor bioavailability^{[18, 19]}. In recent years, the development of nanotechnology has provided new avenues for improving the therapeutic effectiveness of these natural products^[20], which can enhance the biological distribution of existing therapeutic drugs by improving their pharmacokinetics and bioavailability^{[21, 22]}.

Selenium nanoparticles (SeNPs), one of the most widely studied nanoparticles due to their high biocompatibility, bioavailability, and low toxicity^{[23, 24]}, have been utilized for drug delivery of various compounds^[25], such as curcumin^[26], anisomycin^[27], 5-fluorouracil^[28], sorafenib^[29], and cisplatin/siRNA ^[30] et al. Zhao et al. used mesoporous SeNPs as a carrier, which not only decreased the side effects associated with DOX but also enhanced its antitumor activity^[31]. Moreover, SeNPs exhibit glutathione peroxidase-like activity (GPX), capable of scavenging ROS and effectively intervening in inflammatory responses induced by lipopolysaccharide (LPS) or hydrogen peroxide (H₂O₂)^{[6, 32, 33]}. Ouyang et al synthesized hydrogel microbeads with calcium alginate (SA) hydrogel as a protective shell and coated with SeNPs modified with hyaluronic acid, which can inhibit the NF-κB signaling pathway to prominently reduce proinflammatory cytokines secretion to effectively relieve colitis-associated symptoms^[34]. Furthermore, SeNPs are a promising material that can effectively treat sepsis. Chen et al. developed a mesoporous seleno-hyaluronic acid nanase therapy system (MSeNPs) capable of specifically targeting inflammatory macrophages to eliminate ROS, thereby effectively treating local inflammation and sepsis^[33]. Therefore, inspired by these, SeNPs have been employed as a carrier for the targeted delivery of CA, thus effectively treating sepsis.

In this study, BSA@Se-CA nanocomposite system (BSC), as a drug delivery platform, were successfully synthesized through self-assembly and have effectively addressed the issue of poor treatment effect associated with CA. BSC exhibited significant enhancement in ROS clearance induced by lipopolysaccharide (LPS) and inhibition of inflammatory cytokine expression levels compared with the BSA@Se nanoparticles (BS) or CA. Additionally, BSC can also effectively alleviate cell damage caused by oxidative stress and significantly improve the therapeutic effect of sepsis. The in vivo experiments results demonstrate that BSC can increase serum GPX activity, reduce the production of inflammatory factors, improve the survival rate of sepsis mice, and alleviate organ damage caused by sepsis after intraperitoneal injection. Thus, this study provides a new strategy for improving the utilization efficiency of natural products with poor treatment effect and provides a reference for enhancing the therapeutic effect of sepsis.

Synthesis and Characterization of BS and BSC

The BS were synthesized by one-pot method with bovine serum albumin (BSA) as a stabilizer and glutathione (GSH) as a reducing agent (Scheme 1). The transmission electron microscope (TEM) and high-resolution TEM (HRTEM) (Fig. S1) showed that the BS exhibited spheroidal morphology with an average size distribution of approximately 70 nm, which was consistent with the results obtained from Nano Particle Size and Zeta Potential Analysis (Fig. 1C and S2A). The results of Powder X-ray Diffraction (XRD) shows that the synthesized nanoparticles have no apparent lattice structure, which is consistent with the HRTEM observation. (Fig. S3) Moreover, Fourier Transform Infrared Spectroscopy (FT-IR) showed that BS appeared similar to free BSA peaks at 3282 cm^− 1 (O-H), 2930 cm^− 1 (C-H), 1640 cm^− 1 (C-O) and 1392 cm^− 1 (C-N), indicating the presence of BSA in BS and successful synthesis of BS (Fig. 1E).

The synthesis of BSC is similar to that of BS, with calycosin (CA) added to the solution before GSH reduction of sodium selenite (Na₂SeO₃) (Scheme 1). The synthesized BSC also appeared spheroidal morphology which is consistent with BS under TEM and HRTEM (Fig. 1A) and its size was smaller than that of BS with a size of 50 nm, which was consistent with the results of Hydrodynamic Diameter (DLS) (Fig. 1C and S2A). Similarly, XRD was employed to analyze the crystal lattice structure of the synthesized BSC, which is consistent with the BS (Fig. 1D). Zeta Potential Analysis results showed an average Zeta potential value of -30.4 mV for the synthesized BSC which is lower than that of BS alone (Fig. 1C and S2B). Energy Dispersive X-ray Spectroscopy (EDX) images revealed that selenium was the most abundant element in the synthetic nanoparticles, confirming that they were selenium nanoparticles (Fig. 1B and S4). Furthermore, XPS was employed to characterize the valence state of selenium in these particles, demonstrating that elemental selenium has a valence state of zero (Fig. 1J, S5A, and S5B). In addition to BSA-related absorption peaks in BSC in FT-IR, we observed peaks with a similar structure to CA at 1850 cm^− 1 and 1020 cm^− 1 which indicates the existence of CA (Fig. 1E). Furthermore, High-Performance Liquid Chromatography (HPLC) was used for quantitative analysis of CA loading. By analyzing the supernatant ultrafiltrate after centrifugation, it was found that only 50.5% of the initial amount of CA remained in solution, indicating that 49.5% of CA had been loaded onto BS (Fig. 1F). Both BS and BSC have good stability over one week without observing any polymer precipitation in H₂O₂, PBS and DMEM (Figure S6 and S7). Based on the fore results, we successfully fabricated the BSC.

