Effects of fatty acids, fatty amines and propylene glycol on rat stratum corneum lipids and proteins in vitro measured by fourier transform infrared/attenuated total reflection (FT-IR/ATR) spectroscopy.

Fourier transform infrared/attenuated total reflection (FT-IR/ATR) spectroscopy was used to examine the effect of fatty acids, fatty amines and propylene glycol (PG) on the molecular mobility of rat stratum corneum lipids and keratinized proteins, using a hydrophobic solute, indomethacin, and a polar solute, 5- and 6-carboxyfluorescein (CF). Treatment of the skin with either oleic acid or oleylamine resulted in significant CH2 C-H asymmetric stretching band shifts and broadening. The extent of spectral alteration varied with the chemical structure of the penetrant. The penetrants increased the lipophilic indomethacin flux and shortened the lag times through the skin in vitro. The plot of frequency changes vs. indomethacin flux or lag time demonstrated a linear relationship, thus indicating that spectral alteration in CH2 C-H stretching regions of stratum corneum lipids may provide a reliable index for characterizing penetrants. The data also showed that the hydrophilic group which attached to the CH2 group in the penetrant molecules did not play a part in the membrane permeability enhancing action. Oleic acid and oleylamine appeared to induce a conformational alteration of the keratinized proteins from alpha-helix to beta sheet. Such alteration was also observed with PG treatment. Accumulation of CF was significantly increased by the PG pretreatment of the skin, thus suggesting that PG-induced protein conformational changes could be related to the enhancement of CF accumulation.     

Effects of photodynamic therapy with topical application of 5-aminolevulinic acid on normal skin of hairless guinea pigs.

Photodynamic therapy (PDT) is a relatively new approach to the treatment of neoplasms which involves the use of photoactivatable compounds to selectively destroy tumors. 5-Aminolevulinic acid (ALA) is an endogenous substance which is converted to protoporphyrin IX (PpIX) in the synthetic pathway to heme. PpIX is a very effective photosensitizer. The goal of this study was to evaluate the effect of PDT using topical ALA on normal guinea pig (g.p.) skin and g.p. skin in which the stratum corneum was removed by being tape-stripped (TS). Evaluation consisted of gross examination, PpIX fluorescence detection, reflectance spectroscopy, and histology. There was no effect from the application of light or ALA alone. Normal non-TS g.p. skin treated with ALA and light was unaffected unless high light and ALA doses were used. Skin from which the stratum corneum was removed was highly sensitive to treatment with ALA and light: 24 h after treatment, the epidermis showed full thickness necrosis, followed by complete repair within 7 d. Time-dependent fluorescence excitation and emission spectra were determined to characterize the chromophore and to demonstrate a build-up of the porphyrin in the skin. These data support the view that PDT with topical ALA is a promising approach for the treatment of epidermal cutaneous disorders.     

Effects of n-alkyltrimethylammonium on skin permeation of benzoic acid through excised guinea pig dorsal skin

Effects of cetyltrimethylammonium and two other n-alkyltrimethylammoniums on the permeation of benzoic acid through excised guinea pig dorsal skin were examined. Cetyltrimethylammonium markedly increased both the flux and permeability coefficient in the concentration range of 0.5 and 5 mm. However, 50mM cetyltrimethylammonium decreased them. The presence of 2 mm cetyltrimethylammonium, which induced a maximal enhancement effect, also increased the fluxes of 4-methyl, 4-ethyl and 4-n-propyl substituents of benzoic acid, but the enhancement effects were less. Analysis of the free energy of transfer of the methylene group of the substituents from the aqueous phase to skin suggested that cetyltrimethylammonium made the skin relatively more hydrophilic. Electron spin resonance spectra analysis by a long-chain quaternary alkylammonium analog, 4-(N,N-dimethyl-N-pentadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-ox yl iodide (CAT-15), showed that the spin label was present in a nearly solid state in both excised skin and its stratum corneum. This finding suggested the high affinity of the long-chain alkylammoniums to proteins in the stratum corneum and the involvement of the interaction between the cationic surfactants and the proteins in their improvement of the hydrophilic property of the skin, as well as their marked enhancement effects on the skin permeation of relatively hydrophilic drugs.     

