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. 2025 Jul 7;3(11):1311–1319. doi: 10.1021/envhealth.5c00140

Enantioselective Modulation of Inflammatory Response through MAPK Pathway by Wavelike Chiral Nanoparticles

Yijin Tian †, Jinghua Hao †,‡, Chao Lin †, Deming Han †, Nali Zhu †, Zhigang Li †, Lixia Zhao †,‡, Lingxiangyu Li †,*, Yawei Wang †,‡, Guibin Jiang †,‡
PMCID: PMC12645301  PMID: 41306299

Abstract

Infectious inflammation remains a major global health issue, so novel strategies to alleviate inflammatory responses are urgently required. Cinnabar (α-HgS) used in traditional Chinese medicine shows protective effects against inflammation, but low anti-inflammatory efficacies ascribed to poor solubility and bioavailability have hindered extensive applications of cinnabar. Nanoscale chirality provides chances to improve efficacies through enantiomer-dependent interactions between cells and chiral cinnabar nanoparticles (HgSNPs). Herein HgSNPs with intrinsically wavelike chirality were prepared through a one-pot seedless method, exhibiting efficacious alleviation of inflammation in vitro and in vivo by HgSNPs with laevorotatory chirality (l-HgSNPs) rather than those with dextrorotatory chirality (d-HgSNPs). The mitogen-activated protein kinase as a crucial pathway involved in inflammation and immunity was directly blocked by l-HgSNPs through inhibition of p38 phosphorylation, so the levels of pro-inflammatory cytokines were reduced significantly. This study showed an enantiomer-dependent immunological response to chiral HgSNPs, providing a novel protocol to efficaciously modulate health risks from infectious inflammation.

Keywords: Chiral cinnabar nanoparticles, Enantiomer-dependent uptake, Macrophages, Infectious inflammation, Mitogen-activated protein kinase pathway

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Introduction

Global climate change and environmental pollution are two serious challenges for human health; extreme weather events such as heat waves or pollutants can alter immune responses and provoke immunotoxicity. − Previous studies documented that pollutants in mixtures or even at low doses can act as immune disruptors and destabilize the balance between pro- and anti-inflammatory responses in exposed individuals, resulting in persistent inflammation that may increase cancer risk. − Various protocols of anti-inflammatory therapies are thus widely developed. , For example, Li et al. prepared chiral radical trapping polymers by mimicking the structure of the cell membrane with imbedded helical proteins, effectively suppressing ferroptosis and depressing inflammatory cytokines to attenuate uncontrolled inflammation. Traditional Chinese medicine provides attractive opportunities due to the wide range of raw materials and long-term effectiveness against inflammation. , Mercury and mercury-containing agents are used in traditional Chinese medicine, wherein the form of mercury sulfide especially cinnabar (α-HgS) has been applied in clinical practices. There are more than 40 traditional medicines with cinnabar in use, showing pharmacological effects like sedation, anti-inflammatory activity, and the promotion of tissue repair. −

Nevertheless, cinnabar is insoluble in aqueous solutions, reducing its bioavailability and delivery in organisms, which results in low anti-inflammatory efficacies. Nanotechnology offers an attractive opportunity for cinnabar due to the unique set of physiochemical properties of nanoparticles compared to those of their bulk counterparts. , Accordingly, the environmental and health risks of nanoparticulate cinnabar have also attracted rising concern; the neurotoxicity of nanoparticulate cinnabar to Caenorhabditis elegans was reported in a recent study, wherein neurobehaviors in Caenorhabditis elegans were inhibited by nanoparticulate cinnabar, resulting in the suppression of locomotion, defecation, egg-laying, and associative learning behaviors. New protocols to improve the efficacy but reduce the side effects of cinnabar are thus inevitably needed.

Chirality at the nanoscale is denoted as chiral nanomaterials that have documented the ability of enantioselective regulation of many natural or synthetic processes, like biological responses, chemical catalysis, and asymmetric reactions. − Previous studies have shown that chirality can affect the affinity of nanomaterials to biomolecules in cells, thus affecting cells through regulating biomolecular functions. , Imparting chirality to nanoparticulate cinnabar may be a feasible strategy to achieve high efficacies and reduce side effects through enantioselective responses. Surface modifications of mercury sulfide metacinnabar (β-HgS) quantum dots with chiral cysteines improved the water-solubility and cytocompatibility, showing the potential of nanoscale chirality in the regulation of biological responses. In general, the chiral nanomaterials that have been used usually derive the chirality from surface modifications with chiral molecules rather than from the intrinsic chirality of materials themselves. The chiral surface modifications are primarily dictated by molecular-level chirality, while intrinsic chirality is normally controlled by an atomic-level structure. Obviously, intrinsic chirality may provide far deeper insights into the enantiomer-dependent biological processes.

Here cinnabar nanoparticles with intrinsic chirality were prepared, α-HgS nanoparticles with left-handed (i.e., l-HgSNPs) and right-handed (i.e., d-HgSNPs) chiral wavelike lattices, respectively. The chiral HgSNPs with superior cytocompatibility exhibited enantioselective modulation of the inflammatory response both in vitro and in vivo, wherein l-HgSNPs rather than d-HgSNPs efficaciously attenuated the inflammatory response induced by lipopolysaccharide (LPS). l-HgSNPs were internalized more efficiently by cells than d-HgSNPs, wherein the mitogen-activated protein kinase (MAPK) pathway engaged in toll-like receptor (TLR) signaling was blocked by l-HgSNPs through the inhibition of p38 phosphorylation, so the mRNA expression of inflammatory mediators downregulated significantly. Clearly, we illustrated underlying mechanisms for the enantioselective modulation of inflammatory response by chiral HgSNPs, providing a novel strategy to confront the threat of persistent inflammation.

