A label-free non-contact test methodology utilizing fiber-optic-based Fourier Transform Infrared (FTIR) spectroscopy has been developed to identify potential biomarkers in cellular systems, which could be associated with disease processes or therapeutic effectiveness. As a biological in-vitro model, different metabolically active cell lines were considered in this study. FTIR absorption spectra of aggressive human glioblastoma (brain cancer) and human melanoma cell lines were compared to normal human skin fibroblasts. A ~25-cm -1 shift in the absorption peak was observed between normal (~1153 cm -1 ) and malignant cell lines (~1177 cm -1 ). Furthermore, the addition of 30% H 2 O 2 to normal skin cells produced a similar 25-cm -1 shift in the 1153-cm -1 absorption peak from the normal cells to the 1177 cm -1 found in the malignant cells, thus implicating the presence of large endogenous levels of oxidizers such as H 2 O 2 within the cancer cells which could be responsible in producing this particular peak shift. The observed differences in the absorption spectra between normal and cancer cell lines could potentially be used to identify biomarkers within the absorption wavenumber range of 900-1300 cm -1 (7.7-11.1 μm wavelength range).

We recently reported laser-triggered release of photosensitive compounds from liposomes containing dipalmitoylphosphatidylcholine (DPPC) and 1,2 bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC(8,9)PC). We hypothesized that the permeation of photoactivated compounds occurs through domains of enhanced fluidity in the liposome membrane and have thus called them "Pocket" liposomes. In this study we have encapsulated the red light activatable anticancer photodynamic therapy drug 2-(1-Hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH) (Ex/Em410/670 nm) together with calcein (Ex/Em490/517 nm) as a marker for drug release in Pocket liposomes. A mole ratio of 7.6:1 lipid:HPPH was found to be optimal, with >80% of HPPH being included in the liposomes. Exposure of liposomes with a cw-diode 660 nm laser (90 mW, 0-5 minutes) resulted in calcein release only when HPPH was included in the liposomes. Further analysis of the quenching ratios of liposome-entrapped calcein in the laser treated samples indicated that the laser-triggered release occurred via the graded mechanism. In vitro studies with MDA-MB-231-LM2 breast cancer cell line showed significant cell killing upon treatment of cell-liposome suspensions with the laser. To assess in vivo efficacy, we implanted MDA-MB-231-LM2 cells containing the luciferase gene along the mammary fat pads on the ribcage of mice. For biodistribution experiments, trace amounts of a near infrared lipid probe DiR (Ex/Em745/840 nm) were included in the liposomes. Liposomes were injected intravenously and laser treatments (90 mW, 0.9 cm diameter, for an exposure duration ranging from 5-8 minutes) were done 4 hours postinjection (only one tumor per mouse was treated, keeping the second flank tumor as control). Calcein release occurred as indicated by an increase in calcein fluorescence from laser treated tumors only. The animals were observed for up to 15 days postinjection and tumor volume and luciferase expression was measured. A significant decrease in luciferase expression and reduction in tumor volume was observed only in laser treated animal groups injected with liposomes containing HPPH. Histopathological examination of tumor tissues indicated tumor necrosis resulting from laser treatment of the HPPH-encapsulated liposomes that were taken up into the tumor area.

We recently reported on the physical characteristics of photo-triggerable liposomes containing dipalmitoylphosphatidylcholine (DPPC), and 1,2-bis (tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC(8,9)PC) carrying a photo agent as their payload. When exposed to a low-intensity 514 nm wavelength (continuous-wave) laser light, these liposomes were observed to release entrapped calcein green (Cal-G; Ex/Em 490/517 nm) but not calcein blue (Cal-B; Ex/Em 360/460 nm). In this study, we have investigated the mechanism for the 514 nm laser-triggered release of the Cal-G payload using several scavengers that are known specifically to inhibit either type I or type II photoreaction pathways. Liposomes containing DPPC:DC(8,9)PC: distearoylphosphatidylethanolamine (DSPE)-polyethylene glycol (PEG)-2000 (86:10:04 mole ratio) were loaded either with fluorescent (calcein) or nonfluorescent ((3)H-inulin) aqueous markers. In addition, a non-photo-triggerable formulation (1-palmitoyl-2-oleoyl phosphatidylcholine [POPC]:DC(8,9)PC:DSPE-PEG2000) was also studied with the same payloads. The 514 nm wavelength laser exposure on photo-triggerable liposomes resulted in the release of Cal-G but not that of Cal-B or (3)H-inulin, suggesting an involvement of a photoactivated state of Cal-G due to the 514 nm laser exposure. Upon 514 nm laser exposures, substantial hydrogen peroxide (H2O2, ≈100 μM) levels were detected from only the Cal-G loaded photo-triggerable liposomes but not from Cal-B-loaded liposomes (≤10 μM H2O2). The Cal-G release from photo-triggerable liposomes was found to be significantly inhibited by ascorbic acid (AA), resulting in a 70%-80% reduction in Cal-G release. The extent of AA-mediated inhibition of Cal-G release from the liposomes also correlated with the consumption of AA. No AA consumption was detected in the 514 nm laser-exposed Cal B-loaded liposomes, thus confirming a role of photoactivation of Cal-G in liposome destabilization. Inclusion of 100 mM K3Fe(CN)6 (a blocker of electron transfer) in the liposomes substantially inhibited Cal-G release, whereas inclusion of 10 mM sodium azide (a blocker of singlet oxygen of type II photoreaction) in the liposomes failed to block 514 nm laser-triggered Cal-G release. Taken together, we conclude that low-intensity 514 nm laser-triggered release of Cal-G from photo-triggerable liposomes involves the type I photoreaction pathway.

