Rui Shi’s research while affiliated with Ecole Normale Supérieure de Paris and other places

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Publications (3)

Fabrication and characterization of M‐EV and M‐FNP. (a) Schematic diagram for the isolation of extracellular vesicles from raw milk. (b) TEM images of M‐EV. Scale bar: 200 nm and 100 nm. (c) Western blot of extracellular vesicle biomarkers (Alix, HSP70, CD81, and β‐Actin) in M‐EV. Whey was used as a control. (d) Representative HR‐STEM images of M‐FNP. Scale bar: 50 nm. (e) Energy dispersive spectroscopy (EDS) element mapping of C, N, O, P, S, Fe and Co. Scale bar: 50 nm. The particle size (f) and zeta potential (g) of M‐EV, FeCo@G and M‐FNP. (h) Yield of M‐FNP. (j) The magnetic separation procedure of M‐FNP.
Stability of M‐EV in SGF and characterization of its protective effect on proteins. (a) The size variation of M‐EVs in PBS and SGF during 4 hours. (b) The zeta potential variation of M‐EVs in PBS and SGF during 4 hours. (c) Western blot analysis of extracellular vesicle biomarkers (Alix, HSP70, CD81 and β‐Actin) in M‐EV incubated for 2 hours in PBS and SGF. (d) FRET assay in PBS. (e and f) The change of FRET efficiency in PBS and SGF during 6 hours. (g) Co‐localization analysis of M‐GNP fluorescence signal by confocal laser scanning microscop (CLSM). Scale bar: 10 μm. (h) GFP loading efficiency of M‐EV. (i) The Fluorescence change of GFP protein in PBS and SGF during 6 hours. (j) TEM images of M‐FNP in PBS and SGF during 3 hours. Scale bar: 100 nm.
Cellular uptake of M‐EV and magnetic navigation enhances the simulated gastric mucus penetration. (a) A schematic illustration of experimental procedure for cellular uptake of M‐EV. (b) Internalization of Dil‐labeled M‐EV by MGC‐803 cells was determined by CLSM. Scale bar: 50 μm. (c) The change of fluorescence intensity of Dil at different time. (d) A schematic illustration of permeability of M‐EV and M‐FNP stained with Dil in simulated gastric mucus. (e) After penetrating the simulated gastric mucus, the M‐EVs or M‐FNP internalized by MGC‐803 cells determined by CLSM. Scale bar: 50 μm. (f) The fluorescence quantitative data analysis of Figure 3e.
Cytotoxicity of M–DFNP against cancer cells. (a) Schematic diagram of potential mechanism of M–DFNP. (b) DOX loading efficiency of M‐FNP. (c) Position imaging results of DOX and M‐EV by CLSM analysis. (d)The cell viability of M‐EV, M‐FNP, free‐DOX, and M–DFNP of MGC‐803 cells for 5 hours by Calcein‐AM/PI double staining. (e) The effects of M–DFNP on the proliferation of tumor cells (MGC‐803, AGS and HGC‐27) were accessed by CCK‐8 assay.
Iron‐Cobalt alloy@graphene‐engineered milk extracellular vesicles for gastric retentive drug delivery.
Iron‐Cobalt Alloy@Graphene‐Engineered Milk Extracellular Vesicles for Gastric Retentive Drug Delivery
  • Article
  • Publisher preview available

March 2025

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8 Reads

Yijing Wen

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Rui Shi

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Yuqi Cheng

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Oral drug delivery is widely used for treating gastric diseases as it allows drugs to act directly on gastric lesions, thereby improving therapeutic outcomes. However, its efficacy is hindered by the specific gastric environment, such as the gastric mucosal barrier, which limits drug penetration, and the short gastric emptying time, which results in transient residence time. Raw milk‐derived extracellular vesicles (M‐EVs) offer promise as a gastric drug delivery platform. Their high cellular affinity, stability under gastrointestinal conditions, and ability to protect drugs from acidic and enzymatic degradation make them suitable for this purpose. Incorporating mangetic nanoparticles encapsulated in M‐EV provides magnetic navigation and active mucosal penetration capabilities. Herein, we developed a gastric drug delivery system based on iron‐cobalt alloy@graphene (FeCo@G)‐engineered M‐EV (M‐FNP). M‐FNP serves as a versatile drug carrier that can load both small molecules and proteins through simple physical approach. And it demonstrates stability in the simulated gastric fluid system for at least 6 hours. Under magnetic field guidance, it penetrates the simulated mucosal layer and is internalized by cells within 4 hours significantly enhancing cellular drug uptake. M‐FNP is expected to serve as an innovative drug delivery platform with enhanced retention capabilities within the stomach.

