Substrate Stiffness and Particle Properties Influence Cellular Uptake of Nanoparticles and Viruses from the Ventral Side

Abstract

It is a long‐standing challenge to exploit cellular uptake mechanisms to deliver desired cargo into cells, for example, specific drugs or gene editing techniques. This study introduces a bioinspired material approach where nanoparticles are presented at the ventral side of cells adhering to engineered extracellular matrices. The effect of matrix stiffness on cell adhesion and mechanics, as well as on particle internalization by clathrin‐mediated endocytosis (CME), is investigated for varying particle size and surface functionalization. The results presented here show that substrate stiffness affects both cell adhesion and particle internalization, with softer substrates promoting higher levels of particle uptake. However, the activation of the CME pathway, either mechanically by particle size or functionally by receptor binding, regulates the sensitivity of cellular particle uptake to matrix stiffness. Finally, adeno‐associated viruses as the leading platform for therapeutic gene delivery are used as model cargo to showcase the importance of considering multiple components when designing delivery systems. These findings indicate that particle uptake is a multifaceted process that can be improved by the appropriate combination of extracellular environment mechanics and cargo properties.

Impact of mechanical cues on key cell functions and cell-nanoparticle interactions

“…Activation of clathrin-mediated endocytosis reduced the effect of substrate stiffness on particle uptake. Myosin II is important for regulation of the effect of substrate stiffness on particle uptake [ 179 ] HeLa (human cervical cancer); HCT-8 (human large intestine cancer) Polystyrene nanoparticles 100 NA NA ECM stiffness Cellular uptake of nanoparticles decreases on a per-cell-area basis but increases on a per-cell basis with increasing gel stiffness [ 177 ] AsPC-1 (human pancreatic adenocarcinoma); MDA-MB-231 (human breast adenocarcinoma) Polystyrene beads 800; 2400 NA; NA NA; NA ECM stiffness A wave-like (“meandering”) dependence of cell uptake on the substrate stiffness [ 213 ] bEnd.3 (mouse brain endothelial) cells Polystyrene nanoparticles Plain particles 108.07 ± 1.12; RGD-functionalized 114.67 ± 1.53; Scrambled RGD-functionalized 110.33 ± 0.86 − 48.33 ± 0.91; − 36.17 ± 0.61; − 36.13 ± 0.84 0.005 ± 0.003; 0.07 ± 0.02; 0.04 ± 0.02 ECM stiffness Higher stiffness promoted RGD-functionalized nanoparticles uptake by the cells [ 178 ] BeWo b30 cells (choriocarcinoma cell line) PEGylated DOPC liposomes Plain liposomes 48.8 ± 0.3; CSA-conjugated liposomes 93.7 ± 1.2 11.48 ± 4.36; − 25.5 ± 2.81 0.19 ± 0.01; 0.25 ± 0.01 Shear stress Shear stress promoted the cell uptake of liposomes [ 183 ] HUVECs Gold nanoparticles; Liposomes Gold nanoparticles untargeted PEG (5 kDa)-coated; 100; Gold nanoparticles targeted PEG (5 kDa)-coated; UEA-1-modified; ...…”

“…Other research has suggested that the stiffness of the substrate influences both cell adhesion and particle internalization. Softer substrates appear to enhance the levels of particle uptake [ 27 , 179 ]. One can definitely say that cell type predisposes response of cells to external stiffness (Table 2 ).…”

“…6 and Table 2 ). Hence, the activation of clathrin-mediated endocytosis has been observed to mitigate the influence of substrate stiffness on particle uptake [ 179 ]. Additionally, Myosin II plays a crucial role in regulating the impact of substrate stiffness on particle uptake.…”

“…Additionally, Myosin II plays a crucial role in regulating the impact of substrate stiffness on particle uptake. This suggests that both clathrin-mediated endocytosis and Myosin II activity are key factors in modulating how substrate stiffness influences the uptake of particles [ 179 ].…”

“…Current advances in microfluidics, tissue engineering and microfabrication led to the development of sophisticated platforms, including organ-on-chip (OoC) systems, to study effect of shear stress and hydrostatic pressure on living cells [130,132,[134][135][136]. Indeed, by integrating developments in tissue engineering and microfabrication, OoC platforms have ECM stiffness Collagen I-coated polyacrylamide gel substrates Young´s modulus: 1 ± 0.2 kPa; 7 ± 0.7 kPa; 20 ± 1.3 kPa; 25 ± 1.5 kPa [25] ECM stiffness TCPS; fibronectin-coated PDMS substrates Young´s modulus: GPa (TCPS); 2.8 MPa (PDMS); 130 kPa (PDMS) [27] ECM stiffness Collagen I-coated polyacrylamide gel substrates Young´s modulus: 0.25 ± 0.05 kPa; 10.5 ± 0.02 kPa [176] ECM stiffness Fibronectin-coated PDMS substrates Young´s modulus: 0.39 ± 0.03 kPa; 4.5 ± 0.5 kPa; 51.4 ± 4.5 kPa [179] ECM stiffness Fibronectin-coated polyacrylamide gel substrates Young´s modulus: 1.0 ± 0. Tensile forces Flexercell® Tension Plus™ System (Flexcell Int.…”