Shun Lee

Alteration of cell membrane by stress waves in vitro.

Experiments on the biological effects of laser-induced stress waves indicate that there is a transient increase in the permeability of the cell membrane. A cell viability assay (propidium iodide exclusion) shows that mouse breast sarcoma cells are viable after a stress wave. The kinetics of this transient membrane permeability are measured using time-resolved fluorescence imaging. The efflux of a membrane-impermeable fluorescent probe (calcein) following the application of a 300-bar stress wave implies that there is an increase in the membrane permeability. This efflux ceases within 80 s after a stress wave, suggesting that the membrane is no longer permeable to the fluorescent probe. Fitting the observed kinetics to a simple diffusion model yields an average initial diffusion constant of 2.2 +/- 1.3 x 10(-7) cm2/s for mouse breast sarcoma cells following the application of a laser-induced stress wave.

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.

Photomechanical drug delivery into bacterial biofilms

Purpose. To investigate whether photomechanical waves generated bylasers can increase the permeability of a biofilm of the oral pathogenActinomyces viscosus.Methods. Biofilms of Actinomyces viscosus were formed on bovineenamel surfaces. The photomechanical wave was generated by ablationof a target with a Q-switched ruby laser and launched into the biofilmin the presence of 50 μg/ml methylene blue. The penetration depth ofmethylene blue was measured by confocal scanning laser microscopy.Also, the exposed biofilms were irradiated with light at 666 nm. Afterillumination, adherent bacteria were scraped and spread over thesurfaces of blood agar plates. Survival fractions were calculated bycounting bacterial colonies.Results. Confocal scanning laser microscopy revealed that a singlephotomechanical wave was sufficient to induce a 75% increase in thepenetration depth of methylene blue into the biofilm. This significantlyincreased the concentration of methylene blue in the biofilm enablingits photodestruction.Conclusions. Photomechanical waves provide a potentially powerfultool for drug delivery that might be utilized for treatment of microbial infections.

Laser-generated stress waves and their effects on the cell membrane

High-power lasers can generate well characterized stress (pressure) waves. The characteristics of the stress waves can be controlled by the appropriate choice of the laser parameters and the properties of the target material. Laser-generated stress waves can alter the structure and function of cells in vitro. Furthermore, they render the cell membrane permeable. Molecules present in the extracellular medium diffuse into the cytoplasm under the concentration gradient. Subsequently, the plasma membrane reseals, keeping the exogenous molecules inside the cell. Laser-generated stress waves can 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.

Stress-wave-induced injury to retinal pigment epithelium cells in vitro

To determine the survival of in vitro retinal pigment epithelium (RPE) cells subjected to laser‐generated stress transients (shock waves) and compare it to that of other cell lines.

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.