Critical parameters in the cytotoxicity of photodynamic therapy using a pulsed laser

. Photodynamic therapy (PDT) using a pulsed laser is becoming popular, but its cytotoxic effect is still not clear. We therefore studied the cytotoxicity of PDT using a pulsed laser by changing its irradiation parameters and compared the degrees of cytotoxicity with those of PDT using continuous-wave (CW) light sources. Mice renal cell carcinoma cells were incubated with PAD-S31, a water-soluble photosensitiser of which the excitation peak is 670 nm, and were then irradiated with either a tungsten lamp, a CW diode laser, or a nanosecond pulsed Nd:YAG laser-based optical parametric oscillator system. When the PAD-S31 concentration and total light dose were constant (12 μg/ml and 40 J/cm², respectively), the CW laser caused fluence rate-dependent decrease in cellular proliferation until the fluence rate reached 90 mW/cm², at which point inhibition of cellular proliferation was more than 80%. The cytotoxicity then became almost saturated at fluence rates of>90 mW/cm². On the other hand, inhibition of cellular proliferation in samples irradiated with the pulsed laser reached 80% even at the fluence rate of 15 mW/cm², and, interestingly, the cytotoxicity paradoxically decreased with increase in the fluence rate. Moreover, the cytotoxicity in the PDT using the pulsed laser depended on the repetition rate. The inhibition of cellular proliferation by PDT using 30-Hz irradiation was greater than that by PDT using 5-Hz irradiation when the same fluence rates were used. These results suggest that the efficacy of PDT using a pulsed laser depends considerably on fluence rate and repetition rate. Paper received 4 March 2002; accepted after revision 24 May 2002. Correspondence to: Yuji Morimoto, MD, PhD, Department of Medical Engineering, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. Tel: +81-42-995-1596; Fax: +81-42-996-5199; e-mail: moyan@interlink.or.jp     

Comparative study between pulsed and continuous wave lasers for Photofrin® photodynamic therapy

A study was conducted in the normal canine esophagus to compare continuous wave (CW) and pulsed laser light for photodynamic therapy with Photofrin® (4 mg/kg). Forty-eight hours postinjection, 630 nm laser light (CW light from an argon-pumped dye-laser and pulsed light from a KTP/532-pumped dye-laser) was delivered using a 24 mm diameter cylindrical esophageal PDT balloon positioned at either distal or proximal esophagus. A 1.0 cm cylindrical diffuser placed in the center of the balloon delivered 300 J/cm of light at an intensity of 400 mW/cm. Three dogs received CW light proximally and pulsed light distally. Four dogs received CW light distally and pulsed light proximally. The light dose delivered to the esophageal mucosa was measured using three isotropic probes placed on the balloon wall. laser–induced fluorescence technique was used to compare photosensitizer fluorescence intensities at distal and proximal locations. Similar mucosal light doses and drug fluorescence intensities were verified for sites receiving pulsed or CW laser light. Two days after light delivery, the dogs were endoscoped to evaluate the severity of the lesions. While some response variability was observed among different animals, endoscopic examination of the lesions revealed comparable injury from CW and pulsed light in each subject. The animals were then euthanized and necropsies were performed. Based on the gross and histological examination of the lesions, the CW and pulsed laser–induced injuries could not be distinguished. © 1993 Wiley-Liss, Inc.     

Application of lower fluence rate for less microvasculature damage and greater cell-killing during photodynamic therapy

During the process of photodynamic therapy (PDT), problems arise such as stasis or occlusion of microvasculature, tumor oxygen depletion, and photosensitizer bleaching. This study shows that the first problem could be reduced by using a lower fluence rate light source in PDT. Microvasculature damage was studied experimentally in hematoporphyrin derivative–mediated PDT against light fluence rate, and, to some extent, less microvasculature damage was induced under 75 mW/cm² illumination than under 150 mW/cm². Histology of vessels at the end of PDT showed that vessel damage could be observed in both groups, indicating that the microvasculature would eventually be damaged as long as the administration of light fluence was sufficient and regardless of the illuminating fluence rates. Thus microvasculature damage induced by low fluence rate illumination could also be effective in tumor control after PDT. The cell-killing experiment was performed in vitro and designed so that cell-killing rate was influenced only by light characteristics. The higher cell-killing rate caused by 75 mW/cm² illumination indicated that lower fluence rate light could enhance the light absorbency or decrease the bleaching of photosensitizer.An erratum to this article can be found at     

