Photodiagnosis and Photodynamic Therapy - Articles in Press

Photodynamic inactivation requires innovative approach concerning numerous bacterial isolates and multicomponent sensitizing agents - Corrected Proof

Joanna Nakonieczna, Mariusz Grinholc — 2012-05-14

Abstract: It is known that Staphylococcus aureus is susceptible to photodynamic inactivation in general, but the significant variation among particular strains in the response to the treatment exists. However, factors that determine the observed phenomenon remain unclear. This study was aimed to explore the PDI effect of two sensitizers (protoporphyrin diarginate and toluidine blue O) against clinical as well as reference strains of S. aureus. Obtained results indicate that the same isolate could be characterized as highly resistant or highly sensitive to PDI according to a sensitizer used. Moreover, the same sensitizing agent could be successfully used for total eradication of some isolates and could be non-effective in the case of other strains. Additionally, changing the photosensitizer, we are able to reverse the PDI “resistant” phenotype into “sensitive” one. Thus, one could conclude that photoinactivation involving several sensitizing agents and several isolates of the same bacterial species should be undertaken to make antimicrobial photodynamic inactivation reliable.

Comparative characterization of the cellular uptake and photodynamic efficiency of Foscan® and Fospeg in a human prostate cancer cell line - Corrected Proof

Aspasia Petri, Dido Yova, Eleni Alexandratou, Maria Kyriazi, Michail Rallis — 2012-05-07

Summary: Background: m-THPC (Foscan®) is one of the most potent second generation photosensitizers used in photodynamic therapy, photoactivated at higher wavelengths (652nm). However, its strongly hydrophobic nature causes aggregation of the molecules and prevents its unbiased bioavailability in the biological media, resulting in lower accumulation in the tumor cells. Several strategies have been adopted to improve the photodynamic characteristics of the photosensitizer. Among them, very promising seems to be the encapsulation of the molecule into liposomes, due to the superior properties of liposomes as drug carriers.Methods: In this paper the photodynamic characteristics of the PEGylated liposomal formulation of m-THPC, Fospeg, using the human prostate cancer cell line LNCaP, as an in vitro model, were investigated. In addition the spectral characteristics, cellular uptake and localization, dark and light induced cytotoxicity and photodynamic efficacy of Foscan® and Fospeg were compared.Results: Fospeg, compared with Foscan, showed higher intracellular uptake at any concentration and incubation time. Regarding PDT efficacy, Fospeg produced more severe cytotoxicity than Foscan® at any concentration and energy dose. Using Fospeg, the lowest concentration (0.22μM) and energy dose (180mJ/cm2) was adequate to result in the death of 50% of the cells 24h post PDT while an approximately 10 times higher Foscan® concentration (1.8μM) was needed to result in the same cytotoxicity.Conclusions: The use of the PEGylated liposomal formulation of m-THPC resulted in the improvement of its intracellular uptake and the enhancement of its photodynamic activity. Fospeg, compared to Foscan®, proved to be a more advantageous photosensitizer for photodynamic therapy.

Evaluation of diagnostic values of photodynamic diagnosis in identifying the dermal and mucosal squamous cell carcinoma - Corrected Proof

