Panax ginseng-derived Exosome-like Nanoparticles Prevent LPS-induced Septic Shock by Modulating TLR4 Glycosylation in Macrophages

Abstract

Macrophages play a crucial role in the pathophysiology of sepsis, serving as central regulators of both disease onset and progression. Among the therapeutic agents investigated to modulate macrophage-driven inflammation, compounds extracted from the roots of Panax ginseng have been distinguished for their potent anti-inflammatory properties. Recently, attention has shifted toward ginseng-derived exosome-like nanoparticles (GDEs) because of their ability to encapsulate diverse bioactive compounds with high stability and biocompatibility. In this study, we investigated the protective effects of GDEs against lipopolysaccharide (LPS)-induced septic shock, with a particular focus on macrophage-mediated mechanisms. GDEs isolated from Korean Panax ginseng exhibited a spherical morphology, high stability, and effective encapsulation of bioactive ginsenosides, including Rb1, Rg1, and Rg3. GDEs significantly attenuated LPS-induced inflammatory responses in macrophages by reducing toll-like receptor 4 (TLR4) glycosylation, thereby inhibiting LPS binding. This suppression of TLR4 glycosylation led to decreased production of nitric oxide and proinflammatory cytokines (interleukin-1β, interleukin-6, and tumor necrosis factor-α), as well as inhibition of intracellular reactive oxygen species accumulation and NF-κB activation. Furthermore, GDE treatment markedly improved survival and alleviated lung, liver, and spleen damage in an LPS-induced sepsis mouse model. In summary, these findings suggest that GDEs represent a promising nanomedicine strategy for sepsis prevention, offering targeted modulation of macrophage activity without apparent adverse effects.

1. Introduction

Sepsis, along with septic shock, is a life-threatening medical condition characterized by an infection-induced dysregulated host immune response that leads to multiple organ failure and death. Its incidence is not only high but has also been increasing, underscoring its substantial global health burden. The pathophysiology of sepsis is complex and multifaceted, with macrophages playing a pivotal role in their key clinical manifestations. In particular, nitric oxide and various proinflammatory cytokines produced by macrophages, stimulated by bacterial endotoxins, such as lipopolysaccharides (LPS), induce hypotension and severe tissue damage. , Consequently, identifying effective drug candidates that can appropriately modulate macrophage activity during sepsis remains a critical challenge. However, current therapeutic strategies primarily rely on corticosteroids and other nonspecific immunosuppressants, which are associated with significant adverse effects, including an increased risk of secondary infections and hyperglycemia. Therefore, there is a strong unmet need for the development of drugs that precisely target upstream regulators of sepsis-associated inflammatory mediators.

Numerous studies have demonstrated that plant-derived exosome-like nanoparticles (PELNs) contain bioactive molecules with antioxidant, anti-inflammatory, and tissue-protective properties. PELNs represent a promising alternative to animal cell–derived exosomes, offering several advantages, including lower immunogenicity, sustainable and scalable production, the absence of zoonotic risks, and broad therapeutic applicability. Collectively, these attributes make PELNs an attractive platform for the development of novel therapeutics.

Ginseng, the root of Panax ginseng CA Meyer, is widely recognized for containing diverse biologically active compounds, including ginsenosides, polysaccharides, alkaloids, and glycosides. , Several of these components have been reported to alleviate sepsis-associated pathologies through reactive oxygen species (ROS)-scavenging, anti-inflammatory, and organ-protective mechanisms. While most previous studies have focused on the effects of individual ginseng-derived compounds in sepsis, emerging evidence highlights the importance of synergistic interactions among these bioactive constituents. Recently, ginseng-derived exosome-like nanoparticles (GDEs) have gained increasing interest due to their comprehensive encapsulation of ginseng’s biologically active compounds. The isolation process of GDEs can be fully conducted under physiological conditions, which preserve the natural structures of these compounds. Consistent with this, GDEs have demonstrated pronounced immunomodulatory and anticancer activities, including the amelioration of colitis, remission of glioma, and attenuation of ischemia–reperfusion injury. Despite these promising findings, the therapeutic potential of GDEs in the treatment of sepsis has not yet been explored.

This study revealed that GDEs possess remarkable efficacy in suppressing LPS-induced macrophage activation and subsequently alleviating symptoms associated with LPS-induced sepsis by regulating macrophage TLR4 glycosylation, a highest upstream regulator for the macrophage activation.

2. Materials and Methods

2.1. Isolation of GDEs

Seventy grams of ginseng root were ground in a mixer with phosphate-buffered saline (Life Technologies Corp, USA) at medium to high speed for 5 min. After the homogenization, the ginseng mixture was squeezed, and the collected juice was centrifuged at 2,000 × g for 20 min and 10,000 × g for 60 min at 4 °C to remove debris and fibers. The supernatant was gently transferred onto two sucrose layers (27% and 68%) and ultracentrifuged at 100,000 × g for 90 min at 4 °C. The yellow microvesicles between the two sucrose layers were transferred onto a sucrose gradient (8%, 30%, 45%, and 60%) and ultracentrifuged again at 200,000 × g for 90 min at 4 °C. The yellow layer between the 8% and 30% sucrose layers was collected and filtered through a sterilizing-grade 0.2 μm filter membrane (Pall Corp., USA) to obtain nanoscale GDEs. The GDEs were used fresh or stored at – 80 °C. The protein concentration of the GDEs was measured using a BCA assay kit (Thermo Fisher Scientific Inc., USA) according to the manufacturer’s instructions.

2.2. Characterization of GDEs

Nanoparticle tracking analysis (NTA) was performed by NanoSight NS300 (Malvern Panalytical Ltd., Malvern, UK) was used to measure the concentration and size distribution of isolated GDEs. Data were analyzed with NTA software (version 3.4). Further, 1:10 to 1:1000 diluted samples of GDEs were suspended in phosphate-buffered saline (PBS), and the size was determined by repeating the measurement three times. The Zeta potential of GDEs was measured three times at 25 °C with Zetasizer (Malvern, UK) after each freeze–thaw cycle, following the manufacturer’s instructions. Samples were then diluted in PBS by 1:3000.

