Enzyme-like nanoparticle-engineered mesenchymal stem cell secreting HGF promotes visualized therapy for idiopathic pulmonary fibrosis in vivo

Abstract

Stem cell therapy is being explored as a potential treatment for idiopathic pulmonary fibrosis (IPF), but its effectiveness is hindered by factors like reactive oxygen species (ROS) and inflammation in fibrotic lungs. Moreover, the distribution, migration, and survival of transplanted stem cells are still unclear, impeding the clinical advancement of stem cell therapy. To tackle these challenges, we fabricate AuPtCoPS trimetallic-based nanocarriers (TBNCs), with enzyme-like activity and plasmid loading capabilities, aiming to efficiently eradicate ROS, facilitate delivery of therapeutic genes, and ultimately improve the therapeutic efficacy. TBNCs also function as a computed tomography contrast agent for tracking mesenchymal stem cells (MSCs) during therapy. Accordingly, we enhanced the antioxidant stress and anti-inflammatory capabilities of engineered MSCs and successfully visualized their biological behavior in IPF mice in vivo. 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.

Fig. 1.. Schematic illustration of the application of TBNCs@pDNA in gene delivery and dual-modal imaging to track hMSCs in the treatment of IPF.

TBNCs loaded with HGF pDNA (TBNCs@pDNA) were cocultured with human MSCs (hMSCs) that constitutively expressed the luciferase reporter gene to generate multifunctional hMSCs. Subsequently, these multifunctional hMSCs were administered into the lungs of IPF mice via the trachea to facilitate the regeneration of damaged AECs and the reduction of collagen accumulation in the lung interstitium, thereby enhancing the gas exchange function of the alveoli. In addition, CT/BL imaging modalities were used to monitor the in vivo distribution, migration, and viability of the transplanted hMSCs in real time.

Fig. 2.. Synthesis, morphology characterization, and elemental analysis of TBNCs.

(A) Schematic illustration of the preparation of TBNCs. (B) Transmission electron microscopy (TEM) images of TBNCs and (C) the granulometric distribution. (D) Energy-dispersive x-ray spectroscopy mapping of Au, Pt, Co, P, and S elements for TBNCs. Scale bar, 5 nm. (E) X-ray photoelectron spectroscopy of TBNCs and the corresponding resolution spectrum for (F) Au 4f, (G) Pt 4f, and (H) Co 2p regions of TBNCs, respectively.

Fig. 3.. Enzyme-like activity, pDNA loading capacity, and CT imaging performance of TBNCs.

(A) Schematic illustration of enzyme-like activities of TBNCs. (B) Electron spin resonance (ESR) spectra of •HO generated by Fenton reaction with or without TBNC addition. (C) ESR spectra of •O²⁻ generated by xanthine oxidase-xanthine system with or without TBNCs addition. a.u., arbitrary units. (D) Agarose gel electrophoresis of TBNCs/pDNA composites at different mass ratios of TBNCs to pDNA. Naked pDNA was taken as a control. (E) Electrophoretic band intensity at different mass ratios of TBNCs to pDNA. R.F.U., relative fluorescence unit. (F) Zeta potentials and (G) hydrodynamic diameters of TBNCs and TBNCs@pDNA, respectively. (H) Plot of Hounsfield unit (HU) values of TBNCs as a function of the concentration. (I) Transverse CT images of TBNCs at different concentrations. The value is expressed as the means ± SD, with a minimum sample size of 3.

Fig. 4.. Cell labeling ability and gene delivery ability of TBNCs@pDNA and CT imaging performance of their labeled hMSCs.

(A) 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 (DAPI) and the TBNCs@pDNA-labeled hMSCs, respectively. (B) Flow cytometry analysis of the hMSCs labeled with TBNCs@pDNA at different concentrations. (C) Live/dead staining of hMSCs after being treated with TBNCs@pDNA at different concentrations. Red and green fluorescences stand for the dead hMSCs stained with propidium iodide (PI) and alive hMSCs stained with calcein AM. (D) Flow cytometry analysis of the apoptosis of hMSCs with or without TBNCs@pDNA at different concentrations. (E) Immunofluorescence analysis of HGF expression in the hMSCs labeled with TBNCs@pDNA. Red: HGF, Green: EGFP, Blue: DAPI-stained nucleus. (F) Quantification of reverse transcription quantitative polymerase chain reaction data of HGF expression in the hMSCs labeled with TBNCs@pDNA. (G) Calculated HU values as a function of the concentration of TBNCs@pDNA added for cell labeling, and (H) in vitro CT images of the hMSCs labeled with TBNCs@pDNA at different concentrations. The symbol *** indicates a statistically significant difference at P < 0.001. The value is expressed as the means ± SD, with a minimum sample size of 3.