2012-04-18

Summary: Objectives: We conducted a study to determine and compare the efficacy of pathology and photodynamic studies in establishing diagnosis of malignant dermal and mucosal lesions.Methods and patients: This descriptive cross-sectional study was performed on 40 patients suspected of SCC (squamous cell carcinoma). First, in PDD (photodynamic diagnosis) photosensitizing agent was applied to the lesion, and after 4–5h the fluorescence spectrum was detected by laser radiation. Based on fluorescence intensity, normal area was differentiated from malignant area. Also, biopsy samples from these suspected areas were sent to pathology simultaneously.Data were analyzed with SPSS v.16. The distribution of nominal variables was compared using the Chi-square test. Diagnostic index for photodynamic diagnosis were calculated. A two-sided p-value<0.05 was considered to be statistically significant.Results: In 27 cases (90%), results of pathology and photodynamic studies similarly showed malignancy. In 8 cases (80%), results of pathology and photodynamic studies similarly showed non-malignant lesion. But in five cases (12.5%) the results of pathology and photodynamic studies were not the same. This difference was not statistically significant showed by the Chi-square test analysis (p-value>0.05).A sensitivity of 90%, specificity of 80%, accuracy of 87.5%, positive predictive value (PPV) of 93%, negative predictive value (NPV) of 72%, positive likelihood ratio (PLR) of 4.5, negative likelihood ratio(NLR) of 0.125 were found in diagnosing SCC for photodynamic studies.Conclusion: Photodynamic diagnosis is a useful non-invasive initial step in the diagnostic work-up of patients with suspected malignant lesions (SCC). In this work we have studied 40 SCC suspicious patients using PDD method and successfully carried out 27 cases as malignant all of which were matching with pathologic results. This outcome can prove both accuracy and reliability of PDD method for detecting SCC lesions on head and neck regions. Also PDD can fully demarcate the lesion peripheries less invasively as well as preserving much more time and effort. Although PDD method is a bit more expensive that biopsy and pathology but great advantages can easily cover this issue. We recommend PDD as a useful easy technique to visualize and detect the extension of the tumor preoperatively.

Effects of hypericin and a chlorin based photosensitizer alone or in combination in squamous cell carcinoma cells in the dark - Corrected Proof

2012-04-18

Summary: Introduction: The toxic influence of photosensitizers in the dark is poorly investigated. In our study we used the photosensitizers liposomal meso-tetrahydroxyphenyl chlorin derivative (Foslipos®) and hypericin as well as their 1:1 combination on two different head and neck squamous cell carcinoma (HNSCC) cell lines (UMB-SCC 745 and UMB-SCC 969).Materials and methods: We examined uptake, efflux and localization of the photosensitizers with confocal microscopy. Fluorescence quantification was measured with a micro-plate spectrometer. Special interest was given to effects on cell proliferation (BrdU proliferation assay), RNA quality (Bioanalyzer measurements) and DNA damage (comet assays) in the dark.Results: Foslipos® uptake was linear over time and its efflux was not achieved even after 24h while uptake of hypericin reached a plateau after 5h and was almost eliminated after 24h. Localization of Foslipos® was organelle-unspecific. Hypericin was found mainly at membranes and in trans-golgi network. Foslipos® treated cells showed cell toxicity for the highest concentration (10μg/mL). In contrast, hypericin was toxic for all concentrations (10–0.6μg/mL). The photosensitizer combination was non-toxic for all concentrations (10–0.6μg/mL). No changes in RNA quality were monitored. Initial DNA damage was found only in hypericin treated UMB-SCC 745, which recovered after 3h. No significant DNA damage was found for UMB-SCC 969.Conclusion: Our data shows that the combinatorial application decrease photosensitizer toxicity, which can be advantageous in PDT treatments.

Adverse effects associated with photodynamic therapy (PDT) of port-wine stain (PWS) birthmarks - Corrected Proof

Kai-Hua Yuan, Jian-Hua Gao, Zheng Huang — 2012-04-18

Summary: Background: Several Chinese studies suggest that Hemoporfin-mediated photodynamic therapy (PDT) is an alternative treatment for port-wine stain (PWS) birthmarks.Objective: To evaluate treatment responses and adverse effects associated with Hemoporfin PDT for the treatment of PWS and their management.Method: The medical records of 700 patients who underwent PDT treatment in our center were retrospectively examined. Treatment-related reactions and adverse effects were reviewed.Result: Different types of PWS lesions and different individuals showed different immediate responses (e.g. swelling, color change, pain). To certain extents these reactions were a useful indicator of the treatment endpoint. Edema and scabbing were the most common post-treatment responses. Short-term (e.g. blister, eczematous dermatitis, cutaneous photosensitivity) and long-term (e.g. pigmentation change, scar formation) adverse effects were generally caused by the phototoxicity associated with the combination of photosensitizer and light exposure.Conclusion: Although PDT is a safe treatment alternative for PWS birthmarks, treatment parameters must be selected for each individual patient and cutaneous changes must be monitored during light irradiation to minimize the risk of adverse effects. Over estimation of required light dosage or failure to recognize cutaneous changes associated with adverse effects can increase the risk of a poor outcome.

