Photodynamic therapy of human glioma spheroids using 5-aminolevulinic acid

The response of human glioma spheroids to 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) is investigated. A two-photon fluorescence microscopy technique is used to show that human glioma cells readily convert ALA to protoporphyrin IX throughout the entire spheroid volume. The central finding of this study is that the response of human glioma spheroids to ALA-mediated PDT depends not only on the total fluence, but also on the rate at which the fluence is delivered. At low fluences (< or = 50 J cm-2), lower fluence rates are more effective. At a fluence of 50 J cm-2, near-total spheroid kill is observed at fluence rates of as low as 10 mW cm-2. The fluence rate effect is not as pronounced at higher fluences (> 50 J cm-2), where a favorable response is observed throughout the range of fluence rates investigated. The clinical implications of these findings are discussed.     

Highly Selective Targeting of Ovarian Cancer with the Photosensitizer PEG-m-THPC in a Rat Model

Photodynamic therapy (PDT) uses light to activate a photosensitizer that has been absorbed or retained preferentially by cancer cells after systemic administration. The first pegylated photosensitizer, tetrakis-(m-methoxypolyethylene glycol) derivative of 7,8-dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)-21,23-[H]-porphyrin (PEG-m-THPC), was evaluated to target selectively unresectable pelvic ovarian cancer bulks. Our goals were two-fold: (1) to establish an ovarian cancer model suitable for the development of debulking techniques and (2) to characterize the pharmacokinetics and tumor selectivity of PEG-m-THPC by fluorescence microscopy. NuTu-19 ovarian cancer cells were injected into the caudal part of the right psoas muscle of Fisher rats. Five weeks later, 30 mg/kg body weight of PEG-m-THPC was injected intravenously. Necropsy was performed between 4 and 10 days following drug application, and fluorescence of the tumor and various abdominal organs was measured. All rats developed bulky pelvic tumors with an average diameter of 2.6 cm (+/- 0.6 SD). Tumor masses were encompassing and infiltrating pelvic organs in a similar manner to ovarian cancers in humans. Fluorescence of cancer tissue was maximal 8-10 days following drug application. At 8 days, the tumor-to-tissue ratio was 40:1 (+/- 12 SE) for most abdominal organs. We conclude that this tumor model may be used for the study of new pelvic debulking techniques, and that the tumor selectivity of PEG-m-THPC is exceptionally high 8 days after drug application. Based on these data, we are currently developing a PDT-based minimally invasive debulking technique for advanced ovarian cancer.     

THE USE OF EXOGENOUS FLUORESCENT PROBES FOR TEMPERATURE MEASUREMENTS IN SINGLE LIVING CELLS

The fluorescent membrane probes 7-nitrobenz-2-oxa-1,3-diazo1-4-y1 (NBD) and 6-dodeca-noy1-2-dimethylamino-naphthalene (laurdan) have been studied for use as optical thermometers in living cells. The thermal sensitivity of NBD is primarily a consequence of rapid, heat-induced electronic changes, which increase the observed fluorescence decay rate. As a result, fluorescence intensity and lifetime variations of membrane-bound NBD-conjugated phospholipids and fatty acids can be directly correlated with cellular temperature. In contrast, laurdan fluorescence undergoes a dramatic temperature-dependent Stokes shift as the membrane undergoes a gel-to-liquid-crystalline phase transition. This facilitates the use of fluorescence spectra to record the indirect effect of microenvironmental changes, which occur during bilayer heating. Microscope and suspension measurements of cells and phospholipid vesicles are compared for both probes using steady-state and fluorescence lifetime (suspension only) data. Our results show that NBD fluorescence lifetime recordings can provide reasonable temperature resolution (approximately 2°C) over a broad temperature range. Laurdan's microenvironmental sensitivity permits better temperature resolution (0.1-1°C) at the expense of a more limited dynamic range that is determined solely by bilayer properties. The temperature sensitivity of NBD is based on rapid intramolecular rotations and vibrations, while laurdan relies on a slower, multistep mechanism involving bilayer rearrangement, water penetration and intermolecular processes. Because of these differences in time scale, NBD appears to be more suitable for monitoring ultrafast phenomena, such as the impact of short-pulse microirradiation on single cells.     

