Protoporphyrin IX‐Functionalized AgSiO2 Core–Shell Nanoparticles: Plasmonic Enhancement of Fluorescence and Singlet Oxygen Production

Metal‐enhanced processes arising from the coupling of a dye with metallic nanoparticles (NPs) have been widely reported. However, few studies have simultaneously investigated these mechanisms from the viewpoint of dye fluorescence and photoactivity. Herein, protoporphyrin IX (PpIX) is grafted onto the surface of silver core silica shell NPs in order to investigate the effect of silver (Ag) localized surface plasmon resonance (LSPR) on PpIX fluorescence and PpIX singlet oxygen (¹O₂) production. Using two Ag core sizes, we report a systematic study of these photophysical processes as a function of silica (SiO₂) spacer thickness, LSPR band position and excitation wavelength. The excitation of Ag NP LSPR, which overlaps the PpIX absorption band, leads to the concomitant enhancement of PpIX fluorescence and ¹O₂ production independently of the Ag core size, but in a more pronounced way for larger Ag cores. These enhancements result from the increase in the PpIX excitation rate through the LSPR excitation and decrease when the distance between PpIX and Ag NPs increases. A maximum fluorescence enhancement of up to 14‐fold, together with an increase in photogenerated ¹O₂ production of up to five times are obtained using 100 nm Ag cores coated with a 5 nm thick silica coating.     

THE USE OF DIFFUSERS IN THE MEASUREMENT OF TRANSMISSION OF HUMAN EPIDERMAL LAYERS

Abstract— The transmission of the outermost layers of human skin is measured, by use of a diffuser. The sample is transferred to the diffuser and inserted into the measuring beam. The reference beam is also measured after passing through the diffuser. The diffuser transforms the light in both the sample and reference beams into a completely diffuse flux. The radiant flux of light emerging from the diffuser is directly proportional to the radiant flux of the light impinging on the diffuser, but does not depend on the angular distribution of the impinging light. Because of this particular property, a diffuser may be used to measure the transmission of scattering specimens. The analogy between a diffuser and an integrating sphere is pointed out.
Deviations of commercially available diffusers from the perfect behaviour lead to deviations in the measured transmissions that are negligible (< 3%) for epidermal and corneal samples. Spectral transmission data from representative skin samples are presented. It is found that correction for fluorescence is necessary. Due to this correction the epidermal UV-C transmission is lower by a factor of 10–100 than without correction, and the epidermal absorption maximum is shifted from 275 towards 265 nm.     

Laser kinetic spectroscopy of the oxygenation and oxidation of hemoglobin by nitrite in rats exposed to γ-radiation

Hemoglobin molecules were studied by stationary and time-resolved laser absorption spectroscopy. On the basis of these results, the kinetic parameters of oxygenation in the course of methemoglobin formation under the combined action of radiation and sodium nitrite were analyzed. The kinetics of oxygenation of hemoglobin with an initial methemoglobin concentration of 3% or lower is described by a two-exponential function. The rate parameters of the reaction are independent of the radiation history and of the amount of subunits oxidized by sodium nitrite, which is as high as ≈50% relative to the total number of protein subunits     

Redox processes induced in methemoglobin solution by mercury lamp UV light and spark-discharge plasma radiation

The buildup of–SH groups in methemoglobin by irradiation with emission of spark discharge plasma in air and UV light from a low-pressure mercury lamp (λ = 253.7 nm) has been studied. The concentration of–SH groups increases the during spark discharge treatment, with the methionine amino acid residue in the methemoglobin molecule being reduced by nitrous and peroxynitrous acids; the bonds in the methemoglobin molecule are destroyed within 20 minutes of the spark treatment. By the action of UV radiation, methionine is reduced through chromophores (tryptophan, tyrosine) and methemoglobin is reduced by O₂^.- radical ions to oxy- and deoxyhemoglobin.     