Photodynamic therapy for unresectable cholangiocarcinoma: A comparative effectiveness systematic review and meta-analyses - Corrected Proof

2012-04-13

Summary: Background: Photodynamic therapy (PDT) with placement of a biliary stent may improve bile duct patency in patients with cholangiocarcinoma (CCA). We aimed to determine the effectiveness of biliary stenting with PDT compared to biliary stenting alone in the palliative treatment of CCA.Materials and methods: Several databases were searched from inception to December 2011 for prospective studies comparing biliary stenting with PDT vs. biliary stenting only for CCA. Outcomes of interest included patient survival, quality of life (using Karnofsky score), and serum bilirubin levels. The relative risk (RR) for dichotomous outcomes and the weighted mean difference (WMD) for continuous outcomes were estimated using DerSimonian and Laird random-effects model. Inconsistency was quantified using I2 statistics. The extent of publication bias was ascertained by visual inspection of funnel plots and Egger's test.Results: There were six studies that met inclusion criteria. A total of 170 participants received PDT and 157 had biliary stenting only. Compared with biliary stenting, PDT was associated with a statistically significant increase in the length of survival (WMD 265 days; 95%CI: 154–376; p=0.01; I2=65%), improvement in Karnofsky scores (WMD 7.74; 95%CI: 3.73–11.76; p=0.01; I2=14%), and a trend for decline in serum bilirubin (WMD −2.92mg/dL; 95%CI: −7.54 to 1.71; p=0.22; I2=94%). The pooled event rate for biliary sepsis was 15% and was similar between PDT and control groups.Conclusion: Palliative treatment of CCA with PDT is associated with increased survival benefit, improved biliary drainage, and quality of life. However, the quality of this evidence is low.

Successful treatment of 7,12-dimethylbenz(a)anthracene-induced hamster buccal pouch precancerous lesions by topical 5-aminolevulinic acid-mediated photodynamic therapy - Corrected Proof

Yih-Chih Hsu, Deng-Fu Yang, Chun-Pin Chiang, Jeng-Woei Lee, Meng-Kai Tseng — 2012-04-09

Summary: Background: Our previous studies found that topical 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (ALA-PDT) with a light dose of 100J/cm2 is very effective for human oral precancerous lesions.Methods: In this study, 20 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch precancerous lesions were treated by topical ALA-PDT with a light dose of either 75J/cm2 (n=10) or 100J/cm2 (n=10) using a 640-nm light-emitting diode (LED) light to test which light dose could achieve a better clinical outcome.Results: The 10 precancerous lesions treated by 75-J ALA-PDT showed complete response in 8 after an average of 3.4 (range, 2–6) treatments and partial response in 2. The 10 precancerous lesions treated by 100-J ALA-PDT demonstrated complete response in 7 after an average of 4.4 (range, 3–6) treatments and partial response in 3. Fisher exact test showed no significant difference in clinical outcome between these two treatment modalities (p=1.000). One complete-response precancerous lesion in the 75-J ALA-PDT group recurred at the end of 19-week follow-up and another complete response precancerous lesion in the 100-J ALA-PDT group recurred at the end of 16-week follow-up. Both recurrence lesions were treated by the original topical ALA-PDT regimen and demonstrated complete response after 3 PDT treatments. Furthermore, the 5 partial-response precancerous lesions developed into squamous cell carcinomas after 30-week follow-up.Conclusion: Our findings indicate that both the 75-J and 100-J topical ALA-PDT treatment modalities are very effective for DMBA-induced hamster buccal pouch precancerous lesions and no significant difference in clinical outcome between these two treatment modalities.