Methods and Materials

Preparation and Characterization of Chiral HgSNPs

First, 2 mL of l-penicillamine or d-penicillamine (0.09 mol/L, AR) solution was mixed with 10 mL of mercuric nitrate monohydrate (0.018 mol/L, AR) solution, and the mixture was stirred (750 rpm) at room temperature for 15 min. Then, 0.3 mL of a sodium hydroxide (2 mol/L, CP) solution was added into the mixture dropwise, followed by stirring for another 5 min. Subsequently, 1 mL of a thioacetamide (0.18 mol/L, AR) solution was added within 2 min. The mixture was stirred for 3 h at 38 °C in a water bath, followed by centrifugation at 15 300 rpm for 15 min to collect the precipitate and remove excess reactants. The precipitate was collected, washed three times, and dispersed in ultrapure water to prepare a stock solution of the chiral HgSNPs.

The size and morphology of chiral HgSNPs were determined by transmission electron microscopy (TEM) operating at 200 kV (JEM-2100F, Japan). The lattice fringe spacing was also determined by using high-resolution TEM. Generally, an aqueous solution (20 μL) with HgSNPs (50 mg/L) was deposited on a carbon-coated copper grid, followed by drying at room temperature overnight. The zeta potential of chiral HgSNPs was determined by a DLS analyzer (Zetasizer Nano ZS, U.K.). The circular dichroism (CD) spectra of chiral HgSNPs were obtained using a JASCO-815 instrument (Japan) with an optical path length of 1 cm.

Cytotoxicity of Chiral HgSNPs

The cytotoxicity of chiral HgSNPs was evaluated using a Cell Counting Kit-8 (CCK-8) assay. RAW264.7 cells were seeded at a density of 1 × 105 cells per well in a 96-well plate. After an overnight incubation, the medium was replaced with complete medium containing l-HgSNPs or d-HgSNPs at concentrations of 5, 10, 20, 40, and 80 μg/mL, and the cells were cultured for an another 24 h. Cells that were not exposed to HgSNPs served as the control. Following the incubation, 10 μL of CCK-8 reagent was added to each well, and the plate was incubated for another 30 min. Absorbance was measured at 450 nm by using a microplate reader (Infinite 200pro, Switzerland), and cell viability was calculated. The same procedure was applied to other cell lines to evaluate the cytocompatibility of chiral HgSNPs.

Cellular Uptake of HgSNPs

The amount of internalized l-HgSNPs or d-HgSNPs in cells was quantified by using a mercury analyzer (Lumex RA915M). Cells were seeded at a density of 1 × 106 cells per well in a 12-well plate. After an overnight incubation, the medium was replaced with l-HgSNPs or d-HgSNPs (80 μg/mL) in complete medium. Cells were collected at 4, 8, 12, and 24 h, washed three times with phosphate buffered saline (PBS), and counted using a cell counter. Cells were then harvested and digested in aqua regia overnight, and the resulting solution was diluted and analyzed for mercury content using the mercury analyzer. To examine the effect of endocytosis inhibition on nanoparticle uptake, cells were pretreated with 25 μg/mL dynasore for 2 h before exposure to l-HgSNPs or d-HgSNPs (80 μg/mL) for 4 h, followed by mercury content determination as described.

Intracellular Lysosome pH Assay

RAW264.7 cells were seeded in glass-bottom culture dishes at a density of 2 × 106 cells per well. After an overnight incubation, cells were treated with 80 μg/mL l-HgSNPs or d-HgSNPs for 12 h, followed by washing three times with phosphate buffered saline (PBS). Then, 1 μmol/L LysoSensor probe (Yeasen, China) was added for another incubation of 30 min. Cells were then rinsed twice with PBS and imaged with a confocal microscope (STELLARIS 8, Germany). ImageJ software was used to quantify the fluorescence of LysoSensor in cells, wherein at least 30 cells were analyzed for each treatment.

RNA Extraction and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) Analysis

RAW264.7 cells were seeded at a density of 2 × 106 cells per well in a 6-well plate. After an overnight incubation, cells were divided into four treatment groups: control, LPS-treated group, l-HgSNPs-treated group, and d-HgSNPs-treated group. The control group was maintained in complete medium for 24 h. The LPS group was exposed to complete medium for 4 h followed by LPS (1 μg/mL) treatment for 20 h. The HgSNPs groups were pretreated with l-HgSNPs or d-HgSNPs (80 μg/mL) for 4 h, followed by coexposure to LPS (1 μg/mL) for another 20 h. Total RNA was extracted using SparkZol Reagent (SparkJade, China), and its concentration and integrity were examined by a Nanodrop spectrophotometer (ND 2000, U.S.A.). Then, a SPARKscript II All-in-one RT SuperMix for qPCR kit (SparkJade, China) was used for reverse transcription of RNA to cDNA. Real-time qPCR was performed using 2× SYBR Green qPCR Mix on a QuantStudioTM5 system under the following conditions: initial denaturation at 94 °C for 3 min, followed by 40 cycles of 94 °C for 20 s, 55–60 °C for 20 s, and 72 °C for 30 s. GAPDH was used as the reference gene, and relative gene expression was calculated using the 2–ΔΔCt method. Primer sequences are detailed in Table S1.

