Photosensitizer Protoporphyrin IX Fluorescence Research Articles

Photochemical Internalization of Etoposide Using Dendrimer Nanospheres Loaded with Etoposide and Protoporphyrin IX on a Glioblastoma Cell Line.

Glioblastoma multiforme (GBM) is the most common malignant primary neoplasm of the adult central nervous system originating from glial cells. The prognosis of those affected by GBM has remained poor despite advances in surgery, chemotherapy, and radiotherapy. Photochemical internalization (PCI) is a release mechanism of endocytosed therapeutics into the cytoplasm, which relies on the membrane disruptive effect of light-activated photosensitizers. In this study, phototherapy by PCI was performed on a human GBM cell-line using the topoisomerase II inhibitor etoposide (Etop) and the photosensitizer protoporphyrin IX (PpIX) loaded in nanospheres (Ns) made from generation-5 polyamidoamine dendrimers (PAMAM(G5)). The resultant formulation, Etop/PpIX-PAMAM(G5) Ns, measured 217.4 ± 2.9 nm in diameter and 40.5 ± 1.3 mV in charge. Confocal microscopy demonstrated PpIX fluorescence within the endo-lysosomal compartment, and an almost twofold increase in cellular uptake compared to free PpIX by flow cytometry. Phototherapy with 3 min and 5 min light illumination resulted in a greater extent of synergism than with co-administered Etop and PpIX; notably, antagonism was observed without light illumination. Mechanistically, significant increases in oxidative stress and apoptosis were observed with Etop/PpIX-PAMAM(G5) Ns upon 5 min of light illumination in comparison to treatment with either of the agents alone. In conclusion, simultaneous delivery and endo-lysosomal co-localization of Etop and PpIX by PAMAM(G5) Ns leads to a synergistic effect by phototherapy; in addition, the finding of antagonism without light illumination can be advantageous in lowering the dark toxicity and improving photo-selectivity.

Smartphone fluorescence imager for quantitative dosimetry of protoporphyrin-IX-based photodynamic therapy in skin.

.Significance: While clinical treatment of actinic keratosis by photodynamic therapy (PDT) is widely practiced, there is a well-known variability in response, primarily caused by heterogeneous accumulation of the photosensitizer protoporphyrin IX (PpIX) between patients and between lesions, but measurement of this is rarely done.Aim: Develop a smartphone-based fluorescence imager for simple quantitative photography of the lesions and their PpIX levels that can be used in a new clinical workflow to guide the reliability of aminolevulinic acid (ALA) application for improved lesion clearance.Approach: The smartphone fluorescence imager uses an iPhone and a custom iOS application for image acquisition, a 3D-printed base for measurement standardization, an emission filter for PpIX fluorescence isolation, and a 405-nm LED ring for optical excitation. System performance was tested to ensure measurement reproducibility and the ability to capture photosensitizer accumulation and photobleaching in pre-clinical and clinical settings.Results: PpIX fluorescence signal from tissue-simulating phantoms showed linear sensitivity in the 0.01 to 2.0 μM range. Murine studies with Ameluz® aminolevulinic acid (ALA) gel and initial human testing with Levulan® ALA cream verified that in-vivo imaging was feasible, including that PpIX production over 1 h is easily captured and that photobleaching from the light treatment could be quantified.Conclusions: The presented device is the first quantitative wide-field fluorescence imaging system for PDT dosimetry designed for clinical skin use and for maximal ease of translation into clinical workflow. The results lay the foundation for using the system in clinical studies to establish treatment thresholds for the individualization of PDT treatment.

Biosensor device for the photo-specific detection of immuno-captured bladder cancer cells using hexaminolevulinate: An ex-vivo study.

