Enhanced uptake and photoactivation of topical methyl aminolevulinate after fractional CO2 laser pretreatment.

BACKGROUND AND OBJECTIVES: Photodynamic therapy (PDT) of thick skin lesions is limited by topical drug uptake. Ablative fractional resurfacing (AFR) creates vertical channels that may facilitate topical PDT drug penetration and improve PDT-response in deep skin layers. The purpose of this study was to evaluate whether pre-treating the skin with AFR before topically applied methyl aminolevulinate (MAL) could enable a deep PDT-response. MATERIALS AND METHODS: Yorkshire swine were treated under general anesthesia with a fractional CO(2) laser using stacked single pulses of 3 milliseconds, 91.6 mJ per pulse and subsequent topical MAL application for 3 hours (Metvix®). Red light (LED arrays) was then delivered at fluences of 37 and 200 J/cm(2). Fluorescent photography and microscopy was used to quantify MAL-induced porphyrin distribution and PDT-induced photobleaching at the skin surface and five specific depths down to 1,800 µm. RESULTS: Laser-ablated channels were approximately 1,850 µm deep, which significantly increased topical MAL-induced porphyrin fluorescence (hair follicles, dermis, P < 0.0001) and PDT response, both superficially and deep, versus topical MAL application alone. The fraction of porphyrin fluorescence lost by photobleaching was slightly less after 37 J/cm(2) than after 200 J/cm(2) (overall median values 67-90%; 37 vs. 200 J/cm(2), P > 0.05 for all but one comparison). Photobleaching was steady throughout skin layers and did not vary significantly with skin depth at either LED fluence (P > 0.05). CONCLUSIONS: AFR greatly facilitates topical MAL-induced porphyrins and the fraction of photobleached porphyrins is similar for superficial and deep skin. These observations are consistent with AFR-enhanced uptake of MAL, increased porphyrin synthesis, and photodynamic activation of deep porphyrins even at the lower fluence of 37 J/cm(2), widely used in clinical practice. AFR appears to be a clinically practical means for improving PDT deep into the skin. Clinical studies are suggested to evaluate selectivity in targeting dysplastic cell types.

Resonance Raman study of the dark-adapted form of the purple membrane protein.

The resonance Raman spectrum of the dark-adapted form of the purple membrane protein (bacteriorhodopsin) has been obtained and is compared to the light-adapted pigment and model chromophore spectra. As in the light-adapted form, the chromophore-protein linkage is found to be a protonated Schiff base. Electron delocalization appears to play the dominant role in color regulation. The dark-adapted spectrum indicates a conformation closer to 13-cis than the light-adapted spectrum.

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).

Custom designed acoustic pulses.

We have used a tunable, infrared, free-electron laser with a Pockels cell controlled pulse duration to generate photoacoustic pulses with separate variable rise times (from 15 to 100 ns), durations (100-400 ns), and amplitudes (0.005-0.1 MPa). The tunability of the free-electron laser across water absorption bands allows the rise time of the thermal-elastically generated acoustical pulsed to be varied, while a Pockels cell controls the duration and cross polarizers control the pressure amplitude. The mechanical effects of pressure transients on biological tissue can have dramatic consequences. In addition to cell necrosis, carefully controlled pressure transients can also be used for therapeutic applications, such as drug delivery and gene therapy. This technique permits systemic probing of how biological tissue is affected by stress transients. © 1999 Society of Photo-Optical Instrumentation Engineers.

Fluorescence excitation spectroscopy for the measurement of epidermal proliferation.

