Alcian blue staining to track the intracellular fate of hyaluronic-acid-based nanoparticles at transmission electron microscopy

Abstract

The main step in the assessment of nanomaterial safety and suitability for biomedical use is the location and the dynamic tracking of nanoparticles (NPs) inside cells or tissues. To precisely investigate the uptake mechanisms and intracellular fate of NPs, transmission electron microscopy is the technique of choice; however, the detection of NPs may sometimes be problematic. In fact, while NPs containing strongly electron dense (e.g., metal) components do not require specific detection methods at the ultrastructural level, organic NPs are hardly detectable in the intracellular environment due to their intrinsic moderate electron density. In this study, the critical-electrolyte-concentration Alcian Blue method set up by Schofield et al. in 1975 was applied to track hyaluronic-acid-based NPs in muscle cells in vitro. This long-established histochemical method proved to be a powerful tool allowing to identify not only whole NPs while entering cells and moving into the cytoplasm, but also their remnants following lysosomal degradation and extrusion.

A new method for the direct tracking of in vivo lignin nanocapsules in Eragrostis tef (Poaceae) tissues

“…Previously, other stainings have been used for tracking nanoparticles, such as Alcian Blue in hyaluronic-acid-based nanoparticles in muscle cells to be visualized at transmission electron microscopy, 33 while our method appears to be the first used in plants for tracking lignin NCs with a fluorescent microscope in plants.…”

Transmission Electron Microscopy as a Powerful Tool to Investigate the Interaction of Nanoparticles with Subcellular Structures

“…TEM images have also showed that several nanoconstructs are able to escape the endosomes and then occur freely in the cytosol, avoiding lysosomal degradation. This was first observed for cationic nanoconstructs and was explained by the so-called “proton sponge effect” [ 88 , 89 , 90 ] but recently, anionic nanocomplexes [ 91 ] and neutrally charged nanoparticles [ 92 ] have also been proven to efficiently escape endosomes. Endosomal escape has also been observed for gold nanoparticles coated with Listeriolysin O toxin, which is able to perforate the endosomal/lysosomal membrane [ 93 ] and, in prickly nanodiamonds, is able to break the endosome membrane [ 94 ].…”

“…Alternative histochemical methods are, therefore, necessary to unequivocally reveal the nanoconstructs at an ultrastructural level. In a recent work, hyaluronic acid-based nanoparticles [ 163 ] were made recognizable in TEM by adapting the long-established Alcian blue staining [ 91 ] ( Figure 3 ) that was originally proposed to reveal glycosaminoglycans in tissue sections [ 164 ].…”

Imaging techniques in nanomedical research

“… 65 , 66 , 69 , 85 Recently, the Alcian blue staining has been used to detect hyaluronic-acid based NPs 86 at both bright-field microscopy and TEM. 87 Immunocytochemistry allowed detecting chitosan NPs loaded with a synthetic opioid at both FM and TEM, 88 and this technique may simultaneously be performed with photo-oxidation. 89 …”