Predictors and Limitations of the Penetration Depth of Photodynamic Effects in the Rodent Brain

Fluorescence-guided surgery (FGS) is routinely utilized in clinical centers around the world, whereas the combination of FGS and photodynamic therapy (PDT) has yet to reach clinical implementation and remains an active area of translational investigations. Two significant challenges to the clinical translation of PDT for brain cancer are as follows: (1) Limited light penetration depth in brain tissues and (2) Poor selectivity and delivery of the appropriate photosensitizers. To address these shortcomings, we developed nanoliposomal protoporphyrin IX (Nal-PpIX) and nanoliposomal benzoporphyrin derivative (Nal-BPD) and then evaluated their photodynamic effects as a function of depth in tissue and light fluence using rat brains. Although red light penetration depth (defined as the depth at which the incident optical energy drops to 1/e, ~37%) is typically a few millimeters in tissues, we demonstrated that the remaining optical energy could induce PDT effects up to 2 cm within brain tissues. Photobleaching and singlet oxygen yield studies between Nal-BPD and Nal-PpIX suggest that deep-tissue PDT (>1 cm) is more effective when using Nal-BPD. These findings indicate that Nal-BPD-PDT is more likely to generate cytotoxic effects deep within the brain and allow for the treatment of brain invading tumor cells centimeters away from the main, resectable tumor mass.     

Damage Threshold of Normal Rat Brain in Photodynamic Therapy

Normal brain tissue response to photodynamic therapy (PDT) must be quantified in order to implement PDT as a treatment of brain neoplasm. We therefore calculated the threshold for PDT-induced tissue necrosis in normal brain using Photofrin (porfimer sodium, Quadralogic Technologies Inc., Vancouver, BC) as the photosensitizer. The absolute light fluence-rate distribution for superficial irradiation and effective attenuation depth were measured in vivo using an invasive optical probe. Photosensitizer uptake in cerebral cortex was measured with chemical extraction and fluorometric analysis. Photodynamic therapy-induced lesion depths at various drug dose levels were measured as a biological end point. The PDT threshold for normal brain necrosis was calculated as in the magnitude of 10¹⁶ photons/cm³. Thus normal rat brain is extremely vulnerable to PDT damage. This suggests that extra precautions must be exercised when PDT is used in brain.     

Penetration of laser light through red blood cell ghosts

Hemoglobin is the main absorber of visible light in blood and blood-perfused tissues. However, hemoglobin is released from a red blood cell (RBC) during hemolysis. Hemolysis may be caused by a large number of medical conditions, including photodynamic therapy (PDT) and this subsequently can affect passage of light through the treated biological structures. The purpose of the present study was to determine the penetration of a laser beam through a suspension of hemoglobin-free human red blood cells (RBCs) - ghosts. Although hemoglobin has been efficiently removed from the samples used in our experiments, our measurements show that the samples still effectively attenuate the radiant power of penetrating laser light. We established penetration depths of 12.6mm and 15.4mm for two different laser light wavelengths, 532nm and 630nm, respectively. The penetration depth of laser light was about one order of magnitude higher for hemoglobin-free RBC ghosts as compared to intact RBCs [8,10,12]. These results can be important in case of phototherapy or biostimulation, since all photons that penetrate in a biological object may interact with it and evoke biological response.     

Optical properties of human prostate at 732 nm measured in mediated photodynamic therapy

Characterization of the tissue light penetration in prostate photodynamic therapy (PDT) is important to plan the arrangement and weighting of light sources so that sufficient light fluence is delivered to the treatment volume. The optical properties (absorption [mu(a)], transport scattering [mu(s)'] and effective attenuation [mu(eff)] coefficients) of 13 patients with locally recurrent prostate cancer were measured in situ using interstitial isotropic detectors. Measurements were made at 732 nm before and after motexafin lutetium (MLu)-mediated PDT in four quadrants. Optical properties were derived by applying the diffusion theory to the fluence rates measured at several distances (0.5-5 cm) from a point source. mu(a) and mu(s)' varied between 0.07 and 1.62 cm(-1) (mean 0.37 +/- 0.24 cm(-1)) and 1.1 and 44 cm(-1) (mean 14 +/- 11 cm(-1)), respectively. mu(a) was proportional to the concentration of MLu measured by an ex vivo fluorescence assay. We have observed, on average, a reduction of the MLu concentration after PDT, presumably due to the PDT consumption of MLu. mu(eff) varied between 0.91 and 6.7 cm(-1) (mean 2.9 +/- 0.7 cm(-1)), corresponding to an optical penetration depth (delta = 1/micro(eff)) of 0.1-1.1 cm (mean 0.4 +/- 0.1 cm). The mean penetration depth at 732 nm in human prostate is at least two times smaller than that found in normal canine prostates, which can be explained by a four times increase of the mean value of mu(s)' in human prostates. The mean light fluence rate per unit source strength at 0.5 cm from a point source was 1.5 +/- 1.1 cm(-2), excluding situations when bleeding occurs. The total number of measurements was N = 121 for all mean quantities listed above. This study showed significant inter- and intraprostatic differences in the optical properties, suggesting that a real-time dosimetry measurement and feedback system for monitoring light fluences during treatment should be considered for future PDT studies.     

