Darrell B. Tata

Near-IR induced suppression of metabolic activity in aggressive cancers

The role of low light intensity in suppressing metabolic activity of transformed cell lines was investigated through the applications of a 1,552nm wavelength pulsed picosecond laser. Human malignant glioblastoma, human leukemia HL-60, and the NIH 3T3 cell lines were used. The cells were grown in 96 well plates and exposed in their respective growth culture media with 10% (v/v) fetal bovine serum under various fluence exposure conditions ranging from 0.115 - 100 J/cm2. All cell lines were exposed at a constant average intensity value of 0.115 W/cm2; 25 kHz repetition rate with 1.6 micro-joule per pulse; pulse duration = 2.93 picosecond. The human malignant glioblastoma and the HL-60 cell lines exhibited a monotonic decline in metabolic activity (down 50 - 60%) relative to their respective sham exposed control counterparts between the fluence values of 0.115 J/cm2 to 10 J/cm2. The NIH 3T3 cells exhibited a maximum suppression of metabolic activity at the fluence value of 50 J/cm2. Metabolic activity was measured through the colorimetric MTS metabolic assay. Interestingly, for all cell lines the metabolic activity was found to return back to the sham exposed control levels as the fluence of exposure was increased up to 100J/cm2.

Proceedings of Light-Activated Tissue Regeneration and Therapy Conference

The Proceedings of the Light-Activated Tissue Regeneration and Therapy Conference covers the latest advances in the field including measurements that help determine the mechanisms of lightactivated tissue regeneration and therapy. It also covers light sources that include lasers, LEDs, two wavelength sources, broadband sources and the metrology and medical outcomes they produce. These proceedings offer a systematic approach to the field of Light-Activated Tissue Regeneration and Therapy covering theory, basic research and clinical studies.

Fluorometric determination of the redox state and distribution of mitochondria in human malignant glioblastoma cells grown on different culturing substrates.

OBJECTIVES To evaluate the redox state and the spatial distribution of mitochondria in malignant human brain cancer cells grown on different substrates. METHODS Cellular autofluorescence images were obtained through an inverted fluorescence microscope and the redox fluorometric ratio was evaluated (after the subtraction of background) as the net fluorescence signal through the DAPI filter divided by the net fluorescence signal through the FITC filter. Spatial mitochondria distribution patterns were evaluated by division of the cell area at the midpoint between the nuclear and cell membranes. The average fluorescence in the central area (CF) was divided by the average fluorescence from the peripheral area (PF). The CF/PF ratios were compared between cells cultured on either poly-D-lysine or collagen I substrates. RESULTS Glioblastoma cells seeded on the collagen-coated plates were observed to proliferate approximately 33-50% faster than the cells seeded on the poly-D-lysine-coated plates. Consistent with the proliferation findings, the redox ratios were lower for the cells seeded on the collagen-coated plates compared with poly-D-lysine. However, cell size and the percentage of cells with perinuclear mitochondrial distribution were not observed to be different in the cells seeded on the two surfaces. CONCLUSIONS Redox ratio computation by using redox fluorometry is a useful predictor of cellular proliferation.

Near-IR Picosecond Pulsed Laser Induced Suppression of Metabolic Activity in Malignant Human Brain Cancer: An In-Vitro Study

The role of low light intensity in suppressing metabolic activity of malignant human brain cancer (glioblastoma) cell line was investigated through the application of a 1,552 nm wavelength pulsed picosecond laser. Human glioblastomas were grown in T-75 flasks and were utilized when the cells were 50–70% confluent and thereafter transferred into 96 well plates and exposed in their growth culture medium with serum under various energy doses (i.e., fluence) ranging from 0.115–50 J/cm2. All exposure doses were reached with an average intensity of 0.115 W/cm2; 25 kHz repetition rate with 1.6 μJ per pulse; pulse duration = 2.93 ps. The glioblastomas exhibited a maximal decline in the metabolic activity (down 50–60%) relative to their respective sham exposed control counterparts between the fluence dose values of 5.0–10 J/cm2. The cellular metabolic activities for various treatment doses were measured through the colorimetric MTS metabolic assay 3 days after the laser exposure. Interestingly, the metabolic activity was found to return back to the sham exposed control levels as the fluence of exposure was increased up to 50 J/cm2. Addition of (the enzyme) Catalase in the growth medium prior to the laser exposure was found to diminish the laser induced metabolic suppression for all fluence treatment conditions, thus suggesting a functional role of H2O2 in the metabolic suppression. In view of this evidence, a hypothesis is formulated which attributes the classical biphasic response, in part, to the light induced production of H2O2. Furthermore, it was observed that if the glioblastoma cells were allowed to reach 100% confluency within the T-75 flasks the characteristic laser induced metabolic suppression was found to be severely abrogated. Exploratory steps were also undertaken to maximize the suppression in the metabolic activity through repetitive laser dose of exposure every 24 hours for 3 consecutive days. In addition, the efficacy in the metabolic suppression of the 1,552 nm pulsed laser was also compared to a continuous wave broad band continuous wave heating lamp source channeled through a fiber-optic bundle with identical intensity of exposure. Taken together, our findings reveal that near-IR low level light exposures could potentially be a viable tool in reducing the metabolic activity of cancers; however, due to the cellular “biphasic” response to the non-ionizing irradiation, further research needs to be undertaken to determine exposure parameters which would optimize metabolic and cellular growth suppression in-vivo.

In-vitro suppression of metabolic activity in malignant human glioblastomas due to pulsed - low frequency electric potential exposures

The role of pulsed - low repetition frequency electric potential was investigated in suppressing the metabolic activities of aggressive human brain cancer cells. Twenty four hours post exposure the glioblastomas were found to be significantly inhibited in their metabolic activity. The findings herein reveal a near complete inhibition of glioblastomas metabolic activity through selective applications of low frequency pulsed electric potentials.