Necrosis response to photodynamic therapy using light pulses in the femtosecond regime

One of the clinical limitations of the photodynamic therapy (PDT) is the reduced light penetration into biological tissues. Pulsed lasers may present advantages concerning photodynamic response when compared to continuous wave (CW) lasers operating under the same average power conditions. The aim of this study was to investigate PDT-induced response when using femtosecond laser (FSL) and a first-generation photosensitizer (Photogem) to evaluate the induced depth of necrosis. The in vitro photodegradation of the sensitizer was monitored during illumination either with CW or an FSL as an indirect measurement of the PDT response. Healthy liver of Wistar rats was used to evaluate the tissue response. The photosensitizer was endovenously injected and 30 min after, an energy dose of 150 J?cm^?2 was delivered to the liver surface. We observed that the photodegradation rate evaluated via fluorescence spectroscopy was higher for the FSL illumination. The FSL-PDT produced a necrosis nearly twice as deep when compared to the CW-PDT. An increase of the tissue temperature during the application was measured and was not higher than 2.5 °C for the CW laser and not higher than 4.5 °C for the pulsed laser. FSL should be considered as an alternative in PDT applications for improving the results in the treatment of bulky tumors where higher light penetration is required.     

Direct comparison of in-vitro and in-vivo Photofrin-II mediated photosensitization using a pulsed KTP pumped dye laser and a continuous wave argon ion pumped dye laser.

A pulsed KTP pumped dye laser (25 kHz repetition rate and 470 nsec pulse width) has been compared to a continuous wave argon ion pumped dye laser as the source of 630 nm light during in-vitro and in-vivo Photofrin-II mediated photosensitization studies. Individual experiments documented the effectiveness of each laser system on a) photosensitizer induced cytotoxicity and induction of stress protein synthesis using Chinese hamster fibroblasts; b) photobleaching of Photofrin-II in aqueous solution; c) Photofrin II mediated photosensitization of normal mouse skin; d) Photofrin II mediated photodynamic therapy of a mouse mammary carcinoma; and e) tumor temperature levels generated during laser exposure. Comparable results were obtained for both laser systems in all experiments.     

Photodynamic therapy for human oral squamous cell carcinoma and xenografts using a new photosensitizer,PAD-S31

BACKGROUND AND OBJECTIVES: Photodynamic therapy (PDT) is a novel and promising cancer treatment that employs a combination of photosensitizer and visible light. We examined the effect of PDT using a new photosensitizer, PAD-S31, and the 670-nm diode laser in human oral squamous cell carcinomas (SCC). STUDY DESIGN/MATERIALS AND METHODS: SAS and HSC-4 cell lines were used in all the experiments. Cell viability was determined by a modified MTT assay. Two methods were used for the determination of apoptosis in human oral SCC cells: TUNEL assay and detection of fragmented mono- and oligo-nucleosomes by ELISA. Xenografts of human oral SCC cells were generated in KSN S1c nude mice. RESULTS: In vitro PDT using PAD-S31 and the 670-nm diode laser showed cytotoxicity that was a function of laser energy, drug concentration, and time to the SAS and HSC-4 cell lines. On the other hand, PAD-S31 without irradiation had no effect on cell viability. The combinated use of PAD-S31 and the laser irradiation showed excellent anti-tumor activity against tumor xenografts without severe side effects. PDT-mediated cell death occurred predominantly by apoptosis in vitro and in vivo. CONCLUSIONS: The present study demonstrates that PAD-S31 may serve as a potent photosensitizer for PDT. Furthermore, it is expected that this therapy will be clinically useful for the treatment of patients with oral carcinoma.     

Do pulsed lasers produce an effective photodynamic therapy response?

