Cells Adhering to 3D Vertical Nanostructures: Cell Membrane Reshaping without Stable Internalization

Abstract

The dynamic interface between the cellular membrane and 3D nanostructures determines biological processes and guides the design of novel biomedical devices. Despite the fact that recent advancements in the fabrication of artificial biointerfaces have yielded an enhanced understanding of this interface, there remain open questions on how the cellular membrane reacts and behaves in the presence of sharp objects on the nanoscale. Here we provide a multifaceted characterization of the cellular membrane's mechanical stability when closely interacting with high-aspect-ratio 3D vertical nanostructures, providing strong evidence that vertical nanostructures spontaneously penetrate the cellular membrane to form a steady intracellular coupling only in rare cases and under specific conditions. The cell membrane is able to conform tightly over the majority of structures with various shapes while maintaining its integrity.

High‐Aspect‐Ratio Nanostructured Surfaces as Biological Metamaterials

“…Throughout the literature, interactions are variously described as penetrating, piercing, perturbing, impaling, indenting, and mechanically disrupting the cell membrane, which reflects in part the lack of consensus over what is happening. In particular, many reports question whether the cell membrane is spontaneously penetrated by nanostructures, and this topic has been presented as a source of contention within the field. In this context, spontaneous penetration refers to a high‐aspect‐ratio nanostructure piercing the membrane of a cell that has been seeded onto a surface (with minimal applied external force).…”

“…Many electron microscopy studies have illustrated that cell membranes can engulf high‐aspect‐ratio nanostructures, with no clear evidence of membrane rupture (see Figure for some examples) . Electron microscopy is limited to a fixed moment in time, so may fail to capture the highly dynamic nature of the membrane, which can undergo rapid repair .…”

“…Bioelectronic experiments show that nanoelectrodes only measure intracellular potentials after poration techniques have been applied, and rapidly return to measuring extracellular potentials in the absence of further external stimulus, again highlighting the need for an external force to disrupt the membrane. Dipalo et al explored this behavior explicitly using a range of nanopillar geometries using fluorescent and electron microscopy, and electrophysiological measurements . All three methods consistently showed no spontaneous penetration of the membrane, except in a handful cases.…”

“…Tailoring the surface chemistry to promote cell adhesion to nanostructured surfaces consists of either creating a favorable electrostatic interaction between the cell and surface, or by replicating naturally occurring binding sites. Electrostatically charged synthetic polymers such as polylysines and polyornithines have been used to promote the adhesion of cells to diamond nanoneedles, quartz nanopillars, alumina nanostraws, gold nanoelectrodes, silicon nanowires, and polymer nanopillars (Figure E) . Staufer et al claimed that the cell–substrate adhesion created by coating their gold nanoelectrodes with poly‐ l ‐lysine was strong enough to cause widespread spontaneous membrane penetration .…”

Correlating Surface Plasmon Resonance Microscopy of Living and Fixated Cells with Electron Microscopy Allows for Investigation of Potential Preparation Artifacts

“…This makes electron microscopy (EM) an indispensable tool in the field. EM includes multiple different techniques such as transmission electron microscopy (TEM) and scanning (transmission) electron microscopy (S(T)EM) …”