Elucidation of penetration enhancement mechanism of Emu oil using FTIR microspectroscopy at EMIRA laboratory of SESAME synchrotron

Accepted Manuscript Elucidation of penetration enhancement mechanism of Emu oil using FTIR microspectroscopy at EMIRA laboratory of SESAME synchrotron

Randa S.H. Mansour, Alsayed A. Sallam, Imad I. Hamdan, Enam A. Khalil, Ibraheem Yousef PII: DOI: Reference:

S1386-1425(17)30402-X doi: 10.1016/j.saa.2017.05.026 SAA 15168

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date: Revised date: Accepted date:

16 October 2016 11 May 2017 11 May 2017

Please cite this article as: Randa S.H. Mansour, Alsayed A. Sallam, Imad I. Hamdan, Enam A. Khalil, Ibraheem Yousef , Elucidation of penetration enhancement mechanism of Emu oil using FTIR microspectroscopy at EMIRA laboratory of SESAME synchrotron, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2017), doi: 10.1016/j.saa.2017.05.026

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ACCEPTED MANUSCRIPT Elucidation of penetration enhancement mechanism of Emu oil using FTIR microspectroscopy at EMIRA laboratory of SESAME synchrotron Randa SH. Mansour 1*, ALSayed A. Sallam 2, Imad I. Hamdan 1, Enam A. Khalil 1, Ibraheem Yousef 3,4 Faculty of pharmacy, University of Jordan,11942 Amman, Jordan.

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Al-Taqaddom Pharmaceutical Industries Inc., Amman, Jordan.

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SESAME Synchrotron, P.O. Box 7, 19252 Allan, Jordan.

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ALBA Synchrotron, Carrer de la Llum 2-26, 08290 Cerdanyola del Vallès, Barcelona, Spain.

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Current address: Faculty of pharmacy, Philadelphia University, 19392 Amman, Jordan.

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Abstract

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It has been proposed that Emu oil possesses skin permeation-enhancing effect. This study aimed to address its possible penetration enhancement mechanism(s) using IR microscopy, in accordance with LPP theory. The penetration of Emu oil through the layers of human skin was accomplished by monitoring oil-IR characteristic feature at 3006 cm-1. The unsaturated components of Emu oil accumulated at about 270 µm depth of skin surface. The interaction of Emu oil with lipid and protein constituents of SC was investigated in comparison with a commonly used enhancer, IPM. Inter-sample spectral differences were identified using PCA and linked with possible enhancement mechanisms. Emu oil treatment caused a change in the slope of the right contour of amide I band of the protein spectral range. This was also clear in the second derivative spectra where the emergence of a new shoulder at higher frequency was evident, suggesting disorganization of keratin α-helix structure. This effect could be a result of disruption of some hydrogen bonds in which amide C=O and N-H groups of keratin are involved. The low intensity of the emerged shoulder is also in agreement with formation of weaker hydrogen bonds. IPM did not affect the protein component. No conclusions regarding the effect of penetration enhancers on the SC lipids were obtained. This was due to the overlap of the endogenous (skin) and exogenous (oil) CH stretching and scissoring frequencies. The SC carbonyl stretching peak disappeared as a result of IPM treatment which may reflect some degree of lipid extraction.

Keywords

Emu oil, penetration enhancement, stratum corneum, keratin structure, lipid conformation, IR microscopy, chemical mapping.

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ACCEPTED MANUSCRIPT 1 Introduction Transdermal drug delivery is the administration of a therapeutic agent through intact skin for systemic effect. The transdermal route now ranks with oral treatment as the most successful innovative research area in drug delivery [1]. Many advantages are offered by this route of drug delivery compared to conventional administration. These include:

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avoidance of peak and valley levels in the serum, avoidance of first-pass metabolism,

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relatively low skin metabolism, the need of less frequent dosing regimens due to sustainability of zero-order drug delivery, occurrence of less inter-subject variability [2],

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ability to discontinue administration by removal of the system [3], the accessibility of the skin, a relatively large surface area available for absorption in addition to the fact that it

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is non-invasive which allows for more patient compliance [4].

The stratum corneum (SC) is the outermost10-20 µm thick layer of the mammalian skin

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[5] and it presents the primary barrier to the transdermal delivery of drugs [6] due to its unique structure and organization. According to the classic brick and mortar model, the

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SC is composed of keratin-rich corneocytes embedded in a lipid intercellular matrix (ceramides, cholesterol and free fatty acids) organized as lamellar lipid layers [7, 8].

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Thus, the lipids are the mortar and the elongated corneocytes are the bricks. Indeed, limited number of drugs are administered by transdermal route due to the efficient barrier

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provided by the SC, hence transdermal delivery research has been focused to overcome this barrier and increase the transport of drugs through the SC.