In situ COMPARISON OF 665 nm and 633 nm WAVELENGTH LIGHT PENETRATION IN THE HUMAN PROSTATE GLAND

Abstract— The depth of treatment in photodynamic therapy (PDT) of tumors varies with the wavelength of light activating the photosensitizer. New generation photosensitizers that are excited at longer wavelengths have the potential for increasing treatment depths. Tin ethyl etiopurpurin (SnET2), a promising second-generation photosensitizer is maximally activated at 665 nm, which may be significantly more penetrating than 633 nm light currently used with porphyrins in PDT. The penetration of 665 nm and 633 nm wavelength red light in the prostate gland was compared in 11 patients undergoing prostatic biopsies for suspected prostatic cancer. Interstitial optical fibers determined the light attenuation within the prostate gland. Of the 11 patients, 7 had dual wavelength and 4 had single wavelength studies. The mean attenuation coefficients, μ_eff, for 665 nm and 633 nm wavelength light were 0.32 ± 0.05 mm^-1 and 0.39 ± 0.05 mm^-1, respectively, showing a statistically significant difference (P = 0.0003). This represented a 22% increase in the mean penetration depth and at 10 mm from the delivery fiber there was 1.8 times as much 665 nm light fluence than 633 nm. The mean μ_eff at 665 nm for benign and malignant prostate tissue were similar ( P = 0.42), however, there was significant interpatient variation (μ_eff ranging from 0.24 to 0.42 mm^-1) reflecting biological differences of therapeutic importance. The enhanced light fluence and penetration depth with 665 nm light should allow significantly larger volumes of prostatic tissue to be treated with SnET2-mediated PDT.     

In vivo optical properties of normal canine prostate at 732 nm using motexafin lutetium-mediated photodynamic therapy

The optical properties (absorption [mu(a)], transport scattering [mu('s)] and effective attenuation [mu(eff)] coefficients) of normal canine prostate were measured in vivo using interstitial isotropic detectors. Measurements were made at 732 nm before, during and after motexafin lutetium (MLu)-mediated photodynamic therapy (PDT). They were derived by applying the diffusion theory to the in vivo peak fluence rates measured at several distances (3, 6, 9, 12 and 15 mm) from the central axis of a 2.5 cm cylindrical diffusing fiber (CDF). Mu(a) and mu('s) varied between 0.03-0.58 and 1.0-20 cm(-1), respectively. Mu(a) was proportional to the concentration of MLu.Mu(eff) varied between 0.33 and 4.9 cm(-1) (mean 1.3 +/- 1.1 cm(-1)), corresponding to an optical penetration depth (8 = 1/(mu(eff)) of 0.5-3 cm (mean 1.3 +/- 0.8 cm). The mean light fluence rate at 0.5 cm from the CDF was 126 +/- 48 mW/cm2 (N = 22) when the total power from the fiber was 375 mW (150 mW/cm). This study showed significant inter- and intraprostatic differences in the optical properties, suggesting that a real-time dosimetry measurement and feedback system for monitoring light fluences during treatment should be advocated for future PDT studies. However, no significant changes were observed before, during and after PDT within a single treatment site.     