To investigate the relationship between the anti-inflammatory effect of chiral HgSNPs and cell uptake of HgSNPs, cells were pretreated with an inhibitor (i.e., dynasore). Specifically, cells were pretreated with complete medium containing dynasore (25 μg/mL) for 2 h, and the medium was discarded. Cells were then exposed to l-HgSNPs or d-HgSNPs (80 μg/mL) with the inhibitor for 1 h. LPS (1 μg/mL) was added to stimulate cells for another 30 min. Finally, cells were collected for RT-qPCR analysis.

Transcriptomic Analysis

RAW264.7 cells were seeded at a density of 2 × 106 cells per well in a 6-well plate. After an overnight incubation, the control group was maintained in complete medium, while the LPS group was exposed to LPS (1 μg/mL) for 30 min after exposure to the medium for 1 h. The HgSNPs groups were pretreated with l-HgSNPs or d-HgSNPs (80 μg/mL) for 1 h before coexposure to LPS (1 μg/mL) for 30 min. Total RNA was extracted using Trizol reagent. PolyA-tailed mRNA molecules were selectively captured by using Oligo­(dT) magnetic beads. Following library preparation according to the manufacturer’s protocol, quality-verified libraries were pooled and sequenced on the DNBSEQ-T7 platform. Raw image data were converted into raw sequences using base calling, and data quality control was performed using FASTP software, including adapter trimming, low-quality read filtering (Q20 threshold), and N-base removal. Clean data were aligned to the reference genome using STAR, and gene expression was quantified using StringTie2 with FPKM and TPM normalization. Differential expression analysis was conducted using DESeq2, with a significance threshold of adjusted p-value (p adj) ≤ 0.05 and |log2­(fold change)| ≥ 1. Differentially expressed genes were subjected to GO and KEGG pathway enrichment analyses. Gene ontology (GO) is a comprehensive database for describing gene functions, which can be categorized into three domains: biological processes, cellular components, and molecular function. The primary principle of GO functional enrichment analysis is to identify significantly enriched GO terms among differentially expressed genes compared with the genomic background, thereby elucidating the biological functions significantly associated with these differentially expressed genes.

Western Blotting Analysis

Cell treatments for Western blotting analysis were identical to those used for transcriptome sequencing. Cells were lysed on ice for 20 min by using RIPA buffer containing protease and phosphatase inhibitors. Lysates were centrifuged at 12 000 × g for 15 min at 4 °C, and the supernatant was collected. The protein concentration was determined using a BCA (bicinchoninic acid assay) protein assay kit (Beyotime, China). Proteins were mixed with 5× loading buffer (4:1 ratio) and then boiled at 95 °C for 5 min. Protein samples (30 μg) were separated on a 4–20% SDS-PAGE gel. Proteins were then transferred to a poly­(vinylidene fluoride) (PVDF) membrane, blocked with fast-blocking buffer (Share-bio, China) for 30 min, and incubated with primary antibodies at 4 °C overnight. The primary antibodies used in this study were purchased from Cell Signaling Technology (U.S.A.); p38 (1:1000), p-p38 (1:1000), and GAPDH (1:10 000) were obtained from Abcam (U.K.). Membranes were subsequently incubated with HRP-conjugated secondary antibodies (1:8000, Sigma) at room temperature for 1 h. Protein bands were visualized and quantified using a ChemiDoc XRS system (Bio-Rad, U.S.A.), and representative images were cropped for presentation.

LPS-Induced Acute Inflammation Murine Model

The in vivo experiment of acute inflammation was performed on the basis of protocols approved by the Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Institutional Animal Care and Use Committee (Approval number: AEWC-RCEES-2023011). A total of 30 male mice (C57BL/6, 8-week-old) were randomly divided into the following six groups: (1) control group: mice received intraperitoneal (ip) injection of saline for 6 h. (2) LPS group: mice were treated with LPS (10 mg/kg, ip) for 6 h. (3) l-HgSNPs group: mice were pretreated with 5 mg/kg l-HgSNPs intraperitoneally for 1 h before saline injection (ip) for 5 h. (4) d-HgSNPs group: mice were pretreated with 5 mg/kg d-HgSNPs intraperitoneally for 1 h before saline injection (ip) for 5 h. (5) l-HgSNPs + LPS group: mice were pretreated with 5 mg/kg l-HgSNPs intraperitoneally for 1 h before LPS injection (10 mg/kg, ip) for 5 h. (6) d-HgSNPs + LPS group: mice were pretreated with 5 mg/kg d-HgSNPs intraperitoneally for 1 h before LPS injection (10 mg/kg, ip) for 5 h. Mice were then sacrificed, and serum was collected to measure the levels of pro-inflammatory cytokines in serum samples. To assess the histological damage caused by acute inflammation, liver samples were harvested, fixed with 4% paraformaldehyde solution, washed with 75% ethanol, and stained with hematoxylin and eosin by the histology core facility provided by Servicebio Technology Co., Ltd. (China), wherein images were acquired through ImageJ.

Statistical Analysis

Data were presented as average value ± standard deviation. The in vitro experiments were carried out by using three independent experiments with five repetitions under each condition. The mouse in vivo experiments were conducted on the basis of 5 rats for each group. Statistical analysis of the samples was conducted using student’s t test or ANOVA, and a p-value below 0.05 was considered to be significantly different.