TRANSMISSION OF HUMAN EPIDERMIS AND STRATUM CORNEUM AS A FUNCTION OF THICKNESS IN THE ULTRAVIOLET AND VISIBLE WAVELENGTHS

Abstract The dependence of radiation transmission on sample thickness was studied in isolated samples of human stratum corneum and full-thickness epidermis. The investigation also included samples of skin repeatedly exposed to UV-B. Transmission was measured in the ultraviolet and in the visible from 248–546 nm. Two methods, one microscopic and the other mechanical, were used to measure thickness. There was a good correlation between the results.
The dependence of transmission on thickness in these samples could be described satisfactorily by an exponential function, implying that the Lambert-Beer law is approximately valid. Thus, a single parameter, such as the half-value layer ( d _½), is sufficient to characterize absorption in the skin samples.
Water content of the isolated stratum corneum was influenced by maintenance conditions: samples floating on water containing a small amount of NaCl were more hydrated than samples floating on a more concentrated salt solution, or stored in air. Changes in water content of the samples resulted in changes of thickness and, to a lesser extent, of transmission. Approximate in vivo values of d _½ were computed after estimating the in vivo water content of stratum corneum.
Differences found in the shape of the transmission spectra of stratum corneum and full-thickness epidermis may reflect differences in chemical composition. The influence of wetting of the skin on its sensitivity to sunlight is explained in a new way.     

Analysis of electron spin resonance spectra of alkyl spin labels in excised guinea pig dorsal skin, its stratum corneum, delipidized skin and stratum corneum model lipid liposomes

The electron spin resonance (ESR) spectra of alkyl spin labels were observed in the excised guinea pig dorsal skin, its stratum corneum, delipidized skin and stratum corneum model lipid liposomes. The spectrum of 5-doxylstearic acid (5-NS) in the stratum corneum and order parameter obtained from the spectrum, indicated that the spin label was present in highly ordered lipid lamella. On the other hand, the spectrum of methyl ester of 5-NS (5-NMS) and its apparent rotational correlation time calculated from the spectrum, showed only a weakly immobilized component in the stratum corneum as well as in the whole excised skin. The ester spin label seemed to be scarcely present in the rigid lipid lamella, but mainly in the relatively fluid environment. On the other hand, cationic alkyl spin labels showed quite different spectra depending on their alkyl chain lengths. Long-chain 4-(N,N-dimethyl-N,-pentadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl (CAT-15) seemed to be present in the protein region of the stratum corneum as we recently reported, whereas hydrophilic quaternary ammonium spin label 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-oxyl (CAT-1) seemed to be present in the bulk water of the skin, even in delipidized skin. These findings indicated that the different interaction and different localization of the alkyl spin labels depended on their electronic charge as well as their alkyl chain lengths.     

Drug permeation through skin from matrix-type drug delivery systems

Nitroglycerin permeation through hairless mouse skin from various matrix-type transdermal delivery systems has been explained fairly well on the basis of a two-layer skin model. The diffusivity and solubility of the drug in the stratum corneum and in the viable skin have been calculated. The diffusivity in the stratum corneum was found to be affected by the type of delivery system, while that in the viable skin was hardly influenced by the delivery system. The loading dose, diffusivity and solubility of the drug in the delivery system were found to be the important design parameters for skin-control transdermal drug delivery and, therefore, the transdermal delivery system has to be optimized by controlling these three parameters to achieve a maximum rate of skin permeation with minimum drug loading dose.     