BACKGROUND: Photodynamic therapy (PDT) in dermatology is traditionally performed with topical aminolevulinic acid (ALA) and continuous-wave (CW) illumination with blue or red light. Recently, several authors have reported success with laser and other pulsed-light sources for PDT. While the clinical benefits on sun-exposed skin are apparent, no study has demonstrated that the pulsed light sources are responsible for the observed response. STUDY DESIGN: A placebo-controlled study of two pulsed light sources previously reported for PDT: the pulsed dye laser (PDL) or broadband flashlamp filtered intense pulsed light (IPL). Sun-hidden skin was prepared with microdermabrasion and acetone scrub followed by ALA under occlusion. Laser or IPL was delivered under conditions previously reported to produce a clinical response. Control areas were exposed to standardized CW blue light or to no light. A second control area was prepared and received light and the ALA vehicle. RESULTS: IPL and PDL demonstrated a faint dose-response effect on PDT activation, but were less potent than a smaller fluence of CW blue light. Ambient light activated ALA-treated skin. CONCLUSION: Both IPL and PDL are capable of activation of PDT but produce dramatically less PDT reaction than the standard CW blue-light broadband source. Physicians desiring a robust PDT response might select CW sources over pulsed sources. Ambient light may activate a PDT reaction.     

The Effect of Multiple Sequential Light Sources to Activate Aminolevulinic Acid in the Treatment of Actinic Keratoses: A Retrospective Study

There is a lack of research regarding the sequential use of multiple light sources for topical 5-aminolevulinic acid activation in photodynamic therapy for actinic keratosis. This study evaluated 5-aminolevulinic acid-photodynamic therapy for actinic keratosis using blue light combined with red light, pulsed dye laser, and/or intense pulsed light in a retrospective fashion. Field-directed 5-aminolevulinic acid-photodynamic therapy was performed with blue light only, blue light + pulsed dye laser, blue light + intense pulsed light, blue light + pulsed dye laser + intense pulsed light, or blue light + red light + pulsed dye laser + intense pulsed light for nonhyperkeratotic actinic keratoses of face, scalp, or upper trunk. Blue light + intense pulsed light + pulsed dye laser produced greater patient-reported improvement in actinic keratoses than blue light or blue light + intense pulsed light and greater subject-reported improvement in overall skin quality than blue light + intense pulsed light. The addition of red light led to no further benefit in either outcome measure. Photodynamic therapy with multiple, sequential laser and light sources led to greater patient-graded improvement in actinic keratoses than that with a single light source (blue light), without significant differences in post-treatment adverse events. However, the small, widely disparate number of patients between groups and follow-up times between patients, as well as retrospective assessments based on subjective patient recall, severely limit the significance of these findings. Nevertheless, the results raise interesting questions regarding the use of multiple light and laser sources for photodynamic therapy of actinic keratoses and warrant further research with a prospective, randomized, controlled study.Actinic keratoses (AKs) are dysplastic epidermal neoplasms resulting from chronic cutaneous exposure to ultraviolet radiation, commonly found within photodamaged areas of the face, bald scalp, posterior neck, upper trunk, and dorsal upper extremities.1 Risk factors for the development of AKs include older age, male gender, Fitzpatrick I and II skin types, proximity to the equator, immunosuppression, and cumulative exposure to sunlight, tanning beds, and/or psoralen + ultraviolet A light (PUVA).1-3 The fact that 65 to 97 percent of squamous cell carcinomas develop from AKs or areas of field cancerization highlights the need for effective treatment of these lesions.2Numerous options exist for the management of AKs (4 PDT may also have the potential to decrease expression of early markers of cutaneous neoplasia (e.g., Ki-67 and p53), as demonstrated in multiple studies following methyl aminolevulinate PDT (MAL-PDT) using incoherent red light.5,6 Complete response rates with PDT vary based on the area treated, number of sessions required, and the type of exogenous photosensitizer and light or laser source used, ranging from 50 to 90 percent.6-14

TABLE 1

Available treatment options for actinic keratoses NSAIDs, nonsteroidal anti-inflammatory drugs

