Figure 3.

Delivery of Synthetic hGLuc mRNA into Porcine Skin Using Hollow Microneedles and Analysis of Protein Expression

(1) Porcine skin detached from the outer side of pigs’ ears was trimmed and punched into 1-mm-thick pieces with 1.5-cm diameter and disinfected. The structure of the skin is shown schematically. S.c., stratum corneum; E, epidermis; D, dermis. (2) Lipoplexes were generated by incubation of 1.5 μg hGLuc mRNA with 1.5 μl Lipofectamine 2000 in a total volume of 35 μL OptiMEM I reduced serum-free medium for 20 min at RT. The mixture was injected into the skin using MicronJet600 microneedles. (3) After washing with DPBS, the skin was transferred into a ThinCert insert, which served as a permeable barrier between the skin and the surrounding medium. (4) The skin was incubated air-exposed in 1.5 mL human endothelial cell culture medium in one well of a 12-well plate from 24 to 72 hr at 37°C and 5% CO₂. The microinjected lipoplexes can enter the cells via endocytosis. After the release of mRNA into the cytosol, mRNA is translated by ribosomes into protein. (5) After the appropriate incubation time, the produced hGLuc protein is detected by luciferase assay in the surrounding medium as well as in the skin.