Photosensitization of red blood cell hemolysis by lutetium texaphyrin.

Lutetium (III) texaphyrin photosensitizes postirradiation or "delayed" photohemolysis (DPH) of human and bovine red blood cells at 730 nm by a Type-2 pathway mediated by singlet molecular oxygen. The DPH rate increases with increasing incubation temperature and with the second power of the incident fluence. The experimental DPH curves are in good agreement with a multi-hit kinetics model based on target theory.

A note on the analysis of ligand binding by the 'double-logarithmic' plot.

The double-logarithmic plot has been employed for the fluorometric analysis of ligand binding. This analysis is valid only for 1:1 binding of a non-aggregating ligand and if the plot is based on the free, and not the total, ligand concentration.

PDT light dosimetry revisited.

A versatile PDT light dosimetry model is described incorporating the effects of drug photobleaching, drug elimination, and normal tissue damage. The dependence of the necrosis depth (dn) on the incident light dose for the four major modes of PDT light delivery has the form: dn = delta loge(DG), where delta is the optical penetration depth of the tumor tissue, D is the ratio of the incident light dose to the energy fluence at the necrosis threshold, and G is a function of the tissue optical constants. Light dosimetry graphs were calculated for Photofrin at standard conditions.

Sensitization of photohemolysis by hypericin and Photofrin.

Sensitization of post-irradiation (delayed) photohemolysis (DPH) and "during irradiation" (continuous) photohemolysis (CPH) were investigated for sodium hypericin and Photofrin. The photohemolysis rate and relative steepness of the photohemolysis curves were measured for a range of sensitizer concentrations and DPH irradiation times. The data are analyzed by a multihit model based on the assumption that photohemolysis requires thermal activation of light-activated damage. A key result of the model is the photohemolysis rate has a power dependence of 1 or more on the irradiation time and sensitizer concentration for DPH and a power dependence of less than 1 on sensitizer concentration for CPH. The data for sodium hypericin are consistent with the predictions and indicative of a square dependence of photohemolysis rate on the absorbed light dose. The results for Photofrin led to an approximately square dependence of the photohemolysis rate on DPH irradiation time and an apparently anomalous 1.24 power dependence on sensitizer concentration. The model predicts also that the relative steepness of the photohemolysis curves is independent of the light dose and sensitizer concentration for DPH and almost independent of the sensitizer concentration for CPH. This prediction is confirmed to within +/- 10% for all sets of DPH and CPH curves. The relationship of the multihit kinetics model to detailed photohemolysis mechanisms is discussed.

Effects of Photofrin on in vivo skin reflectivity.

Skin reflection spectra were measured before and 24 h after administration of Photofrin to nevoid basal-cell carcinoma syndrome patients. The presence of the drug reduced the reflectivity of uninvolved skin and increased the reflectivity of erythematous basal cell cancers. Data analysis with diffusion approximation and Monte Carlo simulation were employed to estimate the optical constant changes and localized drug concentration.

Photodynamic therapy.

Photodynamic therapy (PDT) is a new treatment for solid tumors utilizing the combined action of light and a photosensitizing drug. Laser-fiber optic delivery systems make it practical to treat superficial and interstitial cancers, including malignancies of the skin, head and neck, esophagus, endobronchial tract, stomach, urinary bladder, female genital tract, and other sites. The putative action mechanism in PDT involves photochemical destruction of tumor tissue membranes mediated by singlet molecular oxygen. Light dosimetry modeling based on tissue optics is applicable for treatment planning. Related research areas include non-invasive optical diagnosis and imaging, development of new PDT drugs and light sources, and the extension of PDT methods to treatment of atherosclerotic vascular disease and blood detoxification.

Diffuse optics determination of hemoglobin derivatives in red blood cells and liposome encapsulated hemoglobin.

High resolution optical absorption spectra of hemoglobin derivatives in red blood cells and phosphatidylcholine liposomes were calculated from diffuse reflection and transmission spectra by means of the one-dimensional diffusion approximation. The numerical technique of singular value decomposition was used to calculate the composition of red cell and liposome mixtures.

Singlet oxygen generation by Photofrin in homogeneous and light-scattering media.