For transmission electron microscope (TEM) imaging of GDEs, a drop of GDEs was deposited onto a Formvar-coated copper grid surface and incubated with 1% uranyl acetate for 2 min followed by washing with absolute ethanol. The samples were then left to dry at room temperature and observed using a TEM (JEM-1400 Flash, JEOL, Japan).

2.3. Liquid Chromatography Coupled with Mass Spectrometry (LC-MS/MS)

The concentrations of Rb1, Rg1, and Rg3 in GDEs were analyzed using LC-MS/MS. Rb1, Rg1, and Rg3 standards were obtained from Merck, Germany. To prepare the samples, 200 μL of GDE sample, 100 μL of BuOH, and 100 μL of DEPC water were mixed, followed by vortexing for 10 min. The mixture was then centrifuged at 13,000 rpm for 10 min. Each sample was dried using a speed vacuum and reconstituted in 10 μL of distilled water, to which 10 μL of internal standard (IS) was added. From the 20-μL mixture, 5 μL was injected into the LC-MS/MS system. The LC-MS/MS system included an Agilent 1200 series high-performance liquid chromatography (HPLC) system (Agilent, Santa Clara, CA) coupled with an API3200 triple-quadrupole mass spectrometer (Applied Biosystems-SCIEX, Concord, Canada). The mobile phases for HPLC consisted of 0.1% formic acid in water (phase A) and acetonitrile (phase B). Analytes were separated on a reversed-phase Atlantis T3 column (100 × 2.1 mm, 3 μm, Waters Corporation, Milford, MA) using gradient elution at a flow rate of 0.3 mL/min.

2.4. Cell Culture

RAW 264.7 cells were obtained from the Korean Cell Line Bank. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Life Technologies Corp., USA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin-streptomycin, in a humidified environment at 37 °C with 5% CO₂. The cells were subcultured every 2–3 days to maintain their viability and growth.

2.5. Cytotoxicity Assay

EZ-Cytox (Daeil Lab Service, Korea) containing WST-1 (water-soluble tetrazolium) was used to evaluate the toxicity of GDEs in RAW 264.7 cells. RAW 264.7 cells were grown at the density of 5 × 10³ cells per well in a 96-well plate. Cells were treated with various GDE concentrations (0–40 μg/mL) for 24 and 48 h after 24 h of incubation. The supernatant of each well was removed and replaced by fresh media and 10% EZ-Cytox after the treatment. Additionally, the plate was incubated in the dark at 37 °C for a further 2 h. Each sample was quantified by a microplate spectrophotometer at an absorbance of 460 nm (Molecular Devices, USA) after the incubation.

A Calcein-AM/Propidium Iodide stain was performed to visualize the cytotoxicity of GDEs in RAW 264.7 cells. Cells were seeded in a 6-well plate at a density of 3 × 10⁵ cells per well. GDEs were treated at concentrations of 0, 1, 5, and 10 μg/mL after 24 h of incubation at 37 °C in a 5% CO₂ environment. The medium was removed after an additional 24 h, and the cells were then gently washed with PBS and stained with a Calcein-AM (Invitrogen-Life Technologies, Carlsbad, CA, USA) and Propidium Iodide (Invitrogen-Life Technologies, Carlsbad, CA, USA) solution, diluted at 1:500 and 1:300 ratios, respectively. Following a 30 min incubation at 37 °C in a 5% CO₂ environment, the dye solution was removed and replaced with DMEM. Samples were then photographed with a stereo microscope (Nikon, Japan).

2.6. Cellular Uptake Profile of GDEs

Vybrant DiI solution (Invitrogen-Life Technologies, Carlsbad, CA, USA) was mixed with GDEs at a volume ratio of 1:200 and incubated at 37 °C in a 5% CO₂ incubator for 30 min to fluorescently label GDEs. Afterward, the mixture was subjected to centrifugation at 4 °C and 100,000 g for 90 min after adding a 2:2:1 ratio of 68% and 27% sucrose solutions. Only the precise middle layer was collected for subsequent experiments after confirming the layer separation. RAW264.7 cells at a density of 3 × 10⁴ cells in a confocal dish for the uptake assay. After 24 h of incubation at 37 °C in a 5% CO₂ environment, GDEs labeled with DiI (Invitrogen-Life Technologies, Carlsbad, CA, USA) were administered at a concentration of 10 μg/mL. After 1, 2, 4, 8, and 12 h post-treatment, cells were gently washed twice with PBS. They were then stained with Calcein-AM (Invitrogen-Life Technologies, Carlsbad, CA, USA), diluted at a ratio of 1:500, and Hoechst33342 (Invitrogen-Life Technologies, Carlsbad, CA, USA), diluted at a ratio of 1:2000 in DMEM for 20 min. After removing the staining solution, the cells were washed gently twice with PBS and imaged with a confocal microscope (LSM 980, Carl Zeiss, Germany) equipped with 63× lens (Carl Zeiss, Germany) and 488, 594, and 647 nm of excitation lasers. Fluorescence intensity was measured with ZEN 3.9 Blue software (Carl Zeiss, Germany).

2.7. LPS Binding Assay

RAW 264.7 cells were pretreated with GDEs at concentrations of 1, 5, and 10 μg/mL for 2 h. Subsequently, the cells were stimulated with 10 μg/mL FITC-conjugated LPS and incubated for 1 h at 37 °C in a 5% CO₂ environment. The cells were washed three times with PBS and stained with Hoechst 33342, following the manufacturer’s protocol. After staining, the cells were gently washed with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. Imaging was performed using a confocal microscope (LSM980, Carl Zeiss, Germany) equipped with a 63× lens and excitation lasers at 488 and 594 nm. The FITC fluorescence intensity of each cell was quantified and normalized against the Hoechst 33342 fluorescence intensity using ZEN 3.9 Blue software (Carl Zeiss, Germany). The normalized data were statistically analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test in GraphPad Prism (MA, USA).