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Among various approaches to overcome the SC barrier properties, chemical penetration enhancers are still the most widely used. These are chemicals that interact with skin

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constituents to promote drug flux [9]. Known chemical penetration enhancers include: water, sulphoxides, azone, pyrrolidones, alcohols, fatty alcohols and glycols, fatty acids, surfactants, urea, essential oils, terpenes and terpenoids, phospholipids and solvents at high concentrations [10]. The fact that no single penetration enhancer is suitable for broad classes of drugs, highlights the need for the discovery of a variety of safe, effective, possibly "broad spectrum" skin penetration enhancers. Hence, the search for penetration enhancers is continued in an attempt to increase the number of therapeutic agents that can be given transdermally. To allow percutaneous penetration of drugs, chemical enhancers change the barrier resistance of the SC through different mechanisms. The lipid-protein2

ACCEPTED MANUSCRIPT partitioning (LPP) theory proposed by Barry is widely accepted and summarizes possible penetration enhancer-SC interactions that imply three main mechanisms of enhancement: (i) interactions with the intercellular lipids, (ii) interactions with the intracellular keratin and (iii) the penetration of high amounts of enhancers or so-called cosolvents into the SC with a resulting improved dissolving capacity of the barrier for drugs and/or co-enhancers [11-13].

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Emu oil is obtained from subcutaneous or retroperitoneal adipose tissue of the Emu [14]

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and considered a valuable product in many branches of industry, cosmetics and nutrition

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[15]. It also has some therapeutic applications [16] as anti-inflammatory, antiviral, and antibacterial as well as being able to aid in the healing of wounds and burns [14]. It has

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also been reported that Emu oil possesses antioxidant, radical scavenging [17] and analgesic properties [16]. Emu oil is predominantly composed of fatty acids. The average

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lipid content of the oil rendered from subcutaneous or retroperitoneal adipose tissue is 98-99% with oleic acid being the major unsaturated component at 43-46 weight

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percentage (wt%), and the major saturated acid component is palmitic acid at 18-19 wt%. The concentration of linoleic acid, the major polyunsaturated fatty acid is 22-23 wt %

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[14]. It has been proposed that preparations of Emu oil contain skin permeationenhancing factors [18] and some transdermal preparations were prepared in which Emu

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oil is incorporated as a penetration enhancer such as insulin Emulgel formulation [19]. Nevertheless, there is no attempt to investigate the mechanism by which it exhibits this

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effect.

The current study compromises the first attempt to address the possible penetration

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enhancement mechanism(s) of Emu oil. Up to our knowledge; no data pertaining to this issue has been published. It is well known that the interaction of chemical enhancers with the lipids and proteins of the SC can be elucidated by FT-IR spectroscopy and thermal analysis of SC [20, 21]. The output of this study demonstrated in a clear illustration that FT- IR microspectroscopic analysis is a powerful tool in the investigation of the three main penetration enhancement mechanisms proposed by LPP theory. Accordingly, the specific objectives addressed here are (i) Visualization of the penetration of the investigated oil through human skin layers and (ii) Investigation of the oil effect on the SC structure by monitoring changes in the SC FTIR spectroscopic behavior 3

ACCEPTED MANUSCRIPT corresponding to lipids and proteins. The effect of Emu oil is also compared to that of isopropyl myristate (IPM), which is a known and very frequently used penetration enhancer. Principal component analysis (PCA), as a type of multivariate statistical analysis, is used to accomplish the second objective. Recently, this statistical approach has been mainly used in IR microspectroscopy for pathological and biomedical investigation purposes of different types of cells and tissues. We believe that this study

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could be the first illustration for application of PCA in IR microspectroscopic

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investigation of penetration enhancers’ effect on the SC biochemical structure and,

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subsequently, on its barrier function.

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2 Materials and methods 2.1 Preparation of human skin sheets

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Human skin from abdominal plastic surgery was obtained from local hospitals immediately after the surgery and the approval of the donors was granted after the

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research objective was explained to them. The skin was immediately defatted using a scalpel, cleaned by tapping with dry wipes and neatly placed on paperboard that was

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wrapped with aluminium foil so that the skin surface is facing upward. The skin was then covered with aluminium foil, kept in zipper plastic bags and subsequently stored at -70°C

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for a maximum period of 6 months. All the experiments were performed on skin samples

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obtained from the same donor.

2.2 FTIR sample preparation

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To prepare the perpendicular skin sections, three circular discs of whole skin 12 mm in diameter were punched out from the frozen skin sheets and left for few minutes to thaw. The first disc was treated with Emu oil (EMU SPIRIT, Australia) and the second with IPM (Supplico Chemicals, Italy) by swabbing the skin surface three times with a cotton swab impregnated with the corresponding oil previously equilibrated at 32 °C. The third disc was left untreated to serve as a control. All the discs were incubated at 32 °C for 24 hr. The excess oil on the skin surface was then removed by pressing the skin surface gently with a filter paper once followed by washing the surface three times with a cotton swab impregnated with distilled water. The skin discs were immediately cryomicrotomed 4

ACCEPTED MANUSCRIPT perpendicular to the skin surface at a thickness of 5µm (Microtome Cryostat microm HM 525, Thermo Fisher scientific USA) and mounted on ZnSe windows (Crystran Ltd, UK). The skin sections were examined using the IR microscope (Thermo Fisher scientific, USA) within not more than one hour after their preparation. For the preparation of isolated SC samples, a circular disc of 12 mm in diameter was punched out from the frozen human skin sheets, left for few minutes to thaw, tape stripped five times to remove

