Photodynamic Therapy Protocol Research Articles

Photodynamic Therapy Using Methylene Blue to Treat Cutaneous Leishmaniasis

The purpose of this study was to show the efficiency and underlying mechanism of action of photodynamic therapy (PDT) using methylene blue (MB) and non-coherent light sources to treat cutaneous leishmaniasis (CL). Systemic treatment can cause severe side effects, and PDT using porphyrin precursors as sensitizers has been used as an alternative to treat CL. MB has been used under illumination or in the dark to treat a wide range of medical conditions, and it exhibits antimicrobial activity against protozoa and viruses. In in vitro tests, the cell viability (via a MTT colorimetric assay) of Leishmania amazonensis parasites was evaluated as a function of MB concentration. In in vivo experiments, we analyzed the treatment of two lesions from a patient with leishmaniasis. The patient received a low dose of pentavalent antimony (SbV), and one lesion was treated with PDT. We observed IC(50) decreases from 100 to 20 μM in response to PDT when MB was used in different concentrations in in vitro tests. Use of SbV in combination with the PDT protocol produced faster wound recovery when compared with the use of SbV alone. The in vitro experiments and the results from the clinical case suggest that the inexpensive PDT protocol that is based on MB and RL50® may be used to treat CL caused by L. amazonensis.

Low dose hypericin-PDT induces complete tumor regression in BALB/c mice bearing CT26 colon carcinoma

Successful tumor eradication with photodynamic therapy (PDT) in vivo depends on the optimal combination of treatment parameters. (Low-dose) PDT may additionally induce antitumoral immune responses. Since the naturally occurring hypericin (Hyp) is a promising photosensitizer for PDT, the aim of the study was to investigate phototoxic and immunologic effects of a low-dose Hyp-PDT on murine tumors in contrast to commonly used Hyp-PDT conditions. BALB/c mice bearing CT26 colon carcinoma received hypericin intravenously and were irradiated with red light 0.5-4h later. Tumor development was recorded. Mice were then re-challenged 60 days after the first tumor cell inoculation to investigate an antitumoral immune response. Different results of tumor/host responses were obtained, ranging from mice exitus over delayed tumor growth to complete tumor regression according to different treatment protocols. PDT with common doses and a 4h drug-light-interval resulted in a four times delayed tumor growth compared to the control groups. PDT with relatively low doses and a drug-light-interval of 0.5h led to 100% tumor eradication. Re-challenge of these mice with CT26 mouse colon carcinoma cells prevented new tumor growth. Not only drug concentrations and light doses seem to determine the efficiency of tumor eradication, but also the localization of hypericin at the time of irradiation. Targets in our low-dose PDT protocol are exclusively the vessels. The advantage of this low-dose PDT beside less drug and light exposure of the animals is reduced skin damage, faster healing of the lesions and induction of an antitumoral immune response.

Sonodynamic therapy enhance Photolon-mediated photodynamic therapy of rat brain tumor model glioma C6

Photodynamic therapy (PDT) has been reported as an excellent option for the treatment of small nodular basal cell carcinomas (nBCC). The standard protocol consists of two sessions, one week apart. Sometimes, returning to the hospital after one week can be impractical for elderly patients, due to comorbidities and mobility issues. Therefore, a new technique performed in one day could be superior for those patients.Evaluate the effectiveness of a PDT Single-visit protocol comparing to the standard protocol, as well as pain and long-term recurrence-free follow-up for nBCC.A total of 120 nBCC were treated through a Standard PDT protocol(two sessions, one week apart), and 120 nBCC were treated through a Single-visit PDT(two sessions in one day). A 30-day-after biopsy was performed in order to evaluate the results after the treatment. The lesions that had successful treatment were clinically and dermoscopically evaluated every 6 months up to 60 months. The pain score was compared between the groups(assessed every 3 min during PDT).A complete response at 30-days-after PDT biopsy was observed in 85% of Standard PDT and in 93.3% of Single-visit PDT. Regarding the pain during the illumination, less pain was observed during the second session of the Single-visit PDT. The recurrence-free follow up showed, after 60 months, an 69.0% cumulative probability of recurrence-free for Standard PDT and 80.6% for Single-visit PDT.The suggested Single-visit PDT protocol resulted in better outcomes at 30-day-after PDT biopsy and in lower recurrence rates than the Standard PDT protocol. A more comfortable and more efficient treatment was offered for the patients, with lower pain.

