DOI: 10.5061/dryad.gmsbcc2wt
title: Enzyme-like nanoparticle engineered-mesenchymal stem cell secreting HGF promotes visualized therapy for idiopathic pulmonary fibrosis in vivo

description: Stem cell therapy has emerged as a potential alternative treatment for
 idiopathic pulmonary fibrosis (IPF). However, its efficacy remains
 challenging due to factors such as ROS and inflammation in fibrotic lungs.
 Moreover, the distribution, migration, and survival of transplanted stem
 cells are still unclear, significantly impeding the clinical advancement
 of stem cell therapy. To tackle these challenges, we fabricate
 trimetallic-based nanocarriers (TBNCs) with enzyme-like activity and
 plasmid loading capabilities, aiming to efficiently eradicate ROS and
 facilitate delivery therapeutic genes and ultimately improve the
 therapeutic efficacy. Simultaneously, TBNCs function as an excellent CT
 contrast agent for tracking mesenchymal stem cells (MSCs), can improve the
 therapy by visualization-guiding. TBNCs was co-incubated with a hepatocyte
 growth factor plasmid gene to create TBNCs@pDNA, and then MSCs were
 modified with TBNCs@pDNA through endocytosis to generate engineered-MSCs,
 which demonstrate enhanced anti-oxidant and anti-inflammatory properties,
 thereby augmenting the therapeutic efficacy of MSCs. Finally, the in vivo
 CT tracking and therapy potential of engineered-MSCs is explored in IPF
 model mice. Overall, this study provides an efficient and forward-looking
 treatment approach for IPF and establishes a framework for a stem
 cell-based therapeutic system aimed at addressing lung disease.
description: # Enzyme-like nanoparticle-engineered mesenchymal stem cell secreting HGF
 promotes visualized therapy for idiopathic pulmonary fibrosis in vivo
 [https://doi.org/10.5061/dryad.gmsbcc2wt](https://doi.org/10.5061/dryad.gmsbcc2wt)  This dataset includes, all additional raw experimental data are presented in the main and supplementary figures of the manuscript. Summary of dataset contents: Three key datasets are included to provide all the data utilized in the manuscript supporting the conclusion that the enzyme-like nanoparticle-engineered mesenchymal stem cell secreting HGF promotes visualized therapy for idiopathic pulmonary fibrosis in vivo. The first and second datasets contain all the original data from the main manuscript (Raw data for main figures) and the supplemental materials (Raw data for supplemental figures), respectively. Initially, the successful synthesis of TBNCs was demonstrated by various characterization methods. Subsequently, the functional regulation and therapeutic visualization of TBNCs engineered MSCs were demonstrated both in vitro and in vivo. The third dataset contains software related to the processing of raw data in the manuscript (Raw data for software), including "CV2.4" for the processing of immunohistochemical section data; "FlowJo_10.8.1" for flow cytometry data processing; "HiscanViewer" for CT imaging data processing; "Image J" for mean fluorescence intensity data statistics; and "Leica" for confocal fluorescence imaging data processing. Together, these datasets provide comprehensive evidence to support the paper's conclusion that TBNCs successfully enhance the antioxidant stress and antifibrotic capabilities of MSCs, as well as real-time visualization of their biological behavior in vivo. #### Description of the data and file structure [https://doi.org/10.5061/dryad.gmsbcc2wt](https://doi.org/10.5061/dryad.gmsbcc2wt)(opens in new window): The data files include three separate zip files labeled as Raw data for main figures, Raw data for supplemental figures, and Raw data for software. Keywords and abbreviations used in the data files: Au: Gold. Pt: Platinum. Co: Cobalt. TBNCs: Trimetallic-based nanocarriers. XPS: X-ray photoelectron spectroscopy. TEM: Transmission electron microscopy. P: Phosphorus. S: Sulfur. EDS: Energy dispersive spectrometer. ESR: Electron spin resonance. •HO: Hydroxyl radicals. •O2− : Superoxide anion. pDNA: Plasmid DNA. CT: Computed tomography. RT-PCR: Reverse transcription polymerase chain reaction. HGF: Hepatocyte growth factor. hMSCs: Human mesenchymal stem cells. PI: Propidium iodide. EGFP: Enhanced green fluorescent protein. ROS: Reactive oxygen species. BEAS 2B: Bronchial epithelium transformed with Ad12-SV40 2B. hMSC-CM: hMSCs conditioned medium. Col I: Collagen I. α-SMA: α-smooth muscle actin. FN: Fibronectin. TGF β1: Transforming growth factor β1 BLM: Bleomycin. BL: Bioluminescence. SOD: Superoxide dismutase. CAT: Catalase. MMP: Matrix metalloproteinase. **Raw Data for main figures** This zip file contains all the source data for the main figures, and each figure has a separate folder with subfolders containing the raw data for each panel. **Figure 