PDT induced bystander effect on human xenografted colorectal tumors as evidenced by sodium MRI - Corrected Proof

2012-04-02

Summary: Background: Previous in vivo studies on photodynamic therapy (PDT)-treated, high cellular density tumors showed evidences of a bystander effect accompanying the therapy, cellular death continuing beyond the limits of the photochemical reactions in time and space. This process is generated by the initially damaged cells on the light pathway. The aim of this study was to determine if the bystander effect may be induced as well in colorectal xenografted tumors (less compact structure) and if the cellular signaling depends primarily on cellular proximity or not.Methods: The photosensitizer was a glycoconjugated, meso substituted porphyrin derivative synthesized at Institut Curie. The longitudinal follow-up of the tumors was carried out by 23Na/1H MRI, ideal imaging modality for mapping the extracellular compartment. Two regimens were followed in order to target either blood vessels alone or blood vessels and cancer cells simultaneously.Results: The antivascular PDT did not succeed to arrest the tumors growth at the end of the follow-up. For double targeting PDT, we managed to stop the tumoral evolution. Sodium MRI evidenced a bystander effect.Conclusion: The results obtained showed that the bystander effect is more difficult to induce for the type of colorectal tumors used in this work. It needs a double treatment, 4 days apart, in order to be promoted.

A basic study on Hypericin-PDT in vitro - Corrected Proof

Nobue Nakajima, Norimichi Kawashima — 2012-03-29

Summary: The effect of photo dynamic therapy (PDT) using hypericin as a photosensitiser and the effect of PDT on intracellular ATP levels using different lamps in a human leukemic monocyte lymphoma cell line (U937) were studied. The time required for hypericin to penetrate into the cancer cells was 1h, and incubation for more than 3h post-irradiation with hypericin-PDT was required to observe effects. Thus, if cancer cell death does not occur immediately following irradiation, it is unnecessary to perform additional irradiation, as most of the cells die via apoptosis during the incubation period post-irradiation. When hypericin-PDT was performed using a Na–Li lamp as a light source, the cell viability decreased approximately 55% immediately following irradiation for 5min; however, after a 5-h post-irradiation incubation, the cell viability approached 0%. Concurrently, intracellular ATP levels increased markedly; thus, irradiation (0.225J/cm2) for 5min provided the best results in terms of the highest degree of cancer cell apoptosis. Similar experiments were performed using three different LED lamps respectively. When cells were treated with the LED lamps, with maximum peaks of 599nm and 595nm, the cell viability approached 0% after incubation for 5h following 15min of irradiation (0.04J/cm2 and 0.099J/cm2, respectively). We confirmed that incubating the cells for more than 3h in a 100× diluted hypericin solution was the most effective for PDT and that a LED lamp of low light intensity led to the highest apoptosis rate in the U937 cells.

Cutaneous penetration of the topically applied photosensitizer Pc 4 as detected by intravital 2-photon laser scanning microscopy - Corrected Proof

2012-02-27

Summary: The fundamental mechanism of photodynamic therapy (PDT)-induced cell death has been characterized, but early critical PDT events in vivo remain incompletely defined. With the recent development in advanced fluorescence imaging modalities, such as intravital 2-photon laser scanning microscopy (2P-LSM), researchers are now able to investigate and visualize biological processes with high resolution in real time. This powerful imaging technology allows deep tissue visualization with single-cell resolution, thus providing dynamic information on the 3-dimensional architectural makeup of the tissue. The main goal of this study was to determine the cutaneous penetration of a topically applied photosensitizer, the silicon phthalocyanine Pc 4, into the skin of live animals and to assess the effective absorption of Pc 4 through the skin barrier. Our 2P-LSM images indicate that Pc 4 penetrates to the epidermal/dermal junction of mouse skin. The data also indicate that the degree of Pc 4 absorption is dose dependent. These findings represent initial steps that may help in improving the clinical utilization of topical Pc 4-PDT.

