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Journal of Peking University (Health Sciences) ›› 2026, Vol. 58 ›› Issue (1): 22-29. doi: 10.19723/j.issn.1671-167X.2026.01.003

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Anti-inflammatory effects of cell membrane vesicle-mediated delivery of small interfering RNA targeting tumor necrosis factor-α on dental pulp stem cells

Ruofan GAO, Tianyu MA, Runkai WANG, Yuchen YIN, Ruidi LI, Dandan WANG*(), Bin XIA*()   

  1. Department of Pediatric Dentistry, Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology, Beijing 100081, China
  • Received:2025-09-19 Online:2026-02-18 Published:2025-11-27
  • Contact: Dandan WANG, Bin XIA
  • Supported by:
    the National Natural Science Foundation of China(82401182); the Natural Science Foundation of Beijing-Haidian Original Innovation Joint Fund(L232110)

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Abstract:

Objective: To evaluate the feasibility of using cell membrane vesicles (CMVs) as a delivery system for small interfering RNA (siRNA) and to assess their performance in a lipopolysaccharide (LPS)-induced inflammatory model of human dental pulp stem cells (DPSCs). Methods: CMVs were generated from cytochalasin B-treated 3T3 cells and characterized for their physicochemical properties, including morphology, size distribution, and zeta potential, using confocal microscopy and dynamic light scattering. To construct CMVs@siTNF-α, saponin-mediated transient permeabilization was used to facilitate siRNA loading, after which encapsulation efficiency was evaluated by flow cytometry and confocal imaging. Intracellular uptake behaviors were examined using flow cytometry in DPSCs. LPS (1 mg/L) was employed to establish a robust in vitro inflammatory microenvironment. DPSCs were subsequently treated with CMVs or CMVs@siTNF-α, and cell viability was assessed via CCK-8 (cell counting kit-8). The expression and secretion of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme linked immunosorbent assay (ELISA) to evaluate both gene-silencing efficiency and downstream anti-inflammatory effects. Results: CMVs exhibited uniform spherical morphology with an average diameter of approximately 903 nm and a zeta potential of -9.39 mV, confirming successful vesicle formation. CMVs efficiently encapsulated FAM-labeled siRNA, as indicated by a pronounced fluorescence shift in flow cytometry and clear colocalization signals in confocal imaging. DPSCs cultured with CMVs@FAM-siTNF-α demonstrated increased intracellular fluorescence, reflecting efficient vesicle uptake and cytoplasmic siRNA release. Importantly, both CMVs and CMVs@siTNF-α displayed negligible cytotoxicity. LPS stimulation significantly elevated TNF-α, IL-1β, and IL-6 expression, validating the inflammatory model. CMVs alone did not affect cytokine levels, indicating biological inertness. In contrast, CMVs@siTNF-α significantly suppressed the LPS-induced upregulation of TNF-α at both mRNA and protein levels., demonstrating potent gene-silencing activity. Furthermore, suppression of TNF-α led to downstream attenuation of IL-1β and IL-6, with both transcription and secretion significantly decreased compared with the LPS group. These findings collectively confirmed that CMVs enabled efficient intracellular siRNA transport and effectively mitigate inflammatory responses in DPSCs. Conclusion: CMVs represent a biocompatible and effective siRNA delivery platform capable of achieving robust TNF-α knockdown and ameliorating inflammatory cytokine production in vitro, highlighting their potential for future nucleic acid-based anti-inflammatory therapies.

Key words: Small interfering RNA, Cell membrane vesicles, Dental pulp stem cells, Tumor necrosis factor

CLC Number: 

  • R78

Table 1

Quantitative real-time PCR primer sequence"

Gene Forward primer (5′ to 3′) Reverse primer (5′ to 3′)
TNF-α AGCAAGGACAGCAGAGGA GGGGAGAGAGGGTGGAG
IL-1β TGTGCTGAATGTGGACTCA ACAAAAGGGCTGGGGAT
IL-6 TGTGTGAAAGCAGCAAAGA ACCAGGCAAGTCTCCTCA
GAPDH GCCAACGTGTCAGTGGTG AAGGTGGAGGAGTGGGTGT

Figure 1

Preparation and characterization of CMVs A, morphological changes of 3T3 cells without treatment and after 30 min of cytochalasin B exposure under a light microscope (scale bar=100 μm); B, alterations of cell membrane and cytoskeleton in untreated and CB-treated (30 min) 3T3 cells observed by confocal microscopy (red: DiI, green: phalloidin, blue: DAPI; scale bar = 40 μm); C, confocal microscopy images of CMVs (red: DiI, green: cFDA-SE, scale bar = 20 μm) and magnified view (red: DiI, green: cFDA-SE, scale bar = 2 μm); D, size distribution of CMVs; E, zeta potential distribution of CMVs. DAPI, 4', 6-diamidino-2-phenylindole; cFDA-SE, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester; DiI, 1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorate; CMVs, cell membrane vesicles; CB, cytochalasin B."

Figure 2

Loading of siTNF-α into CMVs A, flow cytometry analysis of CMVs with or without loading of FAM-siTNF-α; B, confocal microscopy images of CMVs@FAM-siTNF-α (red, DiI; green, FAM; scale bar=2 μm). DiI, 1, 1′-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorate; CMVs, cell membrane vesicles; FAM, fluorescein amidite; siTNF-α, small interfering RNA targeting tumor necrosis factor-alpha."

Figure 3

Intracellular delivery and anti-inflammatory effects of CMVs@siTNF-α A, flow cytometry analysis of DPSCs with or without transfection of CMVs@siTNF-α; B, relative cell viability of DPSCs after treatment with CMVs or CMVs@siTNF-α, measured by CCK-8 assay; C, E, F, mRNA expression levels of TNF-α, IL-1β, and IL-6 in DPSCs under different treatments (blank, LPS, LPS+CMVs, or LPS+CMVs@siTNF-α), determined by qRT-PCR; D, G, H, protein secretion levels of TNF-α, IL-1β, and IL-6, assessed by ELISA. *P < 0.05; * *P < 0.01; * * *P0.001; ns, no significance. CMVs, cell membrane vesicles; siTNF-α, small interfering RNA targeting tumor necrosis factor-alpha; FAM, fluorescein amidite; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-alpha; IL-1β, interleukin-1 beta; IL-6, interleukin-6; DPSCs, dental pulp stem cells; qRT-PCR, quantitative real-time polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay."

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