Permeabilization and recovery of the stratum corneum in vivo: the synergy of photomechanical waves and sodium lauryl sulfate.

BACKGROUND AND OBJECTIVE: Photomechanical waves render the stratum corneum permeable and allow macromolecules to diffuse into the epidermis and dermis. The aim of this study was to investigate the combined action of photomechanical waves and sodium lauryl sulfate, an anionic surfactant, for transdermal delivery. STUDY DESIGN/MATERIALS AND METHODS: A single photomechanical wave was applied to the skin of rats in the presence of sodium lauryl sulfate. The sodium lauryl sulfate solution was removed and aqueous solutions of rhodamine-B dextran (40 kDa molecular weight) were applied to the skin at time points 2, 30, and 60 minutes post-exposure. The presence of rhodamine-B dextran in the skin was measured by fluorescence emission spectroscopy in vivo and fluorescence microscopy of frozen biopsies. RESULTS: The use of sodium lauryl sulfate delayed the recovery of the stratum corneum barrier and extended the time available for the diffusion of dextran through it. CONCLUSION: The combination of photomechanical waves and surfactants can enhance transdermal drug delivery.

Photomechanical transdermal delivery: the effect of laser confinement.

BACKGROUND AND OBJECTIVE: Photomechanical waves can transiently permeabilize the stratum corneum and facilitate the delivery of drugs into the epidermis and dermis. The present study was undertaken to assess the effect of pulse characteristics to the penetration depth of macromolecules delivered into the skin. STUDY DESIGN/MATERIALS AND METHODS: Photomechanical waves were generated by confined ablation with a Q-switched ruby laser. Fluorescence microscopy of frozen biopsies was used to assay the delivery of macromolecules through the stratum corneum and determine the depth of penetration. RESULTS: Photomechanical waves generated by confined ablation of the target have a longer rise time and duration than those generated by direct ablation. Confined ablation required a lower radiant exposure (from approximately 7 J/cm(2) to approximately 5 J/cm(2)) for an increase in the depth of delivery (from approximately 50 microm to approximately 400 microm). CONCLUSIONS: Control of the characteristics of the photomechanical waves is important for transdermal delivery as they can affect the depth of drug penetration into the dermis.

Photomechanical transdermal delivery of insulin in vivo.

BACKGROUND AND OBJECTIVE: Previous studies have shown that photomechanical waves transiently permeabilize the stratum corneum in vivo. The aim of the present work was to investigate the potential of photomechanical waves for systemic drug delivery. STUDY DESIGN/MATERIALS AND METHODS: Photomechanical waves were generated by ablation of a polystyrene target by a Q-switched ruby laser. Systemic insulin delivery in a streptozotocin-diabetic rat model was monitored by measuring the blood glucose level. RESULTS: After photomechanical insulin delivery, the blood glucose decreased 80 +/- 3% and remained below 200 mg/dl for more than 3 hours. Whereas in control experiments (for which insulin was applied without photomechanical waves), there was no dramatic change in the blood glucose (standard deviation of measurements over 4 hours was 7%). CONCLUSION: The application of the photomechanical waves allowed approximately 6-kDa protein molecules (insulin) to pass through the stratum corneum and into the systemic circulation.

Topical drug delivery in humans with a single photomechanical wave.

PURPOSE: Assess the feasibility of in vivo topical drug delivery in humans with a single photomechanical wave. METHODS: Photomechanical waves were generated with a 23 nsec Q-switched ruby laser. In vivo fluorescence spectroscopy was used as an elegant non-invasive assay of transport of 5-aminolevulinic acid into the skin following the application of a single photomechanical wave. RESULTS: The barrier function of the human stratum corneum in vivo may be modulated by a single (110 nsec) photomechanical compression wave without adversely affecting the viability and structure of the epidermis and dermis. Furthermore, the stratum corneum barrier always recovers within minutes following a photomechanical wave. The application of the photomechanical wave did not cause any pain. The dose delivered across the stratum corneum depends on the peak pressure and has a threshold at approximately 350 bar. A 30% increase in peak pressure, produced a 680% increase in the amount delivered. CONCLUSIONS: Photomechanical waves may have important implications for transcutaneous drug delivery.

Wide-band acoustic spectroscopy of biological material based on a laser-induced grating technique.

A laser-induced transient grating technique enables fast noncontact acoustic measurements on transparent biological materials in a frequency range from tens of megahertz to 1 GHz. We have applied this method to the characterization of bovine vitreous and found high-frequency acoustic attenuation values to be close to those of water, with a quadratic dependence on frequency, in contrast to low-frequency data. The potential of the technique for studying other biological materials, such as human stratum corneum, is demonstrated.