Photodynamic therapy in urology: What can we do now and where are we heading? - Corrected Proof

2012-02-23

Summary: Background: Photodynamic therapy (PDT) is an innovative technique in oncologic urology. Its application appears increasingly realistic to all kind of cancers with technological progress made in treatment planning and light delivery associated with the emergence of novel photosensitizers. The aim of this study is to review applications of this technique in urology.Materials and methods: We reviewed the literature on PDT for urological malignancies with the following key words: photodynamic therapy, prostate cancer, kidney cancer, urothelial cancer, penile cancer and then by cross-referencing from previously identified studies.Results: Focal therapy of prostate cancer is an application of PDT. Clinical studies are ongoing to determine PDT efficacy and safety. PDT as salvage treatment after radiotherapy has been tested. Oncologic results were promising but important side effects were reported. Individual dosimetric planning is necessary to avoid toxicity.PDT was tested to treat superficial bladder carcinoma with promising oncologic results. Serious side effects have limited use of first photosensitizers generation. Second generation of photosensitizer allowed reducing morbidity. For upper urinary tract carcinoma and urethra, data are limited. Few studies described PDT application in penile oncology for conservative management of carcinoma in situ and premalignant lesions. For renal cancer, PDT was only tested on preclinical model despite of its potential application. No data is available concerning PDT application for testicular cancer.Conclusion: PDT clinical applications in urology have proved a kind of efficiency balanced with an important morbidity. Development of new photosensitizer generations and improvement in illumination protocols should permit to decrease side effects.

Optical properties of hematoporphyrin monomethyl ether (HMME), a PDT photosensitizer - Corrected Proof

2012-02-10

Summary: We report on some of the optical properties of Hemoporfin® (hematoporphyrin monomethyl ether, HMME), a photodynamic therapy (PDT) photosensitizer that has been in clinical trials in China since the early 1990s. We characterized the photosensitizer on the basis of one- and two-photon absorption and fluorescence emission. The effects of photobleaching were probed to characterize its decay kinetics. Additionally, we determined time resolved fluorescence and thermal effects on fluorescence and absorption properties.

Hyaluronic acid-functionalized mesoporous silica nanoparticles for efficient photodynamic therapy of cancer cells - Corrected Proof

2012-02-01

Summary: Mesoporous silica nanoparticles (MSN) for photodynamic therapy (PDT) were coated with poly-(l-lysine) and hyaluronic acid (HA) by using the layer-by-layer method. HA is able to target cancer cells over-expressing the corresponding CD44 receptor. MSN functionalized with HA (MSN-HA) were more efficient than MSN without the targeting moiety when PDT was performed at low fluence (14Jcm−2) and low dosage of MSN (20μgmL−1) on HCT 116 colorectal cancer cells, known to over-express the CD44 receptor. Incubation of HCT-116 cancer cells with an excess of HA impaired the PDT effect with MSN-HA thus demonstrating that an active endocytosis mechanism was involved in the uptake of MSN-HA by these cells.

Modulation of telomerase and signal transduction proteins by Hexyl-ALA-Photodynamic Therapy (PDT) in human doxorubicin resistant cancer cell models - Corrected Proof

Ellie S.M. Chu, Christine M.N. Yow — 2012-01-30

Summary: Aims: This study employed a doxorubicin resistant (MES-SA-Dx5) human uterine sarcoma cell line and its counterpart (MES-SA), to elucidate the efficacy of aminolevulinic acid-hexylester (hexyl-ALA) mediated PDT at molecular and transcriptional levels.Methods: Hexyl-ALA generated protoporphyrin IX in both cells were determined by molecular probes using Confocal Laser Scanning Microscopy. The hexyl-ALA-PDT induced signal transduction proteins and mode of cell death were quantitated by CASE™ ELISA assays and DAPI staining. The modulation of hTERT mRNA expression and telomerase activity were investigated by TaqMan real-time PCR and ELISA respectively. Hexyl-ALA-PDT mediated cell migratory effect was determined by wound-healing assay.Results: The results demonstrated that mitochondria were the major target of hexyl-ALA. At LD30, hexyl-ALA-PDT significantly provoked an up-regulation of phosphorylated p38MAPK and JNK proteins in both cells. Hexyl-ALA-PDT down-regulated hTERT (a catalytic subunit of telomerase) mRNA expression and showed a strong correlation with diminished telomerase activity in both cells (MES-SA: r2=0.9932; MES-SA-Dx5: r2=0.9775). The suppression of cell migratory effect in both cells was obtained after hexyl-ALA-PDT. Further, 50% and 30% of apoptotic cells were attained at LD50, for wild-type and drug resistant cells respectively. Unlike the wild-type, a higher PDT dose was crucial to induce apoptosis in the drug resistant cells.Conclusions: Our study provides the first evidence that p38MAPK and JNK kinases played a vital role in triggering hexyl-ALA-PDT-induced apoptosis, down-regulated hTERT mRNA expression and telomerase activity in both proposed cells. In vivo studies are worth examining for the benefit of clinical applications in drug resistant cancers and PDT development.