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the sebaceous lipids after which the SC was isolated by incubating the skin disc in 1%

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w/v trypsin (SIGMA-ALDRICH, USA) in distilled water at 32 °C for 24 hr till the

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separation of the SC is observed. The isolated SC was then washed from trypsin by soaking for 2 hr in two consecutive 20 mL portions of distilled water and dried overnight

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in a sealed desiccator. At the time of the experiment, the obtained SC was divided into three pieces. The first piece was treated with Emu oil and the second with IPM by

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swabbing the upper SC surface three times with a cotton swab impregnated with the corresponding oil previously equilibrated at 32 °C. The third piece was left untreated to

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serve as a control. All the SC pieces were incubated at 32 °C for 24 hr. The excess oil from both sides of the SC was then removed by pressing the SC surface gently with a

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filter paper once followed by washing the surface three times with a cotton swab impregnated with distilled water. The control piece was washed similarly. The pieces

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were then mounted on CaF2 windows (Crystran Ltd, UK) and allowed to dry for 30 min in a closed chamber purged with compressed dry air before being immediately analyzed

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by the IR microscope.

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2.3 FTIR microspectroscopy Fourier transform infrared microspectroscopy measurements were performed at EMIRA laboratory of SESAME synchrotron (Jordan). The internal (Globar) source was used for these experiments. The ThermoNicolet 8700 spectrometer coupled with a continuum infrared microscope were used for the measurements. This microscope is equipped with a liquid nitrogen-cooled mercury cadmium telluride detector. The infrared maps from the control and Emu oil-treated sections (map dimensions were 80 µm in width and 400 µm in depth starting from the upper surface of skin) were acquired using the 32X Schwarzschild objective (NA = 0.65) and matching 32x condenser. All spectra were 5

ACCEPTED MANUSCRIPT obtained in transmission mode using OMNIC 9.1software (Thermo Fisher scientific) using a double path single masking aperture size of 20* 20 μm2 at step size of 10 μm, and a spectral resolution of 4 cm-1. For each spectrum, 128 co-added scans were recorded in the mid-infrared range between 4000 and 700 cm−1. Multiple measurements of the SC thickness from each map at different locations were also obtained. The statistical significance of differences in SC thickness values was evaluated by one-way analysis of

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variance at 0.05 level of significance followed by Bonferoni multiple-comparison test

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employing SPSS Statistics 17.0 software.

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For the isolated SC samples, IR microscopic map for each sample was immediately acquired using the same system described above except that the map dimensions were

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150*150 µm2 and 256 co-added scans were recorded. In addition, one pixel spectrum for each neat oil (Emu oil and IPM), equilibrated at 32 °C and swabbed over the windows,

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was obtained using the same system described above.

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2.4 FTIR data treatment and statistical analysis

Concerning the perpendicular skin sections, multiple line maps across the map length of

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the control section were acquired. Chemical image for the Emu oil distribution, based on oil-characteristic peak area, for Emu oil-treated section map was generated. On the other

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hand, the IR maps obtained for the isolated SC (control, Emu oil-treated and IPM-treated samples) were split to yield a data set corresponding to each sample. Each data set

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contained 225 spectra; all of them were of excellent quality and were included in the analysis (The noise level, represented as RMS value, is 0.001calculated in the 2000-2200

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cm-1 spectral range). The resultant raw spectra of all data sets were preprocessed using the Unscrambler 10.3 software (CAMO software) according to the following steps: three ranges of interest in the spectral domain corresponding to lipid CH stretching, lipid CH2 scissoring and protein amide I and II vibrations were identified. Savitsky-Golay second derivatives of all the spectra were then calculated using third polynomial order and 7 smoothening points. Each defined range was then separately range normalized. PCA was performed on each of the predefined spectral ranges in all data sets and the results were represented in the form of score plots. The normalized reduced average spectrum and its corresponding Savitsky-Golay second derivative (third polynomial order and 7 6

ACCEPTED MANUSCRIPT smoothening points) for each data set, in each corresponding spectral range, were obtained. Whenever the PCA captured a variation in the data sets in the defined spectral ranges, the corresponding loadings plot, the average and/or second derivative spectra were carefully examined to find the subtle spectral differences responsible for this variation.

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3 Results and discussion 3.1 The effect of the oil on the SC thickness

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A prerequisite for the interaction of a penetration enhancer with the SC is that the enhancer must be taken up by the SC. To evaluate this, the SC thickness from multiple

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locations of the control, Emu oil-treated and IPM treated perpendicular skin sections was measured. The average values of SC thickness after 24 hr incubation at 32 ˚C are shown

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in table 1. Notably, both Emu oil and IPM treated samples demonstrated higher values compared to the control (P<0.05 in all cases) reflecting the incorporation of both oils

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within the SC layer. Accordingly, both of the investigated penetration enhancers were

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considered to be incorporated within the SC.

