Goblet Cell–Mediated Clearance of Metal Nanoparticles: An In Vivo Pathway Accelerated by Chinese‑Herb‑Induced Diarrhea

Abstract

Despite extensive use of metal nanoparticles in medicine, their in‑vivo clearance remains poorly understood. In this study, we demonstrate that intestinal goblet cells (GCs) and gastric parietal cells (PCs) actively transport a range of metal nanoparticles—triangular silver nanoplates, magnetic nanoparticles, gold nanorods, and gold nanoclusters—into the gut lumen, where they are excreted in feces. Using a mouse model with common bile duct (CBD) ligation to block the hepato‑biliary route, we showed that these nanoparticles accumulate inside GCs throughout the intestinal tract and, for silver nanoplates and gold nanorods, inside PCs. No pathological changes were observed in intestinal tissues. Moreover, the secretion rate of nanoparticles from GCs and PCs was markedly enhanced in a diarrhea model induced by a Chinese‑herb extract, suggesting a modulatory effect of gastrointestinal motility on nanoparticle excretion. These findings uncover a previously unrecognized clearance route that could inform safer design of nanoparticle‑based therapeutics and diagnostics.

Introduction

Nanotechnology has rapidly expanded into pharmaceuticals, diagnostics, and industrial applications, but the health and environmental risks of engineered nanostructures remain incompletely defined. Nanotoxicology, a multidisciplinary field, seeks to map the interactions between nanoparticles and biological systems, assess potential hazards, and establish safety guidelines [1–3]. Metal nanoparticles—such as gold, silver, magnetic, and quantum‑dot variants—have attracted particular interest because of their optical, magnetic, and catalytic properties, enabling targeted imaging, photothermal therapy, and drug delivery [4–10]. However, the same physicochemical traits that grant these materials functionality also hinder their biodegradation and clearance; most particles larger than ~5 nm evade renal filtration, while larger assemblies are sequestered in liver, spleen, or lung tissue [11–12]. Consequently, understanding how the body eliminates these particles is critical for therapeutic safety and risk assessment.

Current knowledge of in‑vivo clearance pathways is fragmented. The hepato‑biliary system (HBS) delivers particles that cannot be degraded by the kidney to feces, whereas the kidney‑urine route handles sub‑5 nm species [16,21,22]. Yet both routes exhibit limited efficiency for many metal nanoparticles, prompting investigation of alternative mechanisms. Recent studies suggest that the intestinal wall can serve as an excretion route: silica nanoparticles were shown to accumulate in the intestinal mucosa and feces in a mouse model, whereas larger particles (up to 500 nm) cleared more rapidly than smaller ones in fish, indicating that the HBS is not the sole pathway [20,23].

Intestinal goblet cells (GCs) are polarized, mucus‑secreting epithelial cells that line the entire gut and protect against mechanical and chemical injury [24–26]. Previous work has shown that GCs can internalize nanoparticles and that intravenously injected particles can be found within GCs, but the dynamics and extent of this process remain unexplored [27–28]. This study aims to elucidate the role of GCs—and, to a lesser extent, gastric parietal cells (PCs)—in the excretion of a representative set of metal nanoparticles, and to assess how gastrointestinal motility, modulated by a Chinese‑herb‑induced diarrhea model, influences this pathway.

Materials and Methods

Synthesis and Characterization of Triangular Silver Nanoplates

Triangular silver nanoplates were synthesized following the protocol of Mirkin et al. with minor adjustments [29,30]. In brief, AgNO₃ (0.1 mM, 100 mL), trisodium citrate (30 mM, 6 mL), PVP (30 kDa, 0.7 mM, 6 mL), and 240 µL H₂O₂ (30 wt %) were mixed at room temperature. After vigorous stirring, 0.8 mL of freshly prepared 0.1 M NaBH₄ was added rapidly, yielding a yellow solution that turned blue within 5 h under light exposure. The final suspension was stored at 4 °C.

UV‑vis spectra were recorded (200–950 nm, 2 nm slit) using a Shimadzu UV‑3600. TEM imaging (JEOL JEM‑200CX) confirmed monodisperse triangular particles with an average edge length of 44.3 nm (Fig. 1b). The characteristic plasmon peak at 648.5 nm indicated successful synthesis of triangular morphology [36].

Preparation of Animal Models with Common Bile Duct Ligation

Female Wistar rats (180–220 g) and Kunming mice (20–22 g) were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. All procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University (No.SYXK2007‑0025). The common bile duct (CBD) was ligated following Lee’s method with minor modifications [34]. Mice were anesthetized with pentobarbital (25 mg kg⁻¹), a mid‑abdominal incision was made, and the CBD was exposed. Two 0.2 mm nylon sutures were placed beneath the duct, creating a sealed segment that was then transected. On day 14 post‑ligation, serum bilirubin and AST were measured to confirm cholestasis (Fig. 2e).

Injection of Nanoparticles

After confirming successful ligation, 12 mice were randomly divided into four test groups (triangular silver nanoplates, magnetic nanoparticles, gold nanorods, gold nanoclusters) and a saline control. Nanoparticles were suspended at 550 µg mL⁻¹ and sonicated for 1 min before intravenous injection (150 µL, tail vein). An additional five mice without ligation served as a second control.

