Separation, retention, and delivery efficiency of effervescent MN patches were evaluated by inserting MN patches loaded with fluorescent dye (Nile red) into stretched porcine skin ex vivo by pressing with thumb against the skin and then leaving the patches on the skin for 50 s to give time for dissolution of the effervescent backing and separation of MNs. The skin was then examined by optical microscopy (Olympus, Tokyo, Japan) to identify detached MNs embedded in the skin. To remove any detached MNs that were partially protruding above the skin surface, a swab was gently and repeatedly scraped across the site of MN patch treatment for 10 s in some cases. Separation and retention efficiency were calculated by dividing the number of colored spots due to the presence of fluorescent MNs in the skin by the number of MNs in the patch (i.e., 100 MNs).

Delivery efficiency of the MN patch was evaluated by using quantitative fluorescence analysis (Microplate Reader, Bio-Rad, Hercules, CA) to measure fluorescence intensity from the dye in the MN patch before and after skin insertion and fluorescence from the dye on the skin surface. The amounts of dye in the residual backing and on the skin surface were subtracted from that in the MN patch before insertion to determine the amount of dye delivered into the skin. To calculate delivery efficiency, the delivered dye in the skin was divided by the amount of dye in the MN patch before insertion. Last, skin was frozen and then cut into 10-μm sections for histological analysis.

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