Photobleaching-based method to individualize irradiation time during interstitial photodynamic therapy

Isolated lung metastases in sarcoma and colorectal cancer patients are inadequately treated with current standard therapies. In Vivo Lung Perfusion, a novel platform, could overcome limitations to photodynamic therapy treatment volumes by using low cellular perfusate, removing blood, theoretically allowing greater light penetration. To develop personalized photodynamic therapy protocols requires in silico light propagation simulations based on optical properties and maximal permissible photodynamic threshold dose of lung tissue. This study presents quantification of optical properties for two perfusates and the photodynamic threshold for 5-ALA and Chlorin e6.Porcine and human lungs were placed on Ex Vivo Lung Perfusion, and perfused with acellular solution or blood. Isotropic diffusers were placed within bronchi and on lung surface for light transmission measurements, from which absorption and light scattering properties were calculated at multiple wavelengths. Separately, pigs were injected with 5-ALA or Chlorin e6, and lung tissue was irradiated at increasing doses. Resultant lesion sizes were measured by CT and histology to quantify the photodynamic threshold.Low cellular perfusate reduced the tissue absorption coefficient significantly, increasing penetration depth of light by 3.3 mm and treatment volumes 3-fold. The photodynamic threshold for lung exposed to 5-ALA was consistent with other malignancies. Chlorin e6 levels were undetectable in lung tissue and did not demonstrate photodynamic-induced necrosis.Light penetration with low cellular perfusate is significantly greater and could enable treatments for diffuse disease. This data aids photodynamic treatment planning and will guide clinical translation of photodynamic therapy protocols in the lung, especially during lung perfusion.

Photodynamic Therapy–Induced Immunosuppression in Humans Is Prevented by Reducing the Rate of Light Delivery

Photodynamic therapy (PDT) of non-melanoma skin cancers currently carries failure rates of 10-40%. The optimal irradiation protocol is as yet unclear. Previous studies showed profound immunosuppression after PDT, which may compromise immune-mediated clearance of these antigenic tumors. Slower irradiation prevents immunosuppression in mice, and may be at least as effective as high-fluence-rate PDT in preliminary clinical trials. The photosensitizers 5-aminolaevulinic acid and/or methyl aminolaevulinate were applied to discrete areas on the backs of healthy Mantoux-positive volunteers, followed by narrowband red light irradiation (632 nm) at varied doses and fluence rates. Delayed type hypersensitivity (Mantoux) reactions were elicited at test sites and control sites to determine immunosuppression. Human ex vivo skin received low- and high-fluence-rate PDT and was stained for oxidative DNA photolesions. PDT caused significant, dose-responsive immunosuppression at high (75 mW cm(-2)) but not low (15 or 45 mW cm(-2)) fluence rates. DNA photolesions, which may be a trigger for immunosuppression, were observed after high-fluence-rate PDT but not when light was delivered more slowly. This study demonstrates that the current clinical PDT protocol (75 mW cm(-2)) is highly immunosuppressive. Simply reducing the rate of irradiation, while maintaining the same light dose, prevented immunosuppression and genetic damage and may have the potential to improve skin cancer outcomes.

Photodynamic Therapy for Variant Central Serous Chorioretinopathy: Efficacy and Side Effects

Background: Retinal pigment epithelial detachment (PED) is a recognised associated finding in central serous chorioretinopathy (CSC). We report our experience in treating patients using photodynamic therapy (PDT) in a variant of chronic CSC that presents with only an isolated PED. We present long-term follow-up data and novel observations on the pattern of retinal pigment epithelium (RPE) changes seen after treatment. Methods: Retrospective case series. Results and Discussion: CSC with subfoveal PED can be associated with a poor visual prognosis especially when the PED becomes chronic. PDT has been used to treat typical CSC with good outcomes. Three eyes in 3 patients were successfully treated with PDT, using the Treatment of Age-Related Macular Degeneration with Photodynamic Therapy protocol. However, marked RPE disturbance was observed after treatment directly overlying the areas of brisk choroidal hyperpermeability.