Fluorescence excitation spectroscopy was used to assess cellular turnover in human skin by monitoring changes of endogenous fluorescence. Epidermal proliferation was induced with alpha-hydroxy acids. Commercially available glycolic acid creams (8 and 4% wt/wt concentration) and a vehicle cream (placebo) were applied in a randomized double blinded fashion on subjects' forearms, twice daily for 21 days. Excitation spectra were recorded (excitation 250-360 nm, emission 380 nm) at days 0, 1, 3, 7, 10, 11, 14, 17 and 21. The 295 nm excitation band (assigned to tryptophan moieties) was used in this study as a marker for cellular proliferation. To further reduce the day-to-day variability of the skin fluorescence the intensity of the 295 nm band was normalized to the 334 nm band (assigned to collagen crosslinks). The fluorescence emission intensity from placebo-treated skin remained practically unchanged over the period of the measurements while the fluorescence intensity measured from the glycolic acid-treated skin increased monotonically with treatment. The rate of increase of the excitation intensity with treatment was found to be dose dependent. The epidermal 295 nm band may be used as a quantitative marker to monitor the rate of proliferation of epidermal keratinocytes noninvasively.

Physical characteristics and biological effects of laser-induced stress waves.

Laser-induced stress waves can be generated by one of the following mechanisms: optical breakdown, ablation, or rapid heating of an absorbing medium. These three modes of laser interaction with matter allow the investigation of cellular and tissue responses to stress waves with different characteristics and under different conditions. The effects of stress waves on cells and tissues can be quite disparate. Stress waves can fracture tissue, kill cells, decrease cell viability and increase the permeability of the plasma membrane. They can induce deleterious effects during medical procedures of high power, short pulse lasers or, alternatively, may facilitate new therapeutic modalities, such as drug delivery and gene therapy. This review covers the generation of laser-induced stress waves and their effects on cell cultures and tissue.

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).

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.

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.

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.

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.

A rapid method to detect dried saliva stains swabbed from human skin using fluorescence spectroscopy.

Saliva on skin is important in forensic trace evidence. If areas where saliva is present can be outlined, this may lead to DNA analysis and identification. This study describes a rapid and non-destructive method to detect dried saliva on the surface of the skin by fluorescence spectroscopy. Eighty-two volunteers deposited samples of their own saliva on the skin of their ventral forearm. A control sample of water was deposited at three different sites on the contralateral arm. Saliva and water control were then allowed to air-dry. Swab samples were taken from dried saliva and control sites and were dissolved in 0.1M KCl solution. Emission spectra were obtained from the solution and were characterized by a principal maximum at 345-355nm with excitation at 282nm. The fluorescence emission intensity was greater than background readings obtained from the control swab site in 80 of 82 volunteers (approximately 97.6%). The fluorescence profile of saliva samples were similar to those obtained from aqueous samples of pure amylase and tryptophan, an endogenous fluorophore in alpha-amylase. The presence of an emission peak at 345-355nm with excitation at 282nm could provide a strong presumptive indication of saliva deposition.

Spectroscopic determination of skin viability. A predictor of postmortem interval.

We have demonstrated that skin viability decreases at a measurable rate following death in an animal model. The decreased skin viability was measured by fluorescein diacetate and ethidium bromide using fluorescence emission spectroscopy. There is significant decrease of the fluorescence intensity of the fluorescein diacetate assay between the 1-4 h, the 6-24 h, and the >40 h time points postmortem. For times between 6-24 h and >40 h postmortem the ethidium bromide assay showed consistent and significant increases in signal. The fluorescence measurements in this study showed that under the experimental conditions the time of death could be determined for <4, 6-24, and >40 hapotmotrem. The application of these assays in the field will require further study of the environmental factors.

Cytoplasmic molecular delivery with shock waves: importance of impulse.

Cell permeabilization using shock waves may be a way of introducing macromolecules and small polar molecules into the cytoplasm, and may have applications in gene therapy and anticancer drug delivery. The pressure profile of a shock wave indicates its energy content, and shock-wave propagation in tissue is associated with cellular displacement, leading to the development of cell deformation. In the present study, three different shock-wave sources were investigated; argon fluoride excimer laser, ruby laser, and shock tube. The duration of the pressure pulse of the shock tube was 100 times longer than the lasers. The uptake of two fluorophores, calcein (molecular weight: 622) and fluorescein isothiocyanate-dextran (molecular weight: 71,600), into HL-60 human promyelocytic leukemia cells was investigated. The intracellular fluorescence was measured by a spectrofluorometer, and the cells were examined by confocal fluorescence microscopy. A single shock wave generated by the shock tube delivered both fluorophores into approximately 50% of the cells (p < 0.01), whereas shock waves from the lasers did not. The cell survival fraction was >0.95. Confocal microscopy showed that, in the case of calcein, there was a uniform fluorescence throughout the cell, whereas, in the case of FITC-dextran, the fluorescence was sometimes in the nucleus and at other times not. We conclude that the impulse of the shock wave (i.e., the pressure integrated over time), rather than the peak pressure, was a dominant factor for causing fluorophore uptake into living cells, and that shock waves might have changed the permeability of the nuclear membrane and transferred molecules directly into the nucleus.