SUBCELLULAR DAMAGE KINETICS WITHIN CO-CULTIVATED WI38 and VA13-TRANSFORMED WI38 HUMAN FIBROBLASTS FOLLOWING 5-AMINOLEVULINIC ACID-INDUCED PROTOPORPHYRIN IX FORMATION

The generation of the photosensitizer protoporphyrin IX (PpIX) in cells can be induced by externally applied 5-aminolevulinic acid (ALA), with that bypassing the feedback control mechanism. The aim of the present study was to investigate the onset of destructive changes in living cocultivated WI38 and VA13-transformed WI38 human fibroblasts following ALA incubation, PpIX production and subsequent irradiation by white halogen light with a dose of 2.2 kJ/m². Specific fluorescence markers such as 3,3′-dihexyloxacarbocyanine iodide for endoplasmic reticulum (ER) staining and dihydrorhodamine for intact mitochondria mapping combined with a low light imaging system are a versatile and sensitive tool to examine the photoinduced destruction of organelles in living cells, while artifacts are minimized. Mitochondria as primary targets of PpIX undergo a condensation under irradiation and are finally destroyed. Photodynamic treatment induces further a significant decomposition of ER, although PpIX localization could not be determined. Initial destabilization and vesiculation of ER is followed by a porous network with large cisternae (indicating the breakdown of cell integrity and cell/nucleus membrane damage). Normal cocultivated lung fibroblasts showed a delay in destruction compared to the transformed WI38-VA13 cells. The observed decomposition pattern resembles the morphological pattern of apoptosis.     

OPTIMIZATION OF MULTIFIBER LIGHT DELIVERY FOR THE PHOTODYNAMIC THERAPY OF LOCALIZED PROSTATE CANCER

The understanding of light distribution within the target organ is essential in ensuring efficacy and safety in photodynamic therapy (PDT). A computer simulator of light distribution in prostatic tissue was employed for optimizing dosimetry for PDT in localized prostatic cancer. The program was based on empirically determined light distributions and optical constants and an assumed Ruence rate differential from fiber source to necrosis periphery. The diffusion theory approximation to the Boltzmann transport equation was the applicable formulation relevant to prostatic tissue, which has a high albedo with forward-scattering characteristics. Solving this equation of diffusive transfer for the appropriate fiber geometry yielded the energy fluence distributions for cleaved fiber and cylindrical diffuser light delivery. These distributions, confirmed by our measurements, show a l/r and l/r dependency (r = distance from light source) of the fluence ø(r) for the cleaved fiber and diffuser, respectively. This manifests itself by the tighter spacing of energy fluence isodoses in the case of the cleaved fiber. It was predicted that for a typical PDT regime a single interstitially placed cleaved fiber would treat 0.05–0.72 cm³, Four parallel fibers improve the uniformity of light distribution and treatment volume, and an interfiber separation of 12 mm would be necessary to provide optimal overlap of PDT necrosis, treating 0.26–3.6 cm³. The cylindrical diffuser, however, could treat larger volumes, and it was predicted that four 3 cm long diffusers at an optimal separation of 25 mm would treat 25–88 cm³ of prostatic tissue.     

Kinetic study of competing first-order processes: simultaneous quantification of hemoglobin and methemoglobin in whole blood

The development and evaluation of a kinetic method for the simultaneous quantification of methemoglobin and hemoglobin is described. A unique feature of the reaction/monitoring system is that the two species produce signals that change in opposite directions. Attempts to optimize the reaction conditions, procedures for preparing reliable standards, a new multiwavelength method used for comparison purposes and results for synthetic standards and blood samples to which known amounts of methemoglobin are added are described. Combined effects of competing directions of signal change, large concentration differences and conditions required for pseudo-first-order behavior results in a relativity narrowly defined set of conditions that permit simultaneous determinations. Even so, results for methemoglobin were in reasonable agreement between the kinetic and spectrophotometric methods (slope 1.05, intercept 0.17 mmol l^?1); difficulties in achieving pseudo-first-order behavior yielded less reliable results for hemoglobin with a least-squares slope of 1.16 for a comparison of results by kinetic and spectrophotometric methods. This latter problem had an adverse effect on comparison of percentages of methemoglobin in samples even though concentration values were in good agreement.     

Defect induced nickel,nitrogen-codoped mesoporous TiO2 microspheres with enhanced visible light photocatalytic activity