We present evidence that real-time optical monitoring of human brain cancer cells through forward Raman spectroscopy can be used as a highly sensitive non-invasive label-free tool in acquiring real-time information on the cellular metabolic activity through the concentration levels of selective biomarkers. In this investigation, we quantified the concentration level of a natural by-product of cellular respiration, hydrogen peroxide (H2O2), through its characteristic Raman signature due to the transitions of O-O vibration energy level, resulting in a sharp Raman scatter frequency shift around 880cm-1. The experimental findings revealed that 3.75´106 cells to have produced a substantial amount (3%) of H2O2.

We have previously reported on a novel class of light-triggerable liposomes prepared from a photopolymerizable phospholipid DC8,9PC (1,2- bis (tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine) and DPPC (1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine)1,2. UV radiation (254 nm) exposure on the liposomes resulted in photopolymerization of DC8,9PC which subsequently resulted in the release of contents (see abstract at this meeting by Puri et al). Treatment of these liposomes by 514 nm laser light is also found to promote the release of encapsulated calcein (Ex/Em 490/517 nm) or (an anticancer drug) Doxorubicin (Ex/Em 490/590 nm); however the onset of this release is dramatically rapid and is found to occur within 1-2 minutes of laser treatment in contrast to greater than 10 minutes after the 254 nm UV exposure.

INTRODUCTION:In this communication we report on a novel non-invasive methodology in utilizing "soft" energy diagnostic X-rays to indirectly activate a photo-agent utilized in photodynamic therapy (PDT): Photofrin II (Photo II) through X-ray induced luminescence from Gadolinium Oxysulfide (20 micron dimension) particles doped with Terbium: Gd_{2}O_{2}S:Tb. Photodynamic agents such as Photo II utilized in PDT possess a remarkable property to become preferentially retained within the tumor's micro-environment. Upon the photo-agent's activation through (visible light) photon absorption, the agents exert their cellular cytotoxicity through type I and type II pathways through extensive generation of reactive oxygen species (ROS); namely, singlet oxygen ^{1}O_{2}, superoxide anion O_{2}^{-}, and hydrogen peroxide H_{2}O_{2}, within the intra-tumoral environment. Unfortunately, due to shallow visible light penetration depth (∼ 2 mm to 5 mm) in tissues, the current PDT strategy has largely been restricted to the treatment of surface tumors, such as the melanomas. Additional invasive strategies through optical fibers are currently utilized in getting the visible light into the intended deep seated targets within the body for PDT. METHODS:X-ray induced visible luminescence from Gd_{2}O_{2}S:Tb particles were spectroscopically characterized, and the potential in-vitro cellular cytotoxicity of Gd_{2}O_{2}S:Tb particles on human glioblastoma cells (due to 48 Hrs Gd_{2}O_{2}S:Tb particle exposure) was screened through the MTS cellular metabolic assay. In-vitro human glioblastoma cellular exposures in presence of Photo II with Gd_{2}O_{2}S:Tb particles were performed in the dark in sterile 96 well tissue culture plates, and the corresponding changes in the metabolic activities of the glioblastoma due to 15 minutes of (diagnostic energy) X-ray exposure was determined 48 Hrs after treatment through the MTS assay. RESULTS:Severe suppression (> 90% relative to controls) in the cellular metabolic activity of human glioblastoma was measured due to the treatment of clinically relevant concentrations of 20 μg/ml Photo II, with Gd_{2}O_{2}S:Tb particles, and (120 kVp) diagnostic X-rays. Taken together, the in-vitro findings herein provide the basis for future studies in determining the safety and efficacy of this non-invasive X-ray induced luminescence strategy in activating photo-agent in deep seated tumors.