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Table 1: The SC thickness (µm) after 24 hr incubation at 32 ˚C. SC Sample Average SC thickness 17.6±3.9

Emu oil-treated

31.0±3.1

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Untreated

24.6±6.9

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IPM-treated

3.2 FTIR analysis 3.2.1 Determination of the penetration depth and the spatial distribution of the oil through the skin layers As expected, and consistent with being composed of 99 % of fatty acids, the measured IR spectrum of Emu oil (Figure 1) exhibited peaks related to vibration of lipids. Notable peaks were identified at 3006, 2956, 2925, 2873, 2854, and 1746 cm-1 corresponding to =C–H stretching, asymmetric CH3 stretching, asymmetric CH2 stretching, symmetric 7

ACCEPTED MANUSCRIPT CH3 stretching, symmetric CH2 stretching and C=O (carbonyl group) stretching vibrations respectively. These vibrations arise from the various hydrocarbon moieties of the lipid components, mostly being fatty acids. CH2 Asym

CH2 Sym

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CH3 Asym

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CH3 Sym

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Fig. 1: Representative raw spectrum of neat Emu oil and expanded part of the spectrum showing the peak at 3006 cm-1 where the vertical line is drawn. In IR microspectroscopy, the spectral features are employed as a native intrinsic contrast mechanism [22] thus in order to monitor the permeation of an exogenous substance

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through skin layers, it is mandatory to possess a characteristic band for this substance that does not overlap with the IR features arising from endogenous skin components. By

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virtue of the presence of a specific IR band, the permeation of a number of compounds, mostly UV filters and preservatives, at different human skin depths was examined using

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synchrotron-based IR miscrospectroscopy [23]. In some cases, a characteristic IR feature for the exogenous substance can be created by deuteration of its acyl chains, the reader is referred to references [24, 25] [26] for further details and specific examples. As mentioned earlier, the IR features of the currently investigated enhancer are related to its fatty acids components so, at first thought, a characteristic IR feature that does not overlap with those of endogenous skin lipids is not expected to be present. Fortunately, taking into consideration that Emu oil contains 43-46 weight % oleic acid and 22-23 weight% linoleic acid [14] which are unsaturated at C9 and at (C9 and C12) respectively,

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ACCEPTED MANUSCRIPT the band located at 3006 cm-1 (Figure 1) corresponding to =C-H stretching can be useful provided it is absent from the skin endogenous IR spectra. A study conducted by IR spectroscopy combined with sequential tape stripping has pointed to the presence of a band arising from =C–H stretch (at ∼3010 cm−1) in the top few spectra of pig SC. Each spectrum was acquired after two tape strips were applied and removed from the SC; therefore each is approximately 1 μm deeper into the SC. This finding was attributed

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either to sebum and/or environmental contamination on the skin, since unsaturated chains

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Deeper into skin

Deeper into skin

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are not present in significant amounts in the SC [25].

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Fig. 2: A) Line map of 400 µm depth extracted from the area map of the untreated skin section and B) Corresponding extracted spectra from top to bottom showing the absence of the peak at 3006 cm-1 outlined by the black rectangular. All the spectra are displayed, and offset for clarity. To confirm the absence of the band at 3006 cm−1 from the skin spectra, multiple line maps were extracted from the control skin section. Complete absence of this band at all the investigated levels (up to 400 µm depth starting from the upper skin surface) was evident. One of the extracted line maps and its corresponding spectra are shown in figure 2. The top spectrum corresponds to the SC and is measured from a pixel of 20*20 µm 2 9

ACCEPTED MANUSCRIPT thus averaged over the SC thickness, and accordingly the aforementioned band, which might be present in the first few micrometers of the SC, is not exhibited. In contrast to the control skin section, Emu oil-treated skin section demonstrated the appearance of the band at 3006 cm-1 (Figure 3). This has enabled visualization of the spatial distribution and determination of the penetration depth of the corresponding

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components through the skin layers (Figure 4).

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Deeper into skin

Fig. 3: A) Line map of 400 µm depth extracted from the area map of the Emu oil-treated skin section and B) Corresponding representative extracted spectra at selective locations from top to bottom showing the presence of the Emu oil characteristic peak at 3006 cm -1 outlined by the black rectangular. The spectra are offset for clarity. 10

ACCEPTED MANUSCRIPT Again, emphasis should be made here that this IR feature belongs to the unsaturated components of Emu oil thus any obtained results actually refer to these components and cannot be inferred to Emu oil as a whole mixture. Indeed, separation and preferential diffusion of the oil components through the skin layers is expected to occur due to differences in the molecular features of the various components such as molecular

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Fig. 4: A) Superimposition of the chemical map, constructed based on the peak area of the characteristic peak of Emu oil at 3006 cm-1, with the corresponding optical image of Emu oil treated perpendicular skin section (the map depth is 400 µm), and B) The intensity of the mentioned peak as a function of the depth (in µm), constructed based on extracted line spectra from the same skin section map. The highest peak intensity is at a depth of about 270 µm

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weight, chain length and molecular geometry.