Nickel, nitrogen-codoped mesoporous TiO₂ microspheres (Ni–N–TiO₂) with high surface area, and an effective direct band gap energy of ∼2.58 eV. Nickel sulfate used as the Ni source and ammonia gas as the N source here. The efficiency of the as-prepared samples was investigated by monitoring the degradation of Rhodamine B under visible light irradiation. The experimental results indicate that Ni-doped mesoporous TiO₂ microspheres show higher photocatalytic activity than mesoporous TiO₂ microspheres under visible light irradiation. It mainly due to that the electron trap level (Ni²⁺/Ni⁺) promoting the separation of charge carriers and the oxygen vacancies inducing the visible light absorption. In addition, Ni–N–TiO₂ shows enhanced activity compared with Ni–TiO₂. Codopants and dopants are found to be uniformly distributed in TiO₂ matrix. Among the all samples the 0.5% molar quantity of Ni dopant and 500 °C 2 h nitriding condition gives the highest photocatalytic activity. The treatment of ammonia gas on Ni–TiO₂ sample induced oxygen vancancies, substitutional and interstitial N. A suitable treatment by ammonia gas also promote separation of charge carriers and the absorption of visible light. The active species generated in the photocatalytic system were also investigated. The strategy presented here gives a promising route towards the development of a metal and non-metal codoped semiconductor materials for applied photocatalysis and related applications.     

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.     

Characterization of Endogenous Protoporphyrin IX Induced by δ-Aminolevulinic Acid in Resting and Activated Peripheral Blood Lymphocytes by Four-Color Flow Cytometry

Lymphocytes treated with δ-aminolevulinic acid (ALA) can accumulate the photoactive, fluorescent heme precursor, protoporphyrin IX (PpIX). With visible light illumination, PpIX can be used in photodynamic therapy (ALA-PDT) to kill or functionally alter cells. The aim of this study was to characterize the effects of ALA and ALA-PDT on resting and activated human peripheral blood T lymphocytes. Accumulation of PpIX depends inversely on the rate of its iron-dependent conversion into heme. Activated, replicating lymphocytes have low intracellular iron levels, with corresponding increases in the transferrin receptor (CD71). Thus, we expected activated lymphocytes would preferentially accumulate PpIX. Using four-color flow cytometry, we examined ALA-induced PpIX levels in T-cell subsets of resting and activated human peripheral blood mononuclear cells and the relationship between CD71 and PpIX. Peripheral blood mononuclear cells stimulated by phytohemagglutinin (PHA) were simultaneously phenotyped for PpIX, CD71 and the T-cell markers CD3 and CD4 or CDS. In activated cells treated with 0-6mM ALA for 4 h, PpIX fluorescence was maximal at 1 mM ALA. On a single cell basis, there was a strong correlation between PpIX ac-cumulation and CD71 expression. The ALA-treated, PHA-stimulated, CD71⁺ lymphocytes had an eight-fold greater mean PpIX fluorescence than nonactivated, CD71- cells. Approximately 87% of the CD4* and 85% of the CD8⁺ T cells accumulated PpIX. The PpIX levels of CDS⁺ cells were about 5% greater than CD4⁺ cells. In addition, mixed lymphocyte reaction-stimulated cells treated with ALA accumulated more PpIX than controls. Thus, activated cells preferentially accumulate endogenous PpIX when exogenous ALA is administered. Cytotoxicity studies showed that the majority of the activated cells following ALA-PDT were killed but resting cells were spared. Also, in examining activation markers by flow cytometry the number of cells that were positive for activation markers CD38 or CD71 dramatically decreased after ALA and light treatment in activated populations. The data suggest a role for ALA-PDT as an immunomodulator or photocytotoxic agent targeting activated lymphocytes.     

激光诱导游离色氨酸对血红蛋白反应动力学影响及机理

血红蛋白活性中心铁卟啉具有环状共轭结构,类似于叶绿素,可以吸收特定波长光,光会诱导铁卟啉发生氧化还原反应。研究中发现,紫外区波长光照射血红蛋白的氧化还原反应情况优于铁卟啉特征吸收波长(406 nm)光照射情况。无游离色氨酸(Trp)时,266 nm激光激发后高铁血红蛋白(metHb)、脱氧血红蛋白(deoxy Hb)、氧合血红蛋白(HbO_2)和碳氧血红蛋白(HbCO)均被激发至各自相应的激发态,其Soret带谱峰衰减至基态的时间大致相同;加入游离Trp后,激发态Trp会转移能量到铁卟啉,在直接和间接光能量双重作用叠加下,激发态铁卟啉衰减时间发生变化。metHb、deoxy Hb和HbCO衰减时间明显延长,但对HbO_2影响相对较小。根据瞬态吸收光谱、动力学曲线和紫外-可见吸收光谱综合分析可知,在加入游离Trp前后,4种形态血红蛋白在被入射光激发后,铁卟啉均反应至具有(或近似具有)一空位的铁六配位平面卟啉结构状态。     