Results and Discussion

Characterization of Wavelike Chiral HgSNPs

Both l-HgSNPs and d-HgSNPs showed similar morphology and size (Figure a–d); an ellipse morphology was observed for l-HgSNPs (Figure a), with the average length and width of 14.5 ± 0.8 and 7.5 ± 0.9 nm (Figure e,f), respectively. Similarly, d-HgSNPs with an ellipse shape showed an average length and width of 14.5 ± 1.1 and 7.5 ± 1.3 nm (Figure c,e,f), respectively. A left-handed wavelike lattice spacing of 0.34 nm was observed for l-HgSNPs (Figure b), corresponding to the interplanar distance between (101) planes of α-HgS. d-HgSNPs exhibited a right-handed wavelike lattice spacing of 0.34 nm (Figure d). Both l-HgSNPs and d-HgSNPs had a negative charge (Figure g); the values of l-HgSNPs and d-HgSNPs were −45.9 ± 2.6 and −47.7 ± 2.8 mV, respectively. The chiroptical properties of the prepared HgSNPs were determined through CD spectra, wherein l-HgSNPs and d-HgSNPs showed corresponding mirror image CD scans with peaks at 244 and 318 nm (Figure h), indicating the occurrence of chirality for the prepared HgSNPs. Given the wavelike lattice of chiral HgSNPs, we synthesized α-HgSNPs with intrinsic chirality.

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Characterization of wavelike chiral α-HgSNPs. (a) TEM image of l-HgSNPs. (b) High-resolution TEM shows left-handed lattice spacing of 0.34 nm for l-HgSNPs. (c) TEM image of d-HgSNPs. (d) High-resolution TEM shows right-handed lattice spacing of 0.34 nm for d-HgSNPs. (e) Length distribution of chiral HgSNPs (n = 97). (f) Width distribution of chiral HgSNPs (n = 97). (g) Zeta potentials of l-HgSNPs and d-HgSNPs. (h) CD spectra of l-HgSNPs and d-HgSNPs.

Enantiomer-Dependent Modulation of Inflammatory Response by Chiral HgSNPs In Vitro and In Vivo

The cytotoxicity of chiral HgSNPs toward macrophages was examined first. Negligible changes in the viability of RAW264.7 cells were observed for either l-HgSNPs or d-HgSNPs even when the concentration of mercury increased to 80 mg/L (Figure S1), being consistent with the general concept of the negligible toxicity of metal sulfide toward organisms. In addition, exposure of chiral HgSNPs to RAW264.7 cells would not cause inflammatory responses (Figure S2). Chiral HgSNPs were thus applied to cells with LPS stimulation to examine the anti-inflammatory activity.

The mRNA expression of TNF-α, IL-1β, IL-6, and iNOS was determined; compared with the control, LPS stimulation caused great upregulation of these inflammatory mediators (Figure ), implying that LPS triggered inflammatory responses. Nevertheless, an application of l-HgSNPs to cells with LPS stimulation can greatly downregulate the mRNA expression of inflammatory mediators (Figure ), showing the superior anti-inflammatory activity of l-HgSNPs. In contrast, d-HgSNPs played a slight or even negligible role in resolving LPS-induced inflammatory responses (Figure ). The great differences in the expression of inflammatory mediators between l-HgSNPs and d-HgSNPs exhibited the enantiomer-dependent modulation of inflammatory responses by chiral HgSNPs. The surface marker cluster of differentiation 86 (CD86), a protein of the immunoglobulin superfamily related to immunological response, also showed the superior anti-inflammatory activity of l-HgSNPs toward macrophages under the condition of LPS stimulation (Figure S3). Clearly, HgSNPs with intrinsically wavelike chirality exhibited enantiomer-dependent regulation of the immunological response in macrophages, implying that l-HgSNPs relative to d-HgSNPs would be a superior anti-inflammatory agent.

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Enantiomer-dependent modulation of inflammatory responses in RAW246.7 cells by chiral HgSNPs, wherein mRNA expression levels of TNF-α, IL-1β, iNOS, and IL-6 were measured by quantitative PCR: (a) TNF-α, (b) IL-1β, (c) iNOS, and (d) IL-6. All data are presented as the mean ± SD, n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant differences compared to the LPS stimulation alone. Different letters indicate significant differences within treatments.

The l-HgSNPs exhibited superior anti-inflammatory activity in vitro. More importantly, l-HgSNPs showed high cytocompatibility; negligible cytotoxicity of l-HgSNPs to mammalian cells, including BV2, A549, NCI-H460, and HepG2, was observed even at 80 mg­(mercury)/L (Figure S4). The anti-inflammatory activity of l-HgSNPs was thus evaluated in vivo (Figure a). Compared with the control group, LPS stimulation increased the expression of pro-inflammatory cytokines significantly (Figure b,c); the levels of TNF-α and IL-1β in serum reached 43.9 ± 1.3 and 3.8 ± 0.5 pg/mL, respectively, indicating the establishment of inflammatory mouse models. l-HgSNPs can significantly reduce the expression of pro-inflammatory cytokines in serum when LPS stimulation was still conducted (Figure b,c), wherein the levels of TNF-α and IL-1β in serum were 24.9 ± 5.8 and 1.7 ± 0.6 pg/mL, respectively, showing the effective suppression of inflammatory responses by l-HgSNPs. However, d-HgSNPs failed in attenuating the inflammatory response induced by LPS stimulation (Figure b,c); the serum levels of TNF-α and IL-1β were still 41.9 ± 8.1 and 3.2 ± 0.6 pg/mL, respectively, even though an injection of d-HgSNPs was performed before LPS stimulation, which are comparable to those from the LPS group.