The antioxidant mechanism of BS and BSC

Selenium, as an essential nutrient, is an important part of GPX which can catalyzes the transformation of H₂O₂ into H₂O and O₂, thus inhibiting the excessive production of ROS. In order to evaluate the GPX-like activity, we used a glutathione peroxidase assay kit to investigate their catalytic activity. We comprehensively characterized the catalytic reaction from multiple perspectives to understand the catalytic mechanism of SeNPs in exerting GPX activity. As depicted in Fig. 1G and Fig. S8, both BS and BSC exhibited GPX activity in a concentration-dependent manner. In further detecting the H₂O₂ scavenging efficiency of BS and BSC at 5 µg/mL was 40.31% and 40.02%, respectively ( Fig. S8 and 1G). These results demonstrated that the capability of H₂O₂ scavenging of BSC mainly depend on Se nanoparticles.

Many studies have been revealed the catalytic mechanism of natural GPX is the Ping-Pong mechanism. The detail catalytic process starts from the reduced state of GPX (E-SeH) is oxidized to selenite (E-SeOH) by peroxide, and then E-SeOH is reduced to the original state (E-SeH) reduced by two molecule of GSH^[35]. Therefore, to further clarify the mechanism of BS and BSC mimics the catalytic activity of GPX, we explored the reaction with various methods. Firstly, supernatant at different time points and concentrations was obtained from the reaction mixture by ultrafiltration, and the selenium content in the supernatant was quantitatively analyzed by ICP-MS. Notably, with the increase in the concentration of BS and BSC, the selenium content in the supernatant increased gradually. The content of selenium element in BS supernatant increased from 0.0712 µg/mL to 0.3881 µg/mL and in BSC supernatant from 0.0727 µg/mL to 0.3771 µg/mL (Fig. 1H and S9A). Moreover, under equivalent concentrations (10 µg/mL) of BS and BSC, selenium content initially rose but then declined over time due to excess GSH re-reducing selenium ions back to elemental selenium after substrate consumption (Fig. 1I and S9B). Selenium concentrations in BS and BSC supernatants increased by 0.238 µg/mL and 0.232 µg/mL, respectively (Figs. 1I and S9B). These results indicate that the valence state of selenium may change from Se⁰ to SeO₃²⁻. Then the valence states of selenium in BSC before and after the reaction were further characterized by XPS analysis (Fig. 1J, 1K, and S5). The results showed that the selenite content of BSC increased, while the elemental selenium content decreased after the reaction (Fig. 1K). From the above results, we could speculate that the catalytic activity of SeNPs stems from the cyclic valence change between Se⁰ and SeO₃²⁻. The possible catalytic mechanism is as follows: In the first step, the low valence Se⁰ component was oxidized to SeO₃²⁻⁻ by H₂O₂. Then, the SeO₃²⁻ intermediate reacted with GSH to produce GSSG and returned to its original state (Fig. 1L). Therefore, it can be concluded that the existence of these two valences of Se components starts and maintains the catalytic reaction process of Se nanozyme.