Nanoparticle tracking analysis (NTA) was performed using the Nanosight NS300 (Malvern Panalytical Ltd., Malvern, UK) to measure the concentration and hydrodynamic size of GDEs in contact with LPS. Data were analyzed using NTA software (version 3.4). GDE samples were diluted 1:1000 and suspended in a 1 μg/mL LPS solution, and size measurements were repeated three times.

2.8. Quantification and Visualization of Intracellular ROS

RAW 264.7 cells were cultured on a confocal dish at a density of 3 × 10⁴ cells. GDEs were treated at various concentrations: 1, 5, and 10 μg/mL, after 24 h of incubation at 37 °C in a 5% CO₂ environment. Cells were stimulated with LPS (1 μg/mL) for 4 h after 2 h of GDE treatment. Subsequently, cells were gently washed twice with PBS and then stained with Hoechst33342 (Invitrogen-Life Technologies, Carlsbad, CA, USA) and Cellrox Deep red (Invitrogen-Life Technologies, Carlsbad, CA, USA) dye, following the manufacturer’s protocol. Cells were imaged with a confocal microscope (LSM980, CarlZeiss, Germany) equipped with a 63 × lens and 488 and 647 nm of excitation lasers. Fluorescence intensity was measured with ZEN 3.9 Blue software (Carl Zeiss, Germany). ImageJ analysis software (National Institutes of Health, USA) was used to obtain relative fluorescence heatmap images.

2.9. Western Blot Analysis

RAW 264.7 cells were seeded at a final concentration of 4 × 10⁵ in 60 mm dishes and incubated for 24 h. The cells were treated with GDEs (0, 1, 5, and 10 μg/mL) for 2 h followed by LPS (1 μg/mL) stimulation for 24 h. The cells were harvested with RIPA buffer (Biosesang, Republic of Korea) containing protease inhibitor cocktails (Sigma-Aldrich, USA) and rested in ice for 30 min. Each supernatant was collected after 12,000 × rpm centrifugation at 4 °C for 20 min. Proteins were then quantified with a bicinchoninic acid assay kit (Thermo Fischer Scientific Inc., USA) with bovine serum albumin as a standard. Protein samples were separated in a 10% sodium salt-polyacrylamide gel and then transferred onto a nitrocellulose membrane (Amersham, Germany). After 1 h of blocking with 5% nonfat milk in TBST at room temperature, the membranes were incubated overnight at 4 °C with antibodies against β-actin (sc-47778; Santa Cruz, USA), iNOS (#13120; Cell Signaling Technology, USA), which were diluted (1:1000) in blocking buffer (Bio-Rad, USA). The membranes were subsequently incubated for 1 h at room temperature with antirabbit (sc-2357; Santa Cruz, USA) and antimouse (sc-516102; Santa Cruz, USA) secondary antibodies diluted (1:2500) with 5% nonfat milk in 1 × TBST after being washed three times with 1 × TBST for 30 min. The membranes were washed three times with 1 × TBST for 30 min, and proteins were detected with an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc., USA). Amersham Imager 600 (GE, USA) was used for protein expression analyses, and protein band densities were measured with image J analysis software (National Institutes of Health, USA).

2.10. Quantitative Real-Time PCR

Total RNA was purified from RAW 264.7 cells stimulated with LPS (1 μg/mL) for 4 h following GDE treatment for 2 h. TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to collect the cell lysates. A total of 1 μg of RNA was reverse transcribed into cDNA using a reverse transcription system with random hexamers (Thermo Fisher Scientific, Inc., USA). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed on a StepOnePlus Real-Time PCR system with Power SYBR-PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc., USA). The PCR program included an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The data were analyzed using the 2–^ΔΔCq method, and the relative expression levels were quantified. Gene-specific primers were synthesized by Bioneer Corp. (Republic of Korea). The sequences of the primers used in the PCR are as follows:

• TNF-α F: 5′-CACCGTCAGCCGATTTGC-3′;

• TNF-α R: 5′-TTGAGGCGAGGAAGAGGTT-3′;

