Response to "Comment on 'Distribution of Nanoparticles in the See-through Medaka (Oryzias latipes)'".

I appreciate Ceyhun’s interest in my early observations (Kashiwada 2006) on the penetration of nanoparticles into neural tissues of Oryzias latipes. Ceyhun finds my conclusions that “[t]hese results suggest that nanoparticles are capable of penetrating the blood–brain barrier and that they eventually reach the brain” and that “[n]anoparticles penetrated to reach the brain, although the amounts of nanoparticles that reached the brain were low” to be misleading. Before 2006 (the year of my publication), there was much less information on the functional structure of the blood–brain barrier (BBB) and the transportation of nanoobjects than is now available. In my study, the intact removed brain was subjected to direct observation. If cross sections of the brain were observed, it should be possible to observe fluorescence from the cut surfaces. However, it would be impossible to directly confirm the details of the nanoobjects’ crossing of the BBB.

It would seem impossible for any solid nanoobject to penetrate the BBB, based on our fundamental understanding of its functional structure. However, nanoobjects have been shown to reach the brain of rats and fish via blood flow in investigations beyond my own (Yamago et al. 1995; Kashiwada 2006; Gonzalez-Carter et al. 2020; Ohta et al. 2020). Further, the ionic nature of particles may be important in determining passage through membranes. Kataoka et al. (2017) reported that water-dispersible single-walled carbon nanotubes coated with a cationic plastic polymer (W-SWCNT) exhibited embryotoxicity to medaka eggs, whereas other hydrophobic polyethylene glycol-functionalized SWCNT and nonfunctionalized SWCNT showed no toxicity. It is unclear how W-SWCNT caused the toxicity and, if the W-SWCNT could pass through biological membranes (e.g., the chorion), whether the pores on such membranes would be large enough to allow the passage of nanoobjects. Like the fluorescent polystyrene nanoparticles used in my early study, W-SWCNT had charged surface cations. Surface charge may control the behavior of nanoobjects in the aquatic ecosystem (Burns et al. 2013). Although some questions remain about the transit of nanoobjects through membranes, my early observation has provided some suggestion that cationic surface charge may be important, potentially in traversing tight junctions.