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Role of chiral HgSNPs in alleviating LPS-induced inflammatory mouse models. (a) Scheme of anti-inflammatory response of chiral HgSNPs in vivo. (b) Levels of TNF-α in serum samples. (c) Levels of IL-1β in serum samples. (d) Number of inflammatory cells in the liver, as calculated by Case Viewer 2.4 software. All data are presented as the mean ± SD, n = 3. *p < 0.05 and **p < 0.01 indicate significant differences compared to the LPS stimulation alone. Different letters indicate significant differences within treatments.

In addition to measuring the serum levels of pro-inflammatory cytokines, hematoxylin and eosin (H&E) staining of liver tissue was performed to observe the anti-inflammatory efficiency of l-HgSNPs, since infectious inflammation can primarily result in liver injury. Compared to the control group, LPS stimulation led to more massive inflammatory cells in the liver tissue (Figure S5), which corresponds to the high levels of pro-inflammatory cytokines in serum. l-HgSNPs significantly attenuated the severity of liver injury compared to the mice receiving LPS injection alone or d-HgSNPs injection in advance (Figure d), wherein the number of inflammatory cells in the liver tissue from mice receiving l-HgSNPs injection was comparable to that from the control group. Taken together, the in vivo data also demonstrated an enantiomer-dependent immunological response to chiral HgSNPs, wherein l-HgSNPs can attenuate the inflammatory response induced by LPS, exhibiting great potential as an anti-inflammatory agent to alleviate inflammation.

Mechanisms for Enantiomer-Dependent Modulation of Inflammatory Response by Chiral HgSNPs

To elucidate the underlying mechanisms for the enantiomer-dependent modulation of inflammatory response by chiral HgSNPs, RNA sequencing of cells was performed. Compared with LPS stimulation alone, an application of chiral HgSNPs to cells prior to LPS stimulation exhibited great differences in gene expression (Figure S6); the introduction of l-HgSNPs induced 405 differentially expressed genes to be changed, wherein 300 and 105 genes were upregulated and downregulated, respectively. GO enrichment analysis of differentially expressed genes was performed to reveal the possible physiological reasons for the enantiomer-dependent immunological response, exhibiting significant changes in the positive regulation of cytokine production between LPS stimulation alone and LPS stimulation with chiral HgSNPs pretreatment (Figure S7). Moreover, great changes in the positive regulation of cytokine production were observed for l-HgSNPs compared to d-HgSNPs, corresponding well with changes in the inflammatory mediators (TNF-α, IL-1β, iNOS, and IL-6). KEGG analysis was then conducted to identify the top 30 enriched pathways (Figure S8), showing that the differentially expressed genes are primarily part of the MAPK signaling pathway (Figure a,b). The MAPK pathway is thus proposed to be closely related to the enantiomer-dependent immunological response by chiral HgSNPs.

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l-HgSNPs target the MAPK pathway to alleviate inflammation. (a) KEGG pathway enrichment analysis of the differentially expressed genes between LPS stimulation and l-HgSNPs pretreatment prior to LPS stimulation. (b) KEGG pathway enrichment analysis of the differentially expressed genes between LPS stimulation and d-HgSNPs pretreatment prior to LPS stimulation. (c) Western blotting analysis revealed the inhibitory effects of chiral HgSNPs on the phosphorylation of p38 in LPS-stimulated cells. GAPDH was used as a loading control. (d) The band density of each blot in Figure c was quantified in a bar graph as the ratio of phosphorylation to total protein under different conditions. (e) Effect of the inhibitor on the cellular uptake of chiral HgSNPs. (f) Western blotting analysis revealed effects of the inhibitor on the phosphorylation of p38 in LPS-stimulated RAW264.7 cells pretreated with chiral HgSNPs. (g) Representative images of control and chiral HgSNPs-treated cells stained with LysoSensor Green DND-189, which accumulated in acidic organelles and showed green fluorescence. (h) The mean intensity of green fluorescence in the cells showed the changes in the lysosomal pH upon the chiral HgSNPs treatment. The data were calculated from 20 cells per condition. All data are presented as the mean ± SD, n = 3 except the denoted number. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant differences between treatments.

In general, the MAPK pathway plays a vital function in TLR signaling and the production of pro-inflammatory mediators. Phosphorylation of p38 is able to induce MAPK activation and subsequently drive inflammation. , Western blotting analysis exhibited that LPS stimulation increased the phosphorylation level of p38 greatly (Figure c,d), suggesting the activation of MAPK by LPS. However, an application of l-HgSNPs to cells prior to LPS stimulation significantly inhibited the phosphorylation of p38, wherein the p-p38 protein almost degraded completely (Figure c,d). In addition, the protein expression of JNK was not activated by chiral HgSNPs (Figure S9). Obviously, l-HgSNPs can regulate MAPK activation through modulation of the phosphorylation of p38 protein. Nevertheless, d-HgSNPs inapparently inhibited the phosphorylation of p38, wherein the expression of p38 phosphorylation was shown clearly (Figure c,d), being far stronger than that in the case of l-HgSNPs.