• IL-1β F: 5′-AGTTGAGGAGACCACCAAAAGAT-3′

• IL-1β R: 5′-GGACACGCAGGTAACAGG-3′

• IL-6 F: 5′-CCACCGGCTTCCCTACTTCC-3′

• IL-6 R: 5′-TTGGGAGTGGTATCCTCTGTGA-3′

• iNOS F: 5′-CCCTTCAATGGTTGGTACATGG-3′

• iNOS R: 5′-ACATTGATCTCGCTGACGAGC-3′

• STT3A F: 5′-GTTGGGTTACCTCTGTGGTGT-3′

• STT3A R: 5′-CATCAATGGGTGGCAACTGA-3′

• STT3B F: 5′-TTACCCTTGTTTCCTGTTTTGTTTT-3′

• STT3B R: 5′-CTCGCTTGTTCTGATTGGCT-3′

• RPN1 F: 5′-TTCTCTTGCCTTCCCAACACT-3′

• RPN1 R: 5′-CTCTCAACAGCAACAGATTCCC-3′

• RPN2 F: 5′-ATCACACATGCAAGGCTCCA-3′

• RPN2 R: 5′-TCCCAGGCTACCCAGATGTT-3′

• DAD1 F: 5′-TTGGCTGTGCTGCTTTCATT-3′

• DAD1 R: 5′-CTGTATTCTCAGGCAAACTGGG-3′

• DDOST F: 5′-GGATAGGCCGAGGGGATGAA-3′

• DDOST R: 5′-AAGTGAGGGGCAGTCCAGTT-3′

• MAGT1 F: 5′-AAGAAGACGTGCTAAATGAACCT-3′

• MAGT1 R: 5′-CTTATTGGCCCATTCCATCAGC-3′

• TUSC3 F: 5′-GTCACCCTCAGCACTCACAAC-3′

• TUSC3 R: 5′-GGCTGGAGTTTGTCGCGT-3′

• KCP2 F: 5′-GGTGTTGTTGCACCGCTTG-3′

• KCP2 R: 5′-CGCCTTCTGATTGACCCTCT-3′

• OSTC F: 5′-CCAGGAGACTAGTGAGGTGTTT-3′

• OSTC R: 5′-ATGGGGACACATAGGAGGAAAA-3′

• GAPDH F: 5′-CTCGTCCCGTAGACAAAATGG-3′

• GAPDH R: 5′-TGACCAGGCGCCCAATA-3′

2.11. Enzyme-Linked Immunosorbent Assay (ELISA)

RAW 264.7 cells (2 × 10⁴ cells/well) were seeded in a 48-well plate and incubated for 18 h. After removing the media, various GDE concentrations (0, 1, 5, and 10 μg/mL) were mixed in prewarmed media for 2 h, and LPS (1 μg/mL) were subsequently treated in each well. The cells were incubated for another 7 and 24 h. The cell medium supernatant from each well was collected and stored at −20 °C for the subsequent analysis. An ELISA was conducted for the quantitative determination of the interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and tumor necrotic factor-alpha (TNF-α) levels, following the manufacturer’s instruction (R&D systems, USA).

2.12. Nitric Oxide Assay

RAW 264.7 cells (2 × 10⁵ cells/well) were cultured in a 6-well plate for 24 h. Culture media in each well was replaced to 100 μM L-NMMA (Sigma-Aldrich, USA) or GDEs (0, 1, 5, and 10 μg/mL). After 2 h, cells were stimulated with LPS (1 μg/mL) for additional 24 h. Cell culture supernatant was obtained to measure the NO level with the NO detection kit (iNtRON biotechnology, Republic of Korea), following the manufacturer’s instructions.

2.13. In Vivo Experiment

8-week male ICR mice were intraperitoneally injected with LPS (Sigma-Aldrich, St. Louis, MO, USA) (27 mg/kg) or same dose of LPS + GDEs (330 μg/kg). The GDE dose was derived by in vitro to in vivo extrapolation (IVIVE), matching the effective in vitro concentration range (5–10 μg/mL total protein) to an estimated initial plasma concentration in mice (apparent distribution volume ≈ 30–50 mL/kg). This calculation yielded approximately 330 μg/kg, ensuring in vivo exposure consistent with the observed in vitro efficacy. 10-fold higher dose was separately used in toxicity assessment to define a safety margin, and no adverse effects were observed, indicating a therapeutic window of at least 10-fold between the effective and nontoxic doses. Subsequently, survival rates were calculated by Kaplan–Meier analysis. The mice were sacrificed 72 h postinjection, and the liver, spleen, lungs, and kidneys were harvested and fixed in 4% PFA. PFA-fixed liver, spleen, lungs, and kidneys were embedded in paraffin wax. For Hematoxylin & Eosin staining, tissues sectioned with 4 μm thickness were stained with general hematoxylin and eosin staining methods. To visualize fibrotic lesion in tissues, trichrome staining was performed by using a Masson trichrome staining kit (Sigma-Aldrich). Prepared tissue section slides were imaged by Leica digital upright fluorescence microscope (Leica Microsystems, Wetzlar, Germany). The Seoul National University Institutional Animal Care and Use Committee approved the animal experiment protocol used in this study (approval number: SNU-240408–3).

2.14. Histopathological Scoring

Fixed and H&E-stained spleen tissues were assessed based on seven criteria: white pulp atrophy, reduced germinal center cellularity, decreased megakaryocyte numbers, red pulp expansion, red pulp congestion, sinusoid dilation and congestion, and the apoptotic body occurrence. Each criterion was scored on a scale from 0 to 2, indicating no, mild, and severe pathologies, respectively, resulting in a total possible score of 0–14.

2.15. Statistical Analysis

Data are expressed as the mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 10 software (GraphPad Software Inc., USA). Data were compared using student’s t test or one-way analysis of variance (ANOVA) followed by appropriate posthoc tests as specified in the figure legends. The sample size for all experiments are reported consistently in the figure legends. Statistical significance for Kaplan–Meier survival curves was assessed using the log-rank (Mantel-Cox) test.

3. Results

3.1. Physicochemical Characterization of GDEs

GDEs were purified from Panax ginseng roots using a modified centrifugation-based isolation method incorporating sucrose cushioning. Following sequential centrifugation steps at increasing speeds, GDEs were collected from the 8%–30% sucrose interface. According to nanoparticle tracking assay (NTA) results, the particle concentration was 1.09 × 10¹² ± 1.19 × 10¹¹ particles/mL and the mean particle size was 176.2 ± 4.6 nm (Figure A). TEM imaging demonstrated that isolated GDEs exhibited a uniform, spherical shape (Figure B). The average zeta potential and hydrodynamic size of GDEs measured by dynamic light scattering (DLS) were approximately −19.45 mV and 163.63 nm, respectively (Figures C-D). The amounts of ginsenosides Rb1, Rg1, and Rg3 encapsulated in GDEs were quantified using LC-MS/MS. The results indicated that 1 mL of GDEs contained 2.88 μg of Rb1, 1.01 μg of Rg1, and 2.88 μg of Rg3 (Figure E). Next, the physicochemical stability of GDEs was subsequently evaluated through three freeze–thaw cycles. No statistically significant changes were observed in the number of particles, zeta potential, particle concentration, and hydrodynamic size following these cycles, indicating that GDEs are highly stable under harsh storage conditions (Figures F-I). Finally, the protein concentration of GDEs was measured by BCA assay. GDE suspensions containing 9.08 × 10⁸ ± 9.91 × 10⁷, 4.54 × 10⁹ ± 4.95 × 10⁸, and 9.08 × 10⁹ ± 9.91 × 10⁸ particles/mL corresponded to concentrations of 1 μg/mL, 5 μg/mL, and 10 μg/mL of protein, respectively.

1.