Referring to figure 4, the penetration of the unsaturated components through the SC and the epidermis tissue is evident. The maximum accumulation of these components at the boundary located at about 270 µm, at the highest peak intensity, is also clear. Two conclusions can be made here; firstly, Emu oil or at least its unsaturated components were taken up by the SC and this is further supported by the increase in the range of the 11

ACCEPTED MANUSCRIPT SC thickness as a result of the oil treatment compared to the control as described earlier. Secondly, the unsaturated components have been diffused across the epidermis, which is typically up to 150 µm thick [25], down to 270 µm. Although this implies accumulation in the upper region of the dermis, which is typically 600–3000 μm thick [25], possible swelling of the epidermis as a result of the oil treatment cannot be ignored. Whether the accumulation boundary is located in the dermis or at the end of a swelled epidermis, it is

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nature thus lower amounts of the oily components been diffused.

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reasonable to conclude that the skin layers below this boundary are more aqueous in

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According to the LPP theory, the penetration of high amounts of enhancers into the SC with improved dissolving capacity for drug is one of the possible mechanisms for the

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chemical penetration enhancement [12, 13]. The above mentioned findings support that

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this can be a possible mechanism for the proposed oil enhancement effect.

3.2.2 Evaluation of the impact of Emu oil on the SC lipid and protein structures in

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comparison with IPM

Many features in the IR spectra of SC arise from lipid or protein molecular vibrations.

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The sensitivity of these features to the structural transitions in the SC is established [2735]; thus providing useful information related to the penetration enhancement

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mechanisms. Accordingly, IR spectroscopy has been a corner stone technique to study the SC barrier [36]. Nevertheless, using the traditional non-spatially resolved IR

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spectroscopy, a single spectrum is collected from each measurement, and so one spectrum for each sample (penetration enhancer-treated and control samples) is obtained.

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After proper treatment of the obtained spectra, spectral comparison is then made to detect variations in peak shapes, frequency and intensity. In some cases, in order to increase the accuracy of data analysis, three measurements are made for each experiment to allow for univariate statistical analysis of the results. These types of analysis are represented as peak area and frequency values ± their corresponding standard deviations. At this point, the advantage of the IR microspectroscopy is obvious, through combining biochemical and spatial information; large number of spectra (hundreds or even thousands) from each measurement of individual small areas on the sample is collected. Subsequently, better

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ACCEPTED MANUSCRIPT characterization of the biochemical composition of samples can be obtained and more accurate comparisons can be made through the use of multivariate statistical analysis.

In this study, IR maps composed from 225 pixels were measured and accordingly 225 IR spectra were obtained for each isolated SC sample (control, Emu oil-treated and IPMtreated samples). The average raw spectra obtained from the control, Emu oil-treated and

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IPM-treated isolated SC samples are depicted in figure 5 showing the IR spectral regions

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discussed through this research.

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Fig. 5: Unit vector normalized average spectra of control (blue), Emu oil-treated (red) and IPM-treated (green) isolated SC samples, showing A) lipid CH stretching, B) lipid carbonyl stretching, C) protein amide I and II peaks, and D) lipid CH2 scissoring regions. The spectra are offset for clarity.

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3.2.2.1 SC protein structure The main IR features of SC that correspond to its protein content are the following: (i) the band at around 1650 cm-1 is termed amide I band and arises mainly from C=O stretching of amide groups of the peptide backbone in proteins and (ii) the second band at around 1550 cm-1 is termed amide II band and arises mainly from N–H bending and C-N stretching vibrations of amide groups of the peptide backbone in proteins. Both bands, particularly the amide I band, are sensitive to protein secondary structure [31-35]. The untreated SC sample exhibited the amide I and amide II peaks at 1655 and 1547 cm-1 respectively (Figure 5 region C). Expanded spectrum showing these peaks is presented in 13

ACCEPTED MANUSCRIPT figure 7A. In human SC, the Amide I band contour arises from keratin with a minor contribution from ceramides and other proteins of the SC and it is sensitive to keratin structural changes [36]. Typically, a shoulder at 1634cm-1 is assigned to β-sheet secondary structures of proteins, while the band at 1655 cm-1 is assigned to α-helix secondary structure [24], thus amide I band of the SC at the latter frequency reflects the predominantly helical secondary structure of keratin [37]. In the untreated SC sample, the

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amide I band was located at 1655 cm-1 which confirms the helical structure of keratin.

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If any of the tested penetration enhancers has the ability to modify keratin structure, it

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would be absolutely evident by changes in the amide I band contour or frequency. Nevertheless, alteration in the secondary structure of keratin is difficult to be manifested

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by analysis the raw spectra; however, it can be deduced by thorough analysis of the second derivative spectra of this band.

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The results of PCA analysis are represented in the form of score plot and corresponding loadings. Each point (score) in the score plot corresponds to one spectrum, thus each of

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the three groups (control, Emu oil treated and IPM treated samples) constitutes 225 points. The score plot corresponding to amide I and amide II region is shown in figure

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6A. Clustering of each of the three groups is clear and 77% of the variability is explained by principal component 1 (PC1). Emu oil-treated group and part of the control group are

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located on the positive side of PC1 while IPM treated group and most of the control group are located in the negative side. This implies that compared to IPM-treated group,

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Emu oil-treated group is spectrally more different from the control; also part of the control group (on the positive side) is spectrally similar to Emu-oil treated group. The