The cellular uptake of nanoparticles is generally considered a vital factor affecting their anti-inflammatory efficiency, , so the uptake of chiral HgSNPs in cells was evaluated. The amount of mercury in cells gradually increased in a time-dependent process, irrespective of l-HgSNPs or d-HgSNPs (Figure S10). For example, the uptake of chiral HgSNPs for 8 h was greater than that for 4 h, whereas significant differences in the amount of mercury were observed between chiral HgSNPs (Figure e). Compared to d-HgSNPs, l-HgSNPs exhibited a high cellular uptake of mercury; the amount of l-HgSNPs in cells was 1.60–1.75 times larger than that of d-HgSNPs (Figure e). Nevertheless, negligible dissolution of chiral HgSNPs was observed in cells irrespective of l-HgSNPs or d-HgSNPs, since ionic Hg2+ was not detected, indicating that the enantiomer-dependent modulation of inflammation by chiral HgSNPs was not attributed to ionic Hg2+. The higher cellular uptake of l-HgSNPs than d-HgSNPs is reasonable, as recent studies have demonstrated that left-handed nanoparticles readily enter into macrophages due to chirality-driven homologous adhesion between left-handed nanoparticles and the macrophage membrane. In our recent study, we also found that gold nanoparticles (AuNPs) with laevorotatory chirality showed superior uptake of AuNPs (l-AuNPs) relative to their dextrorotatory enantiomers (d-AuNPs), wherein l-AuNPs can penetrate the phospholipid molecules by overcoming less energy.

Dynasore, an inhibitor of endocytosis, was applied to cells to examine interactions between chiral HgSNPs and cells. Negligible toxicity of dynasore toward cells was observed when its concentration was not higher than 25 μg/mL (Figure S11), so we chose this dose during inhibitory tests. Dynasore caused a great reduction of chiral HgSNPs uptake in cells, wherein the amount of mercury in cells reduced by almost half (Figure e). Western blotting analysis showed that the inhibition of p38 phosphorylation caused by l-HgSNPs was blocked due to the presence of dynasore (Figure f), demonstrating the vital role of l-HgSNPs in the inhibition of MAPK activation.

Lysosomal damage due to acidification can also affect the phosphorylation of p38 protein. To examine whether chiral HgSNPs caused lysosomal acidification to achieve the inhibition of p38 phosphorylation, a specific probe (LysoSensor Green DND-189) was applied to cells with chiral HgSNPs, showing negligible differences in the intensity of green fluorescence in the cells (Figure g,h). Thus, the high cellular uptake of l-HgSNPs relative to that of d-HgSNPs inhibited the phosphorylation of p38 protein to block MAPK activation directly, downregulating the expression of inflammatory mediators, which results in an enantiomer-dependent immunological response to chiral HgSNPs (Figure ).

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Mechanisms for enantiomer-dependent modulation of immunological response in RAW246.7 cells by chiral HgSNPs.

Conclusions

We reported an enantiomer-dependent immunological response to cinnabar nanoparticles with intrinsically wavelike chirality. Imparting chirality to nanoparticles can provide a novel protocol to efficaciously modulate infectious inflammation by increasing the cellular uptake of nanoparticles, wherein l-HgSNPs compared to d-HgSNPs can readily enter into cells. Accordingly, l-HgSNPs blocked the activation of the MAPK pathway directly through inhibiting the phosphorylation of p38 protein. Moreover, l-HgSNPs exhibited good cytocompatibility, providing an opportunity for modulating inflammatory responses in vivo. Indeed, an application of l-HgSNPs to inflammatory mouse models through intraperitoneal injections alleviated the inflammatory response induced by LPS. This work documented that nanoscale chirality can be used as a novel protocol to fight against health risks arising from infectious inflammation through targeted blocking of inflammatory pathways.

Supplementary Material

eh5c00140_si_001.pdf (1.6MB, pdf)

Acknowledgments

We thank the Natural Science Foundation of Zhejiang Province (LR24D010001), the Research Funds of Hangzhou Institute for Advanced Study, UCAS (2024HIAS-Y001, 2024HIAS-P001, and 2024HIAS-V001), and the National Natural Science Foundation of China (22376045, 22021003, and 21976163) for the financial support.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/envhealth.5c00140.

  • Additional data for the cytotoxicity of chiral HgSNPs, cytocompatibility of chiral HgSNPs, RNA-Seq transcriptome analysis, cellular uptake of chiral HgSNPs, and primer sequence for RAW264.7 cells (PDF)

The authors declare no competing financial interest.