FORWARD SCATTERING PROPERTIES OF HUMAN EPIDERMAL LAYERS

Abstract From an optical point of view the outermost skin layers contain numerous structures by which penetrating radiation may be scattered as well as absorbed. The nature and strength of this scattering may strongly influence the extent of penetration. We illuminated samples of stratum corneum and full-thickness epidermis with collimated radiation and measured the angular intensity distribution of the transmitted radiation; we did this in the ultraviolet for several angles of incidence, and in the visible for perpendicular incidence only. Skin samples were obtained from the skin of the lower back and upper leg of Caucasian volunteers. Epidermis and subsequently stratum corneum were separated by chemical methods. In the case of stratum corneum, the angular intensity distribution of the transmitted radiation peaks strongly at all wavelengths, in approximately the direction of the incident radiation, that has been refracted at the surface of the sample. With full-thickness epidermis, the distribution of the transmitted radiation also peaks, though less strongly than with stratum corneum. These features suggest a forward oriented scattering mechanism. Both in the case of stratum corneum and full-thickness epidermis, the angular distribution flattens towards the shorter wavelengths and with increasing thickness. The wavelength dependence suggests that both scattering and absorption increase towards the shorter wavelengths. The existence of a thickness dependence indicates that volume scattering occurs. Hydration of stratum corneum is found to influence its scattering properties. Dry samples scatter less than hydrated samples. The consequences of our findings for modelling skin optics are briefly discussed.     

Enantiomeric difference in percutaneous penetration of propranolol through rat excised skin.

The percutaneous penetration of R-(+)- and S-(-)-propranolol (PL) through rat excised skin was investigated in vitro. The flux of S-(-)-PL after application to normal skin was high compared with that of R-(+)-PL. On the other hand, in damaged rat skin, the flux of R-(+)-PL was almost equivalent to that of S-(-)-PL. It is suggested that there is an enantiomeric difference between S-(-)- and R-(+)-PL in terms of penetration through rat stratum corneum.     

Topical bioadhesive patch systems enhance selectivity of protoporphyrin IX accumulation

In clinical 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) of skin tumors it is desirable to develop vehicles that minimize the penetration of ALA through normal stratum corneum and maximize it through the compromised stratum corneum of the tumors to improve tumor selectivity. We have designed a bioadhesive patch, which may be able to achieve this aim. It induces levels of protoporphyrin IX (PpIX) in skin overlying tumors similar to those induced by the proprietary cream (Porphin) but at the same time induces less PpIX to form in normal skin and at distant sites. The mechanisms of action of the patch, as compared with that of the cream, were studied by means of Cuprophan barriers that mimic compromised tumor stratum corneum and in a mouse model with transplanted tumors.     

Penetration and growth of DPPC/DHPC bicelles inside the stratum corneum of the skin

The effect of dipalmitoyl phosphatidylcholine (DPPC)/dihexanoyl phosphatidylcholine (DHPC) bicelles on the microstructure of pig stratum corneum (SC) in vitro was evaluated. The physicochemical characterization of these nanoaggregates revealed small disks with diameters around 15 nm and a thickness of 5.4 nm. Upon dilution, the bicelles grow and transform into vesicles. Cryogenic scanning electron microscopy (cryo-SEM) images of the SC pieces treated with this system showed vesicles of about 200 nm and lamellar-like structures in the intercellular lipid areas. These vesicles probably resulted from the growth and molecular rearrangement of the DPPC/DHPC bicelles after penetrating the SC. The presence of lamellar-like structures is ascribed to the interaction of the lipids from bicelles with the SC lipids. The bicellar system used is suitable to penetrate the skin SC and to reinforce the intercellular lipid areas, constituting a promising tool for skin applications.     

Permeation of Cs+ ions across skin barriers

Permeation of metal Cs⁺ ions from water solutions across the intact, stripped and splitted skin of 5- or 9-day old rat was studiedin vitro using vertical diffusion cells. The results showed that the stratum corneum (horny layer) of this new animal skin model represents the principal pentration barrier. The relative importance of the percutaneous absorption of Cs⁺ ions through transepidermal and transfolicular routes has been assessed comparing the penetration across skin without hairs and skin just haired. The results have shown that the shunt diffusion along hair follicles can be of preponderant value.     