Cell loading with laser-generated stress waves: the role of the stress gradient.

PURPOSE: To determine the dependence of the permeabilzation of the plasma membrane on the characteristics of laser-generated stress waves. METHODS: Laser pulses can generate stress waves by ablation. Depending on the laser wavelength, fluence, and target material, stress waves of different characteristics (rise time, peak stress) can be generated. Human red blood cells were subjected to stress waves and the permeability changes were measured by uptake of extracellular dye molecules. RESULTS: A fast rise time (high stress gradient) of the stress wave was required for the permeabilization of the plasma membrane. While the membrane was permeable, the cells could rapidly uptake molecules from the surrounding medium by diffusion. CONCLUSIONS: Stress waves provide a potentially powerful tool for drug delivery.

Photomechanical transcutaneous delivery of macromolecules.

Transcutaneous drug delivery has been the subject of intensive research. In certain situations, rapid transcutaneous delivery is very desirable. A mechanical (stress) pulse generated by a single laser pulse was shown to transiently increase the permeability of the stratum corneum in vivo. The barrier function of the stratum corneum recovers within minutes. The increased permeability during these few minutes allows macromolecules to diffuse through the stratum corneum into the viable epidermis and dermis. Macromolecules (40 kDa dextran and 20 nm latex particles) were deposited into the skin using a photomechanical pulse generated by a single 23 ns laser pulse. This treatment can potentially be utilized in therapies that currently require occlusive dressings for hours or day(s).

Stress-wave-assisted transport through the plasma membrane in vitro.

BACKGROUND AND OBJECTIVE: Laser-induced stress waves have been shown to alter the permeability of the plasma membrane without affecting cell viability. The aim of the work reported here was to quantify the molecular uptake by cell cultures in vitro and determine optimal stress-wave parameters. STUDY DESIGN/MATERIALS AND METHODS: Human peripheral blood mononuclear cells were exposed to laser-induced stress waves in an experimental arrangement that eliminated interference from ancillary effects such as plasma, heat, or cavitation. A radiolabeled compound (tritiated thymidine) was used as the probe. RESULTS: Stress waves enhanced the diffusion of tritiated thymidine by inducing a transient permeabilization of the plasma membrane. Furthermore, maximum intracellular concentration (2 x 10(5) thymidine molecules/cell or 10% of the extracellular concentration) was reached with only 2-3 stress waves. CONCLUSION: Laser-induced stress waves provide an efficient method for delivering molecules through the plasma membrane into the cytoplasm of cells.

Stress-wave-induced membrane permeation of red blood cells is facilitated by aquaporins.

Stress waves generated by lasers and extracorporeal lithotripters have been shown to transiently increase the permeability of the plasma membrane, without affecting cell viability. Molecules present in the medium can diffuse into the cytoplasm under the concentration gradient. Molecular uptake under stress waves correlates with the presence of functioning aquaporins in the plasma membrane.

Basic mechanism of in vitro pulsed-dye laser-induced vasodilation.

Vasodilation of rabbit carotid arteries induced by a pulsed-eye laser was studied in vitro to clarify the underlying mechanism. Artery segments were double cannulated in a pressure-perfusion apparatus which, under physiological conditions, allows for differential application of various solutions, pharmacological agents, and pulsed-dye laser light. Vasoconstriction was activated using both pharmacological and nonpharmacological agonists. Laser energy at a wavelength of either 480 or 575 nm was applied intraluminally in 1-microseconds pulses, which caused dilation of the arteries if hemoglobin was present in the lumen at sufficient concentration. Induced vasodilation did not specifically require the presence of hemoglobin; the same phenomenon could be repeated using an inert dye such as Evans blue as an optical absorber of laser energy. The optical density of the absorber, the number of applied laser pulses, and total amount of applied energy directly influenced the vasodilatory response. Laser-induced vasodilation was possible in both normal vessels and vessels denuded of endothelium. Pulsed-dye laser-induced vasodilation is therefore not a phenomenon mediated through chemical processes, but is rather a purely physical process initiated by the optical absorption of laser energy by the intraluminal medium, which probably induces cavitation bubble formation and collapse, resulting in the vasodilatory response of the vessel.

Physical factors involved in stress-wave-induced cell injury: the effect of stress gradient.

We have studied the biological effects of ablation-induced stress waves in vitro. Mouse breast sarcoma cells (EMT-6) were exposed to stress waves that differed only in rise time. Two assays were used to determine cell injury: incorporation of tritiated thymidine (viability assay), and transmission electron microscopy (morphology assay). We present evidence that the rise time of stress waves can significantly modify cell viability and that cell injury correlates better with the stress gradient than peak stress.

Biological effects of laser-induced shock waves: structural and functional cell damage in vitro.

