To perform this assay, AuNPs@POM@PEG and AuNPs@PEG were previously labelled fluorescently with Alexa647. Before the injection of the nanoparticles, time 0 for the BBB-oC with the cell medium into channels was set, with the corresponding fluorescent settings. Each nanoparticle sample was then resuspended in cell media with a final 2.5 nM concentration (to achieve a good fluorescent signal) and added into the blood channel via independent devices. Then, the BBB-oC was placed into the fluorescent microscope (Nikon Ti2) and fluorescent images were captured until the end point of 1h. The recorded pictures were analyzed using Fiji/ImageJ® software, and the permeability coefficients (cm/s) were determined following the equation from Campisi et al. [77 (link)].
For TEM visualization, AuNPs@POM@PEG were administered in the blood channel and incubated for 24 h at 37 °C and 5% CO2. Next, the channels were washed twice with PBS 1× to remove any nanoparticles that did not cross the BBB. The hydrogel was dissolved adding TRIzol reagent in both channels and incubated at 60 °C for 15 min. After that, the liquid in the main chamber could be collected. Holey carbon grids were prepared via depositing a droplet of the dissolved hydrogel onto them and letting them dry. Finally, the grids were observed with an FEI Magellan 400L XHR SEM, and EDX was performed on the region of interest.
For TEM visualization, AuNPs@POM@PEG were administered in the blood channel and incubated for 24 h at 37 °C and 5% CO2. Next, the channels were washed twice with PBS 1× to remove any nanoparticles that did not cross the BBB. The hydrogel was dissolved adding TRIzol reagent in both channels and incubated at 60 °C for 15 min. After that, the liquid in the main chamber could be collected. Holey carbon grids were prepared via depositing a droplet of the dissolved hydrogel onto them and letting them dry. Finally, the grids were observed with an FEI Magellan 400L XHR SEM, and EDX was performed on the region of interest.
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