Nanoengineered mesenchymal stem cell therapy for pulmonary fibrosis in young and aged mice

Abstract

Pulmonary fibrosis (PF) is an age-related interstitial lung disease that results in notable morbidity and mortality. The Food and Drug Administration-approved drugs can decelerate the progression of PF; however, curing aged patients with severe fibrosis is ineffective because of insufficient accumulation of these drugs and wide necrocytosis of type II alveolar epithelial cells (AEC IIs). Here, we constructed a mesenchymal stem cell (MSC)-based nanoengineered platform via the bioconjugation of MSCs and type I collagenase-modified liposomes loaded with nintedanib (MSCs-Lip@NCAF) for treating severe fibrosis. Specifically, MSCs-Lip@NCAF migrated to fibrotic lungs because of the homing characteristic of MSCs and then Lip@NCAF was sensitively released. Subsequently, Lip@NCAF ablated collagen fibers, delivered nintedanib into fibroblasts, and inhibited fibroblast overactivation. MSCs differentiated into AEC IIs to repair alveolar structure and ultimately promote the regeneration of damaged lungs in aged mice. Our findings indicated that MSCs-Lip@NCAF could be used as a promising therapeutic candidate for PF therapy, especially in aged patients.

Fig. 1.. Illustration of lung-targeting nanoengineered MSCs-Lip@NCAF designed to reverse PF in young and aged mice.

(A) Preparation of MSCs-Lip@NCAF. PC, phosphatidylcholine. (B) MSCs-Lip@NCAF migrated to injured lungs because of their homing ability, and Lip@NCAF was released in response to MMP-2. Then, Lip@NCAF degraded collagen fibers and inhibited fibroblast overactivation. Moreover, MSCs repaired injured AEC IIs in young mice and differentiated into AEC IIs to participate in alveolar reestablishment in aged mice.

Fig. 2.. PF progression in young and aged mice.

(A) Schematic illustration of the study design. H&E staining (B) and IF staining of collagen I (C) in the lungs of young and aged mice challenged with BLM (n = 6). (D) RNA expressions of Col1a1 and Sftpc in the lung of BLM-treated young and aged mice. (E) H&E, Masson, and IF staining of the lungs of young and aged mice treated with NIN (n = 6). WB (F) and qPCR (G) analysis of collagen I and fibronectin (n = 6). Data are means ± SEM. *P < 0.05 and **P < 0.01.

Fig. 3.. Preparation and characterization of MSCs-Lip@NCAF.

The size distribution (A) and zeta potential (B) of Lip@NCAF, MSCs, and MSCs-Lip@NCAF (n = 5). (C) Scanning electron microscopy images of MSCs and MSCs-Lip@NCAF. Fluorescence images of DiO-labeled Lip@CAF and DiD-labeled MSCs, as determined by CLSM (D) and FCM (E). (F) Adipogenic and osteogenic differentiation of MSCs and MSCs-Lip@NCAF. (G) The migration of MSCs and MSCs-Lip@NCAF in the presence of CXCL12 (n = 5). Fluorescence images of DiD-labeled MSCs and released DiO-labeled Lip@CAF in the presence of MMP-2, as determined by CLSM (H) and FCM (I). Data are means ± SD. *P < 0.05.

Fig. 4.. The antifibrotic effects of MSCs-Lip@NCAF in vitro.

(A) Illustration of the collagen barrier model in vitro. (B and C) The fluorescence intensity of y-fibroblasts was measured by FCM after adding different coumarin 6–labeled formulations to the Transwell chambers (n = 5). (D) Cellular uptake in y-fibroblasts, as shown by CLSM. Inhibition of y-fibroblast (E) and a-fibroblast (F) proliferation by Lip@NCAF, as shown by EdU staining (n = 5). (G) IF analysis of collagen I and α-SMA in y-fibroblasts and a-fibroblasts. WB analysis of fibronectin and collagen I in y-fibroblasts (H) and a-fibroblasts (I). (J) Images of collagen contraction mediated by y-fibroblasts and a-fibroblasts. (K) Quantitative analysis of the gel area in (J) (n = 5). (L) IF analysis of vimentin when A549 cells were incubated with MSCs and MSCs-Lip@NCAF in the presence of BLM. The survival (M) and proliferation (N) of A549 cells after incubation with MSCs and MSCs-Lip@NCAF in the presence of BLM (n = 5). Data are means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5.. Homing ability and behavior of MSCs-Lip@NCAF in vivo.

(A) In vivo images of the mice that were intravenously injected with free DiR, DiR-labeled Lip@CAF, MSCs, and MSCs-Lip@CAF (n = 6). (B) Ex vivo images of major organs collected after 12 hours after injection (n = 6). (C) Quantification of the fluorescence intensity of lung/liver (n = 6). (D) Fluorescence imaging of lung sections after the injection of free DiI, DiI-labeled Lip@CAF, MSCs, and MSCs-Lip@CAF for 12 hours. (E) Illustration of the behavior of MSCs-Lip@NCAF in vivo. (F) Fluorescence imaging of the lungs of mice treated with double-labeled MSCs-Lip@CAF (MSCs labeled with DiI and Lip@CAF labeled with DiO) for 1 and 2 hours. (G) IF analysis of lung sections after mice were injected with MSCs-Lip@CA, MSCs-Lip@AF, and MSCs-Lip@CAF (DiO-labeled Lip@CA, Lip@AF, and Lip@CAF, as well as DiI-labeled MSCs) for 4 hours. Data are means ± SD. **P < 0.01.

Fig. 6.. Antifibrotic effects of MSCs-Lip@NCAF on young mice.

(A) Body weight after treatment with different formulations (n = 6). (B) Lung/body weight in the different groups (n = 6). (C) The levels of HYP (n = 6). (D) Morphology and H&E, Masson, and IHC staining of lung sections. RNA levels of Col1a1 (E) and Acta2 (F) (n = 6). (G) WB analysis of collagen I. qPCR (H) and WB (I) of fibronectin (n = 6). (J) The expression of SPC was analyzed by WB. Data are means ± SD (A) to (C) or means ± SEM (E), (F), and (H). *P < 0.05, **P < 0.01, and ***P < 0.001. n.s., no significant difference.

Fig. 7.. Antifibrotic effects of MSCs-Lip@NCAF on aged mice.

(A) H&E, Masson, IHC, and IF staining of lung sections from aged mice treated with different formulations (n = 6). The expressions of fibronectin (B), collagen I (C), and α-SMA (D) by WB (n = 6). RNA levels of Fn1 (E), Col1a1 (F), and Acta2 (G) (n = 6). Data are means ± SEM. *P < 0.05 and **P < 0.01.

Fig. 8.. Antifibrotic mechanisms of Lip@NCAF and MSCs.

(A) Illustration of antifibrotic mechanisms of Lip@NCAF. MEK, mitogen-activated protein kinase kinase. (B) The expressions of p-PDGFR and p-ERK1/2 by WB. RNA levels of Col1a1 (C) and Pdgfrb (D) (n = 5). The levels of TNF-α (E), IL-1β (F), and IL-6 (G) in young and aged mice treated with MSCs and MSCs-Lip@NCAF (n = 6). (H) IF analysis of the lung sections in aged mice injected with MSCs and MSCs-Lip@NCAF expressing GFP for 14 days (n = 6). RNA levels of Cdkn2a (I), Cdkn1a (J), Mmp12 (K), and Mcp1 (L) of lungs in aged mice (n = 6). Data are means ± SD (E) to (G) or means ± SEM (C), (D), and (I) to (L). *P < 0.05 and **P < 0.01.