A new experimental design has been used to study the biological effects of laser-induced shock waves which minimizes or eliminates interference from ancillary effects such as bubble formation, ultraviolet (UV) radiation, or formation of radicals. The effects of these shock waves on human lymphocytes and red blood cells have been investigated. Three assays were used to determine cell injury: electron microscopy, ethidium bromide/fluorescein diacetate (EB/FDA) staining and incorporation of tritiated thymidine. The degree of cell damage was related to the pressure and the number of pulses. Cell damage was quantified and correlated using the three assays. Measurements of gross structural alterations as determined by transmission electron microscopy were less sensitive than assays of structural damage (e.g., EB/FDA assay) which were less sensitive than functional assays (e.g., incorporation of tritiated thymidine).

The melanosome: threshold temperature for explosive vaporization and internal absorption coefficient during pulsed laser irradiation.

The explosive vaporization of melanosomes in situ in skin during pulsed laser irradiation (pulse duration less than 1 microsecond) is observed as a visible whitening of the superficial epidermal layer due to stratum corneum disruption. In this study, the ruby laser (694 nm) was used to determine the threshold radiant exposure, H0 (J/cm2), required to elicit whitening for in vitro black (Negroid) human skin samples which were pre-equilibrated at an initial temperature, Ti, of 0, 20, or 50 degrees C. A plot of H0 vs Ti yields a straight line whose x-intercept indicates the threshold temperature of explosive vaporization to be 112 +/- 7 degrees C (SD, N = 3). The slope, delta H0/delta Ti, specifies the internal absorption coefficient, mua, within the melanosome: mua = -rho C/(slope(1 + 7.1 Rd)), where rho C is the product of density and specific heat, and Rd is the total diffuse reflectance from the skin. A summary of the absorption spectrum (mua) for the melanosome interior (351-1064 nm) is presented based on H0 data from this study and the literature. The in vivo absorption spectrum (380-820 nm) for human epidermal melanin was measured by an optical fiber spectrophotometer and is compared with the melanosome spectrum.

Effects of UVA (320-400 nm) on the barrier characteristics of the skin.

The stratum corneum serves as the major barrier to the entrance of most molecules into the skin. In the studies presented here, the effects of UVA radiation (320-400 nm) on the barrier capacity of human stratum corneum were examined. Penetration of a homologous series of primary alcohols through unirradiated (control) and UVA-irradiated (test) human epidermis was determined in vitro. Permeability constants, kp, were calculated. Mean ratios of permeability constants for UVA-irradiated and unirradiated epidermis (mean kp test)/(mean kp control) ranged from 2.3 to 3.0 for methanol and from 2.2 to 2.5 for ethanol. These mean ratios were determined using different pieces of epidermis from the same piece of skin for test and control samples. When kp control and kp test were determined on the same piece of epidermis on successive days, the ratios (kp test/kp control) were similar to the mean ratios determined on different pieces of epidermis. For other primary alcohols, propanol, butanol, hexanol, and heptanol, UVA radiation did not alter their permeability constants significantly. Partition coefficients, Km, were determined for ethanol and heptanol using UVA-irradiated and unirradiated stratum corneum. For ethanol, irradiation resulted in a 1.5 to 2.6 times increase in Km. For heptanol, irradiation caused no change in Km. These results demonstrate that the barrier capacity of stratum corneum for small, polar, primary alcohols is diminished (permeability increases) and for higher molecular weight less polar alcohols, is unaffected by small doses of UVA radiation. This increased permeability of small polar alcohols through human skin may be due to enhanced partitioning into UVA-irradiated stratum corneum, which was not apparent for a higher molecular weight less polar alcohol.

Laser-induced photoacoustic injury of skin: effect of inertial confinement.

Argon-fluoride (ArF) excimer laser-induced acoustic injury was confirmed by ablating the stratum corneum (s.c.) inertially confined by water in vivo. Hairless rats were irradiated through a quartz chamber with flowing distilled water or air and a 2.5 mm aperture. The laser was adjusted to deliver 150 mJ/cm2 at the skin surface for both conditions. Partial and complete ablation of the s.c. was achieved with 12 and 24 pulses, respectively. Immediate damage was assessed by the transmission electron microscopy. Partial ablation of the s.c. through air produced no damage, whereas partial ablation through water damaged skin to a mean depth of 114.5 +/- 8.8 microns (+/- SD). Full thickness ablation of the s.c. through air and water produced damage zones measuring 192.2 +/- 16.2 and 293.0 +/- 71.6 microns, respectively (P less than 0.05). The increased depth of damage in the presence of inertial confinement provided by the layer of water strongly supports a photoacoustic mechanism of damage. The damage induced by partial ablation of the s.c. provides evidence that photochemical injury is not a significant factor in the damage at a depth because the retained s.c. acts as a partial barrier to diffusion of photochemical products. Combined with our previous studies, these experiments demonstrate that pressure transients are responsible for the deep damage seen with 193 nm ablation and that photoacoustic effects must be considered when using short-pulse, high-peak power lasers.