Uptake of BS and BSC by macrophages

Cellular uptake plays a crucial role in the therapeutic effect of BSC. In this study, we studied the uptake of CA, BS, and BSC at varying concentrations in RAW264.7 cells. After co-incubation with CA, BS, and BSC, aqua regia was used to disrupt the RAW264.7, and then the selenium and CA levels in the supernatant were quantified by ICP-MS and HPLC, respectively. The results showed that the uptake of these materials was found to be concentration-dependent (Fig. 2A, 2B, and S10). The results showed that when the concentration of BS and BSC was 10 µg/mL, the content of selenium in RAW264.7 cells reached 539.1 ng/10⁶ cells and 609.9 ng/10⁶ cells, respectively, which indicated that selenium nanomaterials had high cellular internalization ability (Fig. 2A and S10). To visualize the uptake of nanomaterials by RAW264.7 cells, coumarin 6 (Cy6)-labeled BS and BSC were co-incubated with RAW264.7 cells, followed by observation using Inverted Fluorescence Microscopy at different time points (Fig. 2C and S11). As expected, the fluorescence intensity gradually increased within RAW264.7 cells over time, demonstrating that both materials have good cellular uptake capacity. After that, we detected the biocompatibility of CA, BS and BSC, which is an essential fore step in biological experimentation. Cytotoxicity tests showed that BS, BSC, and CA did not show significant cytotoxicity to RAW264.7 cells when the concentration of BS and BSC was 5µg/ mL^− 1 and CA was 200 µM (Fig. S12A-C). In addition, the hemolytic reaction of BS and BSC did not exceed 5% at high concentrations, indicating that BS and BSC have good blood compatibility (Fig. S13). Those results indicate that BSC with good biocompatibility are suitable for treating sepsis

The antioxidant activity of BSC

Encouraged at the test-tube level, we further explored the antioxidant properties of BSC at the cellular level. To determine the effect of BSC on intracellular GPX activity, we incubated BSC with macrophages (RAW264.7 cells) for 6 hours at different concentrations and inflammatory conditions. The findings revealed a concentration-dependent relationship between GSH elimination and BS as well as BSC concentrations, and the GSH elimination rate increased with the increase of the concentration of the two compounds. indicating that BS and BSC can improve the intracellular GPX activity (Fig. 2F and S14A-C). The clearance rates of BS and BSC at 10 µg/mL were 30.27% and 32.41%, respectively (Fig. 2F and S14A-C). Furthermore, when subjected to inflammatory conditions in RAW264.7 cells activated with LPS (1 µg/mL), the GSH elimination rate was significantly higher in the groups treated with BS (27.86%) and BSC (28.30%) compared to control (2.58%), LPS (3.00%), and CA groups(0.86%) (Fig. 2G and S14D). Hence, our results demonstrate that both BS and BSC can effectively enhance intracellular GPX activity.

Based on this premise, we conducted further investigations into the impact of BSC in intracellular ROS. As shown in Fig. 2E, we used DCFH-DA to detect the total intracellular ROS level. The analysis results showed that BSC had the strongest effect and could effectively reduce the intracellular ROS level. Then we employed a Fluorescent Microplate Reader to quantify intracellular ROS levels. After normalization, the fluorescence intensity of LPS group, CA group, BS group and BSC group were 1.86, 1.79, 1.46 and 1.34, respectively, and the fluorescence intensity in RAW264.7 cells activated with LPS (1 µg/mL) increased compared with untreated cells, but after treatment with CA, BS, and BSC, the fluorescence was decreased. And BSC showed a higher activity, which exhibits the synergistic antioxidant properties of BS and CA (Fig. 2D).

Subsequently, we investigated the protective effect of BSC against ROS-induced oxidative damage in normal cells. H₂O₂ is a pivotal component in ROS production due to its stability and ability to diffuse across cell membranes. Therefore, H₂O₂ was employed as an inducer to provoke damage in RAW264.7 and HUVEC cells, enabling us to assess the protective effects of CA, BS, and BSC. The results demonstrated that CA, BS, and BSC exhibited significant protective effects on both RAW264.7 cells and HUVEC cells (Fig. 2H and 2I). After calculation, Compared with the LPS group (66.33%), the cell viability of RAW264.7 cells treated with CA, BS, and BSC was restored to 75.05%, 80.89%, and 95.45%, respectively (Fig. 2H). Similarly, compared with the LPS group (51.58%), the cell viability of HUVEC cells in the CA group, BS group, and BSC group recovered to 67.19%, 77.19%, and 85.71%, respectively (Fig. 2I). Notably, The protective effect of BSC is higher than that of CA and BS alone, suggesting that BSC have a synergistic protective mechanism. Therefore, it indicated that BSC may be an excellent candidate to protect cells against ROS damage.