The study of the characteristic of photocytotoxicity under high peak power pulsed irradiation with ATX-S10Na(II) in vitro

We studied hydrophilic photosensitizer ATX-S10Na(II) mediated photocytotoxicity against macrophage-like cell under pulsed irradiation. We found that photocytotoxicity suppression under high intensity irradiation was directly induced by a decrease in the Type-II photoreaction. We showed that this decrease was not attributable to absorption saturation with the high intensity irradiation. We found the cell lethality change from 70% to 13% with the pulse peak power density ranging from 0.29 MW/cm² to 1.36 MW/cm², at the light dose of 20 J/cm² and the pulse repetition rate at 40 Hz. To investigate the Type-II reaction, we measured the photobleaching, oxygen consumption and singlet oxygen luminescence of the photosensitizer solution. The transient absorption from the photosensitizer during the irradiation was measured with the pump-and-probe technique. We believe that the photocytotoxicity suppression induced by the high intensity irradiation might be useful for the treatment of depth-controlled photodynamic therapy without the wall damage of a hollow organ.     

Light delivery and light dosimetry for photodynamic therapy

Photodynamic therapy (PDT) has attracted attention because it was considered to be a selective form of cancer treatment causing minimal damage to normal tissues. This is not exactly true, because the ratio between the photosensitizer concentrations in tumour and surrounding normal tissues is not always much more than one. Nevertheless, tumour destruction by PDT with relatively little damage to normal tissue is possible in many cases. This requires sophisticated light delivery and/or light dosimetry techniques. In this respect the limited penetration of light into biological tissues can sometimes be useful. In this paper a qualitative and sometimes quantitative discussion is given of the physical phenomena determining the energy fluence in a biological tissue. Most important is light scattering, the contribution of which depends on the geometrical conditions. Finite beam surface irradiation, irradiation of hollow organs (bladder) and interstitial irradiation are discussed separately. The emphasis is on light dose and light dose distribution. It is emphasized that PDT dosimetry in general is complicated by photosensitizer distribution (which is usually not known), by photobleaching of the sensitizer, by possible effects of hyperthermia, and by changes in optical properties during and as a result of PDT.     

Development of an alternative light source to lasers for photodynamic therapy: 1. Comparative in vitro dose response characteristics

The relative performances of a prototype lamp, a pulsed laser and a continous wave laser, were compared for photodynamic therapy (PDT). Recent advances in short are technology and lamp miniaturization coupled with improvements in the effciency, of optical filter coatings have led to the design and construction of a table-top light source prototype; the first viable and cost-effective alternative to a laser, particularly in the field of PDT. The device can deliver over 1 W directly or 0.5W via a light guide within a 30 nm band centred at any wavelength from the ultra-violet to the near infra-red at fluence rates of over 1 W cm⁻², in excess of that required for PDT. Its relative biological effectiveness (RBE), in vitro, has been proven alongside two PDT laser systems, an argon pumped dye laser and a copper vapour pumped dye laser. These first in vitro tests showed an efficiency of haematoporphyrin derivative, (HPD) induced cellular photoinactivation close to that of the argon/dye laser (RBE 100%), with a mean RBE for the lamp of 87±3% (p<0.05). The lamp proved to be superior, to that of the copper/dye laser system with an RBE of up to 150% at fluence rates above 50 m W cm⁻². Transient photobleaching of the photosensitizer was the probable cause for the relative ineffectiveness of the copper/dye laser for PDT at high fluence rates.     