Fig. 5.. Assessment of the intracellular antioxidative stress capacity of TBNCs in hMSCs.

(A) Intracellular generation of ROS following H₂O₂ stimulation. Significant ROS generation, indicated by green fluorescence, was observed in pure hMSCs, and (B) the cell viability of hMSCs and hMSCs labeled with TBNCs following exposure to H₂O₂ stimulation. (C) TEM images of mitochondrial structure and morphology of hMSCs and hMSCs labeled with TBNCs after H₂O₂ stimulation. (D) Flow cytometry analysis of the apoptosis of hMSCs and hMSCs labeled with TBNCs before and after H₂O₂ stimulation. The symbol *** indicates a statistically significant difference at P < 0.001. The value is expressed as the means ± SD, with a minimum sample size of 3.

Fig. 6.. Evaluation of the capacity of hMSCs labeled with TBNCs to facilitate the repair of damaged BEAS 2B and to impede the transformation of AECs into myofibroblasts in vitro.

(A) Representative images of BEAS 2B wound-healing assays treated with normal medium, hMSC-CM, or labeled hMSC-CM for 12 and 24 hours. (B) Representative images of collagen I (Col I; red), α–smooth muscle actin (α-SMA; red), and fibronectin (FN; red) immunostaining of BEAS 2B after treatment with transforming growth factor β1 (TGF-β1), hMSC-CM + TGF-β1, and labeled hMSC-CM + TGF-β1, respectively. The nuclei were stained with DAPI (blue). (C) The net migration rate of BEAS 2B per well. Quantification of (D) Col I, (E) FN, and (F) α-SMA staining after treatment. The symbols ** and *** indicate a statistically significant difference at P < 0.01 and P < 0.001, respectively. The value is expressed as the means ± SD, with a minimum sample size of 3.

Fig. 7.. Construction of IPF model and visualization of the survival state of TBNC-labeled hMSCs transplanted into IPF mice.

(A) Micro-CT images of mice with and without bleomycin (BLM) injection after 21 days and (B) the percentages of pulmonary CT ventilation ratio. (C) Lung sections from the control and BLM groups were stained using hematoxylin and eosin (H&E) and Masson’s trichrome staining techniques. (D) BL images of the TBNC-labeled hMSCs at various time intervals in IPF mice. (E) Quantitative statistics of the difference between the total BL intensity and the maximum BL intensity. (F) Quantitative statistics pertaining to the mean BL intensity. The value is expressed as the means ± SD, with a minimum sample size of 3.

Fig. 8.. Visualization of biological behavior of TBNC-labeled hMSCs transplanted into IPF mice.

(A) 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 mice, respectively. (B) 3D in vivo CT images of the labeled hMSCs in IPF mice before (left) and after (right) transplantation. (C) Graph illustrating the distribution area of CT signals from labeled hMSCs in relation to the duration of transplantation. (D) 3D CT images depict the labeled hMSCs at 1, 5, 10, 15, and 20 days after transplantation into the lung of IPF mice. (E) CT values of the labeled hMSCs after transplantation into the lung at different time points. The value is expressed as the means ± SD, with a minimum sample size of 3.

Fig. 9.. Expression of HGF and fibrosis markers Col I and α-SMA in the lung of IPF mice.

(A) Laser confocal microscopy images and (B) fluorescence intensity of the expression of HGF in the lung tissue of IPF mice treated with hMSC and labeled hMSC, respectively. Laser confocal microscopy images and corresponding fluorescence intensity of the expressions of (C and D) Col I and (E and F) α-SMA in the lung tissue of IPF mice treated with hMSC and labeled hMSC, respectively. The untreated and BLM-treated mice were used as negative and positive controls, respectively. The symbols ** and *** indicate a statistically significant difference at P < 0.01 and P < 0.001, respectively. The value is expressed as the means ± SD, with a minimum sample size of 3.

Fig. 10.. Efficacy of TBNC-labeled hMSCs in the treatment of pulmonary fibrosis in vivo.

(A) Micro-CT images of the IPF mice treated with hMSC or labeled hMSC and (B) Masson’s 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. (C) The normal aeration area and (D) the poor aeration area were compared between the hMSC- and labeled hMSC–treated groups and the untreated groups. (E) The percentages of pulmonary CT ventilation ratio were compared between the hMSC- and labeled hMSC–treated groups and the untreated groups. The symbol *** denotes a statistically significant difference at a significance level of P < 0.001. The value is expressed as the means ± SD, with a minimum sample size of 3.