The anti-inflammatory activity of BSC

Uncontrollable ROS and sustained oxidative stress will induce irreversible inflammatory injury. Moreover, it initiates the circulatory process that inflammatory response and ROS can promote each other, amplifying oxidative stress throughout the body until it develops into sepsis of systemic inflammation. Therefore, scavenging ROS is an effective approach to inhibit inflammatory signaling. In addition to effectively clearing excess ROS, BS and CA have also been reported to be able to clear inflammation-related cytokines, including NO, IL-6, IL-1β, and TNF-α. Then, we further detected the inflammatory response in macrophages after being treated with CA, BS, and BSC, respectively. The results of our study on the inhibition of NO by different concentrations of BS and CA demonstrated that BS could effectively inhibit the release of NO even at low concentrations (1.25 µM). Both BS and CA exhibited a concentration-dependent inhibition effect on NO production, with higher concentrations resulting in decreased levels of NO (Fig. 3A and 3B). Additionally, we investigated the inhibitory effects of BSC on NO. Compared to the Control group, LPS group, CA group, and BS group, it was observed that BSC exerted a superior inhibitory effect on NO compared to either BS or CA alone (Fig. 3C). The release of NO decreased from 2.78 times (the LPS group) to 1.56 times (the BSC group) (Fig. 3C). Furthermore, as shown in Fig. 3D-F, after activating (the LPS group), the levels of three pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) increased. However, after treatment with CA, BS, and BSC, the levels of pro-inflammatory cytokines were decreased. Notably, BSC displayed superior inhibitory efficacy compared to both BS and CA. However, the synergistic inhibition of TNF-α, IL-6, and IL-1β by BSC is not obvious, which may be due to the difficulty of sustained release of CA at the cellular level. Western blot demonstrated that different experimental conditions did not impact the expression of p65 (Fig. 3G and 3I). However, they did influence the phosphorylation status of the p65 protein(Fig. 3G and 3H). Remarkably, through synergistic inhibition of p65 phosphorylation, BSC effectively mitigated the inflammatory response in RAW264.7 cells.

The therapeutic effect of BSC on mice with sepsis

To further investigate the therapeutic effect of BSC on sepsis, we first studied the toxicity of BSC in healthy mice. After injecting 2 mg/Kg and 4 mg/Kg of BSC into healthy mice, we found that none of the mice died and no significant change in body weight occurred within 15 days. (Fig. S16A and S16B). Blood routine analysis after 15 days showed that BSC did not show obvious toxic side effects on blood cells (Fig. S16C). Encouraged by the above results, then we observed the therapeutic effect of BSC in a well-established mouse model of LPS-induced endotoxemia (Fig. 4A). Mice that received LPS via intraperitoneal (i.p.) administration (7.5 mg/kg) all died within three days. At a dose of 0.5 mg/kg, CA did not enhance the 15-day survival rate of septic mice. Conversely, the survival rates of both BS and BSC were significantly enhanced to 25% and 60%, respectively (Fig. 4B). Although notable body weight loss was initially observed, the mice treated with the BSC all started to recover after day 3 (Fig. 4D).

To gain insights into the distribution and metabolism of the nanomaterial BSC in sepsis mice, we employed ¹⁷⁷Lu for surface labeling of BSC^[36]. Within the initial 2 hours, the nanomaterials were extensively distributed throughout the abdominal cavity, with the discernible presence of BSC in vital organs such as the liver, spleen, lung, kidney, and gastrointestinal tract. Subsequently, over time, circulating ¹⁷⁷Lu-labeled BSC were also detected in brain tissue along with cardiac and select joint tissues (Fig. 4C). In summary, it is clear that metabolic clearance of BSC primarily occurs through fecal and urinary excretion pathways while respiratory elimination is also observed.

In addition, the serum GPX activity of sepsis mice increased four hours after BSC administration. The findings revealed no significant disparity in GPX enzyme activity among the Control, LPS, and CA group. Conversely, a notable elevation in GPX activity was observed in septic mice treated with BS and BSC, increasing by 34.80% and 31.28%, respectively (Fig. 4E and S15). This augmentation of GPX activity in septic mice holds potential benefits for mitigating oxidative stress levels and subsequently controlling the progression of sepsis.

To further evaluate the therapeutic efficacy of the BSC, cytokine levels in the plasma of LPS-treated mice were tested by enzyme-linked immunosorbent assay (ELISA). The concentration of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α, the key factors responsible for the cytokine storm and the high mortality in sepsis mice, were decreased with various degree after BSC administration (Fig. 4F, 4G, and 4H). Compared with the control group, the levels of IL-6, IL-1β and TNF-α in serum of LPS-induced sepsis mice were 11.10 times, 9.54 times and 10.30 times of normal levels, respectively. After treatment with BSC, IL-6, IL-1β and TNF-α were reduced to 2.15, 2.56 and 6.38 times of normal levels, respectively. The expression of multiple pro-inflammatory was decreased, suggesting an improved immune status in these mice. In addition, BSC showed obvious synergistic inhibition on the production of IL-6, IL-1β and TNF-α, which may be due to the continuous decomposition of nanomaterials and the continuous release of CA.