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.

Civilizations in antiquity have endeavored to champion the use of light to treat illnesses in the human body. Although laser light has unique applications in medicine, its mechanism of action for low intensity exposures on cells, tissues, and the body continues to be controversial after nearly 50 years of investigations. This paper presents evidence for visible red and near infrared light to induce (indirect) generation of hydrogen peroxide as an important chemical messenger behind the stimulatory and inhibitory responses observed to low intensity light exposures. It is hypothesized that other ionizing or non‐ionizing modalities can either directly or indirectly induce / or generate H 2 O 2 in an aqueous environment and could also bring about similar stimulatory or inhibitory bio‐effects. One salient parameter which governs the bio‐response is the level of generated H 2 O 2 . The authors discuss the mechanism which enables a small amount of hydrogen peroxide generated by light to produce beneficial effects.

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 glioblastoma's metabolic activity through selective applications of low frequency pulsed electric potentials.

Photodynamic agents such as Photofrin II (Photo II) utilized in photodynamic therapy (PDT) possess a remarkable property to become preferentially retained within the tumor's micro-environment. Upon the photo-agent's activation through visible light photon absorption, the agents exert their cellular cytotoxicity through type II and type I mechanistic pathways through extensive generation of reactive oxygen species (ROS): singlet oxygen 1O2, superoxide anion O2 -, and hydrogen peroxide H2O2, within the intratumoral environment. Unfortunately, due to shallow visible light penetration depth (~2mm to 5mm) in tissues, the PDT strategy currently has largely been restricted to the treatments of surface tumors, such as the melanomas. Additional invasive strategies through optical fibers are currently utilized in getting the visible light into the intended deep seated targets within the body for PDT. In this communication, we report on a novel strategy in utilizing "soft" energy diagnostic X-rays to indirectly activate Photo II through X-ray induced luminescence from Gadolinium oxysulfide (20 micron dimension) particles doped with Terbium: Gd2O2S:Tb. X-ray induced visible luminescence from Gd2O2S:Tb particles was spectroscopically characterized and the ROS production levels from clinically relevant concentration (10 μg/ml) of Photo II was quantified through changes in the Vitamin C absorbance. ROS kinetics through X-ray induced luminescence was found to be similar to the ROS kinetics from red He-Ne laser exposures used in the clinics. Taken together, in-vitro findings herein provide the basis for future studies in determining the safety and efficacy of this non-invasive X-ray induced luminescence strategy in activating photo-agent in deep seated tumors.

Enhanced generation of H2O2, modulations in metabolic activity, and the “by-stander” effect from malignant human brain cancer cells due to red He-Ne laser exposures have been quantified and found to be light exposure dose dependent. The findings herein support evidence for an important role for light induced H2O2 to mediate biomodulations.

Continuous-wave He-Ne laser exposures (Intensity=35 mW/cm2, lambda=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 ldquobystanderrdquo 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 ldquobi-phasicrdquo 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.

Continuous wave He-Ne laser exposures (Intensity = 35 mW/cm(2), lambda= 632.8nm, Fluence range: 1J/cm(2) to 50 J/cm(2)) on non-confluent and fully confluent 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 the laser irradiation. The metabolic activity was found to be dependent on the laser fluence for both cell growth conditions. Furthermore, 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 grown under the non-confluent condition were found to have a significant increase in their metabolic activities, whereas minimal to null response was found for the fully confluent condition. For cells grown under the non-confluent conditions, modulations in the metabolic activities in the non-irradiated cells were found to be laser fluence dependent from the initial laser exposed cells treatment conditions. The results herein support the hypothesis of an important 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.

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.

The role of low visible or near infra-red laser intensity in suppressing metabolic activity of malignant human brain cancer (glioblastoma) cells was investigated through the application of either a continuous wave 633nm HeNe or a pulsed picosecond 1,552nm wavelength laser. Human glioblastomas were exposed in their growth culture medium with serum for several energy doses. For both types of laser exposures the glioblastomas exhibited a maximal decline in the metabolic activity relative to their respective sham control counterparts at 10 J/cm(2). The cellular metabolic activities for various treatment doses were measured through the colorimetric NITS metabolic assay after the laser exposure. Interestingly, 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 fanctional role of H2O2 in the metabolic suppression. Taken together, our findings reveal that visible or near infra-red low level light exposures could potentially be a viable tool in reducing the metabolic activity of cancers; evidence at hand implicates a role of light induced H2O2 in bringing about in part, suppression in the metabolic activity. Due to the cellular "biphasic" response to the laser exposure, further research needs to be undertaken to determine exposure parameters which would optimize metabolic and cellular growth suppression in-vivo.

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.