The action modes of Lippia sidoides (Cham) essential oil as penetration enhancers on snake skin

The aim of this work was to study the effect of Lippia sidoides essential oil (LSEO) on stratum corneum lipids and the permeation of salicylic acid. DSC and FTIR spectroscopy were applied. LSEO 1% (v/v) significantly enhanced salicylic acid flux through snake skin. According to the DSC curves changes in the transition temperature of the lipids were observed indicating that LSEO can interact with stratum corneum. The IR spectrum of skin treated with LSEO showed a decrease in the peak intensity for CH₂ stretchings (2920–2850 cm^–1) however the peak positions did not alter suggesting the extraction of the lipids.     

Improved skin delivery and validation of novel stability-indicating HPLC method for ketoprofen nanoemulsion

Ketoprofen is a non-steroidal anti-inflammatory drug (NSAID) widely used to treat rheumatoid arthritis and other inflammatory diseases. Normally used by oral route, this drug presents numerous side effects related to this administration route, such as nausea, dyspepsia, diarrhea, constipation and even renal complications. To avoid that, topical administration of ketoprofen represents a good alternative, since this drug has both partition coefficient and aqueous solubility suitable for skin application, compared to other NSAIDs. In this study, we describe the production of a nanoemulsion containing ketoprofen, its skin permeation and in vitro release study and a novel validation method to analyze this drug in the permeation samples and a forced degradation study using skin and nanoemulsion samples. The new HPLC method was validated, with all specifications in accordance with validation parameters and with an easy chromatographic condition. Forced degradation study revealed that ketoprofen is sensitive to acid and basic hydrolysis, developing degradation peaks after exposure to these factors. Concerning in vitro release from the nanoemulsion, release curves presented first order profile and were not similar to each other. After 8 h, 85% of ketoprofen was release from the nanoemulsion matrix while 49% was release from control group. In skin permeation study, nanoemulsion enabled ketoprofen to pass through the skin and enhanced retention in the epidermis and stratum corneum, layer on which the formulation presented statistically different values compared to the control group.     

An experimental approach to study the binding properties of vitamin E (alpha-tocopherol) during hairless mouse skin permeation.

An experimental approach to study the binding properties of vitamin E has been developed. Total vitamin E solubility in the skin was determined by a partition study, followed by in vitro skin permeation studies with whole skin and stripped skin. The amount of freely diffusable vitamin E in the diffusion process was determined from the permeation profiles of whole skin and stripped skin by employing a bi-layer model. The concentrations of vitamin E in the stratum corneum and viable dermis were determined separately. By subtracting this amount from the total concentration of vitamin E in the skin, as determined by the solubility study, the amount of bound vitamin E was determined. After skin permeation reached a steady state, the donor solution was removed and the permeation study continued (desorption study). During the entire period of the desorption experiment, the amount of vitamin E in the receptor solution hardly increased and remained constant. After the desorption experiment, vitamin E still remaining in the skin was determined by extracting with tissue solubilizer, SOLABLE, and is considered as the amount of vitamin E strongly bound in the skin. The concentrations of bound vitamin E determined by permeation and desorption studies coincided relatively well. To further investigate skin binding of vitamin E, a differential scanning calorimetry study was performed. Vitamin E-treated stratum corneum showed phase transitions at 76 and 85 degrees C, associated with lipid transitions. The thermal transitions associated with the lipid transition suggested interactions of vitamin E with lipid components of the skin. During skin permeation, vitamin E forms a very strong reservoir in the skin tissue and this amount of vitamin E, about 30%, exists as a bound-form with the lipid components of the stratum corneum.     

Influence of epidermal thickness,pigmentation and redness on skin autofluorescence