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latter finding suggests that some spectral changes brought about by Emu oil may already exist in some areas in the control sample. IPM-treated group is not well separated from the control by PC1 while a little separation by PC2, explaining only 5% of the variance, is noticed. It can be concluded here that, unlike Emu oil, IPM does not induce significant changes on the protein spectral region. In PCA, the spectral origins of the variation which differentiate the data groups according to the wavenumbers are represented in the loadings [38-40] with positive and negative loadings correlate to the positive and negative scores respectively. The loadings are represented in the same shape of data to which PCA was applied, in this case, second derivative spectrum. Nevertheless, 14

ACCEPTED MANUSCRIPT interpretation of loadings spectrum is not an easy task due to its complexity and the fact that it accounts for both intra and intersample variation. For this reason, the loadings here are compared with the samples average spectra to define those related to intersample differences, specifically those related to SC structural alterations which can be linked to

∼1654 cm-1

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possible keratin-penetration enhancer interactions.

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Fig. 6: A) Score plot and B) corresponding PC1 and PC2 loadings of protein region. Score plot color code: Control (blue), Emu oil-treated (red) and IPM-treated (green) isolated SC samples. The PCA was performed in the spectral range of 1700-1848 cm-1. 15

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Before attributing the observed spectral changes to possible keratin-Emu oil interactions, it should be confirmed that these changes are not due to interference arising from exogenous oil peaks with those of endogenous protein. The absence of such interference was confirmed since no oil-related peaks are exhibited in the protein spectral regions both in the raw and second derivative spectra (Figures. 1&7). Looking at the loadings of PC1

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in figure 6B, the positive loadings, which correlate to Emu oil-treated sample, show a

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peak frequency that exactly correlates with emergence of a shoulder in the average

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second derivative spectrum of Emu oil-treated sample (Figure 7B). Second derivative spectrum analysis is used to enhance the resolution of overlapping IR bands and it is one

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of the most popular approaches used for analysis of amide I peak of proteins in solution [41]. This approach allows the identification of various secondary structures present in

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the protein [33] since most of the peak positions can be easily found [41]. The formation of β-sheet secondary structures of keratin is typically associated with appearance of

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another peak at a lower frequency, on the contour of original amide I peak. However, the above described changes are not consistent with this, but they greatly suggest the

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disorganization of the α-helix structure of keratin brought about by the oil treatment. This effect could be a result of disruption of some hydrogen bonds in which amide C=O and

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N-H groups are involved. This explanation is reasonable since the weaker the hydrogen bond involving the amide C=O, the lower the electron density in the C=O group and the

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higher frequency the amide I absorption appears [32]. A shift of the amide I band to a slightly higher frequency has been described as a result of a decrease in hydrogen bond

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strength of the peptide bond brought about by an increase in temperature [36]. The low intensity of the emerged shoulder in Emu oil treated sample is also in agreement with formation of weaker hydrogen bonds, as a result of decreased polarization of the amide C=O. Typically, penetration enhancers that exhibit an impact on keratin do so by either denaturation or secondary structure modification, and it is assumed that the latter is reflected by transition of some of the protein content to β-sheets. Emu oil does not seem to act in exactly the same mechanism because there was no evidence of β-sheet

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Nevertheless, disorganization of the α-helical structure of keratin could

possibly play a role, at least partially, in the enhancement mechanism of Emu oil. Concerning IPM-treated sample, no signs for changes in the amide I peak contour or frequency that can be correlated to its penetration enhancement effect were observed

Amide I

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Fig. 7: A) Unit vector normalized average raw spectra of amide I and II peaks for control , Emu oil-treated and IPM-treated isolated SC samples showing a change in the slope of the left part of amide I peak in Emu oil-treated sample, B) Range normalized average second derivative spectra of amide I and II peaks of control sample, Emu oil-treated sample, IPM-treated sample, neat Emu oil and neat IPM showing a new shoulder at the left side of the amide I peak in Emu oil-treated sample. Color code: Control (blue), Emu oil-treated (red) and IPM-treated (green) isolated SC samples. Neat Emu oil (black) and Neat IPM (purple). The spectra are offset for clarity. 3.2.2.2 SC lipid structure

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Lipid components of the SC exhibit four fine peaks at around 2955, 2920, 2870 and 2850 cm-1 which are assigned to asymmetric CH3 stretching, asymmetric CH2 stretching, symmetric CH3 stretching and symmetric CH2 stretching respectively [27]. These bands arise from the long alkyl chains of the major components of the SC lipids: ceramides, cholesterol and fatty acids. [24, 27, 42, 43] The CH2 stretching frequencies reflect both lipid chain conformational order and packing geometry [27, 28]. Concerning chain conformation, all-trans conformation is characterized by lower wave numbers while introduction of gauche conformers will cause a shift to higher values [4446]. The shift to higher frequencies is associated with higher fluidity, conformational 18

ACCEPTED MANUSCRIPT freedom and flexibility of the alkyl chains as a result of heating [26, 27]. It has been reported that some enhancers may fluidize the SC lipids, as suggested from the shift of CH stretching peaks to a higher wave number and the increase in peak width [27]. The symmetric CH2 stretching band (2850 cm-1) is specific of lipids organization and correlated to cutaneous barrier status and accordingly helps to follow penetration enhancer effect on skin lipids [47, 48].