Colorimetric sensing of cyanide anion in aqueous media with a fluorescein–spiropyran conjugate

A fluorescein–spiropyran conjugate (2) behaves as a receptor for colorimetric sensing of cyanide anion (CN^?) in aqueous media under irradiation of UV light. The compound 2, which exists as a spirocycle-closed (SP) form in the dark condition, is isomerized to the spirocycle-opened merocyanine (MC) form upon irradiation of UV light and shows absorption bands at 467 and 568 nm. Addition of CN^? to the solution leads to a decrease in these bands and an appearance of new absorption band at 512 nm, via a nucleophilic interaction between CN^? and the spirocarbon of MC form. This absorption change occurs selectively with CN^? and enables ratiometric quantification of CN^? by absorption analysis.     

Photodynamic therapy using 5-aminolaevulinic acid for oesophageal adenocarcinoma associated with Barrett's metaplasia

Photodynamic therapy (PDT) is a novel technique for local endoscopic treatment of gastrointestinal neoplasia. Current photosensitisers for PDT may cause prolonged skin phototoxicity. 5-Aminolaevulinic acid (ALA), a precursor of the photosensitiser protoporphyrin IX (PpIX), is more acceptable because of its short half-life and preferential accumulation in mucosa and mucosal tumour. We have treated 12 patients, median age 73 years (range 55-88) with oesophageal adenocarcinoma arising from Barrett's metaplasia (two carcinomas-in-situ, grade 0; 10 carcinomas, grade 1-11A based on endoluminal ultrasound in two and CT scanning in 10 patients). ALA (60 and 75 mg/kg body weight) was given orally in two or five equally divided doses. The PpIX distribution in stomach, normal oesophagus, Barrett's mucosa and carcinoma was measured by quantitative fluorescence photometry. PDT was performed using laser light (630 nm) delivered via a cylindrical diffuser 4-6 h after the first dose of ALA. The patients received one to four sessions of PDT. PpIX accumulation in the mucosa was two to three times that in the lamina propria. The differential distribution between carcinomatous and normal oesophageal mucosa was less marked (carcinoma:normal mucosa ratio = 1.4). Higher doses of ALA increased PpIX accumulation in all tissues but did not increase the differential PpIX distribution between tumour and normal oesophageal mucosa. After PDT using ALA (ALA/PDT), all mucosa showed superficial white necrotic changes and the histology confirmed fibrinoid necrosis. One patient with carcinoma-in-situ had the tumour eradicated after one treatment with no recurrence at 28 months. Another patient with a small T1 tumour required four ALA/PDT treatments, and died of other disease after 36 months. There was no evidence of recurrence. The tumour bulk in the other carcinomas was not significantly reduced. ALA/PDT has a potential for the eradication of small tumours but careful patient selection with endoluminal ultrasound is needed when using ALA/PDT to treat oesophageal cancer.     

The Hydroxypyridinone Iron Chelator CP94 Can Enhance PpIX-induced PDT of Cultured Human Glioma Cells

Photodynamic therapy (PDT) with the pro-drugs 5-aminolevulinic acid (ALA) or methyl aminolevulinate (MAL) utilizes the combined interaction of a photosensitizer, light and molecular oxygen to ablate tumor tissue. To potentially increase accumulation of the photosensitizer, protoporphyrin IX (PpIX), within tumor cells an iron chelator can be employed. This study analyzed the effects of ALA/MAL-induced PDT combined with the iron chelator 1, 2-diethyl-3-hydroxypyridin-4-one hydrochloride (CP94) on the accumulation of PpIX in human glioma cells in vitro. Cells were incubated for 0, 3 and 6 h with various concentrations of ALA/MAL with or without CP94 and the resulting accumulations of PpIX, which naturally fluoresces, were quantified prior to and following light irradiation. In addition, counts of viable cells were recorded. The use of CP94 in combination with ALA/MAL produced significant enhancements of PpIX fluorescence in human glioma cells. At the highest concentrations of each prodrug, CP94 enhanced PpIX fluorescence significantly at 3 h for ALA and by more than 50% at 6 h for MAL. Cells subsequently treated with ALA/MAL-induced PDT in combination with CP94 produced the greatest cytotoxicity. It is therefore concluded that with further study CP94 may be a useful adjuvant to photodiagnosis and/or PpIX-induced PDT treatment of glioma.     

