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Skin application of tape and MN patches
Twenty consecutive D-Squame tape strips (22 mm diameter) were collected from the lesional skin of the patients with AD. Hollow MNs, blank patches, and HA-MNs were also applied to the lesional skin of these patients for 10 min.Upon removal from the skin, HA-MNs were dissolved, and proteins in the skin metabolites were collected for experimental analysis (Figure 1B2,B3).2.4 Protein extraction
To extract proteins from the skin metabolites of the tape, hollow MNs, blank patches, and HA-MNs, each sample was scraped into 1% sodium dodecyl sulfate (SDS) buffer34, 35 (Thermo Fisher Scientific).2.5 Total protein quantification
To quantify the concentration of total proteins in the skin metabolite extracts, a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific) was used according to the manufacturer's protocol.3 RESULTS
3.1 MN patch design
Biocompatible HA-MNs (each 160 array, 1 mm base width, 650 um height) were fabricated using the droplet extension method32 by drying a pharmaceutical-grade HA solution on top of a hydrocolloid patch (Figure 1A).Statistical significance was set at p < 0.05.
Skin application of tape and MN patches
Twenty consecutive D-Squame tape strips (22 mm diameter) were collected from the lesional skin of the patients with AD. Hollow MNs, blank patches, and HA-MNs were also applied to the lesional skin of these patients for 10 min. The collected skin metabolites were stored individually in 60 mm dishes at −80°C prior to processing.
2.4 Protein extraction
To extract proteins from the skin metabolites of the tape, hollow MNs, blank patches, and HA-MNs, each sample was scraped into 1% sodium dodecyl sulfate (SDS) buffer34, 35 (Thermo Fisher Scientific). The protein extracts were then centrifuged to clear the debris and frozen at −80°C for processing.
2.5 Total protein quantification
To quantify the concentration of total proteins in the skin metabolite extracts, a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific) was used according to the manufacturer's protocol.
2.6 Enzyme-linked immunosorbent assay
Human IL-4, IL-13, and IFN-γ enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems) were used to determine the levels of IL-4, IL-13, and IFN-γ in undiluted samples of the skin metabolites following the manufacturer's instructions.
2.7 Statistical analysis
All assays were performed in duplicate, and the data are presented as the mean ± standard deviation. Statistical analyses were conducted using IBM SPSS Statistics 25.0 (Armonk). Pearson's correlation coefficient was used to analyze whether the data were statistically correlated. The data were analyzed by one-way analysis of variance, and the significance of the differences was determined using the unpaired two-tailed t-test or the Tukey–Kramer test. Statistical significance was set at p < 0.05.
3 RESULTS
3.1 MN patch design
Biocompatible HA-MNs (each 160 array, 1 mm base width, 650 μm height) were fabricated using the droplet extension method32 by drying a pharmaceutical-grade HA solution on top of a hydrocolloid patch (Figure 1A). When HA-MNs are applied to the skin at constant pressure, they penetrate the stratum corneum, and skin metabolites around the epidermis and dermis attach to the surface of the MNs (Figure 1B1). Upon removal from the skin, HA-MNs were dissolved, and proteins in the skin metabolites were collected for experimental analysis (Figure 1B2,B3).
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