Enhancement of ALA-PDT Damage by IR-lnduced Hyperthermia on a Colon Carcinoma Model

The interaction of photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) and hyperthermia is not well understood. In the present study, significant enhancement of tumor damage was observed after simultaneous application of ALA-PDT and IR-induced hyperthermia using a broad-band incoherent light source. One hour after systemic administration of ALA at a dose of 200 mg/kg, subcutaneously transplanted C26 colon carcinoma tumors were irradiated with two bands of the VersaLight system, red (R, 580-720 nm) and red plus IR (R + IR, 580-720 nm and 1250-1600 nm). Photoirradiation using the R + IR band at different fluence rates and exposures caused mild heating of the tumor to 39-43 degrees C at a 3 mm depth. Electron microscopy after ALA + R, ALA + R + IR and R + IR treatments showed early mitochondrial swelling that was more pronounced in the ALA + R + IR group. Tumor necrosis assessment, using histology and vital staining, revealed an enhancement of tumor necrosis depth in the ALA + R + IR group compared to ALA + R and R + IR. The results showed that subhyperthermic heating to 39-39.5 degrees C in the ALA + R + IR group decreased the threshold light dose required for 100% tumor necrosis from 210 J/cm2 (observed in the ALA + R group) to 140 J/cm2. A tumor growth delay test, based on tumor volume measurement, also revealed significant enhancement of antitumor effect after application of ALA + R + IR compared to ALA + R.     

EFFECTS OF LIGHT BEAM SIZE ON FLUENCE DISTRIBUTION AND DEPTH OF NECROSIS IN SUPERFICIALLY APPLIED PHOTODYNAMIC THERAPY OF NORMAL RAT BRAIN

The light fluence distributions of 632.8 nm light incident on the exposed surface of normal rat brain in vivo have been measured using an interstitial, stereotactically-mounted optical fiber detector with isotropic response. The dependence of the relative fluence rate on depth and the spatial distribution of fluence were compared for incident beam diameters of 3 and 5 mm. The fluence rate at depth of 1-6 mm along the optical axis within the brain tissue was approximately 70% greater for a 5 mm diameter beam than for a 3 mm beam, at the same incident fluence rate, although the plots of the relative fluence rate vs depth were parallel over the depth range 1-6 mm. The depths of necrosis resulting from photodynamic treatment of brain tissue using the photosensitizer Photofrin and irradiation by 632 nm light with 3 and 5 mm incident beams were also measured. The observed difference in necrosis depths was consistent with the measured difference in fluence. The importance of beam size in photodynamic treatment with small diameter incident light fields is discussed.     

OXYGEN LIMITATION OF DIRECT TUMOR CELL KILL DURING PHOTODYNAMIC TREATMENT OF A MURINE TUMOR MODEL

The relationship between levels of in vivo accumulated photosensitizer (Photofrin II), photodynamic cell inactivation upon in vitro or in vivo illumination, and changing tumor oxygenation was studied in the radiation-induced fibrosarcoma (RIF) mouse tumor model. In vivo porphyrin uptake by tumor cells was assessed by using 14C-labeled photosensitizer, and found to be linear with injected photosensitizer dose over a range of 10 to 100 mg/kg. Cellular photosensitivity upon exposure in vitro to 630 nm light also varied linearly with in vivo accumulated photosensitizer levels in the range of 25 to 100 mg/kg injected Photofrin II, but was reduced at 10 mg/kg. Insignificant increases in direct photodynamic cell inactivation were observed following in vivo light exposure (135 J/cm2, 630 nm) with increasing cellular porphyrin levels. These data were inconsistent with expected results based on in vitro studies. Assessment of vascular occlusion and hypoxic cell fractions following photodynamic tumor treatment showed the development of significant tumor hypoxia, particularly at 50 and 100 mg/kg of Photofrin II, following very brief light exposures (1 min, 4.5 J/cm2). The mean hyupoxic cell fractions of 25 to 30% in these tumors corresponded closely with the surviving cell fractions found after tumor treatment in vivo, indicating that these hypoxic cells had been protected from PDT damage. Inoculation of tumor cells, isolated from tumors after porphyrin exposure, into porphyrin-free hosts, followed by in vivo external light treatment, resulted in tumor control in the absence of vascular tumor bed effects at high photosensitizer doses only.(ABSTRACT TRUNCATED AT 250 WORDS)     