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Schematic illustration of ATCG‐based IPIT against PM in peritoneal cavity. 1. ATCG provides controllable CXCL12 release for recruitment of metastatic DTCs to ATCG surface after thermal‐programmatical alternating magnetic field (AMF). 2. ATCG enables hyperthermic ablation of colonized DTCs for TAA release in peritoneal cavity after hyperthermia AMF. 3. PerC B‐cells are the executors of DTC‐derived TAA presentation and intraperitoneal immune activation. 4. Effector T‐cells acquire cytotoxic molecules and cytokines to kill DTCs against PM in gastric cancer.
IMS as an acceptable ATCG‐based IPIT framework with tunable heating‐up, thermal confinement, good connectivity to enable heat generation and cell loading. a) Schematic illustration for the synthesis of ATCG. b–d) Gross (b, scale bar = 5 mm), SEM (c, scale bar = 50 µm), and micro‐CT image (d) of IMS showing lightness, isotropic porous structure, and good connectivity. e–g) Orientation angle (e) volume/surface area (f) and aspect ratio (g) of pores in the scaffolds showing that most pores exhibited an oblong shape and isotropic structure. h) 3D confocal fluorescence image of GFP‐expressing cells in the scaffold for observation of cell distribution in the pores, scale bar = 200 µm. i,j) Fluorescence image (i) and cell loading ratio (j) of cell‐loaded IMS and P‐IMS after washing in PBS, n = 5. k,l) Stress–strain curves (k) and Young's modulus (l) of plane‐directional scaffolds were evaluated using a compressed force, n = 3. m,n) Magnetothermal response of scaffolds infiltrated with PBS in the open environment under the under different AMF power (340 kHz, 18–27 kA m⁻¹). o) Magnetic hysteresis loop of scaffolds, the MS presented in the picture. p) Function of the major factor χ″ was calculated from MS of scaffolds. The data shown from representative sample and presented as means ± s.d., the asterisk indicates a statistically significant difference, **p < 0.01.
Construction of thermogenetically engineered CXCL12‐expressing cells and optimization of ATCG. a. Schematic illustration of transient transfection with plasmid. b) Flow cytometry of pHSP70‐EGFP‐transfected cells after heat of 45 °C. c) Western blotting of CXCL12 protein expressed in pHSP70‐h/mCXCL12 transfected cells after heat of 45 °C. d) Schematic illustration of stable transfection with lentiviral vectors. e–g) Flow cytometry (e) and fluorescence image (f, scale bar = 100 µm) of GES‐1pHSP70‐EGFP and NIH‐3T3pHSP70‐EGFP after heat of 45 °C, and the quantitative analysis (g) of EGFP intensity, relative intensity fold was normalized to that of the non‐treatment group. h,i) Detection of CXCL12 in engineered cells after different treatment, ple. is the abbreviation of plerixafor. j) Detection of CXCL12 based on count of engineered cells after the application of heat. k) Detection of CXCL12 in conventional BMDCs and engineered cells. l,m). Expression and release dynamics of CXCL12 gene and CXCL12 protein of ATCG after 45AMF treatment. n,o) Viability of engineered cells and CXCL12 release of ATCG after different 45AMF exposure time. The data shown from representative sample and presented as means ± s.d., the asterisk indicates a statistically significant difference, *p < 0.05, **p < 0.01, n.s. not significant.