Increased cutaneous oxygen availability by topical application of hydrogen peroxide cream enhances the photodynamic reaction to topical 5-aminolevulinic acid-methyl ester

Topical 5-aminolevulinic acid (ALA) and methyl aminolevulinate (MAL) photodynamic therapy (PDT) of skin lesions is an accepted treatment for skin tumours but success rates need improvement. The effectiveness of PDT is influenced by availability of oxygen. The aim of this study was to demonstrate, in normal skin, whether a decrease in skin oxygen tension reduces the photodynamic reaction (PDR); and whether the addition of topical hydrogen peroxide can reverse the effect. Topical MAL and red light were administered to the inner forearms of 40 healthy volunteers. Skin oxygen availability was lowered during the illumination phase of the PDT, by applying blanching pressure with a plastic slide. Topical hydrogen peroxide was applied under the pressure slide, immediately prior to illumination, to reverse the effect. Erythema was assessed by naked eye and laser Doppler perfusion imaging (LDPI), at baseline and at 1, 5, 24 and 48 h following illumination. Decreasing oxygen availability by pressure altered the PDR with a larger number of subjects (17.5%) not demonstrating any visible erythema at any time point after plastic slide pressure compared to a PDR Control site (7.5%). The addition of topical hydrogen peroxide during pressure application, restored the number of subjects showing no visible erythema compared to that of PDR Control. LDPI data showed that there was a decrease in mean perfusion after plastic slide pressure when comparing the change from baseline to 24 h (P < 0.05) with the PDR Control. The addition of hydrogen peroxide not only restored but also increased the mean perfusion compared to that of PDR Control when comparing the change from baseline to 5 h and the change from baseline to 24 h (P < 0.001). Increasing oxygen availability increased the PDR in normal skin. The possibility that addition of topical hydrogen peroxide to PDT protocols for non-melanoma skin cancer may increase reactivity and, thus, be relevant for outcomes warrants further study.

Intense pulse light and 5‐ALA PDT: Phototoxic effects in vitro depend on the spectral overlap with protoporphyrine IX but do not match cut‐off filter notations

Successful photodynamic therapy (PDT) requires a light source by which light is absorbed by the photosensitizer. Such absorption is achieved by adapting the emission spectrum of the lamp to the absorption-spectrum of the photosensitizer. Intense pulsed light sources (IPLs) are widely used in dermatology, but a standardized protocol for IPL-PDT is not available. Five different IPLs were chosen to evaluate their efficacy for PDT in vitro and the possibility for developing a standard protocol for PDT. Emission-spectra of IPLs were measured with an optical spectrograph and compared with the absorption spectrum of protoporphyrine IX (PpIX). Keratinocytes were incubated with 5-ALA and illuminated with the IPLs. Cell viability was determined for radiant exposures ranging from 0 to 504 J/cm(2) and pulse durations from 8 to 100 milliseconds. A standard LED light source was used as a reference. Cell viability is less effectively reduced by 5-ALA-PDT with IPLs than by a LED light source. Radiant exposures of the five IPLs ranged between 80 and 311 J/cm(2) to achieve the EC(50) value. This value correlated with the spectral overlap of the respective IPL and the absorption-spectrum of PpIX but not with the cut-off filter notations supplied by the manufacturer. All IPLs assessed emit different spectra because of different filter technologies. Different radiant exposures (J/cm(2) ) were necessary to achieve a photodynamic effect with 5-ALA in vitro depending on these spectra similar to the photodynamic effect of the standard LED light source. IPLs may be applicable in clinical PDT but radiant exposure protocols must be separately evaluated for each single IPL despite similar cut-off filter specifications. Such protocols are highly important for clinical practice to avoid a potential mismatch of excitation wavelengths and to prevent photothermal side effects when light intensities of up to hundreds of W/cm(2) are applied.

Temporary exudative retinal detachment following photodynamic therapy in a patient with retinal capillary hemangioblastoma

A 21-year-old male with von Hippel-Lindau syndrome was treated for a retinal hemangioblastoma with photodynamic therapy (PDT). We employed the standard PDT protocol with doubled treatment duration. Seven days after the procedure, there were some perilesional intra- and subretinal hemorrhages and significant exudative retinal detachment extending inferiorly. Four weeks later, the hemorrhages had mostly cleared, and diminished subretinal fluid and partial regression of the hemangioblastoma were noted. PDT might induce temporary subretinal fluid accumulation in eyes with retinal hemangiomas.