References

  1. Suzuki T., Hidaka T., Kumagai Y., Yamamoto M.. Environmental pollutants and the immune response. Nat. Immunol. 2020;21:1486–1495. doi: 10.1038/s41590-020-0802-6. [DOI] [PubMed] [Google Scholar]
  2. Craig E. A., Lin Y., Ge Y., Wang X., Murphy S. K., Harrington D. K., Miller R. K., Thurston S. W., Hopke P. K., Barrett E. S., O’Connor T. G., Rich D. Q., Zhang J.. Associations of gestational exposure to air pollution and polycyclic aromatic hydrocarbons with placental inflammation. Environ. Health. 2024;2:672–680. doi: 10.1021/envhealth.4c00077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Feng X., Luo Y., Zheng M., Sun X., Shen X.. Independent and combined associations between metals exposure and inflammatory markers among the general U.S. adults. Environ. Health. 2025;3:282–290. doi: 10.1021/envhealth.4c00097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Wang L.-Q., Liu T., Yang S., Sun L., Zhao Z.-Y., Li L.-Y., She Y.-C., Zheng Y.-Y., Ye X.-Y., Bao Q., Dong G.-H., Li C.-W., Cui J.. Perfluoroalkyl substance pollutants activate the innate immune system through the AIM2 inflammasome. Nat. Commun. 2021;12:2915. doi: 10.1038/s41467-021-23201-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Park M., Park S., Choi Y., Cho Y., Kim M. J., Park Y., Chung S. W., Lee H., Lee S.. The mechanism underlying correlation of particulate matter-induced ferroptosis with inflammasome activation and iron accumulation in macrophages. Cell Death Discovery. 2024;10:144. doi: 10.1038/s41420-024-01874-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hassen H. Y., Govarts E., Remy S., Cox B., Iszatt N., Portengen L., Covaci A., Schoeters G., Den Hond E., Henauw S. D., Bruckers L., Koppen G., Verheyen V. J.. Association of environmental pollutants with asthma and allergy, and the mediating role of oxidative stress and immune markers in adolescents. Environ. Res. 2025;265:120445. doi: 10.1016/j.envres.2024.120445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Wang W., Yang C., Wang F., Wang J., Zhang F., Li P., Zhang L.. Does nonsteroidal anti-inflamatory drug use modify all-cause and cause-specific mortality associated with PM2.5 and its components? A nationally representative cohort study (2007–2017) Environ. Health. 2025;3:14–25. doi: 10.1021/envhealth.4c00133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Tabas I., Glass C. K.. Anti-inflammatory therapy in chronic disease: Challenges and opportunities. Science. 2013;339:166–172. doi: 10.1126/science.1230720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Li Y., Wang J., Li Y., Luo J., Liu F., Chen T., Ji Y., Yang H., Wang Z., Zhao Y.. Attenuating uncontrolled inflammation by radical trapping chiral polymer micelles. ACS Nano. 2023;17:12127–12139. doi: 10.1021/acsnano.2c12356. [DOI] [PubMed] [Google Scholar]
  10. Wen Y., Feng C., Chen W., Chen C., Kuang S., Liu F., Tang Q., Chen M.. Effect of traditional Chinese medicine on serum inflammation and efficacy in patients with sepsis: a systematic review and meta-analysis. Ann. Palliat. Med. 2021;10:12456–12466. doi: 10.21037/apm-21-3179. [DOI] [PubMed] [Google Scholar]
  11. Yang J., Yin N., Yang R., Faiola F.. Role of pollution-induced oxidative stress and inflammation in neurodegenerative diseases, and the mechanisms of traditional Chinese medicine’s potential remediation. Rev. Environ. Contam. Toxicol. 2024;262:9. doi: 10.1007/s44169-024-00059-z. [DOI] [Google Scholar]
  12. Zhao M., Li Y., Wang Z.. Mercury and mercury-containing preparations: History of use, clinical applications, pharmacology, toxicology, and pharmacokinetics in traditional Chinese medicine. Front. Pharmacol. 2022;13:807807. doi: 10.3389/fphar.2022.807807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Liu J., Wei L., Wang Q., Lu Y., Zhang F., Shi J., Li C., Cherian M. G.. A review of cinnabar (HgS) and/or realgar (As4S4)-containing traditional medicines. J. Ethnopharmacol. 2018;210:340–350. doi: 10.1016/j.jep.2017.08.037. [DOI] [PubMed] [Google Scholar]
  14. Wang C., Yang Y., Liang R., Wu S., Xuan C., Lv W., Li J.. Preparation and anti-inflammatory effect of mercury sulphide nanoparticle-loaded hydrogels. J. Drug Target. 2024;32:557–569. doi: 10.1080/1061186X.2024.2332729. [DOI] [PubMed] [Google Scholar]
  15. Liu J., Shi J., Yu L., Goyer R. A., Waalkes M. P.. Mercury in traditional medicines: is cinnabar toxicologically similar to common mercurial? Exp. Biol. Med. 2008;233:810–817. doi: 10.3181/0712-MR-336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Wang H., Zhu J.. A sonochemical method for the selective synthesis of alpha-HgS and beta-HgS nanoparticles. Ultrason. Sonochem. 2004;11:293–300. doi: 10.1016/j.ultsonch.2003.06.002. [DOI] [PubMed] [Google Scholar]
  17. Galain I., Maria P. B., Ivana A., Laura F.. Hydrothermal synthesis of alpha- and beta-HgS nanostructures. J. Cryst. Growth. 2017;457:227–233. doi: 10.1016/j.jcrysgro.2016.08.066. [DOI] [Google Scholar]
  18. Li L., Li Y., Zeng K., Wang Q.. Mercuric sulfide nanoparticles suppress the neurobehavioral functions of Caenorhabditis elegans through a Skp1-dependent mechanism. Food Chem. Toxicol. 2024;186:114576. doi: 10.1016/j.fct.2024.114576. [DOI] [PubMed] [Google Scholar]
