Spectroscopic and thermal characterization of alternative model biomembranes from shed skins of Bothrops jararaca and Spilotis pullatus

Recently, there has been an interest in the use of shed snake skin as alternative model biomembrane for human stratum corneum. This research work presented as objective the qualitative characterization of alternative model biomembranes from Bothrops jararaca and Spilotis pullatus by FT-Raman, PAS-FTIR and DSC. The employed biophysical techniques permitted the characterization of the biomembranes from shed snake skin of B. jararaca and S. pullatus by the identification of vibrational frequencies and endothermic transitions that are similar to those of the human stratum corneum.


INTRODUCTION
The intact human skin is considered as a barrier against the penetration and permeation of agents such as particles, chemical substances, radiations and microorganisms (Williams, Barry, Edwards, 1994).It is of utmost importance for the survival of human beings, despite being an obstacle for the action of active substances applied over the skin (Kalia et al., 2004).
Cutaneous penetration and permeation of active substances may be firstly limited by the epidermis, especially the stratum corneum (SC), due to its barrier function and to the fact that it is the first layer of contact with the outer side.The epidermis is divided into two portions: (1) the internal portion, formed by cells that are constantly proliferating; (2) the external portion, formed by the same cells that originate the SC after its keratinization and death (Moser et al., 2001).Skin penetration studies using in vitro evaluation present advantages such as: economy, fast obtainment of results, control of the experimental conditions and the possibility to evaluate a larger number of replicates, among others.The ideal situation would be to use human skin as a model, but the lack of this material, the need to submit the experiment to an Ethics Committee, the storage difficulties and high cost, and the viability of this membrane model limit its usage (Baby et al., 2009;Schmook, Meingassner, Billich, 2001;Rigg, Barry, 1990).
As alternative biomembranes, experimentation animals' skin, synthetic membranes and three-dimensional cultures are used to experimentally simulate epidermis.There is interest in using shed snake skin in alternative models of human skin biomembranes, and researchers have been evaluating its applicability in penetration / permeation studies, obtaining favorable responses.Shed snake skin is composed by pure SC without viable epidermis and follicles (Rigg, Barry, 1990).It offers a barrier similar to human SC and can be obtained abundantly without the death of the animal.Shed snake skin can be easily stored and does not tend to be contaminated nor microbiologically degraded, as it does not present living tissues (Itoh et al., 1990;Baby et al., 2007;Baby et al., 2008a).
Various biophysical methods have been employed to study the morphology and dynamics of the SC, aiming to understand the correlation between its structure and function.Among these methods: X-ray diffraction, electron paramagnetic resonance, nuclear magnetic resonance, DSC (Differential Scanning Calorimetry), FT-Raman, and PAS-FTIR (Lafleur, 2001;Baby et al., 2006a;Baby et al., 2008b).
This research work aimed at characterizing qualitatively alternative models of biomembranes from Bothrops jararaca and Spilotis pullatus shed snake skin through FT-Raman, PAS-FTIR and DSC.It is also worth to mention that the usage of shed snake skin as alternative model of biomembrane to study cutaneous permeation contemplates the aspect of Experimental Ethics in Animals and Human Beings, besides being ecologically correct.

Sample preparation
Ventral portions of Bothrops jararaca and Spilotis pullatus shed snake skin, gently donated by Butantan Institute, São Paulo, were cut and washed in abundance with distilled water.The samples were immersed in distilled water for eight hours in order to hydrate.After samples' hydration, the excess of water was removed with smooth compression using quantitative filter paper (Baby et al., 2006b).The samples were kept between two microscopy laminas and maintained in a desiccator until the time of analysis with FT-Raman, PAS-FTIR and DSC.