Sepsis leads to tissue and organ injuries that can harm the body's overall health. For instance, lung injury resulting from sepsis is often indicated by significantly elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the bloodstream. Therefore, measuring ALT and AST concentrations in serum allows for assessing the extent of lung injury. We further evaluated routine blood and blood biochemical indicators in mice. Our findings reveal that 24 hours after LPS injection-induced sepsis, mice exhibited significantly increased levels of AST and ALT along with lung injury symptoms; however, upon administration of BSC post-injection, both AST and ALT levels were markedly reduced, indicating that BSC can reduce lung injury caused by sepsis (Fig. 5A and 5B). The AST level decreased from 156U/L to 85U/L, and the ALT level from 494U/L to 249U/L (Fig. 5A and 5B). Furthermore, following LPS injection-induced sepsis in mice models, there was a sharp decrease observed in immune cell counts, including white blood cells (WBC), lymphocytes (Lym), monocytes (Mon), and eosinophils (Eso). However, BSC treatment prevented WBC and Lym depletion compared to other treatment groups, and the number of WBC and Lym cells remained at 44.04% and 53.07%, respectively (Fig. 5C and 5D). Moreover, BSC treatment resulted in an 7.52-fold and 0.93-fold increase in Mon and Eso numbers, respectively, compared to the control group, which is beneficial for combating foreign pathogens while preventing immunosuppression compared to the control group (Fig. 5E and 5F).

The sepsis can cause systemic inflammation which severely damage lead to organic failure and death. Therefore, therapy the organs dysfunction inducing by sepsis is urgent. we conducted H&E staining and Tunnel staining analysis on major organs such as the liver, spleen, and kidney from different treatment groups to evaluate the protective effect of BSC. The results showed that the necrosis of liver and kidney cells was significantly reduced after BSC treatment. In addition, the lymphocyte infiltration of liver and kidney tissues and the apoptosis of spleen lymphocytes were also inhibited (Fig. 5G). In short, these comprehensive results confirmed that BSC have an excellent therapeutic effect on sepsis, indicating that it has potential biomedical application value.

In conclusion, we successfully synthesized CA-loaded BSC by one-pot method. The BSC with GPX enzyme activity that acts as an antioxidant and anti-inflammation to eliminate excess ROS production, suppresses inflammatory factor production, mitigates cellular damage, reduces multiorgan dysfunction, and ultimately improves sepsis mouse survival rates. The BSC effectively solved the problem of poor treatment effect of CA, and the synergistic anti-inflammatory and antioxidant properties of BSC offer a promising treatment strategy for sepsis in vivo and may serve as a valuable reference for enhancing therapeutic outcomes.

Materials

Sodium selenite (Na₂SeO₃, Anhydrous) and sodium hydroxide (NaOH, AR) were purchased from Aladdin Chemical Co., China; glutathione (GSH, 98%) was purchased from Macklin Reagent Co., China; methanol (AR) and H₂O₂ (30%) were purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China; calycosin (CA, 98%) was purchased from yuanyeBio-Technology Co., Ltd., Shanghai, China; bovine serum albumin (BSA), CCK-8 and PBS were purchased from Seven Innovation Biotechnology Co., Ltd. Beijing China; lipopolysaccharides (LPS, from Escherichia coli O55:B5) and dimethyl sulfoxide (DMSO, 99.5%) were purchased from Solarbio Science & Technology Co., Ltd., Beijing, China. All the reagents were analytical grade and used without further purification. Human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection. The cells were cultured in a constant temperature incubator with a CO₂ concentration of 5% and a temperature of 37℃. Ultrapure water (18.2 MΩ) was obtained from a Milli-Q water purification system (Millipore, Bedford, USA).

Synthesis of BS and BSC

The synthesis of BS has been reported^[37]. It was synthesized by a one-pot process.307 mg GSH and 825 mg BSA were dissolved in 40 mL ultrapure water and then mixed with 10 mL of 20 mM sodium selenite solution at 1000 rpm/min for 10 min at room temperature. Next, we used NaOH to adjust the pH of the solution to 9 and magnetic stirring for 1 hour. After that, the solution was collected into a dialysis bag for 24 hours and changed the water every four hours. Finally,the BS were washed three times and collected by an amicon ultra-15 centrifugal filter at 3500 rpm/min for 10 min.

The synthesis of BSC is similar to BS. It's worth noting that CA was dissolved in methanol. 307 mg GSH and 825 mg BSA were dissolved in 40 mL ultrapure water and then mixed with 10 mL of 20 mM sodium selenite solution and 1 mL of 8 mM CA at 1000 rpm/min for 10 min. Next, we used NaOH to adjust the pH of the solution to 9 and magnetic stirring for 1 hour. After that, the solution was collected into a dialysis bag for 24 hours and changed the water every four hours. Finally, the BSC were washed three times and collected by an amicon ultra-15 centrifugal filter at 3500 rpm/min for 10 min.

