Facilitating in vitro Tape Stripping: Application of Infrared Densitometry for Quantification of Porcine Stratum Corneum Proteins

Abstract

Infrared (IR) densitometry is a highly practical method recently proposed for protein analysis during in vivo tape stripping. However, this method has not yet been validated for the quantification of porcine stratum corneum (SC) proteins. Therefore, the aim of this study was to establish calibration curves for the analysis of adhesive tapes removed from porcine ear skin. To this end, the protein absorption (as determined via IR densitometry) was correlated with the protein content determined with the Micro BCA™ protein assay after extraction of the tapes. The obtained linear regressions confirm that IR densitometry is suitable for the quantification of not only human, but also porcine, SC proteins. The pattern of protein removal observed with porcine skin differs from that of human skin due to more pronounced corneocyte clustering and deep ‘canyons’, which necessitates specific evaluation of porcine skin samples and a working protocol that takes this into account. The presented data will facilitate future analysis of porcine SC proteins during in vitro tape stripping.

The application of ATR-FTIR spectroscopy and multivariate data analysis to study drug crystallisation in the stratum corneum

“…The absence of 20 crystalline drug in the spectra following delivery in DMSO may be related to the sensitivity of the ATR-FTIR spectroscopy. From the drug extraction profiles ( Figure 7), drug content in each tape strip following application in DMSO is generally below 0.5 µg/cm 2 .…”

“…The amount of sodium hydroxide was four times the quantity previously used to extract protein from porcine SC for an area of ~ 4 cm 2 using D-Squame ® and Corneofix ® tapes [20]. These vials were then shaken at 37ºC for 1 h and sonicated for 30 -60 min at room temperature to fully extract the proteins.…”

“…The total thickness of the SC removed from the 20 tape strips is estimated to be 7.7 ± 1.3 µm. Klang, et al [20] estimated a total thickness of 7.96 ± 3.25 µm for SC removed from porcine skin using 80 -130 tape strips but used a different type of tape.…”

Cutaneous Permeation and Penetration of Sunscreens: Formulation Strategies and In Vitro Methods

“…Recent studies are applying infrared densitometry (IR-D) to in vitro tape stripping using SquameScan TM 850A [3] to verify the endpoint of tape-stripping, i.e., complete SC removal. In fact, the SC depth can be extrapolated from the IR-D data of sequential tape-strips, where the protein content of each tape strip can be indirectly quantified from the tape absorbance [41]; the lower limit of quantification of IR-D indicates the complete removal of the SC (less than 5% of the total SC remaining) and can be used to know the exact numbers of tapes needed. Haque and co-workers [3] investigated the behavior of three UV-filters with different chemical-physical characteristics (BMZ, DHHB and IPMC) using full thickness pig-ear-skin and found that, at the end of the permeation study (12 h), most of the applied dose was recovered in the first five tape strips and none of the sunscreens was detected in the receiving phase.…”

Assessment of Near-Infrared Densitometry for in situ Determination of the Total Stratum Corneum Thickness on Pig Skin: Influence of Storage Time

“…NIR densitometry is an alternative to established methods such as protein quantification on each tape and gravimetric or other optical methods [9,10,11,12]. For human skin in vivo and pig skin in vitro it was established that the NIR pseudoabsorption, measured as the intensity reduction of a circular infrared (IR) laser beam (wavelength 850 nm), is correlated to the amount of protein per area (wt/A) [7,13]:…”