CHRONIC METABOLIC MEASUREMENTS OF NORMAL BRAIN TISSUE RESPONSE TO PHOTODYNAMIC THERAPY

The metabolic response of normal rat brain to photodynamic therapy (PDT) was studied over a 1 week interval using in vivo 31P-NMR spectroscopy. Rats injected with 12.5 mg/kg Photofrin II were submitted to brain photoactivation 48 h after drug administration with either 140 or 70 J/cm2 light (630 +/- 1 nm) from an Argon dye laser. Control studies, animals not given drug or light, animals submitted only to brain illumination without drug, and animals given drug but no light, were also performed. The data revealed a transient metabolic degradation; a decrease in the ratio of beta-nucleotriphosphate to inorganic phosphate (P less than 0.001) at 24 h after PDT treatment was followed by a return to pretreatment spectral values. Brain tissue alkalosis was also noted, with significant (P less than 0.05) differences in brain tissue pH detected at 72 h post treatment between 70 J/cm2 PDT vs control studies and at 1 week post treatment between 140 J/cm2 vs 70 J/cm2, 140 J/cm2 vs no light-no drug and 140 J/cm2 vs drug only. The data suggest that there is no difinitive metabolic marker from 31P-NMR spectroscopy that can identify necrotic brain tissue caused by PDT. Phosphorus-31 NMR data are also presented which suggest that PDT damage to brain is not solely the result of microvascular occlusion causing ischemic necrosis.     

Silkworm-pheophorbide alpha mediated photodynamic therapy against B16F10 pigmented melanoma

In order to apply photodynamic therapy (PDT) to pigmented melanoma, the efficacy of PDT mediated by pheophorbide alpha from silkworm excreta (SPbalpha) and commercial Photofrin against B16F10 melanoma was comparatively studied from the in vivo assay using C57BL/6J mice. From in vitro PDT assay, the proliferation of B16F10 cells treated with SPbalpha (more than 0.5 microg/ml) and light illumination (1.2 J/cm2) were significantly inhibited with the necrotic response. This indicated that the photocytotoxicity of SPbalpha (665 nm) was not influenced by melanin from melanoma. From the assessment of the in vivo photosensitizing activity, the tumor growth was further delayed in groups treated with SPbalpha/PDT compared to that treated with Photofrin /PDT. The survival rate of tumor bearing mice treated with SPbalpha/PDT was closely associated with its photosensitizing effect. In addition, the photosensitizing effect of SPbalpha/PDT showed a dose dependent tendency in light illumination. These results demonstrated that B16F10 melanoma cells were significantly photosensitized by SPbalpha/PDT, regardless of the influence of melanin from melanoma, and SPbalpha/PDT at very low drug dose (1 mg/kg) and light dose (1.2 J/cm2) showed the photosensitizing efficacy surpassing Photofrin against B16F10 melanoma in mice system.     

Tumor blood-flow changes following protoporphyrin IX-based photodynamic therapy in mice and humans

The effects of aminolevulinic acid (ALA)-based photodynamic therapy (PDT) on tumor blood flow are controversial. This study examines the effects of ALA and Photofrin-based PDT on blood flow of Colon-26 tumors implanted in mice as well as the effects of ALA-based PDT on blood flow of human colorectal carcinomas and a carcinoid tumor in situ. Tumors are implanted in both flanks of mice. One tumor of each animal serves as a control. Blood flow is measured using a laser Doppler method. Tumor blood flow in mice not receiving a photosensitizer but treated with three different light fluences (50, 100 and 150 J/cm2) does not differ significantly from blood flow in the untreated tumor in the opposite flank. PDT after ALA administration using the three different light fluences does not significantly affect blood flow. In contrast, PDT after Photofrin administration causes a significant decrease in tumor blood flow with each light fluence, but this change is not as dramatic as reported in other studies. In contrast to mice, six patients who receive ALA prior to surgery all show a decrease in blood flow (mean = 51.8%, p < 0.001) after PDT using 100 J/cm2. Comparison with other published results suggests that it is likely that flow measurement by the laser Doppler method underestimates the effects of PDT on tumor blood flow due to the depth of laser penetration. Nevertheless, the present observations on blood flow suggest that the effects of ALA-based PDT on adenocarcinomas of the colon and rectum as well as an intra-abdominal carcinoid tumor in humans are more pronounced than would be predicated by some animal studies.     