ATCG recruits DTCs in vitro or in vivo and activates intraperitoneal immunity against DTC‐induced metastasis after AMF treatment. a) Schematic illustration of the cell chemotaxis experiment in transwell co‐culture system and representative crystal violet staining image, scale bar = 200 µm. b,c) Quantification of transmigrated MGC‐803 (b) and MFCmCxcr4 (c) cell numbers after different treatment. d) Schematic illustration of recruited DTCs in an experimental peritoneal metastasis model of 8‐week‐old female nude mice. Injection of 10⁶ MGC‐803Luc at 3 days before surgery, implantation of ATCG in the peritoneal cavity, 45AMF treatment at 1 day and 3 days, and monitoring of the scaffold at 7 days. e,f) Luciferase imaging (e) and quantitative radiance (f) of ATCG from peritoneal cavity. g) Schematic illustration of ATCG‐based IPIT to inhibit metastasis in experimental peritoneal metastasis model of 8‐week‐old female BALB/c mice. Injection of 10⁶ 4T1Luc at 3 days before surgery, implantation of ATCG in the peritoneal cavity, 45AMF treatment at 1 day and 3 days for DTC recruitment, and then 55AMF treatment at 5 days and 7 days for DTC‐derived TAA release and intraperitoneal immunity activation. h,i) Luciferase imaging (h) and micro‐CT imaging (i) of mice at 3 days, 7 days, 10 days, and 14 days in different groups. j,k) Quantitative radiance (j) and metastatic site number (k) of mouse abdomen at 3 days, 7 days, 10 days, and 14 days in different groups. l) Percent of Cd4⁺ and Cd8⁺ T‐cells in peritoneal cavity of different groups. The data shown from representative sample and presented as means ± s.d., the asterisk indicates a statistically significant difference, *p < 0.05, **p < 0.01, n.s. not significant.
scRNA‐seq profiles reveal specialized PerC B‐cell subsets for TAA presentation, such as Coro1a‐induced phagosome and immunocyte activation in peritoneal ecosystem. a) Uniform Manifold Approximation and Projection (UMAP) visualization of the main immunocyte types from all samples. Each cluster is colored and annotated according to cell type. NK cells, natural killer cells, SPM, small peritoneal macrophages; LPM, large peritoneal macrophages; cDC1, type 1 conventional dendritic cells; pDC, plasmacytoid dendritic cells. b) Dot plot showing the selected marker genes in each cell types. c) Dot plot and CellPhoneDB analysis showing antigen‐presenting function scores of all cell type and interaction between T‐cells and all cell types from all groups. d) t‐distributed Stochastic Neighbor Embedding (t‐SNE) visualization of PerC B‐cell clusters. e) Dot plot analysis showing B‐cell receptor signaling pathway, B‐cell activation, B‐cell proliferation, and antigen‐presenting function scores of B‐cell clusters. f) Ridge maps showing antigen presentation‐associated gene expression from all groups. g) Monocle Cluster of PerC B‐cell developmental trajectory based on Seurat dimensionality reduction clustering and heatmap of the top 50 DEGs along pseudotime. h) Curve plots showing expression level changes of function‐related genes related to phagosome, B‐cell activation, and antigen‐presentation along pseudotime in PerC B‐cells. i) t‐SNE visualization of T/NK cell clusters. j,k) Dot plot analysis showing naïve, cytokines and effector, inhibitory, costimulatory, Treg, Trm, NK, and proliferation markers and function scores of each T/NK cell cluster. l) Proportion of T/NK cell clusters in each group. In all Dot plot, dot size indicated percent of expressing cells in each cluster, and color represents normalized z‐score of selected gene signatures or the number of interactions.

