Examination of stratum corneum barrier function in vivo by infrared spectroscopy.

It is generally accepted that the stratum corneum (SC) is the least permeable layer of the epidermis. Histologically, though, the SC is a non-uniform, inhomogeneous membrane, and the question "Is barrier function distributed uniformly across the SC thickness?" has been posed. To address this issue, human ventral forearm SC has been studied in vivo by attenuated-total-reflectance Fourier-transform infrared spectroscopy during the course of sequential tape-stripping. Because the intercellular lipids of the SC and the degree of hydration of the membrane have been shown to be crucial determinants of barrier function, attention has been focused on the spectral features, which report specifically on these parameters. The degree of disorder of the SC intercellular lipids has been found to decrease over the outer cell layers (up to three tape-strips) and then to remain essentially constant. The amount of lipids decreases similarly such that a 60% reduction (relative to the "no-strip" baseline) is observed after about four tape-strips. A plausible explanation for these measurements is that the lipids near the surface are a mixture of (a) "true" intercellular lipid (which is expected to be highly ordered), and (b) sebaceous lipid (which contains much greater amounts of low-melting components, such as fatty acids). The sequential infrared (IR) spectra provide at least circumstantial evidence to support this hypothesis. As expected, the IR spectra show that SC hydration increases from the surface towards the SC-stratum granulosum interface. Taken together, the results imply that the SC is indeed non-uniform. The properties of the outer layers (those removed by the first 3-4 tape-strips) change significantly with increasing depth.(ABSTRACT TRUNCATED AT 250 WORDS)

High-frequency sonophoresis: permeation pathways and structural basis for enhanced permeability.

The mechanism of stratum corneum (SC) permeabilization by ultrasound (sonophoresis) is unknown. We studied here permeation pathways, and SC intercellular structural organization following applications of high-frequency sonophoresis to hairless mouse skin. Ruthenium tetroxide post-fixation and tracer solutions of LaNO3 and FITC-dextrans were employed to examine SC lamellar bilayers, lamellar body morphology and subcellular permeation pathways. Sonophoresis disrupted the compact organization of SC bilayers and LB-derived contents at the stratum granulosum (SG)-SC interface, leading to domain separation between 0 and 20 h, reverting by 48 h. Post-sonophoresis, tracers traversed the SC via lacunae within the lamellar bilayers, and via lamellae in sites that displayed domain separation. These studies provide insights about the penetration pathways, permeabilizing mechanisms, and kinetics of sonophoresis on the epidermis.

Far-field potentials generated by action potentials of isolated frog sciatic nerves in a spherical volume.

Previous results in cylindrical volumes have shown that action potentials generate far-field potentials when experimental conditions are such that quadrupolar components of the action potential are reduced to an equivalent dipole. We now show that the same conclusions are also reached within a spherical volume, again recording far-field potentials from isolated bullfrog nerves. A mathematical proof is given that shows that in a sphere, antipodal electrodes primarily detect far-field potentials from dipole generators and not quadrupole generators. A revised conception of the 'far-field' in evoked responses is discussed which equates far-field recordings with dipole detection.

Sonophoresis. I. The use of high-frequency ultrasound to enhance transdermal drug delivery.

Previous attempts to use ultrasound (less than or equal to 1-MHz frequency and 1 to 3-W/cm2 intensity) to enhance transdermal drug delivery (so-called sonophoresis) have produced inconsistent results. Theoretical analysis of ultrasound propagation in tissue predicts that higher-frequency ultrasound (greater than 1 MHz) will increase the concentration of energy deposition in the stratum corneum (SC) (typically, the rate-limiting barrier to percutaneous penetration). This hypothesis was tested by comparing the passive transdermal delivery of salicylic acid with that under the influence of ultrasound at 2-, 10-, and 16-MHz frequency; measurements were performed in vivo in hairless guinea pigs. Total drug absorbed was quantified by determining the amount of salicylic acid (1) present in SC tape strips and (2) eliminated in urine. Sonophoresis for 20 min at 2 MHz caused no significant increase in salicylic acid delivery over passive diffusion; treatment with ultrasound at 10 and 16 MHz, on the other hand, significantly elevated salicylic acid transport, by 4-fold and 2.5-fold, respectively. Kinetic analysis of the sonophoretic data at 10 and 16 MHz also revealed that the diffusion lag time associated with transdermal drug delivery (TDD) was reduced. A shorter period (5 min) of sonophoresis again resulted in enhanced TDD (relative to the corresponding control) at the higher frequencies; the delivered dose, and the level of enhancement, however, were lower than those after the 20-min treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Sonophoresis. II. Examination of the mechanism(s) of ultrasound-enhanced transdermal drug delivery.