Interstitial photodynamic therapy in the Dunning R3327-AT6 prostatic carcinoma

Interstitial photodynamic therapy (PDT) could be an alternative radical treatment for prostate cancer. The ability to predict the depth of necrosis is necessary for light treatment planning using multiple optical fibres. The extent of PDT necrosis was studied in subcutaneously implanted R3327-AT6 Dunning prostate tumours which had similar optical characteristics to human prostate. Tumour-bearing subjects were given 20 mg kg^–1 Haematoporphyrin esters (HPE) and irradiated 24 h later with 630 nm laser light. Five subjects per group were treated with increasing light doses (50–450 J cm^–1) delivered interstitially via a single 2 cm long cylindrical diffuser. After 450 J cm^–1 of irradiation, 4.3±0.8 cm³ [standard error of the mean (s.e.m.)] of tumour tissue was necrosed to a depth of 10.5±0.8 mm around the diffuser. There was an approximately linear correlation between the volume of PDT necrosis around the fibre and prescribed light dose. The mean threshold light dose for PDT effect was 18±2 J cm^–2. In this tumour with a mean photosensitizer concentration of 16±1.5g g^–1, low light doses produced tumour necrosis. PDT using multiple diffusers could destroy a relatively large tumour volume and the diffusion theory model reliably predicted the depth of necrosis.     

Photodynamic efficiency of liposome-administered tetramethyl hematoporphyrin in two human bladder cancer cell lines

The main problems presented by superficial bladder carcinoma, its high recurrence rate and multifocal appearance, require treatment of the bladder as a whole. Photodynamic therapy (PDT) is one such experimental treatment for superficial bladder carcinoma, involving the administration of a photosensitizer that accumulates in the tumor tissue, and subsequent irradiation of the tumor with light. Since the photosensitizers used in PDT suffer from several drawbacks, new photosensitizers are being sought. Drug delivery systems are also being investigated for the administration of hydrophobic photosensitizers and enhancement of photodynamic efficiency and tumor selectivity. In this study we examined a new photosensitizer, tetramethyl hematoporphyrin (TMHP), in two human bladder cancer cell lines. In the first pair of the experiments, TMHP was bound to unilamellar liposomes. Cellular uptake, dark toxicity and photodynamic efficiency were then studied. Fluorescence microscopy showed TMHP localization in the cytoplasm in a perinuclear region, sparing the nucleus. Dark toxicity occurred after incubation of cells with TMHP above a concentration of 20 g/ml. Irradiation was carried out using an argon-pumped dye laser emitting a wavelength of 630 nm at a fluence of 3.6 and 7.2 J/cm². Before irradiation, cells were incubated with TMHP at concentrations of 2.5 and 5 g/ml for 1 h. Cell survival rates after incubation with 5 g/ml TMHP and irradiation at 7.2 J/cm² were 15.7% of control cells for Rec and 4.5% for Waf cells. Uptake studies showed a higher intracellular TMHP concentration in Waf than in Rec cells. This correlates with the higher PDT efficiency seen in Waf cells. Our results show that TMHP can be encapsulated into unilamellar liposomes without losing its photodynamic efficiency. TMHP is taken up by human bladder carcinoma cells after an incubation time of only 1 h. This short incubation time seems to be appropriate for an intravesical instillation of the photosensitizer for PDT in bladder cancer patients. Intravesical instillation might demonstrate higher phototoxic efficiency with reduced side effects. TMHP acts as a potent photosensitizer and shows drug- and light-dose-dependent cell destruction. Thus, TMHP has the potential for use in PDT in bladder cancer.     

Photodynamic inhibition of acetylcholinesterase after two-photon excitation of copper tetrasulfophthalocyanine

Sequential two-photon (2-γ) activated copper tetrasulfophthalocyanine (CuPcS₄) was shown capable of inactivating acetylcholinesterase (ACE). ACE activity was measured photometrically by the Ellman method. Simultaneous irradiation of ACE in the presence of CuPcS₄ with 514 nm (183 mW/cm²) and 670 nm (86 mW/cm²) continuous wave (CW) light induced a 20–50% increase in enzyme inhibition as compared to one-photon (1-γ) irradiation, using either 514- or 670-nm (CW) light at the same fluences. The enzyme activity was not affected by CuPcS₄ or light alone, decreased linearly with the irradiation time, and was shown to be oxygen-dependent. We conclude that the photoinactivation of ACE with sequential 2-γ irradiation involves reactive oxygen species produced by the interaction of the upper excited T_n state of CuPcS₄ with molecular oxygen. As CuPcS₄ shows little activity as a conventional 1-γ photosensitizer, unwanted side effects such as prolonged skin sensitivity are eliminated rendering 2-γ photodynamic therapy advantageous for the treatment of selected medical applications.     