Bevacizumab vs Photodynamic Therapy for Choroidal Neovascularization in Multifocal Choroiditis

To compare the effectiveness of photodynamic therapy (PDT) vs intravitreal bevacizumab injection in patients with subfoveal choroidal neovascularization (CNV) secondary to multifocal choroiditis (MC). Patients affected by subfoveal CNV associated with MC referred for clinical evaluation from March 1, 2005, to July 31, 2008, were considered for this pilot randomized clinical trial. Twenty-seven patients were included in the study and followed up from March 15, 2005, through April 30, 2009. After randomization, patients receiving PDT were treated according to the Treatment of Age-Related Macular Degeneration With Photodynamic Therapy protocol, whereas patients receiving intravitreal bevacizumab injection, after a loading phase of 3 monthly injections, were examined monthly and re-treated on the basis of detection of fluid on optical coherence tomography and/or leakage on fluorescein angiography. The primary outcome measure was the 5- and 15-letter change on the Early Treatment of Diabetic Retinopathy Study charts at 12-month examinations compared with baseline. Secondary outcomes included central macular thickness changes. Thirteen and 14 patients were randomized to PDT and bevacizumab treatment, respectively. At the 12-month examination, 5 of 14 eyes treated with bevacizumab and 0 of 13 eyes treated with PDT experienced a best-corrected visual acuity gain of greater than 3 lines (P = .04). Twelve eyes in the bevacizumab group and 6 eyes in the PDT group gained more than 1 line (P = .04). The central macular thickness showed a progressive reduction in both subgroups without a significant difference compared with the baseline values. Greater beneficial effects can be achieved using intravitreal bevacizumab injection rather than PDT for the treatment of subfoveal CNV secondary to MC. Larger multicenter investigations are needed to confirm our preliminary results. Application to Clinical Practice Currently, there is no precise indication regarding the best therapeutic approach to subfoveal CNV secondary to MC. This investigation was designed to verify whether intravitreal bevacizumab injection has a more beneficial effect with respect to PDT.

Comparative Study of Methylene Blue and Erythrosine Dyes Employed in Photodynamic Therapy for Inactivation of Planktonic and Biofilm-Cultivated Aggregatibacter actinomycetemcomitans

The proposal of this work was to test the efficacy of photodynamic therapy (PDT) by using methylene blue (MB) and erythrosine (ERY) to inactivate Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), the main pathogen in aggressive periodontitis. A. actinomycetemcomitans was cultivated in planktonic cultures and biofilm by using Tryptic Soy Broth medium. The sensibility (dark toxicity) to MB and ERY was determined, and its ideal concentration for PDT protocols was established. An odontologic resin photopolymerizer was used as the light source. The bacterial viability was determined by CFU (planktonic cultures) and microscopic observation (biofilms). The results show that ERY is more efficient at killing bacterial cells of A. actinomycetemcomitans in planktonic (75%) and biofilm (77%) culture compared with MB (50% and 54%, respectively). PDT using MB or ERY as a photosensitizing agent and odontologic resin photopolymerizer as a light source could be an efficient option for pocket decontamination in aggressive periodontal disease.

Mechanisms of Singlet‐Oxygen and Superoxide‐Ion Generation by Porphyrins and Bacteriochlorins and their Implications in Photodynamic Therapy

New halogenated and sulfonated bacteriochlorins and their analogous porphyrins are employed as photosensitizers of singlet oxygen and the superoxide ion. The mechanisms of energy and electron transfer are clarified and the rates are measured. The intermediacy of a charge-transfer (CT) complex is proved for bacteriochlorins, but excluded for porphyrins. The energies of the intermediates and the rates of their interconversions are measured, and are used to obtain the efficiencies of all the processes. The mechanism of formation of the hydroxyl radical in the presence of bacteriochlorins is proposed to involve a photocatalytic step. The usefulness of these photosensitizers in the photodynamic therapy (PDT) of cancer is assessed, and the following recommendations are given for the design of more effective PDT protocols employing such photosensitizers: 1) light doses should be given over a more extended period of time when the photosensitizers form CT complexes with molecular oxygen, and 2) Fe(2+) may improve the efficiency of such photosensitizers if co-located in the same cell organelle assisting with an in vivo Fenton reaction.