Tissue Processing and Imaging

Seven days post‑injection, mice were euthanized, and intestinal and gastric tissues were harvested. Sections (5 µm) were fixed in 10 % formaldehyde, embedded in paraffin, and stained with hematoxylin‑eosin for histology. For ultrastructural analysis, tissues were fixed in 2.5 % glutaraldehyde, dehydrated, embedded in epoxy resin, and examined by high‑resolution TEM (FEI Tecnai G2 Spirit Biotwin). Two‑photon excitation (780 nm) was used to visualize gold nanorods in tissue sections via a Caliper IVIS‑100 system.

Metal Quantification

Feces were collected daily for 7 days, weighed, and digested in aqua regia. Metal content was quantified by ICP‑MS (Agilent 7500a). Similarly, intestinal tissues were digested and analyzed for gold content to assess retention.

Diarrhea Model Induced by Chinese Herbs

A mixture of senna leaf (10 g), rhubarb (2 g), and cannabis fruit (1 g) was boiled in 100 mL water, filtered, and concentrated to 0.3 g mL⁻¹. Mice received 0.1 mL of this extract or saline daily for 7 days via oral gavage to induce diarrhea.

Goblet Cell Analysis

Intestinal segments were embedded in OCT, sectioned (8 µm), and stained with Alcian Blue/Schiff’s reagent to quantify goblet cell density and cavitation. Images were captured with an inverted microscope.

Results and Discussion

Synthesis and Characterization of the Nanoparticles

Triangular silver nanoplates displayed a strong SPR peak at 648.5 nm and a narrow size distribution (average edge 44.3 nm), confirming successful synthesis (Fig. 1a–c). Magnetic nanoparticles (20 nm) and gold nanoclusters (5 nm) were prepared following established protocols [31–33]. Gold nanorods exhibited the expected longitudinal plasmon resonance in the near‑IR region.

Validation of the CBD Ligation Model

CBD ligation produced marked ductal dilatation and increased serum TBIL and AST levels relative to sham controls (Fig. 2d–e), confirming effective isolation of the hepato‑biliary route and induction of cholestasis.

Histological Impact of Nanoparticles

Hematoxylin‑eosin staining revealed no significant inflammation or epithelial damage in any test group compared with controls (Fig. 3). This indicates that the administered nanoparticles did not compromise intestinal barrier integrity.

Localization of Nanoparticles in Goblet Cells

TEM imaging demonstrated that triangular silver nanoplates were present within GCs throughout the small intestine, ileum, and colon, often in both aggregated and dispersed states (Fig. 4). Similar distributions were observed for magnetic nanoparticles, gold nanoclusters, and gold nanorods (Additional File S4–S6). Aggregation likely reflects high local concentrations, whereas dispersion corresponds to lower loading.

Quantitative ICP‑MS of intestinal tissues confirmed that metal levels were higher in CBD‑ligated mice than in controls, whereas fecal metal content was significantly reduced in the ligated group, underscoring the contribution of GCs to excretion (Fig. 8).

Mechanism of Transport into GCs

Nanoparticles were detected within circulating erythrocytes and were observed to cross the vascular wall into the lamina propria, where they entered GCs (Fig. 5). This suggests that particle size and surface chemistry enable passive diffusion across endothelial membranes into the mucosal layer.

Effect of Diarrhea on Nanoparticle Excretion

The diarrhea model increased goblet cell density and the proportion of cavitated cells (Fig. 6), correlating with accelerated secretion of nanoparticles from GCs. Two‑photon imaging of gold nanorods revealed lower tissue retention and higher fecal gold content in diarrheic mice compared to ligated controls (Fig. 7). These findings demonstrate that gastrointestinal motility can modulate the GC‑mediated clearance pathway.

Parietal Cell–Mediated Secretion

Gold nanoclusters and silver nanoplates were also detected within gastric parietal cells (PCs) (Fig. 9). Although the extent of PC involvement appears smaller than that of GCs, these data suggest that PCs may provide an additional excretion route for certain nanoparticle types.

Conclusions

Our study establishes a previously unrecognized, GC‑ and PC‑mediated pathway for the clearance of a diverse set of metal nanoparticles in vivo. By ligating the CBD, we isolated this route from the HBS and demonstrated that nanoparticles are actively transported from circulation into the intestinal lumen via GCs, with PCs contributing to gastric excretion. Importantly, diarrhea induced by a Chinese‑herb extract substantially accelerates this process, highlighting the potential to manipulate gastrointestinal motility to enhance nanoparticle clearance. These insights pave the way for safer design of nanoparticle‑based therapeutics, improved in‑vivo tracking, and more accurate biosafety assessments.

Abbreviations

CBD

Common bile duct

GCs

Goblet cells

GNRs

Gold nanorods

HBS

Hepato‑biliary system

PCs

Parietal cells

TEM

Transmission electron microscopy

ICP‑MS

Inductively coupled plasma mass spectrometry