A Photodynamic System based on Endogenous Bioluminescence for in vitro Anticancer Studies

Photodynamic therapy (PDT) is becoming an important cancer treatment in recent years. However, at present, the therapeutic effect of PDT is limited due to insufficient penetration depth of light. In this study, a new photodynamic system (d ‐Lu)PCN‐224 is constructed by porphyrin‐based metal‐organic framework (MOF) PCN‐224 and bioluminescent molecule d ‐fluorescein (d ‐Lu). The bioluminescence (BL) spectrum of the reaction overlaps with the absorption spectrum of PCN‐224, so it is speculated that bioluminescence resonance energy transfer (BRET) between the MOF and d ‐Lu which indicates inner light can be gained and used for PDT. Confocal imaging analysis and cytotoxicity assays have demonstrated that (d ‐Lu)PCN‐224 can produce singlet oxygen and decrease the cell viability of SKOV‐3. This system provides a possibility of PDT for deep‐level organization without an external light source.     

DRUG AND LIGHT DOSE DEPENDENCE OF PHOTODYNAMIC THERAPY: A STUDY OF TUMOR CELL CLONOGENICITY AND HISTOLOGIC CHANGES

Abstract— The dependence of photodynamic therapy (PDT) on changes in drug and light doses was determined in C3H/HeJ mice bearing the RIF tumor. Measurements of tumor clonogenicity were determined 24 h after PDT over a range of drug and light doses. Representative histological samples were prepared at each of these doses. Both the drug and light dose dependence experiments showed an exponential decrease in clonogenicity after an initial shoulder region. Reciprocity of drug and light dose was established from those clonogenicity curves. Histological examination of tumors gave information concerning the localization of gross damage within tumors. Increases of light dose in PDT were shown to extend the depth of necrosis within tumors. Increases of drug dose produced enlargements in the area of necrotic spots produced by PDT     

Treatment of Malignant Melanoma by High-Peak-Power 1064 nm Irradiation Followed by Photodynamic Therapy

Irradiation of B16 pigmented melanoma subcutaneously transplanted in C57 mice with a single 650 mj pulse (10 ns) of 1064 nm light from a Q-switched Nd: YAG laser caused instantaneous bleaching of the pigmented tissue. Visual and histological examination of the resulting gray-colored tumor revealed the breakdown of melanosomes with no detectable alteration of the normal and tumor-overlying skin. Histological examination of the irradiated tumor showed some degree of vascular damage; the depth of the photodamage was not affected by the successive delivery of three consecutive light pulses. The bleached tumor grew at a modestly slower rate but the high-peak-power (HPP) laser treatment did not affect the tumor concentration of a photodynamic sensitizer Si(IV)-naphthalocyanine (isoBO-SiNc) intravenously injected 24 h before Nd : YAG irradiation. Treatment of the B16 pigmented melanoma by photodynamic therapy (PDT: 1 mg/kg isoBO-SiNc, 300 mW/cm², 520 J/cm²) from a 774 nm diode laser immediately after the 1064 nm irradiation resulted in a 16 day delay of tumor regrowth, which was markedly longer than the delay (ca 6 days) obtained after PDT under identical conditions without the preirradia-tion. Thus, pretreatment of pigmented tumors with HPP 1064 nm light appears to enhance their susceptibility to conventional PDT. The tumor response was further enhanced by repeating the combined HPP/PDT treatment at an interval of 10 days (regrowth delay: 27 days), as well as by applying hyperthermia immediately after HPP/PDT (regrowth delay: ca 34 days).     

Organic Nanoparticles with Persistent Luminescence for In Vivo Afterglow Imaging-Guided Photodynamic Therapy

Optical imaging-guided photodynamic therapy (PDT), with precise localization and non-invasive treatment of tumors, is an emerging technique with great potential for cancer therapy. However, impaired by tissue auto-fluorescence that causes low signal-to-background ratio (SBR), most fluorescence imaging systems show poor sensitivity to tumors in vivo. In this study, we synthesized organic nanoparticles (ONPs) with persistent luminescence and good biocompatibility for afterglow imaging-guided PDT. The ONPs displayed near-infrared light emission with half-life time at minute level, which offered high SBR and good tissue penetration for in vivo afterglow tumor imaging. Taking advantage of their abundant singlet oxygen generation by NIR laser irradiation guided to the tumor sites, the ONPs also enabled imaging-guided PDT for efficient suppression of tumor growth in mice with minimal damage to major organs.     