PAS-FTIR
The samples were adequately cut so as to fulfill the area of the photoacoustic cell in a model MTEC ® 200 spectrometer.In order to avoid the movement of the cell, the samples were covered by a metal device.Then, a flow of helium was used for 2 min to remove water and carbon dioxide molecules.After sealing the cells, vacuum was made in the spectrometer.The 64 co-additions were controlled using the Bomem ® PCDA program, in the spectral range of 4000-400 cm -1 .A rubber composite was used as standard.The experimental conditions were: (1) resolution: 4 cm -1 ; (2) slit: 10; (3) gain: 4; and (4) speed of the mobile mirror: 0.05 cm/s (Baby et al., 2007;Baby et al., 2006a).The measurements were done using three replicates.
The transition temperatures were determined considering the minimum values of the endothermic peaks observed in the heating curves.The DSC curves were built with the values of the heat flow as a function of the temperature.The measurements involved three replicates.
Raman spectra of the SC samples of Bothrops jararaca and Spilotis pullatus shed snake skin (Figures 1 and  2, respectively) presented spectral profiles similar to the ones described in the literature (Williams, Barry, Edwards, 1994).Characteristic vibrational spectral regions of the SC were observed, with intense signal bands between 3100-2700 cm -1 , referring to distensions originated from the lipid carbonic chains (symmetric and non-symmetric CH 3 , symmetric and non-symmetric CH 2 and CH).
There was a band in the region between 1650-1672 cm -1 , related to the distention of the C=O of the α-keratin's amide I and, possibly, β-keratin.Besides, there was a band in the region between 1450-1460 cm -1 , probably originated from the angular deformation from C-H to CH, CH 2 and CH 3 .
When comparing the Raman spectra from the SC of both species, there are similarities.However, the intensity of the signal appears to be greater for Spilotis pullatus, especially as far as the band between 3100-2700 cm -1 is concerned, what indicates differences in the biomembrane surface's morphology among these species.
Raman spectroscopy registers signals due to the spreading light that strikes on the sample's surface.Therefore, differences in signals' intensities are related to alterations in the sample's surface morphology caused by modifications in the molecular density per area unit of the  surface region involved in the spreading of light, and by alterations in its topography (Ingle, Crouch, 1988).
In Figures 3 and 4 (PAS-FTIR spectra), we can observe the typical profiles of the hydrated biological material, with bands in 1650 cm -1 (C=O distension of amide I) and in 1550 cm -1 (C-N distension and N-H deformation of amide II).In the band between 3600-3300 cm -1 , we can observe the presence of water in the species' SC (Golden et al., 1986;Lin et al., 1992).
The usage of DSC enables the study of structure and organization of the SC though the observation of the characteristic temperatures of the endothermic events related to the lipid fraction, e.g.phase transitions, and to the protein fraction of biomembranes that involve dehydration and keratin denaturation phenomena (Lin, Duan, Lin, 1996).
The isolated human SC has four characteristic endothermic transitions.The phase transition of the lipid bilayer from crystalline to gel state is attributed to the temperature of 40 °C.The phase transition of the lipid bilayer from lamellar gel to liquid state is attributed to the temperature of 75-85 °C.The transition that occurs at 105 °C represents the dehydration and denaturation of the protein fraction of the SC and the presence of a certain amount of water in the sample is required for its detection by the technique (Baby et al., 2006b;Golden et al., 1986, Ashton et al., 1992;Leopold, Lipppold, 1995).
In the DSC curve from Bothrops jararaca shed skin (Figure 5), not all the lipids endothermic transition peaks were observed.However, a transition of small magnitude was observed at, approximately, 58 °C.The endothermic peak referring to the transition involving dehydration and keratin denaturation occurred clearly at 130 °C.
Spilotis pullatus shed skin biomembrane presented a DSC curve similar to B. jararaca.The endothermic transi-

CONCLUSIONS
The biophysical techniques FT-Raman, PAS-FTIR and DSC allowed the qualitative characterization of biomembranes alternative models from Bothrops jararaca and Spilotis pullatus shed snake skin, and the identification of the vibrational frequencies and endothermic transitions similar to those of the human SC.