Detection of autofluorescence at the skin surface is highly influenced by melanin and hemoglobin. Epidermal absorption and scattering may also be an influencing factor and is represented in this article as a quantitative parameter, epidermal thickness. To examine this parameter we measured the 370 nm fluorescence in vivo after excitation with 330 nm and the 455 nm fluorescence after excitation with 330 and 370 nm. Measurements were performed on sun-exposed skin at the dorsal aspect of the forearm and shoulder and on nonexposed buttock skin. Skin pigmentation and redness of the same body sites were measured by reflectance spectroscopy. The thickness of the stratum corneum and the cellular part of epidermis was quantified by light microscopy of skin biopsies. Multiple regression analysis was used to find correlations between autofluorescence and the potential influencing factors. We found a highly significant correlation of skin autofluorescence with pigmentation and redness for both emission wavelengths (Em). A small but significant correlation to epidermal thickness was found only for excitation wavelength (Ex) 370 nm and Em455 nm if body site was included in the analysis. No correlation between Ex330:Em370 and Ex330:Em455 and thickness of epidermis was found. For practical use, correction of skin autofluorescence for pigmentation is essential, correction for redness is of less importance and correction for epidermal thickness is unnecessary.     

Keratinized epithelial transport of beta-blocking agents. I. Relationship between physicochemical properties of drugs and the flux across rat skin and hamster cheek pouch.

The maximum fluxes (Jmax) of beta-blockers through keratinized membranes were determined in vitro and compared with their physiochemical parameters such as lipophilicity (log k'0) and melting point (mp). Rat abdominal skin and hamster cheek pouch mucosa were used as the model membranes. Propranolol, metoprolol, timolol, pindolol, nadolol and agenolol were used as beta-blockers with a variety of physicochemical characters. Linear relations of Jmax with either log k'0 or mp were observed both in intact rat skin and in intact hamster cheek pouch, suggesting that the lipophilicity and thermodynamic activity of a drug in the crystal state primarily affect the drug's permeation through these membranes. However, the slope, dJmax/d(log k'0), for cheek pouch mucosa was greater than that for rat skin, corresponding to the lack of appendigeal shunt pathways in cheek pouch. Penetration studies using the delipidized membranes and the isolated stratum corneum sheet of hamster cheek pouch mucosa clarified that the primary rate-limiting barrier function might exist in the lipid layer of the stratum corneum. Jmax values for the tape-stripped and delipidized skins correlated with both the solubilities of drugs in the vehicle and with the mp, suggesting the polar porous characteristics of both model membranes. However, a theoretical approach confirmed that the contribution of an intracellular or aqueous pore route in the intact membrane to the permeation of drugs with positive lipophilic indexes is negligible.     

Zinc analysis in human skin by laser induced-breakdown spectroscopy

The feasibility of using laser induced-breakdown spectroscopy (LIBS) as a quick and simple method to analyze trace elemental concentrations in the stratum corneum of human skin was investigated. A 60 mJ pulse(-1) Nd:YAG laser operating at 1064 nm was used to form the laser induced plasma. Zinc was chosen for this study. By using poly-(methyl methacrylate) (PMMA) solution to coat the slides, zinc standard solutions were dispersed on the slides with the help of a centrifuge. The percent of R.S.D. of the dispersed area size obtained from 17 measurements was 6%. The precision from intra-measurements (precision on one slide) and inter-measurements (precision between slides) was 4-8% and less than 1%, respectively. A calibration curve with a linear correlation coefficient of 0.998 and a detection limit of 0.3 ng cm(-2) were obtained. An aqueous zinc solution or zinc ointment was deposited on the biceps area of the forearms of several volunteers for a time period of 30 min and 2.75 h. Six skin layers of 2-3 mum each in thickness were then removed using cyanoacrylate glue without causing any pain for the volunteers. The results indicated that Zn was absorbed through the skin and the concentration decreased exponentially with depth into the skin. The results indicate that LIBS is a useful tool for trace elemental analysis in human skin.     

Topical application of 5-aminolevulinic acid hexyl ester and 5-aminolevulinic acid to normal nude mouse skin: differences in protoporphyrin IX fluorescence kinetics and the role of the stratum corneum