The singlet oxygen quantum yield (phi delta) for Photofrin solubilized by Triton X-100 was measured in homogeneous and light-scattering media using the photosensitized inactivation of lysozyme as an internal actinometer. Higher values of phi delta at 630 nm than at 514 nm are attributed to the formation of a far-red-absorbing photosensitizing photoproduct.

Hypericin photosensitization in aqueous model systems.

Photosensitization of lysozyme, liposomes, ghosts and intact red blood cells (RBC) was investigated for aqueous hypericin. The effects of azide ion, 1,4-diazabicyclo(2.2.2)octane, and superoxide dismutase on photosensitized inactivation of lysozyme in 0.5% Triton X-100 indicate that singlet oxygen is the major inactivating intermediate with a contribution from superoxide. The singlet oxygen quantum yield (phi delta) scaled to methylene blue is 0.49 +/- 0.06 at monochromatic wavelengths from 514 to 600 nm. Relative values of phi delta based on lysozyme inactivation for different vehicles are: 0.5% Triton X-100 (1.13), human serum albumin (0.65), Cremophor-EL (0.76), Cremophor-RH40 (0.98), egg phosphatidylcholine (EPC) liposomes (0.04), hydrogenated soy phosphatidylcholine (HSPC) liposomes (< 0.01). Hypericin photosensitized lipid peroxidation of EPC liposomes and RBC ghosts. Extensive cross-linking of ghost membrane proteins occurred during the initial stages of lipid peroxidation. Prompt photohemolysis was used as the assay of RBC membrane damage. The photohemolysis curves are modeled with multihit target theory based on the "hit number" (n) and the target cross section (v). The values of v and the conventional "1/t50" parameter are equivalent determinants of the photohemolysis rate. The photohemolysis curves are in good agreement with n = 15 for incubation in phosphate-buffered saline at different hypericin concentrations and with additives. The measurements for other vehicles led to n = 19 for Cremophor-EL and n = 3 for EPC and HSPC liposomes, indicating that the kinetics of photohemolysis depend on the conditions of incubation.

Clinical experiences with photodynamic therapy for recurrent malignancies of the lower female genital tract.

Photodynamic therapy (PDT) with Photofrin II was administered over a 7-year period to 17 patients with recurrent carcinoma in situ (CIS) of the vulva, vagina, and perianum. Ten patients were treated two or three times after average intervening periods of 23 and 19 months, respectively. A histologically complete response at 3 months after the PDT session was achieved for 27 of 38 (71%) anatomic sites. Ten patients of this group remain free of recurrences for periods of 2-7 years. Condylomata acuminata associated with CIS in 16 of 17 patients recurred rapidly in 7 patients after PDT. Only short-term palliative results were achieved for 4 patients treated with PDT for invasive carcinoma. Fifteen patients experienced a significant skin reaction to direct sunlight during a 7-week period post-PDT.

Photosensitization of aqueous model systems by hypericin.

Absorption and fluorescence measurements of purified hypericin (HY) were made in various media. Photosensitization of two aqueous systems was investigated: resealed red blood cell membranes (ghosts) and hen lysozyme (Lys). Solubilization of HY by ghost membranes was shown by means of diffuse reflectance spectroscopy. Visible light irradiation of the ghosts incorporating HY led to lipid peroxidation with evidence of singlet oxygen involvement. A binding model applicable for insoluble ligands is indicative of strong HY binding to HSA. The HY-HSA complex photosensitized inactivation of Lys. The pseudo-first-order reaction kinetics with protection by azide ion are consistent with a Type II mechanism mediated by singlet oxygen. The results are discussed in the context of the HY photodynamic and antiretroviral activities.

Reflection and transmission coefficients in plane-parallel layers with refractive-index mismatch.

A ray-tracing calculation that uses reflection and transmission coefficients for layers with refractive-index matched boundaries leads to the corresponding coefficients for refractive-index mismatch. The model is compared with Monte Carlo calculations for a range of layer parameters. The absorption by a mismatched layer is higher than the corresponding layer with matched boundaries and relatively insensitive to the extent of scattering anisotropy. The model should be useful for practical calculations on biological tissues where refractive-index mismatch is usually present.

