A novel method to estimate cellular internalization of nanoparticles into gram-negative bacteria: Non-lytic removal of outer membrane and cell wall.

Bacteria efficiently take up small organic molecules and ions. However, the internalization of particulate forms, specifically nanoparticles (NPs) has been understudied and is a newly-emerging area of interest. However, determination of true cellular internalization is challenging owing to the difficulty of separating the aqueous phase from bacteria-associated NPs and, more importantly, of differentiating between internalized and NPs sorbed on bacteria surfaces. In this work, we developed and validated an extraction method which can operationally estimate internalization of metal NPs into Gram-negative bacteria. The outer cell membrane and cell wall, collectively called the periplasm, was successfully removed from bacteria using ethylenediaminetetraacetic acid (EDTA) at an optimized exposure period and concentration, without lysis of bacteria. This was followed by standard digestion and metal measurements. Verification of each step of the methodology was conducted by assessing both cellular and metal behavior. Specifically, the combined approaches of live/dead staining of bacteria, optical density measurements, transmission electron microscopy (TEM) and metal analyses of the supernatant indicated that the method operationally separated externally-sorbed NPs from those internalized actually localized within the bacterial cytoplasm. However, this new method is ideally used alongside other methods in a multi-method approach, to provide improved data quality. Therefore, it should be used with CSLM, FACS, TEM and other available methods.