Characterization of GDEs (A) Hydrodynamic size distribution of GDEs measured by NTA (Nanoparticle Tracking Analysis). (B) TEM image of GDEs (white scale bar = 250 nm, yellow scale bar = 100 nm). (C) Hydrodynamic size distribution of GDEs measured by DLS. (D) Zeta potential of GDEs. Three independent replicates are plotted in different colors. (E) Rb1, Rg1, and Rg3 levels in GDEs quantified by LC-MS/MS (Liquid Chromatography–Mass Spectrometry/Mass Spectrometry). (F) Hydrodynamic size distributions (measured by NTA), (G) Zeta potentials, (H) average particle number, and (I) hydrodynamic size distribution (measured by DLS) of GDEs after the first, second, and third freeze–thaw cycles (n = 3). One-way ANOVA is conducted with Dunnett’s multiple comparisons test. Significance is set to ns: nonsignificant (p > 0.05).

3.2. Cytotoxicity and Cellular Uptake Kinetics of GDEs in RAW 264.7 Cells

First, the cytotoxicity and cellular uptake of GDEs in RAW 264.7 macrophages were evaluated. Cell viability and proliferation were assessed using the WST-1 assay and Calcein-AM/propidium iodide staining. The WST-1 assay revealed that GDEs did not exhibited significant cytotoxicity across a concentration range of 0.1–30 μg/mL (GDEs of 0.1, 0.5, 1, 5, 10, 20, and 30 μg/mL) following 24 h of incubation with RAW 264.7 cells. Notably, treatment with 5, 10, 20, and 30 μg/mL GDEs significantly enhanced proliferation, with the maximal effect observed at 10 μg/mL (Figure A). To confirm whether these enhanced WST-1 absorbance values were due to enhanced proliferation or metabolic rate, cells were directly visualized with Calcein-AM/PI staining. Calcein-AM/PI results showed that the treatment of those three GDE concentrations for 24 h did not demonstrate any cytotoxicity, since all observable cells were positive for Calcein-AM and negative for PI staining (Figure B). Notably, a greater number of cells were detected in the GDE-treated groups, indicating that enhanced cell proliferation, rather than an increased metabolic rate, caused the increase in coloration in the WST-1 assay.

2.

Cytotoxicity and Cellular Uptake Kinetics of GDEs in RAW 264.7 cells (A) Relative cell viabilities of RAW 264.7 cells 24 h after treatment of different GDE concentrations (n = 3, GDE-untreated group is set to 100%). (B) Live and dead assay on RAW 264.7 cells 24 h after treatment of different GDE concentrations. Live cells are labeled with Calcein-AM (green) and dead cells are labeled with Propidium Iodide (red). The scale bar indicates 100 μm. (C) Time-dependent GDE uptake to RAW 264.7 cells. Cells are labeled with Calcein-AM (green), nuclei are stained with Hoechst 33342 (blue), and GDEs are labeled with Dil (red). Scale bar indicates 10 μm. (D) Relative fluorescent intensities of Dil within Calcein-AM-positive cells are quantified based on Figure C. The CTRL group is set as 100% (n = 6–31). One-way ANOVA is conducted with Dunnett’s multiple comparisons test. Significance is set to *: p < 0.05, ***: p < 0.001, ****: p < 0.0001, ns: nonsignificant (p > 0.05).

To investigate the uptake kinetics of GDEs, DiI-labeled GDEs were incubated with Calcein-AM-loaded RAW 264.7 cells for 1, 2, 4, 8, and 12 h. Confocal live-cell imaging revealed that intracellular uptake of GDEs became detectable at 2 h and steadily increased up to 12 h (Figures C and D). Based on these findings, a 2 h preincubation period with GDEs was selected prior to LPS stimulation in subsequent experiments.

3.3. GDEs Prevent LPS Binding to Macrophage TLR4 by Reducing Its Degree of Glycosylation

The LPS-induced inflammatory response in macrophages involves several sequential events, including the binding of LPS to membrane-localized TLR4, the elevation of intracellular ROS, activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and the excessive production of NO and inflammatory cytokines, ultimately driving toward pro-inflammatory macrophage polarization. To initially assess the effect of GDEs on the interaction between LPS and TLR4, an LPS-binding assay was performed. FITC-conjugated LPS (LPS-FITC) was incubated with RAW 264.7 cells pretreated with or without GDEs (1, 5, and 10 μg/mL), followed by washing after 1 h. The remaining LPS-FITC bound to the RAW 264.7 cells was then imaged and quantified. Pretreatment with GDEs significantly inhibited the binding of LPS to RAW 264.7 cells in a dose-dependent manner, indicating a potential interference with the interaction between LPS and TLR4 (Figures A-B).

3.

GDEs prevent LPS binding to macrophage TLR4 by reducing the glycosylation degree (A) Images of FITC-labeled LPS (green) bound on RAW 264.7 cells (nucleus stained with Hoechst 33342, indicated in blue) pretreated with different GDE concentrations (upper panel). Relative FITC signals are expressed in the colored heatmap. Scale bar indicates 20 μm). (B) Intensities of FITC signals are quantified based on Figure A (n = 10). (C) Average hydrodynamic size distributions of LPS, GDEs, and GDEs incubated with LPS (GDE + LPS) measured by NTA (n = 3). (D) Average hydrodynamic sizes of GDEs and GDEs incubated with LPS (n = 3). Unpaired t test is conducted. Significance is set to ns: nonsignificant (p > 0.05). (E) Western blot image of glycosylated TLR4 (Toll-like receptor 4) (110 kDa), nonglycosylated TLR4 (81 kDa), and beta-actin (42 kDa) expressed in RAW 264.7 cells after treatment of different GDE concentrations (n = 3). Apparent band positions were observed around 130 kDa and 90 kDa. (F) Quantification of total TLR4 expression normalized to beta-actin based on Figure E (n = 3). (G) Glycosylation ratio of TLR4 quantified according to Figure E (n = 3) (H) Western blot image of glycosylated TLR4 (110 kDa), nonglycosylated TLR4 (81 kDa), and beta-actin (42 kDa) expressed in RAW 264.7 cells after LPS and different GDE concentration treatment (n = 3). Apparent band positions were observed around 130 kDa and 90 kDa. (I) Quantification of total TLR4 expression normalized to beta-actin based on Figure H (n = 3). (J) Glycosylation ratio of TLR4 quantified according to Figure H (n = 3). One-way ANOVA is conducted with Dunnett’s multiple comparisons test. Significance is set to *: p < 0.05, ****: p < 0.0001, ns: nonsignificant (p > 0.05).