He-Ne laser enhanced cellular hydrogen peroxide production and induced modulations in metabolic activity in malignant human brain cancer: Evidence for a “by-stander” effect

Continuous-wave He-Ne laser exposures (Intensity=35 mW/cm2, λ=632.8nm, Fluence range: 1J/cm2 to 50 J/cm2) on non-confluent and actively dividing human malignant glioblastoma cells was found to increase the cellular production levels of H2O2. Modulations in the cellular metabolic activity were detected (through the MTS assay) three days after laser irradiation. The metabolic activity was found to be dependent on the laser dose of exposure (i.e., fluence). In addition, three days after the laser exposure, the potential laser induced “bystander” effect was tested through the transfer of growth media from laser irradiated cells onto non-irradiated cells. After two additional days of incubation (5 days post exposure), the non-laser irradiated cells were found to have a significant increase in their metabolic activities. Modulations in the metabolic activities in the non-irradiated cells were found to be fluence dependent from the initial laser exposed cells treatment conditions. The results herein support the hypothesis of an important functional role for light enhanced cellular H2O2 generation to yield bio-modulatory effects locally and at a distance. The classical “bi-phasic” modulation response of cells to light irradiation is hypothesized to depend upon the quantity of light-enhanced H2O2 molecules generated from the mitochondria and the number of cells which interact with the H2O2 molecules.

Independent Applications of Near-IR Broadband Light Source and Pulsed Electric Potential in the Suppression of Human Brain Cancer Metabolic Activity: An In-Vitro Study

The roles of two different non-conventional techniques in suppressing the metabolic activity of a malignant human brain cancer (glioblastoma) cell line were explored through the application of (i) pulsed electric field, or (ii) independent application with a continuous wave broadband near infrared light channeled through a fiber-optic bundle exposing cancer cells within growth medium. Human glioblastomas were grown in T-75 flasks and were utilized when the cells were 50–70% confluent. The cells were either transferred into 96 well plates for exposures through a fiber bundle, or into 1.4 ml sterile eppendoff tubes for exposures to pulsed electric potential. The glioblastomas within wells were light exposed through a fiber bundle at an average intensity of 0.115 W/cm2 from the underside of the well, with the light dose (fluence) values ranging from 0.115—50 J/cm2. Glioblastomas exhibited a maximal decline in the metabolic activity (down 80%) relative to their respective sham exposed control counterparts between the fluence dose values of 5.0–10 J/cm2. The cellular metabolic activities for various treatment doses were measured through the colorimetric MTS metabolic assay 3 days after the broadband near infrared light exposure. Interestingly, the metabolic activity was found to return back to the (sham exposed) control levels as the fluence of exposure was increased up to 50 J/cm2. Glioblastomas in suspension within sterile eppendoff tubes were exposed to pulsed electrical potential fluctuations: rectangular pulse width = 250 ms with pulse amplitude = 100 V, with 8 square pulses per burst, 2 bursts per second. A time course study of treatment exposure revealed a complete obliteration of glioblastomas for in-vitro treatment duration beyond 7 min.

Analysis of biomodulative effects of low-intensity laser on human skin fibroblast cells using fiber optic nano-probes

Over the past few decades, many efforts were devoted to study low power laser and cellular interaction. Some of the investigations were performed on cell populations. In this work fiber-optic based nano-probe is used for the precise delivery of laser light on to a single cell and the mechanism of light interaction with the cell during irradiation was studied. A human skin fibroblast cell line was utilized in this investigation. The human fibroblasts were irradiated under two different schemes of exposure: (1) entire cell population was irradiated within a Petri dish using a fan beam, (2) laser energy was precisely delivered on to a single cell using fiber-optic nano-probe. Studies were conducted by variation of laser intensity, exposure time, and the energy dose of exposure. Proliferative effect of laser irradiation was determined through cell counting for both exposure schemes. Enhancement of the rate of proliferation was observed to be dependent on laser parameters and method of laser delivery. Variation of total energy dose had greater effect on the enhancement of the rate of cellular proliferation compared to that of laser intensity. The photobiostimulative effect was also observed to have a finite life-time. Fluorescent life-time imaging of reactive oxygen species (ROS) was performed during the single cell exposure method. ROS generation was found to depend strongly on both laser energy doses and irradiation time. It is demonstrated in this communication that by using specially engineered nano-probes, laser light can be precisely delivered on to a targeted single cell.

Non-Thermal Dental Ablation Using Ultra-Short Pulsed Near Infrared Laser

Ultra-short pulsed lasers are known for their ability to precisely machine materials including human hard and soft tissues while minimizing the amount of thermal energy deposited to the surroundings. Non-thermal ablation produced by ultra-short pulsed lasers in the femtosecond to picosecond range is very effective for dental applications. As shown in Figure 1, most decay occurs in the dentin, which is found between the outer surface (enamel) and the inner region containing the nerve endings (pulp) [1]. Caries removal and the preparation of cavities in dentistry are primarily performed by the use of mechanical drills. The current techniques are invasive and cause patient discomfort. Due to the vibrations of the drills it is necessary to use local anesthetic for the majority of dental procedures. A continuous water spray is used in conjunction with the drills to balance the temperature rise produced by mechanical vibrations. Drills are somewhat limited in precision causing a large amount of healthy tooth to be lost during any restoration process. Replacing a multitude of mechanical tools with one non-invasive, accurate and painless laser treatment will be a huge advancement to the current dental techniques.Copyright