An important limitation of topical 5-aminolevulinic acid (ALA)-based photodetection and photodynamic therapy is that the amount of the fluorescing and photosensitizing product protoporphyrin IX (PpIX) formed is limited. The reason for this is probably the limited diffusion of ALA through the stratum corneum. A solution to this problem might be found in the use of ALA derivatives, as these compounds are more lipophilic and therefore might have better penetration properties than ALA itself. Previous studies have shown that ALA hexyl ester (ALAHE) is more successful than ALA for photodetection of early (pre)malignant lesions in the bladder. However, ALA pentyl ester slightly increased the in vivo PpIX fluorescence in early (pre)malignant lesions in hairless mouse skin compared to ALA. The increased PpIX fluorescence is located in the stratum corneum and not in the dysplastic epidermal layer. In the present study, ALA- and ALAHE-induced PpIX fluorescence kinetics are compared in the normal nude mouse skin, of which the permeability properties differ from the bladder. Application times and ALA(HE) concentrations were varied, the effect of a penetration enhancer and the effect of tape stripping the skin before or after application were investigated. Only during application for 24 h, did ALAHE induce slightly more PpIX fluorescence than ALA. After application times ranging from 1 to 60 min, ALA-induced PpIX fluorescence was higher than ALAHE-induced PpIX fluorescence. ALA also induced higher PpIX production than ALAHE after 10 min of application with concentrations ranging from 0.5 to 40%. The results of experiments with the penetration enhancer and tape stripping indicated that the stratum corneum acts a barrier against ALA and ALAHE. Use of penetration enhancer or tape stripping enhanced the PpIX production more in the case of ALAHE application than in the case of ALA application. This, together with the results from the different application times and concentrations indicates that ALAHE diffuses more slowly across the stratum corneum than ALA.     

Confocal Raman microspectroscopy for skin characterization: a comparative study between human skin and pig skin

The present paper provides a spectral comparison between abdominal human skin (Transkin) and pig ear skin using confocal Raman microspectroscopy at 660 nm. Pig ear skin is usually utilized as a substitute for human skin for active ingredients assessment in dermatological and cosmetics fields. Herein, the comparison is made at the level of the stratum corneum (SC), the SC/epidermis junction and the viable epidermis. The 660 nm excitation source appears to be the most appropriate wavelength for such skin characterization. From Raman signatures of both skin types, a tentative assignment of vibrations was performed in the fingerprint and the high wavenumber spectral regions. Significant differences were highlighted for lipid content in in-depth spectra and for hyaluronic acid (HA) and carotenoid in SC spectra. Marked tissular variability was also revealed by certain Raman vibrations. These intrinsic molecular data probed by confocal Raman microspectroscopy have to be considered for further applications such as cutaneous drug permeation.     

Curcumin-loaded layer-by-layer folic acid and casein coated carboxymethyl cellulose/casein nanogels for treatment of skin cancer

Targeted drug delivery systems using natural polysaccharide/protein biopolymer for tumor cells are an attractive platform for enriching the therapeutic effects and reducing the side effects of the drug. Carboxymethyl cellulose (CMC) and casein (CA) nanogels (NGs) loaded with curcumin (CUR) were prepared by self-assembly method and fabricated with folic acid (FA) and casein using layer-by-layer (LbL) technique for skin cancer drug delivery. The prepared samples were characterized by techniques like zeta potential, FTIR, XRD, TGA and Cryo-SEM. Both the swelling and in vitro drug release was performed in acidic pH (4.5 and 6.8) and physiological pH 7.4. Hemolysis assays demonstrated that the drug carriers are hemocompatible. Confocal microscope studies indicate facilitated uptake of 2-FA/CA/CUR@CMC-CA NGs in MEL-39 melanoma cancer cell line, which in turn result in a higher potential for apoptosis. Compared to pure CUR and CUR@CMC-CA NGs, the 2-FA/CA/CUR@CMC-CA NGs has lower IC50 value and superior cytotoxicity in MEL-39 cells because of folate-receptor mediated endocytosis evaluated by the cellular viability quantification using MTT assay and optical microscope images. Finally from in vitro skin permeation experiments, 2-FA/CA/CUR@CMC-CA NGs showed 3.47 ± 0.03 to 4.15 ± 0.25 μg/ml CUR concentrations at the stratum corneum, epidermal and dermal layers. Overall, our results put forth 2-FA/CA/CUR@CMC-CA NGs as an aspiring candidate to achieve enhanced anticancer effects against melanoma skin cancer.