To determine whether this inhibitory effect resulted from direct sequestration of LPS by GDEs, changes in the average particle size of GDEs following incubation with LPS were analyzed using nanoparticle tracking analysis. The NTA results showed that there was no change in the average particle size even after mixing with LPS (Figures C-D), indicating that the interference was not caused by direct interaction between GDEs and LPS.

Based on these observations, we hypothesized that GDEs may alter the binding affinity of TLR4 for LPS. TLR4 glycosylation is known to be essential for its membrane localization and ligand recognition. Western blot analysis of RAW 264.7 cells showed glycosylated and deglycosylated TLR4 bands at 110–130 kDa and 81 kDa, respectively, with endogenously expressed TLR4 predominantly present in the glycosylated form (Figure E). However, treatment with 5 and 10 μg/mL of GDEs caused a shift toward the deglycosylated form of TLR4 without changing the total amount of TLR4 expression (Figures F and G). Similarly, upon LPS stimulation, GDE pretreatment significantly reduced glycosylated TLR4 expression in a concentration-dependent manner (Figure H). At 10 μg/mL of GDEs, TLR4 remained largely in the deglycosylated form while total TLR4 levels were unchanged (Figures I and J).

To validate that the observed conversion of TLR4 to its deglycosylated form by GDEs is indeed due to the inhibition of N-glycosylation, we employed tunicamycin, a well-characterized glycosylation inhibitor. Western blot analysis demonstrated that tunicamycin treatment successfully reproduced the effect seen with GDEs, significantly reducing the abundance of glycosylated TLR4 while simultaneously promoting the accumulation of the deglycosylated 81 kDa form (Figure S1). The concordance between the effects of GDEs and tunicamycin strongly reinforces the conclusion that GDEs specifically exert their action by suppressing TLR4 glycosylation, thereby lending substantial support to our mechanistic hypothesis.

To further investigate the effect of GDEs on TLR4 deglycosylation, we analyzed changes in the expression levels of oligosaccharyltransferase (OST) complex components, which mediate N-glycosylation within the endoplasmic reticulum. The results showed that GDE treatment significantly reduced the mRNA expression of STT3A, which is the catalytic subunit of the OST complex, by 58.8% compared with the control group. Additionally, mRNA levels of structural OST subunits were markedly decreased after GDE exposure: RPN2 (22.6%), MGAT1 (52.7%), DDOST (26.4%), TUSC3 (41.8%), KCP2 (22.0%), and OSTC (39.0%) (Figure S2). To verify whether the downregulation of OST subunits also occurs at the protein level, a Western blot analysis was performed using STT3A as a representative catalytic component of the OST complex. Quantitative analysis revealed that STT3A expression in the GDE-treated group was reduced to approximately 17.5% of that in the control group (Figures S3A-S3B). These findings suggest that GDEs may suppress TLR4 glycosylation by downregulating OST complex expression, potentially reducing TLR4 affinity for LPS.

Finally, to determine whether the reduction in STT3A mRNA expression was primarily driven by individual ginsenosides or by the synergistic effect of the GDE nanoformulation, we treated RAW 264.7 cells with Rb1, Rg1, and Rg3 at concentrations equivalent to those present in 10 μg/mL GDEs. The results showed that Rb1 and Rg1 had minimal impact on STT3A mRNA levels compared to the control, while Rg3 induced a moderate reduction of approximately 44.5% (Figure S4). In contrast, GDE treatment reduced STT3A expression by approximately 96% under the same conditions (Figure S4). These findings suggest that the transcriptional repression of STT3A is not solely attributable to individual ginsenosides, but rather to the combinatorial effect of GDE components, including lipids, major and minor ginsenosides, which are delivered in the exosome-like nanoformulation.

3.4. Suppression of LPS-Induced Intracellular ROS Spike and NF-κB Activation by GDEs

An increase in intracellular reactive oxygen species (ROS) following LPS binding to TLR4 is a critical step in the macrophage inflammatory signaling cascade. To determine whether reduced LPS-TLR4 interaction by GDEs attenuates the subsequent intracellular ROS burst, RAW 264.7 cells pretreated with 1, 5, and 10 μg/mL of GDEs were stimulated with LPS, and their intracellular ROS levels were visualized by CellRox DeepRed staining. LPS-treated cells demonstrated approximately 2.6-fold higher intracellular ROS signal compared to the naïve cells (Figures A and B). However, GDE pretreatment effectively decreased LPS-induced intracellular ROS in a dose-dependent manner (Figures A and B). Pretreatment with 5 and 10 μg/mL GDEs significantly decreased intracellular ROS levels. Moreover, the intracellular ROS level of the 10 μg/mL GDE-treated group was comparable to that of the CTRL group (naïve RAW 264.7 cells) (Figures A and B). We next investigated whether the prevention of the LPS-induced spike in intracellular ROS sequentially reduced NF-κB activation. As expected, LPS treatment significantly increased NF-κB phosphorylation at p65, an indicative marker for NF-κB activation, and GDE pretreatment decreased the LPS-induced activation of NF-κB dose-dependent manner (Figures C and D).

4.