Cell selectivity to laser-induced photoacoustic injury of skin.

Cell selectivity to photoacoustic injury induced by argon-fluoride excimer laser (193 nm) was studied. Rats were irradiated through air or water and a 2.5 mm aperture. The laser was adjusted to deliver 150 mJ/cm2 at the skin surface with 12 and 24 pulses. Immediate damage was assessed by transmission electron microscopy. Cell selectivity was observed in dermis and epidermis. Fibroblasts showed alteration of nuclear chromatin and cytoplasmic organelles, while some of the migratory cells adjacent to fibroblasts did not. Similar difference of damage was observed between keratinocytes and Langerhans cells in epidermis. Considering the relationship between cells and their microenvironment in tissue, this selectivity may be due to the difference of acoustical coupling of propagation of acoustic waves rather than to differential sensitivity of the cells to damage.

Enhancing the carotenoid content of atherosclerotic plaque: implications for laser therapy.

Selective laser ablation of human atherosclerotic plaque is possible because endogenous carotenoid pigments found in atherosclerotic plaque confer a twofold preferential absorption of laser radiation at 450 to 500 nm. In this study, patients with carotid endarterectomy were pretreated with oral beta carotene to determine if the carotenoid content and therefore laser selectivity of plaque could be increased in vivo. Beta carotene-treated patients had a significant, nearly twofold increase in their plaque carotenoid concentration, which increased from 0.22 to 0.40 microgram beta carotene/mg cholesterol. These results suggest that selective ablation of atherosclerotic plaque may be enhanced by pretreating patients with doses of oral beta carotene for short periods of time.

Putative photoacoustic damage in skin induced by pulsed ArF excimer laser.

Argon-fluoride excimer laser ablation of guinea pig stratum corneum causes deeper tissue damage than expected for thermal or photochemical mechanisms, suggesting that photoacoustic waves have a role in tissue damage. Laser irradiation (193 nm, 14-ns pulse) at two different radiant exposures, 62 and 156 mJ/cm2 per pulse, was used to ablate the 15-microns-thick stratum corneum of the skin. Light and electron microscopy of immediate biopsies demonstrated damage to fibroblasts as deep as 88 and 220 microns, respectively, below the ablation site. These depths are far in excess of the optical penetration depth of 193-nm light (1/e depth = 1.5 micron). The damage is unlikely to be due to a photochemical mechanism because (a) the photons will not penetrate to these depths, (b) it is a long distance for toxic photoproducts to diffuse, and (c) damage is proportional to laser pulse intensity and not the total dose that accumulates in the residual tissue; therefore, reciprocity does not hold. Damage due to a thermal mechanism is not expected because there is not sufficient energy deposited in the tissue to cause significant heating at such depths. The damage is most likely due to a photoacoustic mechanism because (a) photoacoustic waves can propagate deep into tissue, (b) the depth of damage increases with increasing laser pulse intensity rather than with increasing total residual energy, and (c) the effects are immediate. These effects should be considered in the evaluation of short pulse, high peak power laser-tissue interactions.

Controlled removal of human stratum corneum by pulsed laser.

A new method is presented for controlled removal of the stratum corneum of human skin. An excimer laser (193 nm wavelength, 14 ns pulsewidth) was used to remove stratum corneum from in vitro human skin samples by an ablative process. The tritiated water (3H2O) permeability constant and electrical resistance of skin samples were measured in a diffusion chamber apparatus to quantify the enhancement of skin permeability. Each laser pulse ablates about a micrometer of stratum corneum, which allows controlled removal of tissue. The maximum specific enhancement of the 3H2O permeability constant obtained after complete stratum corneum removal depends on the laser pulse energy used. The most gentle laser ablation, achieved with a radiant exposure of 70 mJ/cm2 per pulse, produced a 124-fold enhancement, which is comparable to that achieved after stratum corneum removal by tape-stripping or removal of epidermis by mild heat treatment. Rapid tissue ablation occurred at higher radiant exposures of 170-480 mJ/cm2 per pulse, but only a 45-fold enhancement of permeability was achieved. The precision with which stratum corneum can be ablated using excimer laser pulses may allow further basic research on the internal structure of stratum corneum and on the re-epithelization in controlled wounds. The technique may prove useful clinically to enhance percutaneous transport in applications such as topical delivery of drugs, patch testing, and percutaneous blood gas monitoring.