2, this folder contains the following subfolders:** Figure 2B, the source data of the TEM images of TBNCs. Figure 2C, the source data for the granulometric distribution of TBNCs. Figure 2D, the source data of the EDS-mapping of Au, Pt, Co, P, and S elements for TBNCs. Figures 2E-H, the source data for the XPS of TBNCs and the corresponding resolution spectrum for Au 4f, Pt 4f, and Co 2p regions of TBNCs, respectively. **Figure 3, this folder contains the following subfolders:** Figure 3B, the source data for the ESR spectra of •HO generated by Fenton reaction with or without TBNCs addition. Figure 3C, the source data for the ESR spectra of •O2− generated by xanthine oxidase-xanthine system with or without TBNCs addition. Figure 3D, the source data for the agarose gel electrophoresis of TBNCs/pDNA composites at different mass ratios of TBNCs to pDNA. Naked pDNA was taken as control. Figure 3E, the source data for the electrophoretic band intensity at different mass ratios of TBNCs to pDNA Figures 3F-G, the source data for the zeta potentials and hydrodynamic diameters of TBNCs and TBNCs@pDNA, respectively. Figure 3H, the source data for the plot of hounsfield units (HU) values of TBNCs as a function of the concentration. The value is expressed as the mean ± SD, with a minimum sample size of 3. Figure 3I, the source images of the transverse CT images of TBNCs at different concentrations. **Figure 4, this folder contains the following subfolders:** Figure 4A, the source data for the laser confocal microscopy images of the hMSCs labeled with TBNCs@pDNA at different concentrations; blue and red fluorescences stand for the nucleus stained with 4′,6-diamidino-2-phenylindole and the TBNCs@pDNA-labeled hMSCs, respectively. Figure 4B, the source images of the flow cytometry analysis of the hMSCs labeled with TBNCs@pDNA at different concentrations. Figure 4C, the source data for the live/dead staining of hMSCs after treated with TBNCs@pDNA at different concentrations. Red and green fluoresences stand for the dead hMSCs stained with PI and alive hMSCs stained with caclein AM. Figure 4D, the source images of the flow cytometry analysis of the apoptosis of hMSCs with or without TBNCs@pDNA at different concentrations. Figure 4E, the source data for the immunofluorescence analysis of HGF expression in the hMSCs labeled with TBNCs@pDNA. Red: HGF, Green: EGFP, Blue: DAPI-stained nucleus. Figure 4F, the source data for the quantification of RT-PCR data of HGF expression in the hMSCs labeled with TBNCs@pDNA. Figure 4G, the source data for the calculated HU values as a function of the concentration of TBNCs@pDNA added for cell labeling. Figure 4H, the source images of the in vitro CT images of the hMSCs labeled with TBNCs@pDNA at different concentrations. **Figure 5, this folder contains the following subfolders:** Figure 5A, the source data for the intracellular generation of ROS following H2O2 stimulation. Significant ROS generation, indicated by green fluorescence, was observed in pure hMSCs. Figure 5B, the source data for the cell viability of hMSCs and hMSCs labeled with TBNCs following exposure to H2O2 stimulation. Figure 5C, the source images of the TEM images of mitochondrial structure and morphology of hMSCs and hMSCs labeled with TBNCs after H2O2 stimulation. Figure 5D, the source data for the flow cytometry analysis of the apoptosis of hMSCs and hMSCs labeled with TBNCs before and after H2O2 stimulation. **Figure 6, this folder contains the following subfolders:** Figure 6A, the source data for the representative images of BEAS 2B wound healing assays treated with normal medium, hMSC-CM, or labeled hMSC-CM for 12 h and 24 h. Figure 6B, the source images of the representative images of Col I (red), α-SMA (red), and FN (red) immunostaining of BEAS 2B after treatment with TGF β1, hMSC-CM＋TGF β1 and Labeled hMSC-CM＋TGF β1, respectively. The nuclei were stained with DAPI (blue). Figure 6C, the source data for the net migration rate of BEAS 2B for per well. Figures 6D-F, the source data for the quantification of Col I, FN and α-SMA staining after treatment. The symbols ** and *** indicate a statistically significant difference at p &lt; 0.01 and p &lt; 0.001, respectively. The value is expressed as the mean ± SD, with a minimum sample size of 3. **Figure 7, this folder contains the following subfolders:** Figure 7A, the source data for the micro-CT images of mice with and without BLM injection after 21 days. Figure 7B, the source data for the percentages of pulmonary CT ventilation ratio. Figure 7C, the source images of the lung sections from the Control and BLM group were stained using H&E and Masson's trichrome staining techniques. Figure 7D, the source images of the BL images of the TBNCs labeled hMSCs at various time intervals