SINGLET OXYGEN GENERATION OF PORPHYRINS, CHLORINS, AND PHTHALOCYANINES

The production of singlet oxygen was measured indirectly for three classes of photosensitizers: porphyrins (Photofrin II, TPPS4), chlorins (MACE, DACE), and a phthalocyanine (CASPc). Buffered solutions of sensitizers and singlet oxygen acceptors were irradiated with a CW dye laser and the oxygen depletion was monitored electrochemically with a Clark-type microelectrode. A comparison of oxygen-depletion rate constants and quantum efficiencies yields the order of efficiency of the sensitizers: TPPS4 greater than MACE greater than PII greater than DACE greater than CASPc. For singlet oxygen acceptors the order was: furfuryl alcohol greater than imidazole greater than tryptophan. CHO cell suspensions were also used as acceptors. Here the order of efficiency (per absorbed photon) was PII greater than MACE approximately CASPc. Expressed in terms of oxygen depletion per cell the order was CASPc approximately PII greater than MACE. When performing cell clonogenicity studies the order of efficiencies, expressed as percentage cell kill per unit weight of sensitizer, was CASPc greater than PII greater than MACE approximately DACE. The discrepancy between the efficiencies of sensitizers to generate singlet oxygen and their cytotoxicity was explained in terms of photodegradation (for the chlorins), intracellular localization (for PII), and contributions from a Type I mechanism (for CASPc).     

Monitoring tumor response during photodynamic therapy using near-infrared photon-migration spectroscopy.

Benzoporphyrin-derivative (BPD)-monoacid-ring A photodynamic therapy (PDT) was performed on subcutaneous tumor implants in a rat ovarian cancer model. In order to assess PDT efficacy the tumor and normal tissue optical properties were measured noninvasively prior to and during PDT using frequency-domain photon migration (FDPM). FDPM data were used to quantify tissue absorption and reduced scattering properties (given by the parameters mu a and mu's, respectively) at four near-infrared (NIR) wavelengths (674, 811, 849 and 956 nm). Tissue physiologic properties, including the in vivo concentration of BPD, deoxy-hemoglobin (Hb), oxy-hemoglobin (HbO2), total hemoglobin (TotHb), water (H2O) and percent tissue hemoglobin oxygen saturation (%StO2), were calculated from optical property data. PDT efficacy was also determined from morphometric analysis of tumor necrosis in histologic specimens. All the measured tumor properties changed significantly during PDT. [Hb] increased by 9%, while [HbO2], [TotHb] and %StO2 decreased by 18, 7 and 12%, respectively. Using histologic data we show that long-term PDT efficacy is highly correlated to mean BPD concentration in tumor and PDT-induced acute changes in [HbO2], [TotHb] and %StO2 (correlation coefficients of 0.829, 0.817 and 0.953, respectively). Overall, our results indicate that NIR FDPM spectroscopy is able to quantify noninvasively and dynamically the PDT-induced physiological effects in vivo that are highly correlated with therapeutic efficacy.     

Fluorescence resonance energy transfer: FRET studies of ligand binding to cell surface receptors

We describe a simple optical system employing fluorescence resonance energy transfer (FRET) to identify potential binding domains on the macrophage scavenger receptor for the ligand maleylated bovine serum albumin (mal-BSA). Using a plasma membrane vesicle system, we placed donor probes on the ligand and acceptor probes in the membrane to determine the distance of bound ligand from the cell surface. Two donors and three acceptors were employed. Transfer between ligand covalently modified with multiple dansyl molecules and hexadecanoylaminoeosin in the membrane yielded a distance of 46.5 ± 7.5 å; transfer from the same type of donors to octadecylrhodamine B in the membrane gave a distance of 58.5 ± 3.0 å. No transfer was observed between ligand mono-labeled with fluorescein and l,l′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanineperchlorate in the membrane. This suggests that the orientation of mal-BSA bound to the receptor places the fluorescein probe too far from the lipid surface to experience energy transfer. The distance information identifies a potential location for the binding site, which can be compared to structural information about the receptor and used to extract a binding sequence.     

IMAGING: Focusing light in scattering media

Combining ultrasonic modulation and optical phase conjugation allows light to be tightly focused in a scattering medium, providing benefits for studies of photophysical, photochemical and photobiological processes.     

Imaging subcellular scattering contrast by using combined optical coherence and multiphoton microscopy

The structural origin of scattering contrast from single cells is examined by using a combined optical coherence and multiphoton microscope based on a 12 fs Ti:sapphire source and a 0.95 NA objective. High-resolution coherence-gated scattering images from single cells are coregistered and compared with two-photon-excited fluorescence images. Scattering contrast is observed from mitochondria, plasma membrane, actin filaments, and the boundary between cytoplasm and nucleus. There is little contribution to scattering from regions inside the nuclear core. These results confirm that light scattering signals from specific subcellular structures can be visualized by using coherent reflectance geometry.     

Modulated imaging: quantitative analysis and tomography of turbid media in the spatial-frequency domain

Experiments performed on turbid phantoms demonstrate that spatially modulated illumination facilitates quantitative wide-field optical property mapping and tomographic imaging in turbid media.