The Effect of Substrate Properties on Cellular Behavior and Nanoparticle Uptake in Human Fibroblasts and Epithelial Cells

Abstract

The delivery of nanomedicines into cells holds enormous therapeutic potential; however little is known regarding how the extracellular matrix (ECM) can influence cell-nanoparticle (NP) interactions. Changes in ECM organization and composition occur in several pathophysiological states, including fibrosis and tumorigenesis, and may contribute to disease progression. We show that the physical characteristics of cellular substrates, that more closely resemble the ECM in vivo, can influence cell behavior and the subsequent uptake of NPs. Electrospinning was used to create two different substrates made of soft polyurethane (PU) with aligned and non-aligned nanofibers to recapitulate the ECM in two different states. To investigate the impact of cell-substrate interaction, A549 lung epithelial cells and MRC-5 lung fibroblasts were cultured on soft PU membranes with different alignments and compared against stiff tissue culture plastic (TCP)/glass. Both cell types could attach and grow on both PU membranes with no signs of cytotoxicity but with increased cytokine release compared with cells on the TCP. The uptake of silica NPs increased more than three-fold in fibroblasts but not in epithelial cells cultured on both membranes. This study demonstrates that cell-matrix interaction is substrate and cell-type dependent and highlights the importance of considering the ECM and tissue mechanical properties when designing NPs for effective cell targeting and treatment.

Keywords: epithelial cells; extracellular matrix; fibroblasts; mechanobiology; nanofibers; nanoparticle uptake; stiffness.

Figure 1

Characterization of non-aligned and aligned polyurethane electrospun fibrous membranes. Scanning electron microscopy images and fiber orientation of non-aligned (A) and aligned fibers (B). Tables presenting information regarding the Young’s modulus, average fiber diameter, and membrane thickness (C). Data are presented as the mean ± standard error of the mean. II—fibers along the strain direction; T—fibers perpendicular to the strain direction.

Figure 2

Cell interaction with different substrates. Confocal laser scanning microscopy (CLSM) images showing the morphology of the attached A549 lung epithelial cells (top row) and MRC-5 lung fibroblasts (bottom row) on glass, non-aligned polyurethane (PU) fibers, and aligned PU fibers. Nuclei are in cyan and f-actin is in magenta. Arrows indicate cell/fiber orientation.

Figure 3

Scanning electron microscopy (SEM) images of lung epithelial cells (A549) and lung fibroblasts (MRC5) cultured on glass and aligned and non-aligned electrospun polyurethane (PU) membranes.

Figure 4

Impact of fibrous polyurethane (PU) membranes on the cytotoxicity and metabolic activity of lung epithelial cells (A549) and fibroblasts (MRC-5). Bar graphs, at the top, represent the release of lactate dehydrogenase relative to tissue culture plastic (TCP) from A549 (A) and MRC-5 (B) cells. Bar graphs, at the bottom, represent the reduction in WST-1 relative to TCP from A549 (C) and MRC-5 (D) cells. Absorbance values for the WST-1 assay were normalized against the total protein content. Data are presented as the mean ± standard error of the mean (n = 3). Statistical significance was determined via one-way ANOVA and Dunnett’s post hoc test for multiple comparisons and is represented by **** p ≤ 0.0001. ns: not significant.

Figure 5

Impact of fibrous polyurethane (PU) membranes on the inflammatory response of lung epithelial cells (A549) and fibroblasts (MRC-5). Bar graphs represent the release of interleukin (IL)-6 and IL-8 from A549 (A,B) and MRC-5 (C,D) cultured on tissue culture plastic (TCP) and non-aligned and aligned PU. Values were normalized against the total protein content. Data are presented as the mean ± standard error of the mean (n = 3). Statistical significance was determined via one-way ANOVA and Tukey’s post hoc test for multiple comparisons and is represented by * p ≤ 0.05 and ** p ≤ 0.01. ns: not significant.

Figure 6

Immunofluorescence staining for α-smooth muscle actin (α-SMA). Confocal laser scanning microscopy (CLSM) images showing the immunostaining of MRC-5 fibroblasts with α-SMA cultured on glass, non-aligned and aligned polyurethane (PU) fibers, and glass + transforming growth factor beta (TGF-β). TGF-β was added for 24 h to MRC-5 cells at a concentration of 5 ng/mL. Nuclei are in cyan and α-SMA is in magenta.

Figure 7

Impact of fibrous polyurethane (PU) membranes on SiO₂ nanoparticle (NP) uptake in lung epithelial cells (A549) and fibroblasts (MRC-5). Bar graphs show the median fluorescence intensity (MFI) values obtained from flow cytometry measurements upon 24 h of exposure of 20 µg/mL SiO₂ NPs to A549 (A) and MRC-5 cells (C) cultured on tissue culture plastic (TCP) and non-aligned and aligned PU fibers. Data are presented as the mean ± standard error of the mean (n = 3). Statistical significance was determined via one-way ANOVA and Dunnett’s post hoc test for multiple comparisons and is represented by ** p ≤ 0.01. ns: not significant. Confocal laser scanning microscopy (CLSM) images showing the internalization of SiO₂ NPs in A549 (B) and MRC-5 cells (D) cultured on glass and non-aligned and aligned PU fibers. Nuclei are in cyan, F-actin is in magenta, and SiO₂ NPs are in yellow. Arrows indicate the intracellular localization of NPs.