# Raman Spectroscopy to Monitor the Delivery of a Nano-Formulation of Vismodegib in the Skin
## 3 Results and Discussion
### VDG Determination by Raman Spectroscopy  
As can be seen in Figure 3, the Raman signals of the drug exhibited higher intensity in the superficial layers of the human SC than in the pig SC, and it showed partial agreement with the spectroscopic UV-visible assay. On the other hand, concerning the distribution of VDG in the VED shown in Figure 4, the Raman signals for both the carrier and the drug were detected more intensely in pig skin, whereas they were barely distinguishable from the background noise in the human samples. It was also coincident with the higher VDG signal in the UV-visible analysis for pig skin.  
In Figures 3 and 4, the Raman spectra obtained from the different regions of human and pig skin are shown for empty transfersomes (T) and transfersomes loaded with VDG (T + VDG). In the cases detailed in the previous paragraph for Figure 4, a peak at 1434 cm\({}^{-1}\) corresponding to transfersomes, previously observed in the SC [24], was found. Additionally, a peak at 770 cm\({}^{-1}\) was observed in the VDG-loaded transfersomes in Figures 3 and 4, which coincides with a value previously reported through theoretical calculations [10]. Another peak reported for transfersomes at 1150 cm\({}^{-1}\) was also observed in pig VED, as well as other peaks corresponding to VDG at 350 cm\({}^{-1}\) and 415 cm\({}^{-1}\), were pointed out in  
Figure 2: VDG recovered from the different sections of human and pig skin incubated with same amounts of T + VDG for 1 h at 35 \({}^{\circ}\)C (\(n=4\)). VDG was expressed as the percentage recovered from skin relative to the initial concentration of the incubated drug. Data are shown as mean \(\pm\) SD.  
Figure 3. On the other hand, in Figure 5, a fourth derivative version of the Raman spectra of human skin in the VED is shown for better observation of the VDG presence, because the original signal presented very low intensity. Derivative analysis is commonly used to detect small signals hidden in the original spectra. Mathematically, if the original spectrum has a small peak (our case with the Raman Signal of VDG), the fourth derivative will put this in evidence. The first derivative of the original signal will be a line passing through zero, the second-order derivative will be an inverted peak with respect to the original signal. Finally, the fourth-order derivative is the inverse signal of the second-order derivative. This is the case of the Raman Spectra of VDG shown in Figure 5[25]. In sum, these results could also confirm that VDG reaches the VED through this transport system.  
Figure 4: Raman spectra of VED from the remaining skin after tape stripping. The upper part shows the results from human skin samples, while the lower part corresponds to pig skin samples. In both cases, empty transfersomes (T) and VDG-loaded transfersomes (T + VDG) are shown. Arrows show the peaks found corresponding to the Raman signal of VDG, or from the lipid matrix of the transfersomes (T).  
Figure 3: Raman spectra of tapes from tape stripping. In the upper part, the results from human skin samples are shown, while the lower part corresponds to pig skin samples. The controls were tapes stripped from untreated skin samples. Arrows show the peaks found corresponding to the VDG Raman signal.