Apostolos G. Doukas

Fractional CO2 laser‐assisted drug delivery

Ablative fractional resurfacing (AFR) creates vertical channels that might assist the delivery of topically applied drugs into skin. The purpose of this study was to evaluate drug delivery by CO2 laser AFR using methyl 5‐aminolevulinate (MAL), a porphyrin precursor, as a test drug.

Photodynamic treatment of endodontic polymicrobial infection in vitro

We investigated the photodynamic effects of methylene blue on multispecies root canal biofilms comprising Actinomyces israelii, Fusobacterium nucleatum subspecies nucleatum, Porphyromonas gingivalis, and Prevotella intermedia in experimentally infected root canals of extracted human teeth in vitro. The 4 test microorganisms were detected in root canals by using DNA probes. Scanning electron microscopy showed the presence of biofilms in root canals before therapy. Root canal systems were incubated with methylene blue (25 microg/mL) for 10 minutes followed by exposure to red light at 665 nm with an energy fluence of 30 J/cm(2). Light was delivered from a diode laser via a 250-microm diameter polymethyl methacrylate optical fiber that uniformly distributed light over 360 degrees. Photodynamic therapy (PDT) achieved up to 80% reduction of colony-forming unit counts. We concluded that PDT can be an effective adjunct to standard endodontic antimicrobial treatment when the PDT parameters are optimized.

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.

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.

Phototargeting Oral Black-Pigmented Bacteria

ABSTRACT We have found that broadband light (380 to 520 nm) rapidly and selectively kills oral black-pigmented bacteria (BPB) in pure cultures and in dental plaque samples obtained from human subjects with chronic periodontitis. We hypothesize that this killing effect is a result of light excitation of their endogenous porphyrins. Cultures of Prevotella intermedia and P. nigrescens were killed by 4.2 J/cm2, whereas P. melaninogenica required 21 J/cm2. Exposure to light with a fluence of 42 J/cm2 produced 99% killing of P. gingivalis. High-performance liquid chromatography demonstrated the presence of various amounts of different porphyrin molecules in BPB. The amounts of endogenous porphyrin in BPB were 267 (P. intermedia), 47 (P. nigrescens), 41 (P. melaninogenica), and 2.2 (P. gingivalis) ng/mg. Analysis of bacteria in dental plaque samples by DNA-DNA hybridization for 40 taxa before and after phototherapy showed that the growth of the four BPB was decreased by 2 and 3 times after irradiation at energy fluences of 4.2 and 21 J/cm2, respectively, whereas the growth of the remaining 36 microorganisms was decreased by 1.5 times at both energy fluences. The present study suggests that intraoral light exposure may be used to control BPB growth and possibly benefit patients with periodontal disease.

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.

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

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.

Non-invasive determination of shock wave pressure generated by optical breakdown

Shock waves generated by a laser-induced plasma were investigated using a pump-and-probe technique. Both 7-ns and 40-ps laser pulses at 1.06 μm were employed to initiate breakdown in water. Two He-Ne laser beams were used as a velocity probe, allowing the accurate measurement of the shock velocity around the plasma. The maximum shock pressure was determined from the measured shock velocities, the jump condition and the equation of state for water. The conservation of the total momentum of the shock front was used to derive expressions for the shock velocity, particle velocity and shock pressure vs. the distance (r) from the center of the plasma. For a shock wave of spherical symmetry, the shock pressure is proportional to 1/r2. Our work shows that the expanding plasma initially induces a shock wave; the shock wave dissipates rapidly becoming an acoustic wave within 300–500 μm.

Shock wave-mediated molecular delivery into cells

A single shock wave generated by a shock tube is able to effectively deliver macromolecules such as fluorescein isothiocyanate-dextran into the cytoplasm of living cells without causing cytotoxicity. We report on the effect of varying the molecular weight of the dextran and the number of shock waves on the efficiency of delivery into a cancer cell line. The fraction of cells permeabilized and the total fluorescence delivered were measured by flow cytometry, and the cellular viability by a tetrazolium assay on adherent cells and these values were compared to cell permeabilization using digitonin. Shock waves can deliver molecules of up to 2000000 molecular weight into the cytoplasm of cells without toxicity and may have applications in gene therapy.