in IPF mouse. Figure 7E, the source data for the quantitative statistics of the difference between the total BL intensity. Figure 7F, the source data for the quantitative statistics pertaining to the mean BL intensity. The value is expressed as the mean ± SD, with a minimum sample size of 3. **Figure 8, this folder contains the following subfolders:** Figures 8A-D, the source data for in vivo micro-CT images (indicated by yellow and red arrows) of the labeled hMSCs at 1, 5, 10, 15, and 20 days after transplantation into the lung of IPF mouse, respectively; 3D in vivo CT images of the labeled hMSCs in IPF mouse before (left) and after (right) transplantation; 3D CT images depict the labeled hMSCs at 1, 5, 10, 15, and 20 days after transplantation into the lung of IPF mouse. Figure 8C, the source data for the graph illustrating the distribution area of CT signals from labeled hMSCs in relation to the duration of transplantation. Figure 8E, the source data for the CT values of the labeled hMSCs after transplantation into the lung at different time points. The value is expressed as the mean ± SD, with a minimum sample size of 3. **Figure 9, this folder contains the following subfolders:** Figure 9A, the source data for the laser confocal microscopy images of the expression of HGF in the lung tissue of IPF mouse treated with hMSC and Labeled hMSC, respectively. Figure 9B, the source data for the fluorescence intensity of the expression of HGF in the lung tissue of IPF mouse treated with hMSC and Labeled hMSC, respectively. Figure 9C, the source data for the laser confocal microscopy images of the expressions of Col I in the lung tissue of IPF mouse treated with hMSC and Labeled hMSC. Figure 9D, the source data for the corresponding fluorescence intensity of the expressions of Col I in the lung tissue of IPF mouse treated with hMSC and Labeled hMSC, respectively. Figure 9E, the source data for the laser confocal microscopy images of the expressions of α-SMA in the lung tissue of IPF mouse treated with hMSC and Labeled hMSC. Figure 9F, the source data for the corresponding fluorescence intensity of the expressions of α-SMA in the lung tissue of IPF mouse treated with hMSC and Labeled hMSC, respectively. **Figure 10, this folder contains the following subfolders:** Figure 10A, the source data for the micro-CT images of the IPF mouse treated with hMSC or Labeled hMSC. Figure 10B, the source data for the masson trichrome staining of the lung tissues from the IPF mice treated with hMSC or Labeled hMSC. The untreated mouse served as the negative control, while the BLM-treated mouse served as the positive control. Figure 10C, the source data for the normal aeration area were compared between the hMSC and Labeled hMSC treated groups and the untreated groups. Figure 10D, the source data for the poor aeration area were compared between the hMSC and Labeled hMSC treated groups and the untreated groups. Figure 10E, the source data for the percentages of pulmonary CT ventilation ratio were compared between the hMSC and Labeled hMSC treated groups and the untreated groups. The symbols *** denote a statistically significant difference at a significance level of p &lt; 0.001. The value is expressed as the mean ± SD, with a minimum sample size of 3. **Raw data for supplemental figures** This zip file contains all the source data for the supplementary figures, and each figure has a separate folder containing the raw data for all panels. **Figure S1, this folder contains the following subfolders:** Figures S1B-C, the source data for the SOD and CAT enzyme activities of TBNCs. **Figure S4, this folder contains the following subfolders:** Figure S4A, the source data for the laser confocal microscopy images of the hMSCs labeled with TBNCs@pDNA at different time points. Blue and red fluorescences stand for the nucleus stained with 4′,6-diamidino-2-phenylindole and the TBNCs@pDNA-labeled hMSCs, respectively. Figure S4B, the source data for the laser confocal microscopy images of the hMSCs labeled with TBNCs@pDNA at different time points. Blue and red fluorescences stand for the nucleus stained with 4′,6-diamidino-2-phenylindole and the TBNCs@pDNA-labeled hMSCs, respectively. **Figure S5, this folder contains the following subfolders:** Figure S5, the source data for the flow cytometry analysis of the cell labeling efficiency of hMSCs after co-incubation with 50 μg/mL TBNCs@pDNA for 4 h. **Figure S6, this folder contains the following subfolders:** Figure S6, the source data for the intracellular fluorescence intensity of hMSCs after uptake of RB@TBNCs@pDNA for 24 h, 48 h and 72 h. The symbols *, ** and *** indicate a statistically significant difference at p&lt; 0.05, p&lt; 0.01 and p&lt; 0.001, respectively. Mean ± SD, n=3. **Figure S7, this