# Raman Spectroscopy to Monitor the Delivery of a Nano-Formulation of Vismodegib in the Skin
## Materials and Methods
### Skin Experiments  
Human or pig skin samples with a diameter of 24 mm were excised and placed onto Teflon devices, known as the Saarbrucken Penetration Model (SPM) [12]. These devices consist of a flat-bottomed cylindrical cavity measuring 25 mm in diameter and 5 mm in depth. Prior to placement, a filter paper disk soaked in 200 \(\mu\)L of PBS was positioned on the cavity floor to create a highly humid environment, replicating the moisture gradient across the _stratum corneum_ (SC). Each skin disk was evenly distributed with 20 aliquots of 2.5 \(\mu\)L (totaling 50 \(\mu\)L) of T + VDG suspension and incubated at 33 \({}^{\circ}\)C for 1 h. All experiments were conducted in quadruplicate. Subsequently, the skin was affixed to a polystyrene plate using pins, and _tape stripping_ was performed using 20 consecutive adhesive tapes loaded with a weight of 2 kg for 10 s, after which they were rapidly removed, taking along a layer of the SC.  
For the spectrometry assays, the tapes from each sample of human or pig skin were grouped into tubes corresponding to the shallow (tapes 1-5), middle (6-10), and deep (11-20) SC regions. Additionally, the remaining skin disk, consisting of viable epidermis and dermis (referred to as VED), was mechanically disrupted, and placed in an extra tube. Then, the samples were extracted for 1 h under stirring, using the same solvent used for the calibration curve, followed by the determination of the presence of VDG in each sample as in Section 2.2.  
For the Raman assays, representative adhesive tapes (0.04 mm) of the different human and pig skin portions (tapes number 1, 9, and 15 from the SC, as well as the intact portion of VED with a thickness of 3.62 mm and 3.15 mm for human and pig, respectively), were collected from each sample. The tapes were lifted using tweezers and stretched over a microscope slide, securing them in a manner that the adhesive surface which was in contact with the SC faced upwards. A _Horiba Yovin Ivon_ confocal Raman microscope (Horiba Ltd., Kyoto, Japan), equipped with a 785 nm laser emitting at a maximum power of 150 mW, was utilized to analyze the VDG signal. The system is equipped with an objective of 20\(\times\), with a working distance of 1.2 mm, an effective focal length of 0.9 mm, and a numerical aperture of 0.4. This objective has a focal volume of 0.2 mm\({}^{3}\).The microscope employed a _Cross Czerny Turner_ spectrometer, with a grating of 600 lines/mm as its detector, with an optical resolution of 1 cm\({}^{-1}\). The detector in this monochromator was a CCD cooled to \(-60\)\({}^{\circ}\)C. The obtained data were subsequently processed using Labspec software10.4 (Dilor) to generate Raman spectra. The entire thickness of the sample could not be completely estimated, due to limitations of the equipment.  
Blanks of untreated skin were prepared and subjected to the same procedures, to determine whether the skin or the adhesive tape generate Raman or UV-Visible signals that could act as interferences for the spectra of interest. Empty transfersomes were also used as another control.