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PerC B‐Cells Activation via Thermogenetics‐Based CXCL12 Generator for Intraperitoneal Immunity Against Metastatic Disseminated Tumor Cells

January 2025

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43 Reads

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Ling Li

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During cancer peritoneal metastasis (PM), conventional antigen‐presenting cells (dendritic cells, macrophages) promote tumorigenesis and immunosuppression in peritoneal cavity. While intraperitoneal immunotherapy (IPIT) has been used in clinical investigations to relieve PM, the limited knowledge of peritoneal immunocytes has hindered the development of therapeutic IPIT. Here, a dendritic cell‐independent, next‐generation IPIT is described that activates peritoneal cavity B (PerC B) cell subsets for intraperitoneal anti‐tumor immunity via exogenous antigen presentation. The PerC B‐cell‐involved IPIT framework consists of an isotropic‐porous, cell‐fitting, thermogenetics‐based CXCL12 generator. Such nanoscale thermal‐confined generator can programmatically fine‐tune the expression of CXCL12 to recruit disseminated tumor cells (DTCs) through CXCL12‐CXCR4 axis while avoiding cytokine storm, subsequently release DTC‐derived antigen to trigger PerC B‐cell‐involved immunity. Notably, antigen‐presenting B‐cell cluster, expressing the regulatory signaling molecules Ptpn6, Ms4a1, and Cd52, is identified playing the key role in the IPIT via single‐cell RNA sequencing. Moreover, such IPIT availably assuages peritoneal effusion and PM in an orthotopic gastric cancer and metastatic model. Overall, this work offers a perspective on PerC B‐cell‐involved antigen‐presenting in intraperitoneal immunity and provides a configurable strategy for activating anti‐DTC immunity for next‐generation IPIT.

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Schematic of the CTC removal by vascular‐like ITD. 1) HA‐MVS comprising the PLLA fibers encapsulated with FeCo@G and the crosslinked HA was grafted into blood vessel. 2) HA‐MVS construct the adhesive sites for capture of CD44⁺ CTC. 3) Integrated HA‐MVS generate hyperthermia under AMF for ablation of the captured CD44⁺ CTC.
Characterization of HA‐MVS. a) Morphology of HA‐MVS was measured using optical microscopy and scanning electron microscopy. The white double‐headed arrow indicated the fiber orientation. Black scale bar = 1 mm and white scale bar = 50 µm. b) The stress–strain curves, c) Young's modulus, d) ultimate tensile strength, and e) elongation at failure of circumferential and axial HA‐MVS were evaluated using a tensile force applied to scaffold, n = 3. f) Weight loss of HA‐MVS with or without crosslinking after immersion in PBS at 37 °C, n = 5. g) FTIR spectrum of FeCo@G PLLA fiber, crosslinked HA, and abluminal/luminal surface of HA‐MVS. h) The cobalt ions concentration was measured in HA‐MVS and PLLA at simulated physiological environment, n = 3. i,j) Magnetothermal response of HA‐MVS in the air at various FeCo@G concentrations under AMF (340 kHz, 22.5 kA m⁻¹), scale bar = 5 mm. k) Real‐time temperature curves of HA‐MVS and PLLA in the air under six‐times on/off cycles. All data shown from representative sample and presented as means ± s.d., the asterisk indicated a statistically significant difference, **p < 0.01.
Capturing CD44⁺ cancer cells in vitro. a) Schematic illustration of the HA‐MVS in a closed‐loop circulation set‐up, consisting of a peristaltic pump, catheter, a plastic tube, and cells‐containing fluid. b) CD44 expression of MDA‐MB‐231 and MCF‐7 was confirmed by confocal laser scanning imaging, scale bar = 20 µm. c) Macroscopic bioluminescence imaging after a labelling experiment with 1.3 × 10⁴ cells mL⁻¹ in DMEM and blood showed that, in the luminal surface of HA‐MVS and PLLA, the cells were infected with luciferase‐lentivirus; scale bar = 2 mm. d) Capture efficiencies for both CD44⁺ and CD44⁻ cells in a closed‐loop circulation set‐up with DMEM, n = 3. e) Capture efficiencies for CD44⁺ cells in DMEM, blood, and CD44 antibody blocked cells, n = 3. f) Effect of recycle numbers on the capture efficiency of the HA‐MVS, n = 3. g) Re‐capture test of HA‐MVS in DMEM after trypsin treatment, n = 3. h) Capture efficiencies in DMEM at different cells density, n = 3. All data shown from representative sample and presented as means ± s.d.; the asterisk indicates a statistically significant difference, **p < 0.01.
Magnetothermal ablation of CD44⁺ cancer cells in vitro. a,b) Magnetothermal response of HA‐MVS in a closed‐loop circulation set‐up at various FeCo@G concentrations under AMF (340 kHz, 22.5 kA m⁻¹); scale bar = 1 cm. c) SEM image of cells on the HA‐MVS after AMF treatment; scale bar = 50 µm. d) Real‐time temperature curves of HA‐MVS in a closed‐loop circulation set‐up under six‐times on/off cycles. e,f) Representative fluorescence images of e) the live/dead stained cells captured by HA‐MVS, scale bars = 100 µm (e), and cell survival in different treatment, n = 6 (f). All data shown from representative sample and presented as means ± s.d.; the asterisk indicates a statistically significant difference, **p < 0.01.
Removal of CD44⁺ CTC in vivo. a) Schematic illustration of HA‐MVS with capture and magnetothermal ablation of CTC in the rat abdominal aorta under AMF. b) Blood flow speed of rat abdominal aorta after different treatment, n = 6. c) Color Doppler ultrasound image of rat abdominal aorta after transplanted scaffold and AMF treatment. d,e) Macroscopic bioluminescence imaging of transplanted scaffold after a capturing experiment and different treatments, scale bar = 5 mm (d), and quantification of cell radiance (e); the cells were infected with luciferase‐lentivirus, n = 4. f) Quantification curve of cell count, g) the capture efficiencies, and h) the cell survival for luciferase‐labeled cells in rat abdominal aorta after 1 and 3 h, n = 4. i) Magnetothermal response of HA‐MVS in rat abdominal aorta under AMF (340 kHz, 31.5 kA m⁻¹); scale bar = 2 cm. All data shown from representative sample and presented as means ± s.d.; the asterisk indicates a statistically significant difference, *p < 0.05, **p < 0.01.