The Cy6-labeled BS / BSC are synthesized similarly to BS / BSC. In the preparation of BS / BSC, a small amount of Cy6 was added to the solution. During the formation of BS / BSC, Cy6 was doped into the nanomaterials.

Characterization of BS / BSC

TEM images were obtained using a Tecnai G2 spirit BioTwin (FEI, USA) electron microscope operated at 120 kV. HRTEM and EDX analyses were performed using a Tecnai F20 microscope (FEI, USA) operated at 200 kV. The phase and crystallinity of the synthesized materials were determined by powder X-ray diffraction (PXRD) on a Bruker D8 Advance diffractometer equipped with Cu Kα radiation (λ = 1.5406 Å). The X-ray photoelectron spectroscopy (XPS) analyses were carried out with a Thermo ESCALAB 250Xl spectrometer (Thermo-Fisher Scientific, USA) using a monochromatic Al Kα X-Ray source (1486.6 eV). The size and charge of the nanoparticles were obtained using Zetasizer Nano ZS90 (Malvern, UK) at room temperature. The selenium contents in solution were detected by inductively coupled plasma mass spectrometry (ICP-MS).

CA content detection

In order to determine the CA content of the load on BSC, the remaining CA content in the supernatant was detected by High-Performance Liquid Chromatograph (HPLC). In the analysis, mobile phases were A, ultrapure water and B, methanol. The gradient condition was 0–10 min, 25%~45% B; 10–20 min, 45%-60% B; 20–22 min, 60%-25% B; and 22–25 min, 25%-25% B. Other chromatographic conditions were as follows: column temperature, 40°C; flow rate, 1 mL/min; detection, 254 nm.

Cytotoxicity Assays

Cell viability of RAW264.7 cells was measured by CCK-8 reagent. Cells were inoculated on a 96-well plate and cultured for 12 h. CA, BS, or BSC with different concentration gradients were added and incubated in the incubator for another 24 h. Finally, cell viability was measured at 450 nm in a Microplate Reader (Epoch2, Bio-Tek, USA).

Glutathione peroxidase (GPX) like activity assay

We used a glutathione peroxidase assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) to detect the GPX activity of BS/ BSC. We configured BS / BSC with different concentrations (0, 1.25, 2.5, 5, 10 µg/mL). 0.5 mL BS and BSC solution was mixed with 0.4 mL GSH solution and bathed in water at 37 ℃ for 15 min. Then H₂O₂ (0.1 mL) was added, and the mixture was bathed at 37 ℃ for 15 min. Then a color development agent was added for color development. The GPX activity was measured at 405 nm in a Microplate Reader (Epoch2, Bio-Tek, USA).

The selenium element content of BS and BSC supernatants at different time points was detected under the same concentration condition. 0.5 mL (10 µg/mL) BS and BSC solution was mixed with 0.4 mL GSH solution and bathed in water at 37 ℃ for 15 min. Then H₂O₂ (0.1 mL) was added, and supernatant was obtained by using ultrafiltration centrifuge tubes at different time points (0, 5, 10, 20, 30, 40, 50, 60, 120 min). The selenium element content was detected by ICP-MS (iCAP RQ, Thermo, USA).

The valence states of selenium in BSC after exerting GPX activity were detected. 0.5 mL (10 µg/mL) BSC solution was mixed with 0.4 mL GSH solution and bathed in water at 37 ℃ for 15 min. Then H₂O₂ (0.1 mL) was added, and the mixture was bathed at 37 ℃ for 15 min. Then a color development agent was added for color development. The valence states of selenium in BSC were characterized by XPS (Thermo-Fisher Scientific, USA).

Cellular uptake detection

We observed the uptake of Cy6-labeled BS and BSC by RAW264.7 cells at different time points using inverted fluorescence microscopy(ECLIPSE Ts2, Nikon, Japan). RAW264.7 cells with a density of 1×10⁶ cells / well were inoculated on a 6-well plate and cultured for 12 h. Then Cy6-labeled BS / BSC were added and obtained the concentration was 20 µg/mL. After incubation for 0.5, 1, 2, 4 and 6 h, the cells were washed with PBS for three times and stained with Hoechst for 15 min. The intracellular fluorescence was observed by an inverted fluorescence microscope (Ex/Em = 450/505 nm).

We detected the uptake of BS / BSC at different concentrations in RAW264.7 cells. RAW264.7 cells with a density of 1×10⁶ cells / well were inoculated on a 6-well plate and cultured for 12 h. Subsequently, the cells were treated with different concentrations of BS / BSC (0, 5, 10, 20 µg/mL) for 6 h. Then, the cells were washed with PBS for three times and dissolved with aqua regia. The intracellular selenium content was detected by ICP-MS (iCAP RQ, Thermo, USA).