This study investigate the relationship between different regulatory factors (time, concentration, temperature and addition of a fluorescent dye) and Hexaminolevulinate (HAL)-mediated photodynamic diagnosis of bladder cancer (PDD) in vitro. We examine the natural photosensitizer Protoporphyrin IX (PpIX) fluorescence induced by HAL in several human bladder cancer cell lines and one non-cancer foreskin fibroblast cell line and identify the processing conditions that maximise the difference in fluorescence intensity between malign and benign cell types. The detection of HAL induced fluorescence at a single cell level by a selective cancer cell capture platform is also tested. Experiments were performed on cultured monolayer cells and cells in suspension. The cell lines examined included the transitional epithelium carcinoma cell lines HT1197, HT1376, EJ138 and RT4, and the non-cancer foreskin fibroblasts HFF. Cells were incubated with HAL in various doses, time and temperature settings. We also used the nuclear red as a tool to study the PpIX subcellular localization. PpIX fluorescence intensities were measured and analysed using fluorescence microscope software. Finally, we evaluated the possibility of using HAL to discriminate between cancer and non-cancer cells from a mixed cell population using a newly developed immunofunctionalized microfluidic platform. The accumulation of PpIX in bladder cancer cells was significantly higher than in non-cancer cells, both cultured monolayer cells and cells in suspension. Effectively, the fluorescence intensity was 4.5 times brighter in bladder cancer cells than non-cancer foreskin fibroblast cells when incubated in the optimum condition, in which the nuclear stain adjuvant acted as a fluorescence enhancer. Cancer cells displayed PpIX accumulated mainly in mitochondria but none or very little PpIX was observed in non-cancer cells. HAL-induced fluorescence could be used to correctly identify bladder cancer cells within the EpCAM conjugated POx based microfluidic sensor with an 88% capture selectivity rate. These findings prove that the application of HAL-induced PpIX fluorescence can successfully distinguish between cancer and non-cancer cells in vitro. This test can provide advanced second level of confidence on the cancerous nature of cells captured by the immunofunctionalized bladder cancer diagnostic platform.

Efficacy of 5-Aminolevulinic Acid in Photodynamic Detection and Photodynamic Therapy in Veterinary Medicine.

5-Aminolevulinic acid (5-ALA), a commonly used photosensitizer in photodynamic detection (PDD) and therapy (PDT), is converted in situ to the established photosensitizer protoporphyrin IX (PpIX) via the heme biosynthetic pathway. To extend 5-ALA-PDT application, we evaluated the PpIX fluorescence induced by exogenous 5-ALA in various veterinary tumors and treated canine and feline tumors. 5-ALA-PDD sensitivity and specificity in the whole sample group for dogs and cats combined were 89.5 and 50%, respectively. Notably, some small tumors disappeared upon 5-ALA-PDT. Although single PDT application was not curative, repeated PDT+/−chemotherapy achieved long-term tumor control. We analyzed the relationship between intracellular PpIX concentration and 5-ALA-PDT in vitro cytotoxicity using various primary tumor cells and determined the correlation between intracellular PpIX concentration and 5-ALA transporter and metabolic enzyme mRNA expression levels. 5-ALA-PDT cytotoxicity in vitro correlated with intracellular PpIX concentration in carcinomas. Ferrochelatase mRNA expression levels strongly negatively correlated with PpIX accumulation, representing the first report of a correlation between mRNA expression related to PpIX accumulation and PpIX concentration in canine tumor cells. Our findings suggested that the results of 5-ALA-PDD might be predictive for 5-ALA-PDT therapeutic effects for carcinomas, with 5-ALA-PDT plus chemotherapy potentially increasing the probability of tumor control in veterinary medicine.

Matrix mediated fluorescence enhancement — why nanoparticles and PPIX make a friendly couple

The hexyl ester of 5-aminolevulinic acid (HAL) is an efficient inducer of the endogenous photosensitizer protoporphyrin IX (PPIX) in neoplastic tissue and approved for the fluorescence photodetection of bladder cancer. It is, however, limited to topical application due to unsuitable pharmacokinetic and pharmacodynamic properties following systemic administration. In this study we sought to prepare HAL laden polymeric nanoparticles made from polylactic acid (PLA) as possible means to improve the systemic bioavailability of HAL. Nanoparticles were prepared by nanoprecipitation. Particle size and morphology of freshly prepared and dehydrated nanoparticles were assessed and the ability to generate PPIX fluorescence was tested on a T24 human bladder carcinoma cell line. Fluorescence intensities of cells incubated with nanoencapsulated HAL were significantly higher than in presence of free HAL. Interestingly, the effect was — at least partly — provoked by the direct interaction of exogenously induced PPIX with the nanoparticles.