Suppression of LPS-induced intracellular ROS spike and NF-κB activation by GDEs (A) Intracellular ROS levels of RAW 264.7 cells treated with LPS and different GDE concentrations imaged with CellRox DeepRed (red); the nucleus is stained with Hoechst33342 (blue) (left column). Scale bar indicates 10 μm. Relative intracellular ROS (Reactive Oxygen Species) levels are expressed in the colored heatmap (right column). (B) Relative intracellular ROS levels (CellRox DeepRed signal intensity normalized by Hoechst33342 signal intensity) quantified based on Figure A. The CTRL group is set to 100% (n = 11–19). (C) Western Blot image of phosphorylated NF-κB­(65 kDa) and beta-actin (42 kDa) in RAW 264.7 cells treated with LPS and different GDE concentrations. (D) Quantification of phosphorylated NF-κB expression normalized to beta-actin based on Figure C. One-way ANOVA is conducted with Dunnett’s multiple comparisons test. Significance is set to *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001, ns: nonsignificant (p > 0.05).

3.5. GDEs Suppress NF-κB’s Downstream Target Gene Expressions

The activation of NF-κB initiates the transcription of multiple downstream proinflammatory genes. To further evaluate the inhibitory effects of GDEs on NF-κB-dependent gene expression, mRNA and protein levels of inducible nitric oxide synthase (iNOS), IL-1β, IL-6, and TNF-α were quantified at 6 and 24 h following LPS stimulation in RAW 264.7 cells pretreated with increasing concentrations of GDEs.

Both RT-qPCR and Western blot data for iNOS indicated that LPS-induced mRNA and protein-level upregulation of iNOS expressions were significantly decreased in 5 and 10 μg/mL GDE-pretreated cells (Figures A and B). NO production in LPS-stimulated RAW 264.7 cells was also significantly reduced by GDE treatment at 1, 5, and 10 μg/mL (Figure C). Notably, pretreatment with 10 μg/mL GDEs demonstrated a NO reduction rate comparable to that of cells pretreated with 100 μM L-NMMA, a highly potent iNOS inhibitor (64.59% and 69.76% reduction from the nontreated group, respectively) (Figure C).

5.

GDEs suppress NF-κB’s downstream target gene expressions (A). Relative mRNA expression levels of iNOS (inducible Nitric Oxide Synthase) gene in RAW 264.7 cells treated with LPS (Lipopolysaccharide) and different GDE concentrations. The CTRL group was considered 1 (n = 3). (B) Western blot image of iNOS (130 kDa) and beta-actin (42 kDa) after treatment of LPS and different GDE concentrations. (C) Amount of NO (Nitric Oxide) produced in RAW 264.7 cells treated with LPS, GDEs, and L-NMMA (L-NG-Monomethyl arginine) of 100 uM (n = 3). (D-F) Western blot images of (D) IL-1β (25 kDa), (E) IL-6 (20 kDa), (F) TNF-α (20 kDa), and β -actin (42 kDa) in RAW 264.7 cells treated with LPS and different GDE concentrations. (G-I) ELISA (Enzyme-linked Immunosorbent Assay) assay of secreted (G) IL-1β, (H) IL-6, and (I) TNF-α proteins in supernatant. One-way ANOVA is conducted with Dunnett’s multiple comparisons test. Significance is set to **: p < 0.01, ***: p < 0.001, ****: p < 0.0001, ns: nonsignificant (p > 0.05).

The expression and secretion of proinflammatory cytokines were next examined 24 h after LPS stimulation. Western blot analysis showed that LPS markedly increased IL-1β, IL-6, and TNF-α expression, whereas pretreatment with 5 and 10 μg/mL GDEs progressively reduced protein levels (Figures D-F). Consistently, the ELISA assay revealed that LPS-induced oversecretion of IL-1β, IL-6, and TNF-α was all inhibited by GDE pretreatment at 5 and 10 μg/mL. However, GDE pretreatment at 1 μg/mL exhibited no significant inhibitory effect (Figures G-I).

3.6. Effect of GDEs on LPS-induced Sepsis Model

Finally, the therapeutic efficacy of GDEs was evaluated in an in vivo sepsis model. To assess the in vivo toxicity of GDEs, 8-week-old male ICR mice were intraperitoneally administrated with GDEs at doses of 330 μg/kg and 3,300 μg/kg. Body weight was monitored for 14 days, and major organs were harvested for histological examination. Neither GDE-treated group exhibited significant changes in body weight throughout the observation period (Figure A). Moreover, histological examination revealed no notable abnormalities in the liver, kidney, lung, spleen, or small intestine in either group, indicating that administration of GDEs was well tolerated in vivo (Figure B).

6.

Effect of GDEs on LPS-induced Sepsis model (A) Body weight changes monitored for 14 consecutive days following the intraperitoneal injection of PBS, low-dose GDEs (330 μg/kg), or high-dose GDEs (3300 μg/kg). Body weight measured on the first day is set to 100% (n = 5). (B) Haematoxylin and Eosin (H&E) staining images of the lung, liver, spleen, kidney, and small intestine 14 days after the intraperitoneal injection of PBS, low-dose GDEs (330 μg/kg), or high-dose GDEs (3300 μg/kg). Scale bar indicates 600 μm. (C) Kaplan–Meier survival curve of LPS-injected (n = 20) and LPS/GDE coinjected (LPS + GDE, n = 10) mice. (D) H&E staining and Masson–Trichrome (M-T) staining images of the lung, liver, and spleen harvested from mice injected with PBS (n = 3), LPS (n = 2), or LPS + GDE (n = 6). Erythrocyte infiltrations in the liver tissue are marked with a yellow arrow. Tingible body macrophages are marked with a black arrow. Yellow scale bar indicates 200 μm. White scale bar indicates 50 μm (magnified inset). (E) Histopathological scoring of the spleen based on the image of Figure D (n = 3). One-way ANOVA is conducted with Tukey’s multiple comparisons test for A and E. A log-rank (Mantel-Cox) test is performed for C. Significance is set to *: p < 0.05, **: p < 0.01, ****: p < 0.0001, ns: nonsignificant (p > 0.05).