We detected the uptake of CA at different concentrations in RAW264.7 cells. RAW264.7 cells with a density of 1×10⁶ cells / well were inoculated on a 6-well plate. After 12 h, the cells were treated with different concentrations of CA (0, 25, 50, 100, 200, 400 µM) for 6 h. Then, the cells were washed with PBS for three times and dissolved with Cell lysis buffer. Finally, 150 µL methanol was added to the mixture. After centrifugation at 12000 rpm/min for 10 min, CA content in the supernatant was detected by HPLC (LC-2030, Shimadzu, Japan).

The intracellular GPX activity detection

We detected the effects of different concentrations of BS / BSC (0, 2.5, 5, 10 µg/mL) on the GPX activity of normal RAW264.7 cells and the effects of the same concentration of BS / BSC (10 µg/mL) on the GPX activity of RAW264.7 cells in inflammatory condition. RAW264.7 cells treatment is consistent with the above. LPS (1 µg/mL) was used to induce inflammation in RAW264.7 cells. After treatment, the cells were washed with PBS for three times and dissolved with cell lysis buffer. Finally, the supernatant was collected, and a glutathione peroxidase assay kit was used to detect the activity of GPX.

Intracellular ROS detection

RAW264.7 cells with a density of 4×10⁴ cells / well were inoculated on a 96-well plate and cultured for 12 h. Then, the cells were treated with the same concentration of BS and BSC (2.5 µg/mL) containing LPS (1 µg/mL) for 12 h. After incubation, the cells were washed with PBS for one time and stained with DCFH-DA for 30 min. After washing again with PBS, the fluorescence intensity in cells was measured by Microplate Reader (Ex/Em = 488/525 nm). The image of intracellular fluorescence was observed by an inverted fluorescence microscope

Inflammatory factor detection

The release of NO was measured by the NO kit (Beyotime). RAW264.7 cells with a density of 4×10⁴ cells / well were inoculated on a 96-well plate and cultured for 12 h. Then, the cells were treated with the different concentration of BS (0, 1.25, 2.5, 5 µM)、 CA (0, 25, 50, 100 µM) or BS /BSC (2.5 µM) containing LPS (1 µg/mL) for 24 h. After co-incubated, the supernatant was collected for detection of NO content.

TNF-α, IL-6, and IL-1β were detected by using an enzyme-linked immunosorbent assay (ELISA) kit (Elabscience). RAW264.7 cells with a density of 4×10⁴ cells / well were inoculated on a 96-well plate and cultured for 12 h. Then, the cells were treated with BS / BSC (1.25 µg/mL) containing LPS (1 µg/mL) for 12 h. After co-incubated, the supernatant was collected for TNF-α, IL-6, and IL-1β content detection.

Protective effect of BSC

We used H₂O₂ to induce the generation of cell damage models. RAW264.7 cells with a density of 6×10⁴ cells / well or HUVECs with a density of 8000 cells / well were inoculated on a 96-well plate and cultured for 12 h. Then, the cells were treated with BS / BSC (5 µM) for 6 h. Then, H₂O₂ (0.5 µM) was added and incubated for 2 h. Finally, cell viability was measured by CCK-8 reagent at 450 nm.

Western blotting

The total protein was extracted from RAW264.7 cells. Then, the total protein was divided into different target proteins via 10% sodium-dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE). Next, these target proteins were transferred onto PVDF membranes (EMD-Millipore Co., Ltd.). These membranes were then incubated with primary antibodies for > 8h at 4℃, followed by incubation with secondary antibodies for 1h at room temperature. Finally, a scanner and an advanced chemiluminescence solution were used to detect these target protein bands. The following primary antibodies were used: anti-NF-κB p65(phosphos536)(CAT NO. ab76302; 1:1000; Abcam), anti-NF-κB p65 (CAT NO. ab16502; 1:1000; Abcam), Actin β polyclonal Antibody (CAT NO.YT0099; 1:1000; Immunoway). The following secondary antibodies were used: goat anti-rabbit-IgG (CAT NO. RS0002; Immunoway). TheImage-Lab (version 4.0.1) software was used to analyze the data of the target proteins’ bands.

Animals and animal models

In this experiment, female C57BL/6 mice (8 weeks, ~ 18 g) were used, which were purchased from Beijing Huafukang Biotechnology Co., LTD. The animal experiments involved in this paper are carried out in accordance with the operating rules and regulations of the Experimental Animal Center of Shanxi Medical University, Grant no. SYXK (JIN) 2019-0007. Mice were housed under a 12-hour light/dark cycle with a temperature of approximately 25°C and humidity around 55%. Standard laboratory diet and water was freely available.