Oxygen saturation and perfusion changes during dermatological methylaminolaevulinate photodynamic therapy

Methylaminolaevulinate (MAL)-photodynamic therapy (PDT) is a successful topical treatment for a number of (pre)cancerous dermatological conditions. In combination, light of the appropriate wavelength, the photosensitizer protoporphyrin IX (PpIX) and tissue oxygen result in the production of singlet oxygen and reactive oxygen species inducing cell death. This study investigates real-time changes in localized tissue blood oxygen saturation and perfusion in conjunction with PpIX fluorescence monitoring for the first time during dermatological MAL-PDT. Oxygen saturation, perfusion and PpIX fluorescence were monitored noninvasively utilizing optical reflectance spectroscopy, laser Doppler perfusion imaging and a fluorescence imaging system, respectively. Patients attending for standard dermatological MAL-PDT were recruited to this ethically approved study and monitored prior to, during and after light irradiation. Significant reductions in mean blood oxygen saturation (P < 0·005) and PpIX fluorescence (P < 0·001) were observed within the first minute of irradiation (4·75 J cm(-2) ) while, in contrast, perfusion was observed to increase significantly (P < 0·01) during treatment. The changes in oxygen saturation and PpIX fluorescence were positively correlated during the initial phase of treatment (r(2) = 0·766). Rapid reductions in the localized blood oxygen saturation have been observed for the first time to occur clinically within the initial minutes of light irradiation and positively correlate with the concurrent PpIX photobleaching. Furthermore, perfusion increases, suggesting that the microvasculature compensates for the PDT-induced oxygen depletion.

An In Vitro Comparison of the Effects of the Iron‐Chelating Agents, CP94 and Dexrazoxane, on Protoporphyrin IX Accumulation for Photodynamic Therapy and/or Fluorescence Guided Resection

Photodynamic therapy (PDT) utilizes the combined interaction of a photosensitizer, light and molecular oxygen to ablate tumor tissue. Maximizing the accumulation of the photosensitizer protoporphyrin IX (PpIX) within different cell types would be clinically useful. Dermatological PpIX-induced PDT regimes produce good clinical outcomes but this currently only applies when the lesion remains superficial. Also, as an adjuvant therapy for the treatment of primary brain tumors, fluorescence guided resection (FGR) and PDT can be used to highlight and destroy tumor cells unreachable by surgical resection. By employing iron chelators PpIX accumulation can be enhanced. Two iron-chelating agents, 1,2-diethyl-3-hydroxypyridin-4-one hydrochloride (CP94) and dexrazoxane, were individually combined with the porphyrin precursors aminolevulinic acid (ALA), methyl aminolevulinate (MAL) and hexyl aminolevulinate (HAL). Efficacies of the iron-chelating agents were compared by recording the PpIX fluorescence in human squamous epithelial carcinoma cells (A431) and human glioma cells (U-87 MG) every hour for up to 6 h. Coincubation of ALA/MAL/HAL with CP94 resulted in a greater accumulation of PpIX compared to that produced by coincubation of these congeners with dexrazoxane. Therefore the clinical employment of iron chelation, particularly with CP94 could potentially increase and/or accelerate the accumulation of ALA/MAL/HAL-induced PpIX for PDT or FGR.

Uptake and kinetics of 5-aminolevulinic acid in oral squamous cell carcinoma

Photodynamic diagnosis (PDD) is a form of cancer detection based on the administration of an exogenous photo-activated compound that accumulates in malignant cells, followed by appropriate photo irradiation. The authors describe a spectroscopic study of 5-aminolevulinic acid (5-ALA)-generated photosensitizer protoporphyrin IX (PpIX) fluorescence in human oral squamous cell carcinoma (OSCC) cell lines to validate its clinical use. 5-ALA-induced PpIX fluorescence intensity was measured in the presence and absence of deferoxamine mesylate (DFO). Two, one and two cell lines produced poorly, moderately and well differentiated carcinomas, respectively, on transplantation in scid mice. The fluorescence intensity was high in the poorly differentiated cell lines, followed by the moderately differentiated cell line; the intensity of the well differentiated cell lines was low and not significantly different from that of normal keratinocytes in the absence of DFO. It was elevated to the level of poorly differentiated cell lines following DFO treatment. This elevation was not observed in normal keratinocytes. The results indicate that DFO enhances the photodynamic sensitivity of 5-ALA in non-responsive carcinoma cells as a result of increased cellular accumulation by inhibiting haeme biosynthesis. This PDD system can be applied clinically to the detection of OSCC irrespective of the degree of differentiation.

