Promoting intracellular delivery of sub-25 nm nanoparticles via defined levels of compression

Many investigations into the interactions between nanoparticles and mammalian cells entail the use of culture systems that do not account for the effect of extracellular mechanical cues, such as compression. In this work, we present an experimental set-up to systematically investigate the combined effects of nanoparticle size and compressive stress on the cellular uptake and intracellular localization of poly(ethylene glycol)-coated gold nanoparticles (Au-PEG NPs). Specifically, we employ an automated micromechanical system to apply defined levels of compressive strain to an agarose gel, which transmits defined amounts of unconfined, uniaxial compressive stress to a monolayer of C2C12 mouse myoblasts seeded underneath the gel without compromising cell viability. Notably, uptake of Au-PEG NPs smaller than 25 nm by compressed myoblasts is up to 5-fold higher than that by uncompressed cells. The optimal compressive stress for maximizing the cellular uptake of sub-25 nm NPs monotonically increases with NP size. With and without compression, the Au-PEG NPs enter C2C12 cells via energy-dependent uptake; they also enter compressed cells via clathrin-mediated endocytosis as the major pathway. Upon cellular entry, the Au-PEG NPs more readily reside in the late endosomes or lysosomes of compressed cells than uncompressed cells. Results from our experimental set-up yield mechanistic insights into the delivery of NPs to cell types under extracellular compression.