folder contains the following subfolders:** Figure S7A, the source data for the calcein AM was used to stain hMSCs, and the growth state of hMSCs was observed by laser confocal microscopy. Figure S7B, the source data for the cell healing rate of hMSCs and labeled hMSCs was assessed on consecutive days (Day 1, Day 2, Day 3, and Day 4) during the growth period. Mean ± SD, n=3. **Figure S8, this folder contains the following subfolders:** Figure S8A, the source data for the Oil Red O staining of the adipogenic cells. Figure S8B, the source data for the Alizarin Red S staining of the osteogenic cells. **Figure S9, this folder contains the following subfolders:** Figure S9A, the source data for in vitro CT imaging of agarose phantoms containing various numbers of hMSCs labeled with 50 μg/mL of TBNCs@pDNA. Figure S9B, the source data for the CT values of agarose phantoms containing various numbers of hMSCs labeled with 50 μg/mL of TBNCs@pDNA. The symbols *, ** and ***indicate a statistically significant difference at p&lt; 0.05, p&lt; 0.01 and p&lt; 0.001, respectively. Mean ± SD, n=3. **Figure S10, this folder contains the following subfolders:** Figure S10A, the source data for the laser confocal microscopy images of the apoptosis of myofibroblasts induced by hMSC-CM and Labeled hMSC-CM in vitro. Untreated myofibroblasts were used as control. Figure S10B, the source data for the flow cytometry analysis of the apoptosis of myofibroblasts induced by hMSC-CM and Labeled hMSC-CM in vitro. Untreated myofibroblasts were used as control. **Figure S11, this folder contains the following subfolders:** Figures S11A-B, the source data for the expression of MMP-9 and MMP-2 in myofibroblast induced by hMSC-CM and labeled hMSC-CM were determined by ELISA. Untreated myofibroblast served as controls. The symbols *, ** and *** indicate a statistically significant difference at p&lt; 0.05, p&lt; 0.01 and p&lt; 0.001, respectively. Mean±SD, n=3. **Figure S12, this folder contains the following subfolders:** Figure S12, the source data for the Fluorescence images of Luc labeled hMSCs; green image and blue image indicate the EGFP-expressing hMSCs and the nucleus stained with DAPI, respectively. **Figure S13, this folder contains the following subfolders:** Figure S13, the source data for the biodistribution of Au in major organs (heart, liver, spleen, lung, and kidney). The mice treated with the TBNCs@pDNA labeled hMSCs were sacrificed on the 1th and 45th day and Au concentration was detected by ICP-MS, respectively. Mean±SD, n=3. **Figure S14, this folder contains the following subfolders:** Figure S14, the source data for the biosafety evaluation. HE staining for major organs (heart, liver, spleen, lung, and kidney). The mice treated with the TBNCs@pDNA labeled hMSCs were sacrificed on the 45th day. Age-matched healthy mice without treatment were used as a control group. **Figure S15, this folder contains the following subfolders:** Figure S15, the source data for the visualization of biological behavior of TBNCs labeled hMSCs transplanted into IPF mouse. In vivo micro-CT images (indicated by yellow and red arrows) and 3D images of the labeled hMSCs at 30, 35, and 45 days after transplantation into the lung of IPF mouse, respectively. **Figure S16, this folder contains the following subfolders:** Figure S16, the source data for the TBNCs@pDNA was co-located with hMSCs in vivo. Fluorescence images of frozen lung sections from the IPF mouse at 45 d post-transplantation of the labeled hMSCs. The hMSCs were pre-tagged with DiO before RB@TBNCs@pDNA labeling; green and red fluorescence refer to the DiO-stained cytomembrane and RB@TBNCs@pDNA, respectively. **Figure S17, this folder contains the following subfolders:** Figure S17, the source data for the Labeled hMSCs inhibited EMT and induced apoptosis of myofibroblasts. Immunohistochemical analysis of NLRP3, α-SMA, and TUNEL. **Figure S18, this folder contains the following subfolders:** Figure S18, the source data for the efficacy of TBNCs labeled hMSCs in the treatment of pulmonary fibrosis in vivo. H&E staining of the lung tissues from the IPF mice treated with hMSC or Labeled hMSC. The untreated mouse served as the negative control, while the BLM treated mouse served as the positive control. **Raw data for software** This zip file contains all the source data for the software. CV2.4, this software is used to analyze tissue sections for immunohistochemical data analysis. FlowJo_10.8.1, this software is used to analyze flow cytometry data. HiscanViewer, this software is used to analyze CT imaging data. Image J, this software is used to statistical average fluorescence intensity statistics for confocal fluorescence imaging. Leica, this software is used to analyze confocal fluorescence imaging data.