We have shown previously that high-frequency ultrasound (sonophoresis) can significantly enhance the transdermal delivery of a topically applied drug in vivo and that the augmentation of transport was caused by the action of the ultrasound on the skin. However, these earlier experiments did not reveal (i) the mechanism of sonophoresis, (ii) the pathway of drug permeation under the influence of ultrasound, and (iii) any potentially detrimental effects of the enhancement procedure on skin structure and morphology. In the study reported here, these three key issues have been addressed using electron microscopy to follow the penetration of an electron-dense, colloidal tracer (lanthanum hydroxide; LH). Experiments have again been performed using the hairless guinea pig animal model. Colloidal LH suspensions were applied to skin sites, which were then immediately exposed to ultrasound (at 10 or 16 MHz) for 5 or 20 min. Passive transport of LH under identical conditions (but without ultrasound) provided the control measurements. Tissue processing after the treatment periods utilized standard electron microscopy staining procedures. We found the following: (1) LH does not permeate the skin by passive diffusion; under the influence of ultrasound, on the other hand, it penetrates through the stratum corneum (SC) and the underlying viable epidermal cell layers via an apparently intercellular route. (2) LH transports through the epidermis to the upper dermis, even after only 5 min of ultrasound treatment, a remarkable and unexpected finding.(ABSTRACT TRUNCATED AT 250 WORDS)

Validation of reflectance infrared spectroscopy as a quantitative method to measure percutaneous absorption in vivo.

Attenuated total-reflectance infrared (ATR-IR) spectroscopy has been used to follow the penetration of a model compound (4-cyanophenol; CP) across human stratum corneum (SC) in vivo, in man. CP was administered for periods of 1, 2, or 3 hr, either (a) as a 10% (w/v) solution in propylene glycol or (b) in an identical vehicle which also contained 5% (v/v) oleic (cis-9-octadecenoic) acid. At the end of the treatment periods, SC at the application site was progressively removed by adhesive tape-stripping. Prior to the removal of the first tape-strip, and after each subsequent tape-strip, an ATR-IR spectrum of the treated site was recorded. The presence of CP, as a function of position in the SC, was monitored spectroscopically via the intense C = N stretching absorbance at 2230 cm-1. The absolute amount of CP, as a function of SC depth, was determined by "spiking" the applied solutions with 14C-labeled compound and subsequent liquid scintillation counting of the removed tape-strips. The presence of oleic acid in the applied formulation significantly increased the rate and extent of CP delivery as evaluated by either spectroscopy or radiochemical analysis. Furthermore, the ATR-IR and direct 14C analysis of CP as a function of SC position were highly correlated. These data strongly support, therefore, the validation of ATR-IR as a quantitative tool to assess percutaneous penetration in vivo.

Effect of electroporation on transdermal iontophoretic delivery of luteinizing hormone releasing hormone (LHRH) in vitro.

Electroporation, the creation of transient, enhanced membrane permeability using short duration (microseconds to millisecond) electrical pulses, can be used to increase transdermal drug delivery. The effect of an (electroporative) electric pulse (1000 V, tau = 5 msec) on the iontophoretic transport of LHRH through human skin was studied in vitro. Fluxes achieved with and without a pulse under different current densities (0- 4 mA/cm2) were compared. The results indicated that the application of a single pulse prior to iontophoresis consistently yielded higher fluxes (5-10 times the corresponding iontophoretic flux). For example, at 0.5 mA/cm2 fluxes were 0.27 +/- 0.08 and 1.62 +/- 0.05 micrograms/hr/cm2 without and with the pulse, respectively. At each current density studied, the LHRH flux decreased after iontophoresis, approaching pre-treatment values. The results show that electroporation can significantly and reversibly increase the flux of LHRH through human skin. These results also indicate the therapeutic utility of using electroporation for enhanced transdermal transport.