To evaluate the protective efficacy of GDEs against sepsis, mice were intraperitoneally administered LPS (27 mg/kg), followed immediately by GDE injection (330 μg/kg). Survival rates were monitored for 3 days, and organs were harvested on day 3 for histological assessment. Mice receiving combined LPS and GDE treatment exhibited a 60% survival rate, compared with only 15% survival in mice treated with LPS alone, demonstrating that administration of GDEs significantly improved survival following septic challenge (Figure C). Histopathological analysis revealed that LPS treatment induced significant tissue damage in multiple organs, including the lung, liver, kidney, and spleen. In the lungs, LPS-treated mice exhibited pronounced alveolar septal thickening and fibrotic lesions, indicative of reduced functional alveolar surface area (Figure D). In contrast, mice cotreated with LPS and GDEs exhibited only mild septal thickening and minimal fibrotic changes, comparable to the control group (Figure D). In the liver, LPS treatment caused marked erythrocyte infiltration and disorganized tissue architecture, whereas these pathological features were largely absent in the LPS/GDE cotreated group, which showed only mild structural disruption without detectable erythrocyte infiltration (Figure D, yellow arrow). LPS exposure also resulted in splenic pathology characterized by expansion of the white pulp and extensive infiltration of tingible body macrophages actively phagocytosing damaged cells (Figure D, black arrow). However, spleens from the LPS/GDE cotreated groups demonstrated histological features comparable to those of the control groups, with no noticeable expansion of the white pulp area or increased tingible body macrophage infiltration. Consistently, histopathological scoring demonstrated a significant increase in spleen injury in the LPS-treated group compared with controls (P < 0.0001), whereas cotreatment with GDEs significantly reduced the injury score relative to the LPS group (P = 0.0012) (Figure E).

In summary, immediate administration of GDEs following LPS-induced septic shock significantly improved survival and effectively mitigated organ damage in vivo.

4. Discussion

Ginseng has been used in pharmaceutical applications for thousands of years. In this study, we demonstrated that Ginseng-Derived Exosome-like nanoparticles (GDEs), enriched with diverse ginsenosides, demonstrated anti-inflammatory effects and may serve as potential alternatives or adjuncts to corticosteroid-based therapies. The GDEs used in this study were produced with high purity and yield without heat or toxic chemicals, and their protein and RNA contents were stable for up to 60 days at room temperature. In addition, their physicochemical properties were preserved after repeated freeze–thaw cycles, confirming excellent stability under diverse storage conditions.

The pathophysiology of sepsis is multifactorial, with macrophages playing a central role in initiating and propagating systemic inflammation. Macrophages, as primary responders to pathogens derived signals, release proinflammatory cytokines that recruit and activate other immune cells. Throughout the progression of sepsis, macrophages propagate inflammation to the systemic level and release NO and other mediators, thereby further increasing vasodilation and vascular permeability leading to a cytokine storm and impaired tissue perfusion. These pathological changes can affect the lungs, liver, spleen, kidney, and other organs, ultimately resulting in multiple organ failure. In this study, we demonstrate that GDEs isolated from Panax ginseng C.A. Meyer effectively suppress these key macrophage-mediated pathogenic processes in sepsis.

We found that the suppressive effects of GDEs on LPS-induced macrophage activation are closely associated with the inhibition of TLR4 N-linked glycosylation. Notably, GDEs treatment led to a transcriptional downregulation of multiple subunits of the oligosaccharyltransferase (OST) complex, including STT3A, RPN1, and DAD1. It indicates that GDEs exert a broader regulatory effect on the host glycosylation machinery rather than a TLR4-specific action. Our results suggest that GDEs may serve as novel regulators of OST subunit expression in immune cells. As a functional consequence, GDE treatment markedly reduced the fully glycosylated 110–130 kDa TLR4 band while generating a strong 81 kDa band corresponding to fully deglycosylated TLR4, indicating a near-complete inhibition of TLR4 glycosylation. Given that human TLR4 contains nine N-linked glycosylation sites essential for proper membrane trafficking and LPS recognition, disruption of this glycosylation process can significantly impair downstream signaling.

These findings align with previous reports showing that ginsenoside Rg3, a major component of GDEs, induces deglycosylation of PD-L1 by downregulating glycosylation-related enzymes such as B3GNT3 and MGAT5 in cancer cells. Collectively, these observations suggest that ginsenosides may broadly modulate glycosylation pathways, and our results expand this concept to immune regulation by demonstrating nanoformulated GDEs-mediated inhibition of the OST complex in macrophages.

In addition to glycosylation-dependent effects, other ginsenosides encapsulated within GDEs may contribute to the overall anti-inflammatory activity. Ginsenoside Rb3 has been reported to directly interfere with LPS-TLR4 binding by occupying the hydrophobic pocket of the TLR4/MD2 complex, while ginsenoside Re exhibits modest ROS-scavenging activity and enhances antioxidant enzyme expression through Nrf2 and HO-1 related pathways. These complementary mechanisms may account for the pronounced reduction in intracellular ROS observed following GDE treatment. Consistent with this, we found that GDEs attenuated NF-κB p65 phosphorylation and suppressed downstream expression of inflammatory mediators including iNOS, TNF-α, IL-6, and IL-1β, which is consistent with previous reports of NF-κB inhibition by various ginsenosides such as Rb1 and Rg3.

In summary, GDEs markedly attenuated macrophage-derived proinflammatory mediators and improved survival in a murine sepsis model, supporting their potential as therapeutic candidates for acute inflammatory conditions. Nevertheless, further studies are needed to clarify the broader mechanisms through which GDEs modulate key pathways in sepsis pathophysiology in order to optimize their efficacy and ensure their biosafety for future clinical applications.

5. Conclusion

GDEs effectively prevent LPS-induced hyperactivation of macrophages through a combination of mechanisms, including interference with LPS-TLR4 interaction via TLR4 deglycosylation, ROS level reduction, and NF-κB inactivation. Additionally, GDEs have demonstrated success in improving survival rates and mitigating organ damage in an LPS-induced sepsis model. These results indicate that GDEs hold significant promise as a potential nanomedicine for preventing the fatal manifestations of sepsis without inducing side effects.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00113.

• Supplemental results of molecular works (PDF)

§.

Youngjoon Kim, Youn Kyung Kim, and Sunwoo Lee contributed equally as first authors.

The authors declare no competing financial interest.