Picosecond grating spectroscopy for characterizing the acoustic properties of biological material.

We present measurements of the ultrasound attenuation and sound velocity of a number of liquids, transparent biological materials (the vitreous and lens of the bovine eye), and biological fluids (whole blood) at frequencies between 925 and 1020 MHz by using a picosecond thermal grating. Sound velocity and attenuation measurements of liquids (e.g., methanol and ethanol) agree very well with those reported in the literature. The sound velocity in the biological materials studied also agrees with the reported values in the literature. In contrast, the attenuation coefficients measured for biological materials, 2000-5000 dB/cm, are much higher than would be extrapolated from published low-frequency data.

Rapid allergen delivery with photomechanical waves for inducing allergic skin reactions in the hairless guinea pig animal model.

BACKGROUND: Patch testing is the confirmatory procedure for allergic contact dermatitis. The test requires the application of chemicals under occlusion for approximately 48 hours to maximize penetration, although it can also produce irritation. Photomechanical waves (PW) have been shown to render the stratum corneum transiently permeable and facilitate the delivery of macromolecules into the epidermis. This alternative might reduce prolonged occlusion of the skin to minimize irritancy, while retaining the sensitivity of the test. OBJECTIVE: PW was used to facilitate the delivery of an allergen into the skin in vivo. METHODS: The allergic skin reaction using PW delivery was compared with 5-minute and 21-hour occlusion in a sensitized hairless albino guinea pig model. The pigs were sensitized by intradermal injection of (0.01%) dinitrochlorobenzene (DNCB) and topical administration (0.1%, 1 week later) of the hapten. One month later, testing for the allergic response was performed by the administration with PW of 10 microL of 0.1% DNCB. RESULTS: Our results show that there was an allergic reaction for the 24 hour occlusion or PW delivery of the antigen. In contrast, no response was observed for the 5-minute occlusion with the antigen. CONCLUSION: The rapid delivery of antigens with PW can improve the test for the diagnosis of contact dermatitis.

Fluorescence relaxation kinetics from rhodopsin and isorhodopsin.

The fluorescence kinetics of bovine rhodopsin and isorhodopsin excited with a single picosecond laser pulse have been measured with a streak camera. The rise and the decay time of the intrinsic fluorescence emission from rhodopsin and isorhodopsin are found to be <12 ps.

Photomechanical drug delivery into bacterial biofilms.

PURPOSE: To investigate whether photomechanical waves generated by lasers can increase the permeability of a biofilm of the oral pathogen Actinomyces viscosus. METHODS: Biofilms of Actinomyces viscosus were formed on bovine enamel surfaces. The photomechanical wave was generated by ablation of a target with a Q-switched ruby laser and launched into the biofilm in the presence of 50 microg/ml methylene blue. The penetration depth of methylene blue was measured by confocal scanning laser microscopy. Also, the exposed biofilms were irradiated with light at 666 nm. After illumination, adherent bacteria were scraped and spread over the surfaces of blood agar plates. Survival fractions were calculated by counting bacterial colonies. RESULTS: Confocal scanning laser microscopy revealed that a single photomechanical wave was sufficient to induce a 75% increase in the penetration depth of methylene blue into the biofilm. This significantly increased the concentration of methylene blue in the biofilm enabling its photodestruction. CONCLUSIONS: Photomechanical waves provide a potentially powerful tool for drug delivery that might be utilized for treatment of microbial infections.

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.

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.