Fractional resurfacing aiding photodynamic therapy of a recalcitrant plantar verruca: a case report and review of the literature

Fractional resurfacing has become a very popular laser modality in recent years, and photodynamic therapy (PDT) has become a mainstay of many practices treating a wide array of clinical entities. In this case report, we describe a recalcitrant verrucous lesion on the foot that is unresponsive to cryotherapy, pulsed dye laser, and pulsed dye laser with PDT. The lesion did, however, respond very well to the use of a fractional laser to enhance the penetration of the PDT photosensitizer and then responded to pulsed dye laser with PDT. Fractional resurfacing prior to PDT may be a novel dermatologic treatment approach, making PDT an even better treatment option in the future.     

Biofilms of Candida albicans serotypes A and B differ in their sensitivity to photodynamic therapy

Candida albicans is classified into different serotypes according to cell wall mannan composition and cell surface hydrophobicity. Since the effectiveness of photodynamic therapy (PDT) depends on the cell wall structure of microorganisms, the objective of this study was to compare the sensitivity of in vitro biofilms of C. albicans serotypes A and B to antimicrobial PDT. Reference strains of C. albicans serotype A (ATCC 36801) and serotype B (ATCC 36802) were used for the assays. A gallium-aluminum-arsenide laser (660 nm) was used as the light source and methylene blue (300 μM) as the photosensitizer. After biofilm formation on the bottom of a 96-well microplate for 48 h, each Candida strain was submitted to assays: PDT consisting of laser and photosensitizer application (L?+?P+), laser application alone (L?+?P?), photosensitizer application alone (L?P+), and application of saline as control (L?P?). After treatment, biofilm cells were scraped off and transferred to tubes containing PBS. The content of the tubes was homogenized, diluted, and seeded onto Sabouraud agar plates to determine the number of colony-forming units (CFU/mL). The results were compared by analysis of variance and Tukey test (p?C. albicans serotype B were more sensitive to PDT.     

Selective occlusion of choroidal neovascularization by photodynamic therapy with a water-soluble photosensitizer, ATX-S10

BACKGROUND AND OBJECTIVE: To determine the optimal treatment parameters for selective occlusion of choroidal neovascularization (CNV) by photodynamic therapy (PDT) by using the photosensitizer ATX-S10 and a diode laser (wavelength = 670 nm). MATERIALS AND METHODS: Experimental CNV was induced in rat fundi by argon laser photocoagulation. The distribution of ATX-S10 in the chorioretina was analyzed by fluorescence microscopy, and the optimal treatment parameters for selective occlusion of CNV were investigated by changing the dosage and timing of laser irradiation. CNV closure and resulting damage of the surrounding tissue were documented by fluorescein angiography and light and electron microscopies. RESULTS: Fluorescence of ATX-S10 was observed to be localized in the vascular lumen of the retina and choroid within 5 min after dye injection and increased in intensity in CNV up to 2-6 h and decreased rapidly in normal tissue. Laser irradiation with radiant exposures of 7.4 J/cm2 applied immediately after dye injection or with 22.0 J/cm2 at 2-4 h later effectively occluded the induced CNV without causing significant damage to normal retinal capillaries and large choroidal vessels. CONCLUSIONS: PDT using ATX-S10 can selectively occlude CNV. ATX-S10 is a potentially useful photosensitizer for the treatment of CNV.     

Near infra-red diode laser for photodynamic tumour therapy using bacteriochlorina

Clonogenic cell survivals were performed in order to assess the feasibility of tumour cell kill with an experimental diode laser emitting 250 mW of light at λ = 779 nm using the photosensitizer bacteriochlorina(BCA). The AlGaAs diode laser is based on organometallic vapour epitaxial crystal growth technology. The electrical to optical conversion efficiency amounts to 21% and the beam divergence is 47° by 7.0° full width at half maximum. BCA was proved to be an effective non-toxic photosensitizer in vitro and in vivo. It has a major absorption peak at 760 nm where tissue penetration of light is optimal. Clonogenic T24 human bladder carcinoma cell survivals were photosensitizer concentration and light dose dependent. A 0.1% survival rate was obtained with an illumination intensity of 50 mWcm⁻² for 90 s (4.5 Jcm⁻²) and a BCA concentration of 6 μgml⁻¹. Illumination without BCA at energy levels exceeding the PDT levels with a factor 10, or BCA alone without illumination had no effect on the cells in the clonogenic cell survivals. The combination of BCA with a near infra-red diode laser is most promising for photodynamic tumour therapy as a result of the reliability, compactness and relatively low price of the illumination device, the high transmittance of near infra-red light in tissue and the tumour killing potential of BCA.     