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On the other hand, CH2 scissoring vibrations of acyl chains of lipids appear in the region

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1480–1460 cm-1 [29], and provide information on the acyl chain packing of the SC lipids

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[25]. The band at ∼1468 cm-1 represents hexagonal packing of the lipids, whereas orthorombic chain packing is indicated by two components at ∼1472 cm-1 and ∼1464 cm[49]. These characteristics of CH2 scissoring region were identified in the second-

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derivative of the spectra collected from human SC [29]. Thermotropic transitions of the

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human SC shows that as the temperature increases, the two orthorhombic-indicating components approach each other to the point where the splitting collapses leaving only

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the hexagonal characteristic central component [25]. In this work, the PCA results of the lipid spectral regions (CH stretching and scissoring)

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are distinctive for both Emu oil- and IPM-treated samples and the clustering patterns imply that, compared to the control, IPM-treated sample has more spectral differences

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than Emu oil-treated sample (Figure 8). The loadings plot of CH stretching (not represented) show multiple frequencies responsible for the variation. The comparison of

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the symmetric and asymmetric CH2 stretching peaks in the average raw spectra shows a shift to a higher frequency in both of the treated samples (Table 2). In all cases, the shifts

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were of higher magnitude in the IPM treated sample.

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Fig. 8: Score plots of A) CH stretching and B) scissoring of SC lipids. Color code: Control (blue), Emu oil-treated (red) and IPM-treated (green) isolated SC samples.

Due to the overlap of the exogenous peaks of Emu oil/IPM with that of the endogenous lipids at this spectral range, the peak frequency will be a weighted average for the groups undergoing stretching at this frequency, i.e. a contribution of both endogenous (SC) and 20

ACCEPTED MANUSCRIPT exogenous (Emu oil/IPM) groups. It is difficult to decide whether the observed shifts are simply due to averaging of the endogenous and exogenous peaks or a combination of this effect with SC lipid fluidization effect of Emu oil/IPM.

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Table 2: The symmetric and asymmetric CH2 stretching peaks frequencies (cm-1) obtained from unit vector normalized average raw spectra of the SC samples and neat penetration enhancers. CH2 asymmetric stretching CH2 symmetric stretching 2918.51

2850.15

Emu oil treated SC

2920.00

2850.82

IPM treated SC

2923.03

Neat Emu oil

2924.75

Neat IPM

2924.41

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Untreated SC

2852.34

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2853.75

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2854.59

Concerning the second derivative spectrum of the CH2 scissoring region (Figure 9), the

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control sample showed two components at about 1471.5 and 1465 cm-1 corresponding to orthorhombic lipid chain packing. These two components are not well resolved and they

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do not reach the base line from the right and left contours respectively. Instead, their contours merge at 1467.7 cm-1. This overlap could be obscuring the central peak of the

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hexagonal packing component. On the other hand, CH3 bending modes associated mostly with keratin also occur in the range of the CH2 scissoring region [29] which could be

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another reason for the obscured hexagonal packing component. The identification of the scissoring peaks and splitting pattern is usually accomplished by procedures based on

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difference spectra [50-52], spectrum deconvolution [53], and curve fitting [52, 54]. Another approach utilizing the width of the second derivative spectra in the region ~1475–1460 cm-1 was also described [29]. In both treated samples, the presence of the orthorhombic packing indicators is less obvious while the peak indicating the hexagonal packing is intensified and these effects are more prominent in IPM treated sample. On the other hand, both neat Emu oil and IPM have clear peak at about 1467 cm-1 (not shown), indicating their own hexagonal packing. Therefore, as with CH2 stretching, it is not clear if this effect is due to overlap of the endogenous and exogenous lipid CH2 peaks, or partially contributed from orthorhombic-hexagonal transition as a result of the treatment. 21

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Fig. 9: Range normalized average second derivative spectra of CH scissoring for control (blue), Emu oil-treated (red) and IPM-treated (green) isolated SC samples. The spectra are offset for clarity.

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The literature includes several studies in which IR spectral behavior was used to investigate the effect of some penetration enhancers on the SC lipids. In some of these studies, the observed signs of lipid disordering were inferred to the interaction of the

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enhancer (oleic acid [29] and IPM [55]) with the lipids ignoring the fact that these

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enhancers have IR features that are similar to the endogenous lipid components .On the other hand, in some studies deuteration of the enhancer acyl chains was performed (oleic acid [26], palmitic and myristic acids [24]) since, as mentioned earlier, CD2 stretching

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vibrations are located in a range where endogenous IR features are not exhibited (20002200 cm-1) and far away from CH2 stretching vibrations (2800-3000 cm-1) [24, 25], thus it was possible to independently monitor the phase behavior of both endogenous lipids and exogenous enhancer. The use of perdeuterated enhancer seems to be attractive, but in the case of Emu oil this approach is not feasible since it is obtained from natural origin and composed of a mixture of compounds. The carbonyl group (C=O) stretching vibration is another IR feature of lipids which generates a band near 1740 cm-1 [35]. Since this band arises from the head groups of lipid

