Daniel J. McAuliffe

THE MELANOSOME: THRESHOLD TEMPERATURE FOR EXPLOSIVE VAPORIZATION AND INTERNAL ABSORPTION COEFFICIENT DURING PULSED LASER IRRADIATION

Abstract— —The explosive vaporization of melanosomes in situ in skin during pulsed laser irradiation (pulse duration <1 μs) 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°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°C (SD, N = 3). The slope, ∂H 0/∂T i, specifies the internal absorption coefficient, μa, within the melanosome: μa = −ρC/(slope(1 + 7.1R d)), where ρC is the product of density and specific heat, and Rd is the total diffuse reflectance from the skin. A summary of the absorption spectrum (μa 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.

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).

Topical Drug Delivery in Humans with a Single Photomechanical Wave

AbstractPurpose. Assess the feasibility ofin 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 ∼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.

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.

Cell Loading with Laser-Generated Stress Waves: The Role of the Stress Gradient

AbstractPurpose. 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.

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.

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

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.

SENSITIVITY OF MONONUCLEAR CELLS TO UV RADIATION: EFFECT ON SUBSEQUENT STIMULATION WITH PHYTOHEMAGGLUTININ

The ability of peripheral blood mononuclear cells to incorporate 3[H] thymidine into nuclear DNA following stimulation by phytohemagglutinin is reduced by prior exposure to UV radiation in vitro: the reduction is dose and wavelength dependent. The doses required to affect this function of mononuclear cells are higher than the doses required to reduce trypan blue dye exclusion, so that following exposure to radiation populations of cells that are unable to exclude trypan blue dye are still capable of responding to phytohemagglutinin. This finding indicates that trypan blue dye exclusion may not accurately reflect the viability of cells after exposure to UV radiation.

Nuclear Transport by Laser-Induced Pressure Transients

AbstractPurpose. Control of the transport of molecules into the nucleus represents a key regulatory mechanism for differentiation, transformation, and signal transduction. Permeabilization of the nuclear envelope by physical methods can have applications in gene therapy. Laser-induced pressure transients can produce temporary aqueous pores analogous to those produced by electroporation and that the cells can survive this procedure. In this study, we examine the role of the pressure transients in creating similar pores in the nuclear envelope. Methods. The target human peripheral blood mononuclear cells in a 62 μM 72 kDa fluoresceinated dextran solution were exposed to the pressure transients generated by laser ablation. An in vitro fluorescence confocal microscope was used to visualize and quantify the fluoresceinated dextran in the cytoplasmic and nuclear compartments. Results. In contrast to electroporation, the pressure transients could deliver 72 kDa fluoresceinated dextrans, which are normally excluded by the nucleus, across the nuclear envelope into the nucleus. In addition to creating pores in the plasma membrane, temporary pores were also created in the nuclear envelope following exposure to pressure transients. Conclusion. The production of temporary nuclear pores could provide a unique resource for drug-delivery and gene therapy.

Laser-induced enhancement of drug cytotoxicity: a new approach to cancer therapy

A new approach to drug delivery has been developed at the Wellman Laboratories of Photomedicine that is analogous to photodynamic therapy except that it utilizes high pressure impulse waves to increase the effectiveness of a variety of drugs rather than light activated drugs. This therapeutic modality offers a generic technology that can be used in a variety of conditions including infections, abscesses, and cancer.