Application of lower fluence rate for less microvasculature damage and greater cell-killing during photodynamic therapy

During the process of photodynamic therapy (PDT), problems arise such as stasis or occlusion of microvasculature, tumor oxygen depletion, and photosensitizer bleaching. This study shows that the first problem could be reduced by using a lower fluence rate light source in PDT. Microvasculature damage was studied experimentally in hematoporphyrin derivative–mediated PDT against light fluence rate, and, to some extent, less microvasculature damage was induced under 75 mW/cm² illumination than under 150 mW/cm². Histology of vessels at the end of PDT showed that vessel damage could be observed in both groups, indicating that the microvasculature would eventually be damaged as long as the administration of light fluence was sufficient and regardless of the illuminating fluence rates. Thus microvasculature damage induced by low fluence rate illumination could also be effective in tumor control after PDT. The cell-killing experiment was performed in vitro and designed so that cell-killing rate was influenced only by light characteristics. The higher cell-killing rate caused by 75 mW/cm² illumination indicated that lower fluence rate light could enhance the light absorbency or decrease the bleaching of photosensitizer.The online version of the original article can be found at     

Photofrin-mediated photodynamic therapy of rat palatal mucosa: Normal tissue effects and light dosimetry

Photodynamic therapy (PDT) is a treatment modality with potential application for premalignant lesions and squamous cell carcinoma of the oral mucosa. PDT in principle has dual selectivity. This may result from a preferential retention of the photosensitizer in target tissue. In addition, the photodynamic activity will be limited to the irradiated area because PDT will not affect tissues in the absence of excitation light. The specificity of PDT is limited by the fact that normal tissues also retain the photosensitizer to some degree, which makes these tissues susceptible to PDT damage. To optimize PDT for oral malignancies, a study was undertaken on normal tissue to investigate the responses in rat palatal mucosa and surrounding anatomical structures. Eighty male Wistar rats were used in the study. Photofrin was administered i.v. at four doses (0, 2.5, 5 or 10 mg kg^–1 body weight). Irradiation for PDT was performed 24 h later. An argon pumped dye laser system was used to produce light of two different treatment wavelengths (514.5 and 625 nm), and various energy density levels (0, 25, 50, 100 or 200 J cm^–2). Early effects of PDT were studied at 2 days and late effects at 2 months after treatment. Twenty-four hours after i.v. administration of Photofrin, it was found that PDT affects normal tissues of the oral cavity both macroscopically and microscopically. Combinations of photosensitizer doses 5 mg kg^–1 and light doses100 J cm^–2 caused severe and permanent damage to the palatal mucosa and adjacent normal structures such as palatal bone and dentition.Light scattering and internal reflection usually raise the fluence rate in tissue above the irradiance of the incident beam. In an additional study using six male Wistar rats, the energy fluence rate at two treatment wavelengths (514.5 and 625 nm) was measured ex vivo in the palatal mucosa and adjacent anatomical structures. As expected, the energy fluence rates were wavelength, tissue and depth dependent. At the air-mucosa boundary, light of 625 nm was found to have a three-times higher fluence rate than the primary incident beam. Under similar conditions, the fluence rate of 514.5 nm was found to be less, but still twice as high as the primary incident beam. At deeper levels of the rat maxilla, fluence rates were still elevated compared with the incident beam. For 625 nm light, this phenomenon was observed up to the level of the nasal cavity. These increased fluence rates could largely explain the pattern of damage to normal mucosa and surrounding anatomical structures.