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ACCEPTED MANUSCRIPT acyl chains, it is not related to chain conformation or packing geometry as with the previously discussed features. In the current study, the treated samples demonstrated obvious changes in the mentioned band which cannot be neglected (Figure 10). Emu oiltreated sample exhibited the peak at (1745.3 cm-1) which is intermediate between those of control SC (1744 cm-1) and neat Emu oil (1746 cm-1) indicating a contribution from both. The appearance of a new shoulder at lower frequency (1728 cm-1) in the same sample is

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not fully understood, it cannot be ascertained if it implies a limited interaction with the

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polar heads of the SC lipid chains which is typically suggested as a penetration

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enhancement mechanism for hydrophilic enhancers while Emu oil is a hydrophobic one. An interesting finding is the disappearance of the original SC lipids peak (1744 cm-1) in

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IPM-treated sample accompanied by appearance of a high intensity peak (1735.6 cm-1) at the same location of neat IPM (1735.6 cm-1) and this could be a sign of some endogenous

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lipid extraction by IPM. In the control SC the intensity of the lipid carbonyl peak is originally lower than those of lipid CH stretching peaks accordingly, disappearance of the

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carbonyl peak is expected to be accompanied by reduction in CH stretching peaks intensity, but in the contrary the intensity of these peaks has increased due to summation

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of the remaining endogenous lipids and exogenous IPM accumulated in the SC. This

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could be a core difference in enhancement mechanism between the two enhancers.

Fig. 10: Peaks of carbonyl stretching of lipids and their corresponding frequency values for control sample (blue), Emu oil-treated sample (red), IPM-treated sample (green), neat Emu oil (black) and neat IPM (purple), showing the appearance of a new shoulder at lower frequency in Emu oil-treated sample and disappearance of the original SC lipids 23

ACCEPTED MANUSCRIPT peak in IPM-treated sample accompanied by appearance of a peak at the same location of neat IPM. The spectra are offset for clarity.

4 Conclusions IR microspectroscopy technique was demonstrated to be a useful tool for intrinsic visualization of the penetration and spatial distribution of exogenous substances through

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the layers of the skin. Equipped with multivariate analysis, this technique was also

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demonstrated to abstract valuable information concerning the barrier properties of the SC

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and possible mechanisms of penetration enhancement. According to this study, the penetration enhancement effect of Emu oil could be attributed, at least partially, to its

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ability to disorganize the keratin structure in the SC. Another possible mechanism is the enhanced partitioning of penetrants into the SC since Emu oil was proven to be taken up

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by the skin and penetrate through the epidermis layer. The effect of Emu oil on the lipid domain of the SC was not confirmed in this study. Nevertheless, this effect is highly anticipated based on the fact that Emu oil is mostly composed of fatty acids. There is a

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wide consensus in literature that fatty acids exhibit their penetration enhancement action

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by affecting SC lipids. For example, proposed modes of action of oleic acid include SC lipid fluidization [26] [56] , lipid phase separation [26],[57-59] and increased partitioning

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of the drug into the SC [60].

As confirmed by the IR microscopy study in this project, IPM did not affect the protein

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component of the SC. On the other hand, its effect on the SC lipid domain was not confirmed. Nevertheless, it has been proposed by many researchers that IPM partitions in

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the ordered lipid domains of the SC [61, 62] according to this, direct disruption of the lipid assembly or even extraction of certain SC lipids into a separate phase will occur [63]. The overall effect will be liquefaction of the SC lipids and lowering the diffusion resistance of the SC. Barry (1991) reported that the Lipid-Protein-Partitioning theory of skin penetration enhancement suggests that skin penetration enhancers usually act by one or more of three main mechanisms; they can alter the intercellular lipid or intracellular protein domains of the horny layer and they may also increase partitioning into the skin of a drug, a coenhancer, water or any combination of these [13]. Thus our findings with Emu oil as 24

ACCEPTED MANUSCRIPT penetration enhancer agrees well with LPP theory. Furthermore, to have a boarder insight on the possible enhancement mechanisms, the effect of Emu oil on the transdermal diffusion of model penetrants must be studied. The diffusion behavior of penetrants having different hydrophilicities in the presence and absence of Emu oil, in correlation with the results of the present study, could be beneficial to clarify the situation more.

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Indeed, we have performed these experiments and the results will be published later.

Acknowledgments

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We would like to thank the SESAME synchrotron for the granted beam time. We acknowledge the Richard Lounsbery Foundation, the SESAME Synchrotron, the

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University of Jordan and Scientific Research Support Fund for funding this project. We also aknowledge M.D. Ali Alkhader at Jordan University Hospital for his assistance with

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the cryomicrotoming of the skin samples.

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Graphical abstract

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A) Penetration of Emu oil through the skin layers. B) Effect of Emu oil on the protein constituent of stratum corneum (red) in comparison to untreated (blue) and isopropylmyristate treated (green) stratum corneum samples.

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The possible penetration enhancement mechanisms of Emu oil are addressed.

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The unsaturated components of Emu oil accumulate at about 270µm deep within the skin. PCA results suggest disorganization of the α-helix structure of keratin by Emu

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oil.

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