Compartmental Targeting for mTHPC-Based Photodynamic Treatment In Vivo: Correlation of Efficiency, Pharmacokinetics, and Regional Distribution of Apoptosis

The present study investigates the efficacy of compartmental targeting in xenografted tumors treated by meta-tetra(hydroxyphenyl)chlorin (mTHPC)-mediated photodynamic therapy (PDT). The therapeutic efficacy was, furthermore, related to a regional photoinduced distribution of apoptosis and an mTHPC biodistribution profile. Mice bearing EMT6 tumors were subjected to a single irradiation (10 J/cm(2)) of red laser light (652 nm) at different intervals after a single- (0.3 mg/kg or 0.15 mg/kg) or double-intravenous (2 × 0.15 mg/kg) injection(s) of mTHPC. Efficiency of the treatment was evaluated by monitoring tumor regrowth. mTHPC pharmacokinetics were assessed by high-performance liquid chromatography analysis of excised organs. The regional distribution of apoptosis in tumor sections was investigated with a newly developed colabelling immunohistochemistry technique. A fractionated double-injection protocol of mTHPC with 24-h and 3-h drug-light intervals (DLI) yielded 100% tumor cure, with tumors presenting a massive apoptosis of neoplastic cells along with a distortion of vessels. The best efficiency for a single injection (0.3 mg/kg) was about 54% tumor cure and corresponded to a DLI of 3 h. At this DLI, tumors showed apoptosis of endothelial cells in residual vessels. Concentrations of mTHPC observed in plasma and tumor for the fractionated injection were not statistically different and were less than the total drug dose in each compartment. The present work suggests that clinical PDT protocols with mTHPC could be greatly improved by fractionation of the drug administration. Time points should be chosen based on the intratumoral spatiotemporal drug distribution.

P25 Devices for phototherapy based on modern nanotechnologies

Photodynamic therapy (PDT) is an adjunctive treatment that aims to inactivate microorganisms through an oxidative reaction produced by irradiating a photosensitizing agent. The quest for improved root canal disinfection has sought supplementary methods when performing chemomechanical procedures. From this perspective, PDT protocols were proposed as an auxiliary approach in endodontics. Thus, the aim of this study was to investigate publication metrics and research trends related to this scope.This review is reported in accordance with the PRISMA 2020 recommendations. Two blinded and independent reviewers systematically searched five electronic databases until December 2021. The acquired bibliometric parameters were analyzed through descriptive statistics and graphical mappings with VOSViewer software.The search retrieved 342 studies from 84 journals originating from 33 countries. About 85% of the included studies were published over the last decade. Most of the available evidence is laboratory-based (74.5%), and the main clinical outcomes evaluated were microbiological load reduction and postoperative pain. Mayram Pourhajibagher is the researcher with the most publications as the first author (n = 16). Tehran University of Medical Sciences carried out the highest number of studies (n = 29), and Photodiagnosis and Photodynamic Therapy is the journal that most published on the theme (n = 111).This bibliometric analysis mapped and discussed the scientific progress and publication metrics in PDT in endodontic research. Additionally, future perspectives were highlighted and should focus on discovering new photosensitizer agents, standardizing optimal photoactivation protocols, and conducting more clinical-oriented research.

Successful treatment of an early invasive oral squamous cell carcinoma with topical 5-aminolevulinic acid-mediated photodynamic therapy

Our previous studies showed successful treatment of a series of 36 oral verrucous hyperplasia lesions and of an extensive oral verrucous carcinoma with a topical 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (topical ALA-PDT) protocol (with a fluence rate of 100 mW/cm 2 and a light exposure dose of 100 J/cm 2 ) using a 635-nm light-emitting diode (LED) light source. In this case report, we tested whether an enhanced topical ALA-PDT protocol (with a fluence rate of 200 mW/cm 2 and a light exposure dose of 200 J/cm 2 ) could be used to treat an early invasive oral squamous cell carcinoma (OSCC) with a verrucous appearance of the left lower posterior edentulous alveolar mucosa of a 67-year-old male former areca-quid chewer and ex-smoker. The main verrucous lesion showed complete regression after eight treatments with PDT. However, 10 extra treatments were needed to eradicate the multiple residual leukoplakia lesions on the edentulous alveolar mucosa. Moderate to severe post-PDT pain was noted during the initial eight treatments, and the patient needed analgesics (codeine phosphate, 30 mg three times daily) to control the pain. No recurrence of the OSCC lesion was found after a follow-up period of 4 years. We suggest that our enhanced topical ALA-PDT protocol may have good potential to be used as a treatment of choice for a superficially invasive OSCC without regional or distant metastasis before the commencement of other effective therapies.