Monitoring PDT by means of superficial reflectance spectroscopy

Monitoring of relevant parameters during photodynamic therapy (PDT) and correlating these with treatment response is necessary to guarantee optimal and reproducible treatment outcome. In this paper we study the correlation between changes in the local tissue optical properties (absorption and scattering coefficients) during ALA-PDT and changes in PpIX fluorescence. The optical properties are measured extremely superficially by employing a single fiber for the delivery and collection of white light to and from the tissue. The measured reflectance spectrum is modeled in terms of four relevant parameters: blood saturation, relative blood volume fraction, scattering intensity and wavelength dependence of the scattering. All these parameters, except the relative blood volume fraction, are shown to correlate with the rate of photobleaching of PpIX, which in turn has previously been shown to correlate with the response of tissues to PDT. These results yield valuable insight in the behavior of these parameters during PDT and their suitability to predict PDT-response for other photosensitizers for which monitoring through photobleaching is not possible.     

Virus photoinactivation in stroma-free hemoglobin with methylene blue or 1,9-dimethylmethylene blue

Photoinactivation of vesicular stomatitis virus (VSV) in stroma-free hemoglobin (SFH) was carried out using methylene blue (MB) or 1,9-dimethylmethylene blue (DMMB). The VSV was more sensitive to inactivation by 660 nm light with 1 microM DMMB than with the same concentration of MB. Under conditions that inactivated 6 log10 of VSV, the methemoglobin content (Met-Hb[%]) and P50 of hemoglobin were changed by 1 microM MB phototreatment but were not changed by 1 microM DMMB phototreatment. The migration of hemoglobin during electrophoresis and the activity of superoxide dismutase were not changed by MB or DMMB phototreatment. In contrast to the results obtained with DMMB at 660 nm, 580 nm irradiation of SFH with DMMB resulted in a significant increase of Met-Hb(%) under conditions that only inactivated 1.19 log10 VSV. The 580 nm irradiation primarily activates the dimer and higher-order aggregates of the dyes, while 660 nm irradiation primarily activates the monomer. These results indicate that the monomer form of DMMB can effectively inactivate viruses without damage to SFH.     

HepG2 human hepatocarcinoma cells: an experimental model for photosensitization by endogenous porphyrins

Endogenous protoporphyurin IX (PpIX) synthesis after δ-aminolaevulinic acid (ALA) administration occurs in cancer cells in vivo; PpIX, which has a short half-life, may thus constitute a good alternative to haematoporphyrin derivative (HPD) (or Photofrin). This study assesses the ability of the human hepatocarcinoma cell line HepG2 to synthesize PpIX in vitro from exogenous ALA, and compares ALA-induced toxicity and phototoxicity with the photodynamic therapy (PDT) effects of HPD on this cell line.

ALA induced a dose-dependent dark toxicity, with 79% and 66% cell survival for 50 and 100 μg ml⁻¹ ALA respectively after 3 h incubation; the same treatment, followed by laser irradiation (λ = 632 nm, 25 J cm⁻²), induced a dose-dependent phototoxicity, with 54% and 19% cell survival 24 h after PDT. Whatever the incubation time with ALA, a 3 h delay before light exposure was found to be optimal to reach a maximum phototoxicity.

HPD induced a slight dose-dependent toxicity in HepG2 cells and a dose- and time-dependent phototoxicity ten times greater than that of ALA-PpIX PDT. After 3 h incubation of 2.5 and 5 μg ml⁻¹ HPD, followed by laser irradiation (λ = 632 nm, 25 J cm⁻²), cell survival was 59% and 24% respectively at 24 h.

Photoproducts induced by light irradiation of porphyrins absorb light in the red spectral region at longer wavelengths than the original porphyrins. The possible enhancement of PDT effects after HepG2 cell incubation with ALA or HPD was investigated by irradiating cells successively with red light (λ = 632 nm) and light (λ = 650 nm). The total fluence was kept constant at 25 J cm⁻². For both HPD and ALA-PpIX PDT, phototoxicity was lower when cells were irradiated for increased periods with λ = 650 nm light than with λ = 632 nm light alone. This suggests that any photoproducts involved either have a short life or are poorly photoreactive.

Not all cell lines can synthesize PpIX after ALA incubation. HepG2 cells, which can synthesize enzymes and precursors of endogenous porphyrin synthesis, represent a good in vitro model for experiments using ALA-PpIX PDT. In addition, ALA-PpIX PDT may represent a new, specific treatment for hepatocarcinomas.     