5.02] Preliminary results: In vitro and in vivo evaluation of photodynamic techniques for the experimental treatment of human hepatoblastoma and neuroblastoma

Objective Hepatoblastoma and neuroblastoma are cancers which occur in early childhood. Intraoperative fluorescence guidance or post-surgical photodynamic therapy (PDT) might improve treatment outcome. In this study, human cell lines derived from these cancers were studied in vitro and in an animal model for their responsiveness to photodynamic methods using 5-aminolevulinic acid (5-ALA) as a precursor of the fluorescent photosensitizer protoporphyrin IX (PpIX). Material and methods Cell cultures of human hepatoblastoma (HuH6) and neuroblastoma (MHH-NB-11) were incubated in 96-well plates with 5-ALA (medac, Wedel, Germany) at increasing concentrations and incubation times to determine optimal incubation parameters by measurement of PpIX-fluorescence. Human fibroblast cells (N1) served as controls. Cell survival following PDT with increasing light doses was tested with the CTB assay (Promega, Madison, WI, USA) at 150 μg/ml 5-ALA for 4 h. HuH6-cells were also studied in vivo after laparoscopic implantation of 3×106 cells between peritoneum and abdominal wall of immunoincompetent nude rats. After 7 weeks of tumor growth, fluorescence kinetics (tumor, adjacent peritoneum, liver, colon) was assessed by fiber-based measurements following i.p. injection of 5-ALA at 500 mg/kg bodyweight (10 animals) and on further 24 animals, laser irradiation was performed with a light dose of 50 J/cm2 at 25 or 100 mW/cm2. Two days later, rats were again injected with 5-ALA and irradiated tissues were excised and studied by conventional and fluorescence microscopy. Results In vitro, PpIX fluorescence approached saturation at 150 μg/ml 5-ALA-concentration, whereas there was no saturation effect observed with respect to incubation time, 4 h was set as practically feasible. HuH6 and NB-11-cells showed comparable fluorescence intensities, whereas N1 fluorescence was low. After PDT, cell survival was lowest for HuH6-cells (LD50 at 1.5 J/cm2) compared to MHH-NB-11 (LD50 at 6 J/cm2) and N1-cells (LD50>20 J/cm2). In vivo, tumor fluorescence was highest at around 200 min post injection with a maximum contrast to the surrounding peritoneum at approx. 150 min. The mean contrast of tumor fluorescence versus fluorescence measured in peritoneum and liver was 22 at this time (not corrected for optical parameters), whereas versus colon it was 5. PDT response was visible in irradiated tumor and liver tissue. The depth of necrosis could be assessed by observation of fluorescence on cryosections of excised tissue. While peritoneum exposed to PDT remained unaffected, irradiation of the liver led to superficial necrosis and sensitized tumor showed deep necrosis. Conclusion The human hepatoblastoma and neuroblastoma cells were susceptible to selective PpIX accumulation and PDT effect. In vivo, HuH6-tumors showed high and selective PpIX accumulation and extended necrosis after PDT. The possibility of a PDT-induced necrosis of normal liver tissue must be considered for clinical trials.

Protoporphyrin IX Fluorescence Photobleaching and the Response of Rat Barrett's Esophagus Following 5-aminolevulinic Acid Photodynamic Therapy