Light dosimetry model for photodynamic therapy treatment planning.

A light dosimetry model for photodynamic therapy is described leading to the dependence of necrosis depth on incident light dose. Semiempirical dosimetry relations are derived for four modes of light delivery. Results for Photofrin II at 630 nm are summarized in graphical form. Numerical values of the parameters and necrosis depth calculations are compared with literature data.

Photodynamic therapy in the treatment of squamous cell carcinoma of the head and neck.

Photodynamic therapy is an experimental modality for tumor treatment based on the combined action of the tumor-localizing agent, ie, hematoporphyrin derivative, and red light. From 1985 through 1989, 26 patients were treated using hematoporphyrin-derived drugs and 630-nm light delivered by a tunable dye laser. All patients had biopsy-proved squamous cell carcinoma of the head and neck, and they had either failed the traditional treatment modalities or refused conventional therapies. Histological complete responses were achieved in 20 (77%) of 26 patients and partial responses in 5 (19%) of 26 patients for periods up to 48 months. Only minimal toxic reaction was noted in the group. As a guide to treatment planning for a patient group with large tumors, we used an optical dosimetry model based on tissue optics. The rate of complete responses to this treatment was 8 (73%) of 11. Our data indicate that photodynamic therapy is capable of inducing significant clinical and histological responses in the majority of those treated, and in some patients a prolonged response is produced. In certain select head and neck malignancies, photodynamic therapy has an important role as a treatment modality.

Modeling of tissue heating with a pulsed Nd:YAG laser.

Temperature profiles were measured in large sections of bovine muscle and bovine liver exposed to single pulses from a clinical Nd:YAG laser. The data were analyzed with a photothermal model applicable to an incident gaussian beam including the effects of beam spread and heat flow. Closed form expressions for the radial average initial temperatures and thermal relaxation were derived and compared with the measurements.

Gaussian beam spread in biological tissues.

Flux density distributions were measured in large tissue sections illuminated with 633- and 1064-nm laser radiation delivered by an optical fiber. The results were modeled by solving the 2-D diffusion approximation for an incident Gaussian beam and fitting the data with nonlinear regression. It is shown that the radial average flux density is exponentially attenuated for an arbitrary incident irradiance profile.

Photosensitization tissue model.

Photosensitization in a light scattering matrix was investigated with a tissue model consisting of polystyrene microsphere scattering particles, in the presence of a Photofrin II as a photosensitizing agent, and subtilisin Carlsberg as an enzyme target. The photodynamic rate constant for irradiation at 435 nm, 545 nm, and red light was measured at different microsphere concentrations. The reaction rate was almost independent of the microspheres, ruling out a significant effect of light scattering on the integrated photosensitization efficiency. Modeling with the one-dimensional diffusion approximation showed that increasing scatterer concentrations led to lower transmission and higher diffuse reflection, such that the fractional absorption was almost constant. The quantum efficiency of enzyme inactivation in the light-scattering systems was 0.0014 +/- 0.0003. In terms of incident dose, red and green light had approximately the same effectiveness and blue light was two-fold more efficient.

Applications of the 1-D diffusion approximation to the optics of tissues and tissue phantoms.

Optical constants of animal and plant tissues were measured over a wide spectral range using an integrating sphere spectrophotometer. The data were analyzed with two formulations of the 1-D diffusion approximation differing in the phase function. The accuracy of the similarity transformation was examined with tissue phantoms incorporating known chromophores in light scattering media.

Nd:YAG laser-induced hyperthermia in a mouse tumor model.

Hyperthermic tumor response induced by 1,064-nm radiation from an Nd:YAG laser was investigated in DBA/2J female mice bearing the SMT-F mammary carcinoma. The measured temperature-depth profiles indicate that hyperthermic temperatures can be achieved in tumors ranging from 3 to 8 mm thick at power inputs on the order of 1 W. For small tumors, a 5-week complete response rate exceeding 50% required 45 minutes at 45.0 degrees C. Control of large tumors (6-8 mm thick) was not achieved. The observed tumor response rates are consistent with semiempirical time-temperature relations based on other heating modalities.