In vivo TESTS OF THE CONCEPT OF PHOTODYNAMIC THRESHOLD DOSE IN NORMAL RAT LIVER PHOTOSENSITIZED BY ALUMINUM CHLOROSULPHONATED PHTHALOCYANINE

In its simplest form, the photodynamic therapy (PDT) threshold dose model states that tissue necrosis due to PDT will occur if the number of photons absorbed by the photosensitizer per unit volume of tissue exceeds a critical value. This threshold is given by the product of photon fluence, photosensitizer concentration and specific absorption coefficient. To test the validity of this concept for PDT of normal rat liver sensitized with aluminum chlorosulphonated phthalocyanine (AISPC), all three of these parameters were varied by changing the injected AISPC dose, the wavelength of excitation and the irradiation geometry. The extent of necrosis caused by the treatment was consistent with the threshold model, except when the concentration of AISPC in the liver exceeded 20 micrograms g-1. For this animal model, we estimate the threshold to be (3.8 +/- 0.2) x 10(19) photons cm-3.     

Choice of optimal wavelength for PDT: the significance of oxygen depletion

We have investigated the role of tissue oxygenation on light penetration into tissue at different wavelengths. As a field of application we have chosen aminolevulinic acid-photodynamic therapy (ALA-PDT). To calculate efficiency spectra of PDT on human skin one needs to know the excitation spectrum of the photosensitizer of interest and the relative fluence rate as a function of depth in the tissue. We measured the former and computed the latter with an accurate radiative transfer algorithm. In this way we determined the efficiency spectra as functions of depth for different types of basal cell carcinomas (BCC). Our results suggest that ALA-PDT works best for nodular BCC at a wavelength of 630 nm, whereas it works best for pigmented superficial BCC at a wavelength of 390 nm. At 630 nm the light penetration into a tumor depends strongly on the oxygenation of the blood. Below a 2 mm thick, well-oxygenated, nodular BCC, we find the efficiency to be an order of magnitude larger than below a poorly oxygenated tumor. At 390 nm, the light penetration into a tumor does not depend on the oxygenation of the blood.     

Hitherto-Unexplored Photodynamic Therapy of Ag2S and Enhanced Regulation Based on Polydopamine In Vitro and Vivo

Given their superior penetration depths, photosensitizers with longer absorption wavelengths present broader application prospects in photodynamic therapy (PDT). Herein, Ag₂S quantum dots were discovered, for the first time, to be capable of killing tumor cells through the photodynamic route by near-infrared light irradiation, which means relatively less excitation of the probe compared with traditional photosensitizers absorbing short wavelengths. On modification with polydopamine (PDA), PDA-Ag₂S was obtained, which showed outstanding capacity for inducing reactive oxygen species (increased by 1.69 times). With the addition of PDA, Ag₂S had more opportunities to react with surrounding O₂, which was demonstrated by typical triplet electron spin resonance (ESR) analysis. Furthermore, the PDT effects of Ag₂S and PDA-Ag₂S achieved at longer wavelengths were almost identical to the effects produced at 660 nm, which was proved by studies in vitro. PDA-Ag₂S showed distinctly better therapeutic effects than Ag₂S in experiments in vivo, which further validated the enhanced regulatory effect of PDA. Altogether, a new photosensitizer with longer absorption wavelength was developed by using the hitherto-unexplored photodynamic function of Ag₂S quantum dots, which extended and enhanced the regulatory effect originating from PDA.     

In vitro and in vivo photodynamic activity of core-modified porphyrin IY69 using 690 nm diode laser

Core-modified porphyrins have been explored as the second-generation photosensitizers due to their excellent photophysical properties. IY69 [(5-phenyl-10,15-bis(4-carboxylatomethoxyphenyl)-20-(2-thienyl)-21,23-dithiaporphyrin] was developed from the structure optimization guided by in vitro phototoxicity, showing potent activity (IC(50)=80 nm, broadband at 5 J cm(-2), R3230AC cells). The present study demonstrates in vivo photodynamic therapy (PDT) efficacy of IY69 using a murine tumor model (colon 26 cells on BALB/c mice) and 690 nm diode laser. In vitro phototoxicity of IY69 with the diode laser was compared with that with broadband light against colon 26 cells. Attenuation of the laser light by tissue samples was determined to estimate actual power density at targets. Biodistribution in various organs 24, 48, 72 h after i.p. administration was determined. Even though IY69 phototoxicity with the diode laser was less effective than that with the broadband light, the diode laser was quite effective in vitro (IC(50)=0.1 μm, 10 J cm(-2), colon 26 cells). Concentration and light dose-dependent phototoxicity was observed. A significant light attenuation of 95% and 99% was observed by skin and 3 mm muscle with skin. IY69 PDT showed significant damage on tumor and delay in tumor growth in a dose-dependent manner.