Investigating the efficiency of novel metallo-phthalocyanine PDT-induced cell death in MCF-7 breast cancer cells - Corrected Proof

Tamarisk Kerry Horne, Heidi Abrahamse, Marianne J. Cronjé — 2012-01-16

Summary: Background: Cancer cells possess an innate resistance to inducers of the death program. Novel phthalocyanines with improved physiochemical properties harbor the potential for use in photodynamic therapy (PDT); a rising treatment alternative preferred for its mostly asymptomatic application and unique mechanism of action.Methods: This study aimed to determine whether in vitro PDT with two new metallo-phthalocyanines (metallo-Pcs), AlPcSmix and GePcSmix, are similarly effective in overcoming resistance to cell death in MCF-7 cells with a brief comparison to an established chemotherapeutic agent, etoposide. Optimum induction of cell death in these cancer cells was initially determined via measurement of cellular respiration and energy production levels. Indications of cytotoxicity and cell stress were evaluated and resultant levels compared to those in cells treated with etoposide.Results: Initial findings report AlPcSmix is predominantly more detrimental to cellular function and homeostasis when excited via red light irradiation of 15J/cm2. It appears GePcSmix requires higher dosage administrations to achieve similar responses within identical populations. However, due to the mechanism of PDT application, our findings indicate a greater toxic effect with both phthalocyanines when compared to etoposide of higher dosage within MCF-7 cells.Conclusion: Both phthalocyanines, despite similarity in structure, indicate induction of different cell death response pathways based on their toxicity potential. These therefore show great promise as potential PDT agents for the treatment of cancer.

Pivotal roles of peptide transporter PEPT1 and ATP-binding cassette (ABC) transporter ABCG2 in 5-aminolevulinic acid (ALA)-based photocytotoxicity of gastric cancer cells in vitro - Corrected Proof

2012-01-05

Summary: Background: Recently, 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) is being widely used in cancer therapy owing to the tumor-specific accumulation of photosensitizing protoporphyrin IX (PpIX) after the administration of ALA. In the present study, by focusing on genes involved in the porphyrin biosynthesis pathway, we aimed to explore biomarkers that are predictive for the efficacy of ALA-PDT.Methods: We used five lines of human gastric cancer cells to measure the ALA-based photocytotoxicity. ALA-induced production of PpIX in cancer cells was quantified by fluorescence spectrophotometry. To examine the potential involvement of PEPT1 and ABCG2 in the ALA-PDT sensitivity, stable cell lines overexpressing PEPT1 were established and ABCG2-specific siRNA used.Results: We observed that three cell lines were photosensitive, whereas the other two cell lines were resistant to ALA-based photocytotoxicity. The ALA-based photocytotoxicity was found to be well correlated with intracellular PpIX levels, which suggests that certain enzymes and/or transporters involved in ALA-induced PpIX production are critical determinants. We found that high expression of the peptide transporter PEPT1 (ALA influx transporter) and low expression of the ATP-binding cassette transporter ABCG2 (porphyrin efflux transporter) determined ALA-induced PpIX production and cellular photosensitivity in vitro.Conclusion: PEPT1 and ABCG2 are key players in regulating intracellular PpIX levels and determining the efficacy of ALA-based photocytotoxicity against gastric cancer cells in vitro. Evaluation of the expression levels of PEPT1 and ABCG2 genes could be useful to predict the efficacy of ALA-PDT. Primers specific to those target genes are practical and useful biomarkers for predicting the photo-sensitivity to ALA-PDT.