LNG-loaded effervescent MN patches were applied to adult female Sprague-Dawley rats (~200 g) while under isoflurane anesthesia. The rats’ dorsal skin was shaved before application of one MN patch per rat, taking care not to damage skin during shaving. Animal studies were performed with approval by the Georgia Institute of Technology Institutional Animal Care and Use Committee (IACUC), and IACUC guidelines were followed in all studies with animal subjects.

To investigate detachment of PLGA/LNG MNs in rats, effervescent MN patches containing Nile red were administered to the rats using the methods described above for ex vivo MN patch application to porcine skin. The administration sites on the skin were imaged by fluorescence microscopy (Olympus).

To study pharmacokinetics of LNG release from MNs in skin, a group of 10 rats received LNG-loaded effervescent MN patches. A power analysis indicated that a sample size of 10 rats per group would be sufficient to distinguish pharmacokinetic profiles in animals receiving LNG from those without application of patches with 95% confidence. The primary endpoint of the animal study was LNG plasma concentration above the human therapeutic threshold level (0.2 ng/ml in plasma) for 1 month. The secondary endpoint was irritation at the site of MN patch administration. All data collected in this study were retained; no outliers were excluded.

Blood samples (~500 μl) were drawn from the tail vein 0, 12, and 24 hours and 3, 7, 10, 14, 17, 21, 24, 28, 31, 35, 38, 42, 45, 49, 52, 55, and 60 days after MN patch application. Plasma was separated by centrifuging blood samples at 2000g for 15 min at 4°C, and LNG concentration in the plasma was analyzed by enzyme-linked immunosorbent assay (Thermo Fisher Scientific) following the manufacturer’s instruction. Biocompatibility of LNG delivery from effervescent MN patches was evaluated by euthanizing rats by CO2 asphyxiation at the end of the study (i.e., 60 days after MN patch application) and excising tissue surrounding the patch application site. This tissue was fixed in 10% neutral-buffered formalin for 2 days at 4°C and then embedded in paraffin after complete dehydration, cut into sections of 5-μm thickness, and stained using hematoxylin and eosin for histological analysis.

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