Barrett's esophagus (BE) can experimentally be treated with 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT), in which ALA, the precursor of the endogenous photosensitizer protoporphyrin IX (PpIX) and subsequent irradiation with laser light are applied to destroy the (pre). malignant tissue. Accurate dosimetry is critical for successful ALA-PDT. Here, in vivo dosimetry and kinetics of PpIX fluorescence photobleaching were studied in a rat model of BE. The fluence and fluence rate were standardized in vivo and PpIX fluorescence was measured simultaneously at the esophageal wall during ALA-PDT and plotted against the delivered fluence rather than time. Rats with BE were administered 200 mg kg-1ALA (n = 17) or served as control (n = 4). Animals were irradiated with 633 nm laser light at a measured fluence rate of 75 mW cm-2and a fluence of 54 J cm-2. Large differences were observed in the kinetics of PpIX fluorescence photobleaching in different animals. High PpIX fluorescence photobleaching rates corresponded with tissue ablation, whereas low rates corresponded with no damage to the epithelium. Attempts to influence tissue oxygenation by varying balloon pressure and ventilation were shown not to be directly responsible for the differences in effect. In conclusion, in vivo dosimetry is feasible in heterogeneous conditions such as BE, and PpIX fluorescence photobleaching is useful to predict the tissue response to ALA-PDT.

Protoporphyrin IX Fluorescence Photobleaching and the Response of Rat Barrett's Esophagus Following 5-aminolevulinic Acid Photodynamic Therapy

Barrett's esophagus (BE) can experimentally be treated with 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT), in which ALA, the precursor of the endogenous photosensitizer protoporphyrin IX (PpIX) and subsequent irradiation with laser light are applied to destroy the (pre)malignant tissue. Accurate dosimetry is critical for successful ALA-PDT. Here, in vivo dosimetry and kinetics of PpIX fluorescence photobleaching were studied in a rat model of BE. The fluence and fluence rate were standardized in vivo and PpIX fluorescence was measured simultaneously at the esophageal wall during ALA-PDT and plotted against the delivered fluence rather than time. Rats with BE were administered 200 mg kg(-1) ALA (n = 17) or served as control (n = 4). Animals were irradiated with 633 nm laser light at a measured fluence rate of 75 mW cm(-2) and a fluence of 54 J cm(-2). Large differences were observed in the kinetics of PpIX fluorescence photobleaching in different animals. High PpIX fluorescence photobleaching rates corresponded with tissue ablation, whereas low rates corresponded with no damage to the epithelium. Attempts to influence tissue oxygenation by varying balloon pressure and ventilation were shown not to be directly responsible for the differences in effect. In conclusion, in vivo dosimetry is feasible in heterogeneous conditions such as BE, and PpIX fluorescence photobleaching is useful to predict the tissue response to ALA-PDT.

Lipid cubic phases for improved topical drug delivery in photodynamic therapy

We have evaluated the efficacy of lipid cubic phases, highly ordered self-assembly systems on the nanometer level, as drug delivery vehicles for in vivo topical administration of δ-aminolevulinic acid (ALA) and its methyl ester (m-ALA) on nude mice skin. ALA, a precursor of heme, induces the production of the photosensitizer protoporphyrin IX (PpIX) in living tissue. Measuring the PpIX fluorescence at the skin surface, after topical administration, makes indirect quantification of the penetration of ALA into the tissue possible. Cubic phases were formed of lipid (monoolein or phytantriol), water and drug. In some cases, propylene glycol was included in the cubic phase as well. The drug concentration was 3% (w/w, based on the total sample weight) in all investigated vehicles. When the formulations were applied for 1 h, the monoolein cubic systems and the three-component phytantriol sample showed higher fluorescence compared to the standard ointment during the 10 h of measurement. Both ALA and m-ALA yielded similar results, although the differences between the investigated vehicles were more pronounced when using m-ALA. For the 24-h applications, the monoolein cubic systems with m-ALA showed faster PpIX formation than the standard ointment, implying higher PpIX levels at short application times (less than 4 h). The systemic PpIX fluorescence of ALA was elevated by using the lipid cubic formulations. Notably, a small systemic effect was also observed for the monoolein cubic sample with m-ALA. These results imply improved PpIX formation when using the lipid cubic systems, most probably due to enhanced drug penetration.

Fluorescence detection of superficial skin cancers

We present a simple fluorescent diagnosis system for the demarcation of skin cancers. 5-aminolaevulinic acid (ALA) cream is applied to the suspicious area resulting in the accumulation of the photosensitizer Protoporphyrin IX (PpIX) in the cancerous tissue. When excited at 400 nm using a filtered mercury arc lamp, the PpIX fluoresces at 635 nm. Our imaging system acquires four spectrally specific images at 635, 540, 470 and 400 nm for the PpIX fluorescence, natural skin autofluorescence and illumination respectively. Subsequent image processing combines these images into a single display showing clearly the localization and extent of the tumour.