The mouse model of sepsis was established by intraperitoneal injection of LPS (7.5 mg/kg)for 1 h. The septic mice were then treated by injection of BSC (0.5 mg/kg). After 12 h of BSC injection, mouse blood was collected for subsequent inflammatory factors (TNF- α, IL-6, and IL-1β ), and GPX activity was measured four hours after injection of BSC. Finally, mice were euthanized using isoflurane anesthesia for subsequent experiments.

The distribution of BSC in vivo

We used ¹⁷⁷Lu labeled BSC and stirred at 650 rpm/min at room temperature for 2 h. The marked BSC are collected and washed by centrifugation in an ultrafiltration tube. Mice were injected with ¹⁷⁷Lu-labeled BSC (150 µg) by intraperitoneal injection. The distribution of BSC in mice was observed at different time points (0, 1, 2, 4, 8 h).

Blood compatibility assay

Fresh blood from healthy mice was collected in an anticoagulant tube and then centrifuged at 1500 rpm/min for 20 min to collect red blood cells. The obtained red blood cells were suspended using PBS (49:1) and centrifuged at 3000 rpm/min for 10 min until the supernatant showed no red color. 20 uL red blood cells were taken and mixed with 980 µL BS / BSC (200, 100, 50, 25, 12.5, 6.25, 3.12, 1.6, 0.8, 0.4 µg/mL). Ultra-pure water and PBS were used as positive and negative controls, respectively. Separate BS and BSC solutions (200, 100, 50, 25, 12.5, 6.25, 3.12, 1.6, 0.8, 0.4 µg/mL) were used as blank controls. After co-incubation at room temperature for 2 h, the supernatant was collected for blood compatibility assay. The hemolysis rate was detected at 540 nm by Microplate Reader (Epoch2, Bio-Tek, USA). If the hemolysis rate does not exceed 5%, it can be regarded as blood compatibility well.

Blood analysis and Pathological analysis in mice

The Blood samples of mice were obtained at 24 h after BSC administration for subsequent aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood routine, and blood biochemical detection. Meanwhile, the major organs were obtained, fixed in 4% polyformaldehyde, and stained with H&E or Tunnel for pathological analysis.

Statistical Analysis:

All data are expressed as mean ± standard deviation (SD) and are based on at least three independent experiments. The data were processed using GraphPad Prism 8 software. Significance was determined by a Student’s two-tailed t tests and denoted by an asterisk (*, set at *p < 0.05, **p < 0.01, ***p < 0.001, ns indicates no significant difference.).

Data availability

No datasets were generated or analysed during the current study.

Acknowledgements

We are grateful to the National Atomic Energy Agency nuclear technology (Nonclinical evaluation of radiopharmaceuticals) research and Development Center, CNNC Key Laboratory on Radiotoxicology and Radiopharmaceutical Preclinical Evaluation, Shanxi Key Laboratory of Drug Toxicology and Preclinical Study of Radiopharmaceuticals.

Funding

This work was supported by Four “Batches” Innovation Project of Invigorating Medical through Science and Technology of Shanxi Province (No. 2023XM036), Fundamental Research Program of Shanxi Province (202303021221223). Fund Program for the Scientific Activities of Selected Returned Overseas Professionals in Shanxi Province (20240054). Research Project Supported by Shanxi Scholarship Council of China (2024-144).

Data Availability statement

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no conflict of interest.

Ethics statement

This work was approved by the ethics committee of Shanxi Medical University. All procedures of animal experiment in accordance with ARRIVE guidelines.

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No competing interests reported.

Scheme1.png
Scheme 1. Schematic illustration of BSC synergistic therapy for sepsis.
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Selenium nanoparticles combined with Calycosin treated sepsis through synergistic anti-inflammatory and antioxidant effects

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Abstract

Figures

Introduction

Results

Synthesis and Characterization of BS and BSC

The antioxidant mechanism of BS and BSC

Uptake of BS and BSC by macrophages

The antioxidant activity of BSC

The anti-inflammatory activity of BSC

The therapeutic effect of BSC on mice with sepsis

Discussion

Materials and methods

Materials

Synthesis of BS and BSC

Characterization of BS / BSC

CA content detection

Cytotoxicity Assays

Glutathione peroxidase (GPX) like activity assay

Cellular uptake detection

The intracellular GPX activity detection

Intracellular ROS detection

Inflammatory factor detection

Protective effect of BSC

Western blotting

Animals and animal models

Blood compatibility assay

Blood analysis and Pathological analysis in mice

Statistical Analysis:

Data availability

Declarations

References

Additional Declarations

Supplementary Files

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