Investigating the Internalization of the Polyelectrolytes Microcapsules Using Scanning Ion Conductance Microscopy

Introduction:Polyelectrolyte multilayer microcapsules represent one of the promising strategies for intracellular drug delivery. However, the underlying mechanism of cellular internalization remains poorly understood due to... [ view full abstract ]

Introduction:

Polyelectrolyte multilayer microcapsules represent one of the promising strategies for intracellular drug delivery. However, the underlying mechanism of cellular internalization remains poorly understood due to limited availability of specific fluorescent markers and blockers of the processes involved in the internalization. Here we employ an emerging scanning probe microscopy technique called Scanning Ion Conductance Microscopy (SICM) to study these complex processes at nanoscale resolution.

Methods:

In SICM, an electrolyte-filled glass nanopipette, vertically positioned above a sample, serves as the scanning probe using a small reduction (≈0.3 %) in the ionic current flowing out of the nanopipette to detect proximity of the sample surface. To record topography of the sample surface, the nanopipette periodically approaches the surface at selected x-y coordinates and records the relative height of a sample. To estimate elasticity of the sample simultaneously with topography, the nanopipette is allowed to continue closer to the sample surface at each imaged point until current drops by 1 - 3%. Changes in the sample height in response to stress exerted by the nanopipette tip at these higher current drops is used to estimate elasticity.

Results:

We were able to obtain high resolution topographical recordings of 12 cases of single capsule internalization events with mean duration of internalization of 46 minutes (ranging from 17 to 71 minutes). The recordings provide a first direct proof that capsules landing on the cell membrane trigger formation of membrane protrusions in its immediate vicinity. The protrusions gradually formed a complex and rather irregular mesh enveloping the capsule and finally pulling the capsule into the intracellular space (Fig. 1a). Simultaneous measurement of elastic modulus revealed quick build-up of membrane engulfing the capsule, followed by relatively long plateau, and surprisingly abrupt sinking of the capsule deep into the cell (Fig. 1b-c).

Discussion:

According to our knowledge these are the first live recordings of complex topographical changes during the internalization process of microcapsules demonstrating the response triggered by capsules and the importance of membrane protrusions. Furthermore, our data show that mapping of elastic modulus has a potential to provide new insights into the mechanobiology of cellular uptake of microcapsules.