The fabrication of novel optical diffusers based on UV-cured polymer dispersed liquid crystals

Polymer dispersed liquid crystals (PDLCs) with different sizes of the LC droplets are prepared based on the ultraviolet (UV) light curable acrylate monomers/LCs composites to fabricate the optical diffuser films. To acquire light diffusers with high optical performance, the effects of the monomer structure and the UV light intensity on the micro-structure of the PDLC films are studied. Results show that the PDLC films could exhibit a strong light scattering at the premise of maintaining high transmittance in the visible region. As the LC droplets are spherically dispersed in the polymer networks, when the size of LC droplets is about 3.0 μm, the haze can reach 88.5% and the transmittance is nearly 90.0%, which can be used as a bottom diffuser film. While when the size of LC droplets is about 10.0 μm, the haze and transmittance are 39.2% and 90.2%, respectively; hence, it can be a good choice for a top diffuser film. With the advantages of simple preparation, roll-to-roll industrial production and tunable optical properties, it is supported that the films based on UV-cured PDLC films can be applied as outstanding optical diffuser films in the liquid crystal display industry.     

The effect of air cooling pain relief on protoporphyrin IX photobleaching and clinical efficacy during dermatological photodynamic therapy

Methyl aminolevulinate photodynamic therapy (MAL-PDT) is utilized to successfully treat licensed indications (e.g. actinic keratosis (AK), superficial basal cell carcinoma (sBCC) and Bowen's disease (BD)) in the UK. Air cooling devices (ACD) are commonly utilized as a method of pain relief, however the effect of this on treatment outcome has never been extensively investigated. This non-randomized, retrospective observational controlled study investigated whether the application of the ACD limited photosensitiser (protoporphyrin IX - PpIX) photobleaching during irradiation and/or subsequent clinical outcome. Patients utilizing the ACD throughout treatment were observed to undergo significantly less PpIX photobleaching than the control group (P<0.001) and complete clinical clearances observed at 3 months were also reduced within the ACD group. Separate analysis of the different lesion types indicated that significantly less photobleaching occurred in AK lesions with ACD and all lesion types failed to fully utilize the accumulated PpIX when ACD was employed. The application of the ACD as pain relief during light irradiation therefore resulted in lower PpIX photobleaching which corresponded to a reduction in the efficacy of PDT treatment. Whilst the ACD is an effective method of dermatological PDT analgesia it should be utilized as sparingly as possible to minimize any deleterious effects on treatment outcome.     

Monitoring ALA-induced PpIX photodynamic therapy in the rat esophagus using fluorescence and reflectance spectroscopy

The presence of phased protoporphyrin IX (PpIX) bleach kinetics has been shown to correlate with esophageal response to 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) in animal models. Here we confirm the existence of phased PpIX photobleaching by increasing the temporal resolution of the fluorescence measurements using the therapeutic illumination and long wavelength fluorescence detection. Furthermore fluorescence differential pathlength spectroscopy (FDPS) was incorporated to provide information on the effects of PpIX and tissue oxygenation distribution on the PpIX bleach kinetics during illumination. ALA at a dose of 200 mg kg(-1) was orally administered to 15 rats, five rats served as control animals. PDT was performed at an in situ measured fluence rate of 75 mW cm(-2) using a total fluence of 54 J cm(-2). Forty-eight hours after PDT the esophagus was excised and histologically examined for PDT-induced damage. Fluence rate and PpIX photobleaching at 705 nm were monitored during therapeutic illumination with the same isotropic probe. A new method, FDPS, was used for superficial measurement on saturation, blood volume, scattering characteristics and PpIX fluorescence. Results showed two-phased PpIX photobleaching that was not related to a (systematic) change in esophageal oxygenation but was associated with an increase in average blood volume. PpIX fluorescence photobleaching measured using FDPS, in which fluorescence signals are only acquired from the superficial layers of the esophagus, showed lower rates of photobleaching and no distinct phases. No clear correlation between two-phased photobleaching and histologic tissue response was found. This study demonstrates the feasibility of measuring fluence rate, PpIX fluorescence and FDPS during PDT in the esophagus. We conclude that the spatial distribution of PpIX significantly influences the kinetics of photobleaching and that there is a complex interrelationship between the distribution of PpIX and the supply of oxygen to the illuminated tissue volume.