  19. Xu L., Wang X., Wang W., Sun M., Choi W. J., Kim J., Hao C., Li S., Qu A., Lu M., Wu X., Colombari F. M., Gomes W. R., Blanco A. L., de Moura A. F., Guo X., Kuang H., Kotov N. A., Xu C.. Enantiomer-dependent immunological response to chiral nanoparticles. Nature. 2022;601:366–373. doi: 10.1038/s41586-021-04243-2. [DOI] [PubMed] [Google Scholar]
  20. Li S., Xu X., Xu L., Lin H., Kuang H., Xu C.. Emerging trends in chiral inorganic nanomaterials for enantioselective catalysis. Nat. Commun. 2024;15:3506. doi: 10.1038/s41467-024-47657-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mishra K., Guyon D., San Martin J., Yan Y.. Chiral perovskite nanocrystals for asymmetric reactions: a highly enantioselective strategy for photocatalytic synthesis of N-C axially chiral heterocycles. J. Am. Chem. Soc. 2023;145:17242–17252. doi: 10.1021/jacs.3c04593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wang W., Zhao J., Hao C., Hu S., Chen C., Cao Y., Xu Z., Guo J., Xu L., Sun M., Xu C., Kuang H.. The development of chiral nanoparticles to target NK cells and CD8+ T cells for cancer immunotherapy. Adv. Mater. 2022;34:2109354. doi: 10.1002/adma.202109354. [DOI] [PubMed] [Google Scholar]
  23. Shi B., Zhao J., Xu Z., Chen C., Xu L., Xu C., Sun M., Kuang H.. Chiral nanoparticles force neural stem cell differentiation to alleviate Alzheimer’s disease. Adv. Sci. 2022;9:2202475. doi: 10.1002/advs.202202475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Yang F., Gao G., Wang J., Chen R., Zhu W., Wang L., Ma Z., Luo Z., Sun T.. Chiral β-HgS quantum dots: Aqueous synthesis, optical properties and cytocompatibility. J. Colloid Interface Sci. 2019;537:422–430. doi: 10.1016/j.jcis.2018.11.057. [DOI] [PubMed] [Google Scholar]
  25. Levard C., Hotze E. M., Colman B. P., Dale A. L., Truong L., Yang X. Y., Bone A. J., Brown G. E., Tanguay R. L., Di Giulio R. T., Bernhardt E. S., Meyer J. N., Wiesner M. R., Lowry G. V.. Sulfidation of silver nanoparticles: Natural antidote to their toxicity. Environ. Sci. Technol. 2013;47:13440–13448. doi: 10.1021/es403527n. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tang H., Zhang Y., Yang T., Wang C., Zhu Y., Qiu L., Liu J., Song Y., Zhou L., Zhang J., Wong Y. K., Liu Y., Xu C., Wang H., Wang J.. Cholesterol modulates the physiological response to nanoparticles by changing the composition of protein corona. Nat. Nanotechnol. 2023;18:1067–1077. doi: 10.1038/s41565-023-01455-7. [DOI] [PubMed] [Google Scholar]
  27. Ahmed O., Robinson M. W., O’Farrelly C.. Inflammatory processes in the liver: divergent roles in homeostasis and pathology. Cell. Mol. Immunol. 2021;18:1375–1386. doi: 10.1038/s41423-021-00639-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Arthur J. S. C., Ley S. C.. Mitogen-activated protein kinases in innate immunity. Nat. Rev. Immunol. 2013;13:679–692. doi: 10.1038/nri3495. [DOI] [PubMed] [Google Scholar]
  29. Zarubin T., Han J.. Activation and signaling of the p38 MAP kinase pathway. Cell Res. 2005;15:11–18. doi: 10.1038/sj.cr.7290257. [DOI] [PubMed] [Google Scholar]
  30. Kim S. Y., Tang M., Chih S. Y., Sallavanti J., Gao Y., Qiu Z., Wang H., Li W.. Involvement of p38 MAPK and MAPKAPK2 in promoting cell death and the inflammatory response to ischemic stress associated with necrotic glioblastoma. Cell Death Dis. 2025;16:12. doi: 10.1038/s41419-025-07335-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Bachmaier K., Stuart A., Singh A., Mukhopadhyay A., Chakraborty S., Hong Z., Wang L., Tsukasaki Y., Maienschein-Cline M., Ganesh B. B., Kanteti P., Rehman J., Malik A. B.. Albumin nanoparticle endocytosing subset of neutrophils for precision therapeutic targeting of inflammatory tissue injury. ACS Nano. 2022;16:4084–4101. doi: 10.1021/acsnano.1c09762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zhou M., Tang Y., Lu Y., Zhang T., Zhang S., Cai X., Lin Y.. Framework nucleic acid-based and neutrophil-based nanoplatform loading baicalin with targeted drug delivery for anti-inflammation treatment. ACS Nano. 2025;19:3455–3469. doi: 10.1021/acsnano.4c12917. [DOI] [PubMed] [Google Scholar]
  33. Zhang Y., Cui T., Yang J., Huang Y., Ren J., Qu X.. Chirality-dependent reprogramming of macrophages by chiral nanozymes. Angew. Chem., Int. Ed. 2023;62:e202307076. doi: 10.1002/anie.202307076. [DOI] [PubMed] [Google Scholar]
  34. Hao J., Tian Y., Tang J., Zhu N., Li Z., Li L., Wang Y., Jiang G.. Enantiomer-dependent uptake of chiral nanoparticles in macrophages modulates the inflammatory response through the NF-κB pathway. Environ. Sci. Technol. 2025;59:6428–6439. doi: 10.1021/acs.est.4c12577. [DOI] [PubMed] [Google Scholar]
  35. Tremblay C. S., Chiu S. K., Saw J., McCalmont H., Litalien V., Boyle J., Sonderegger S. E., Chau N., Evans K., Cerruti L., Salmon J. M., McCluskey A., Lock R. B., Robinson P. J., Jane S. M., Curtis D. J.. Small molecule inhibition of dynamin-dependent endocytosis targets multiple niche signals and impairs leukemia stem cells. Nat. Commun. 2020;11:6211. doi: 10.1038/s41467-020-20091-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Gallagher E. R., Oloko P. T., Fitch T. C., Brown E. M., Spruce L. A., Holzbaur E. L. F.. Lysosomal damage triggers a p38 MAPK-dependent phosphorylation cascade to promote lysophagy via the small heat shock protein HSP27. Curr. Biol. 2024;34:5739–5757. doi: 10.1016/j.cub.2024.10.061. [DOI] [PMC free article] [PubMed] [Google Scholar]

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