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Implantable Magnetic Vascular Scaffold for Circulating Tumor Cell Removal in vivo

November 2022

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408 Reads

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17 Citations

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Rui Shi

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Xin Xia

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An integrated trapped device (ITD) capable of circulating tumor cell (CTC) removal can assuage or even prevent metastasis. However, adhesion repertoires are ordinarily neglected in the design of ITD, possibly leading to the omission of highly metastatic CTC and treatment failure. Here, we present a vascular-like ITD with adhesive sites and wireless magnetothermal response to remove highly metastatic CTC in vivo. Such a vascular-like ITD comprises circumferential well-aligned fibers and artificial adhesion repertoires and is optimized for magnetothermal integration. Continuous and repeated capture in a dynamic environment increases capture efficiency over time. Meanwhile, the heat generation of the ITD leads to the capture of CTC death owing to cell heat sensitivity. Furthermore, the constructed bioinspired ultrastructure of the ITD prevents vascular blockage and induces potential vascular regeneration. Overall, our work defines an extendable strategy for constructing adhesion repertoires against intravascular shear forces, provides a vascular-like ITD for reducing CTC counts, and is expected to alleviate the risk of cancer recurrence. This article is protected by copyright. All rights reserved.

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Citations (1)

... [21] In IMS, FeCo@G was selected as the heat source for highly effective magnetothermal transfer based on its high degree of saturation magnetization (M S ) at 197.2 emu g −1 , good specific loss power (SLP) at ≈534.1 W g −1 and good physicochemical stability compared with conventional superparamagnetic iron oxide nanoparticles. [6,22] IMS infiltrated with PBS was exposed to an AMF in the open environment, and the temperature was monitored using an IR thermal imaging camera (Figure 2m). The results showed a tunable heating up of IMS under AMF powers from 18 to 27 kA m −1 and FeCo@G concentration of 1.25-10 mg mL −1 , as well as good thermostability after seven on/off cycles ( Figure 2n; Figure S11, Supporting Information). ...

Reference:

PerC B‐Cells Activation via Thermogenetics‐Based CXCL12 Generator for Intraperitoneal Immunity Against Metastatic Disseminated Tumor Cells
Implantable Magnetic Vascular Scaffold for Circulating Tumor Cell Removal in vivo

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Rui Shi

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Xin Xia

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