&lt;I&gt;In Vitro&lt;/I&gt; Phototoxicity of Liposomes and Nanocapsules Containing Chloroaluminum Phthalocyanine on Human Melanoma Cell Line

In this study, the photodynamic action of liposomes (LP) and nanocapsules (NC) containing Chloroaluminum phthalocyanine (CIAIPc), on the human melanoma cell (WM 1552C), was assessed. The light source was setup at 672 nm, which corresponds to the maximum absorption wavelength of the CIAIPc. Both colloidal carriers presented size in nanometric scale as well as negative zeta potential. The cellular damage was light dose dependent ranging from 30% of cell death at 70 mJ x cm-2 to 90% of death at 700 mJ x cm(-2). However, the photocytotoxic effect of LP at 70 mJ x cm(-2) was slightly more efficient to induce cellular death than NC formulation. At 140 mJ x cm(-2), and 700 mJ x cm(-2) both nanocarriers were equally efficient to induce cellular damage. Therefore, in the present work, the maximum phototoxic effect was obtained with 700 mJ x cm(-2) of light dose, in combination with 0.29 microg x mL(-1) of CIAIPc encapsulated into LP and NC. The cells were also positive to annexin V, after the PDT treatment with LP and NC, showing that one of the mechanisms of cellular death involved is apoptosis. In summary, the potential of LP and NC as a drug delivery system, in Photodynamic Therapy (PDT) against melanoma, has been confirmed using a lower concentration of the photosensitizer and lower light doses than that applied in current protocols. This is an innovative proposal to treat melanoma cell lines that until now have not received the benefit of the PDT protocol for treatment.

Photodynamic Therapy with Two Different Photosensitizers as a Supplement to Instrumentation/Irrigation Procedures in Promoting Intracanal Reduction of Enterococcus faecalis

Introduction This in vitro study aimed to investigate the antibacterial effects of photodynamic therapy (PDT) with methylene blue (MB) or toluidine blue (TB) (both at 15 μg/mL) as a supplement to instrumentation/irrigation of root canals experimentally contaminated with Enterococcus faecalis. Methods Seventy extracted teeth had their root canals contaminated with an endodontic strain of E. faecalis for 7 days, instrumented with nickel-titanium instruments and irrigated either with 2.5% NaOCl or with 0.85% NaCl, and then randomly distributed into four experimental groups: MB/NaOCl (PDT with MB and NaOCl as the irrigant), TB/NaOCl (PDT with TB and NaOCl as the irrigant), MB/NaCl (PDT with MB and NaCl as the irrigant), and TB/NaCl (PDT with TB and NaCl as the irrigant). For PDT, the photosensitizer remained in the canal for 2 minutes before exposed to red light emitted from a diode laser for 4 minutes. Samples were taken before and after instrumentation/irrigation and following the specific PDT procedure for each group, plated onto Mitis-salivarius agar and the colony forming units counted. Results Regardless of the irrigant used (NaOCl or NaCl), instrumentation significantly reduced bacterial counts in comparison to the baseline (p < 0.001). NaOCl as the irrigant was significantly more effective than NaCl, and this difference persisted after PDT, irrespective of the photosensitizer used (p < 0.05). PDT with either MB or TB did not significantly enhance disinfection after chemomechanical preparation using NaOCl as irrigant (p > 0.05). No significant differences were observed between the two photosensitizers (p > 0.05). Conclusion These in vitro results suggest that PDT with either MB or TB may not exert a significant supplemental effect to instrumentation/irrigation procedures with regard to intracanal disinfection. Further adjustments in the PDT protocol may be required to enhance predictability in bacterial elimination before clinical use is recommended.

The effect of the first application of verteporfin photodynamic therapy on lesion growth in choroidal neovascularisation and its potential impact on combination therapy