Cell cycle phase influences tumour cell sensitivity to aminolaevulinic acid-induced photodynamic therapy in vitro.

Photodynamic therapy (PDT) is a form of cancer treatment based on the destruction of cells by the interaction of light, oxygen and a photosensitizer. Aminolaevulinic acid (ALA) is the prodrug of the photosensitizer protoporphyrin IX (PpIX). ALA-induced PDT depends on the rate of cellular synthesis of PpIX, which may vary with cell cycle phase. This study has investigated the relationship between cell cycle phase, PpIX generation and phototoxicity in synchronized and unsynchronized bladder cancer cells (HT1197). In unsynchronized cells, relative PpIX fluorescence values (arbitrary units) were significantly different between cell cycle phases after a 1-h ALA incubation (G1 24.8 +/- 0.7; S-phase, 32.7 +/- 0.8, P < 0.05; G2 35.4 +/- 0.8, P < 0.05). In synchronized cells after a 1-h ALA incubation, cells in G1 produced less PpIX than those in S-phase or G2 [6.65 +/- 1.1 ng per 10(5) cells compared with 15.5 +/- 2.1 (P < 0.05), and 8.1 +/- 1.8 ng per 10(5) cells (not significant) respectively] and were significantly less sensitive to ALA-induced PDT (% survival, G1 76.2 +/- 8.3; S-phase 49.7 +/- 4.6, P < 0.05; G2 44.2 +/- 2.4, P < 0.05). This differential response in tumour cells may have implications for clinical PDT, resulting in treatment resistance and possible failure in complete tumour response.

The importance of fluorescence distribution and kinetics of ALA-induced PpIX in the bladder in photodynamic therapy

Photodynamic therapy (PDT) is a new anticancer technique directed at the selective destruction of neoplastic tissue. Aminolevulinic acid (ALA), a precursor in the biosynthesis of heme, induces the production of the endogenous photosensitizer protoporphyrin IX (PpIX). In this study the fluorescence distribution of ALA-induced PpIX was investigated in the rat bladder wall by fluorescence microscopy. The fluorescence studies showed that PpIX concentrates in bladder mucosa and that the highest fluorescence levels are achieved after four hours of 300 mg/kg ALA administration. A clear trend in difference between mucosa and muscularis layers was found in all samples taken after 1, 2, 4 and 6 hours of ALA administration. Our results sugges that to get the highest PpIX fluorescence intensity in bladder mucosa it is best to use 300 mg/kg ALA administration. Four hours is the time point when the highest difference fluorescence between mucosa and muscularis is reached.

Detection of early stages of carcinogenesis in adenomas of murine lung by 5-aminolevulinic acid-induced protoporphyrin IX fluorescence.

Administration of the heme precursor 5-aminolevulinic acid (ALA) leads to the selective accumulation of the photosensitizer protoporphyrin IX (PpIX) in certain types of normal and abnormal tissues. This phenomenon has been exploited clinically for detection and treatment of a variety of malignant and nonmalignant lesions. The present preclinical study examined the specificity of ALA-induced porphyrin fluorescence in chemically induced murine lung tumors in vivo. During the early stages of tumorigenesis, ALA-induced PpIX fluorescence developed in hyperplastic tissues in the lung and later in early lung tumor foci. In early tumor foci, maximum PpIX fluorescence occurred 2 h after the administration of ALA and returned to background levels after 4 h. There was approximately a 20-fold difference in PpIX fluorescence intensity between tumor foci and the adjacent normal tissue. The specificity of ALA-induced fluorescence for hyperplastic tissues and benign tumors in lung during tumorigenesis suggests a possible use for this fluorochrome in the detection of premalignant alterations in the lung by fluorescence endoscopy. Two non-small cell lung cancer cell lines developed ALA-induced PpIX fluorescence in vitro. These lines exhibited a light-dose-dependent phototoxic response to ALA photodynamic therapy (PDT) in vitro. Because PpIX is a clinically effective photosensitizer for a wide variety of malignancies, these results support the possible use of ALA-induced PpIX PDT for lung cancer.