Purpose: To evaluate the impact of lesion size on the observed growth of the choroidal neovascularisation (CNV) following the first application of photodynamic therapy (PDT).Methods: A retrospective study of consecutive patients with subfoveal classic CNV or predominantly classic CNV undergoing first verteporfin photodynamic therapy (VPDT) according to treatment of age‐related macular degeneration with photodynamic therapy (TAP) protocol between June 2005 and September 2005. Patients were classified into two groups according to baseline greatest linear dimension (GLD) at the time of VPDT monotherapy. Group 1 comprised patients with lesion GLD less then 2000 µm and Group 2, patients with lesion GLD 2000 µm or larger. Difference between the mean GLD at baseline and at three months post‐treatment were investigated for clinical significance using the paired t‐test.Results: Group 1 (n = 16) showed a mean change in GLD from 1380.25 µm to 2031.25 µm, while Group 2 (n = 26) showed a mean change of 2909.26 µm to 3023.07 µm. Data of mean percentage change in GLD for Group 1 showed a 51.97 per cent increase in the lesion size [95% CI, 22.93 ‐ 81.01%] in comparison to Group 2, which showed only 5.8 per cent increase in the lesion [95% CI, −9.65 ‐ 21.61%]. Comparison of the mean percentage change in GLD between the two groups showed a statistically significant difference (p = 0.003).Conclusions: Our study demonstrates that following the first application of VPDT, smaller lesions enlarge disproportionately more than larger lesions. As we have entered into the era of using angiostatic agents in combination with VPDT, it may be important to evaluate this effect with regards to the timing of VPDT commencement.

Monitoring Singlet Oxygen and Hydroxyl Radical Formation with Fluorescent Probes During Photodynamic Therapy

Singlet oxygen (1O2) is the primary oxidant generated in photodynamic therapy (PDT) protocols involving sensitizers resulting in type II reactions. 1O2 can give rise to additional reactive oxygen species (ROS) such as the hydroxyl radical (*OH). The current study was designed to assess 3'-p-(aminophenyl) fluorescein (APF) and 3'-p-(hydroxyphenyl) fluorescein (HPF) as probes for the detection of 1O2 and *OH under conditions relevant to PDT. Cell-free studies indicated that both APF and HPF were converted to fluorescent products following exposure to 1O2 generated by irradiation of a water-soluble photosensitizing agent (TPPS) and that APF was 35-fold more sensitive than HPF. Using the 1O2 probe singlet oxygen sensor green (SOSG) we confirmed that 1 mm NaN3 quenched 1O2-induced APF/HPF fluorescence, while 1% DMSO had no effect. APF and HPF also yielded a fluorescent product upon interacting with *OH generated from H2O2 via the Fenton reaction in a cell-free system. DMSO quenched the fluorogenic interaction between APF/HPF and *OH at doses as low as 0.02%. Although NaN3 was expected to quench *OH-induced APF/HPF fluorescence, co-incubating NaN3 with APF or HPF in the presence of *OH markedly enhanced fluorescence. Cultured L1210 cells that had been photosensitized with benzoporphyhrin derivative exhibited APF fluorescence immediately following irradiation. Approximately 50% of the cellular fluorescence could be suppressed by inclusion of either DMSO or the iron-chelator desferroxamine. Combining the latter two agents did not enhance suppression. We conclude that APF can be used to monitor the formation of both 1O2 and *OH in cells subjected to PDT if studies are performed in the presence and absence of DMSO, respectively. That portion of the fluorescence quenched by DMSO will represent the contribution of *OH. This procedure could represent a useful means for evaluating formation of both ROS in the context of PDT.

Regression of the murine LM3 tumor by repeated photodynamic therapy withmeso-tetrakis-(4-N,N,N-trimethylanilinium)porphine

Photodynamic therapy (PDT) of cancer is based on the cytotoxicity induced by a photosensitizer (PS) in the presence of oxygen and visible light, with formation of reactive oxygen species which cause cell death and tumor destruction. This work describes the response of the murine mammary adenocarcinoma, LM3, to repeated PDT using meso-tetrakis(4-N,N,N-trimethylanilinium)porphine (TMAP), a PS that has been overlooked for PDT applications. Intradermal LM3 tumors in BALB/c mice (controls) were left untreated, only treated with light, only injected with 0.9% NaCl solution or with TMAP alone (10 μg/0.1 ml). For PDT, the intratumoral PS injection was followed 1 h later by blue-red light irradiation (290 J.cm-2). In all cases, control and PDT treatments were performed on the depilated and glycerol-covered skin which covers the tumor of anesthetized animals, and repeated 4 times (every 2 days). No significant differences were found in the growth rate of all control tumors. PDT-treated tumors showed complete and long-term regression in 4 out of 5 mice, and cure in one animal. The survival of PDT-mice was significantly longer than that of controls (TMAP alone), showing a lower number of tumor-draining lymph node metastasis. The PDT protocol applied in the present work (intratumoral PS injection, repeated photosensitizing treatments, and reduction of tissue light scattering by glycerol) could be a useful strategy in studies on PDT of cancer.