Exploration of surfactin production by newly isolated Bacillus and Lysinibacillus strains from food-related sources.

As a lipopeptide (LP), surfactin exhibits properties, such as emulsifying and dispersing ability, which are useful in food industry. Discovery of new LP-producing strains from food sources is an important step towards possible application of surfactin in foods. A total of 211 spore-forming, Gram-positive, and catalase-positive bacterial strains were isolated from fermented African locust beans (iru) and palm oil mill effluents in a screening process and examined for their ability to produce surfactin. This was achieved by a combination of methods, which included microbiological and molecular classification of strains, along with chemical analysis of surfactin production. Altogether, 29 isolates, positive for oil spreading and emulsification assays, were further identified with 16S rDNA analysis. The strains belonged to nine species including less commonly reported strains of Lysinibacillus, Bacillus flexus, B. tequilensis, and B. aryabhattai. The surfactin production was quantitatively and qualitatively analysed by high-performance thin-layer chromatography and liquid chromatography-mass spectrometry (LC-MS). Confirmation of surfactin by MS was achieved in all the 29 strains. Highest surfactin production capability was found in B. subtilis IRB2-A1 with a titre of 1444·1 mg L-1 .

Photophysical properties and in vitro photocytotoxicity of disodium salt 2.4-di(alpha-methoxyethyl)-deuteroporphyrin-IX (Dimegine).

Photophysical and in vitro photocytotoxicity studies were performed for the photosensitizer Dimegine, a disodium salt 2.4-di(alpha-methoxyethyl)-deuteroporphyrin-IX with very low systemic toxicity. The singlet oxygen and luminescence quantum yield were ΦΔ = 0,65 ± 0,06, and Φƒ=0,11 ± 0,01 respectively, and were independent of the excitation wavelength. The photobleaching coefficients for Dimegine dissolved in phosphate buffer (pH 7.4), and DMEM medium at concentration 2 µM/l and in phosphate buffer (pH 7.0) at concentration 10 µM/l were 16·10-5, 9·10-5 and 2·10-5 respectively. In vitro cellular distribution and photocytotoxicity was studied in two human (U87 - primary glioblastoma and HT1376 - bladder cancer) and two rat cell lines (RG2 - glioma, and AY27 - bladder carcinoma). Fluorescence microscopy analysis shows primary Dimegine accumulation as small fluorescent inclusion bodies around the nuclei, suggesting an apoptotic over a necrotic cell death mechanism. The PDT efficacy was slightly higher for the rat cell lines over the human-derived cell lines, with LD50 values of 2,5 µM/l, 2.8 µM/l, 4.5 µM/l, 2.8 µM/l using 530 nm excitation wavelength for AY27, RG2, HT1376 and U87 respectively, and 1.8 µM/l, 2 µM/l, 5 µM/l, 2.4 µM/l using 625 nm excitation wavelength for AY27, RG2, HT1376 and U87 respectively. Comparison to literature data showed that Dimegine demonstrated improved phototherapeutic characteristics comparing to PpIX-mediated PDT.

Energy-partition diagnostic for measuring time-resolved scattering and absorption in burst-mode laser ablation.

We describe an energy-partition diagnostic based on integrating sphere principle for measuring absorption and scattering in plasma-mediated ablation by a high repetition-rate (133 MHz), pulsetrain-burst ultrafast-pulse laser. The system time-resolves the partition of elastically scattered laser light into specular reflection, diffuse reflection, and transmission, giving access to per-pulse absorption dynamics. Physical events such as optical breakdown and incubation effects in glass and aluminum are illustrated.

In vivo quantification of fluorescent molecular markers in real-time by ratio imaging for diagnostic screening and image-guided surgery.

Future applications of "molecular diagnostic screening" and "molecular image-guided surgery" will demand images of molecular markers with high resolution and high throughput (~ > or =30 frames/second). MRI, SPECT, PET, optical fluorescence tomography, hyper-spectral fluorescence imaging, and bioluminescence imaging do not offer such high frame rates. 2D optical fluorescence imaging can provide surface images with high resolution and high throughput. The ability to accurately quantify the fluorescence in vivo is critical to extract functional information of the disease state, however few methods are available. Here, a ratiometric 2D quantification method is introduced. Through mathematical modeling the performance was evaluated using optical properties that resembled biological tissues with the fluorescent marker Protoporhyrin IX. Experimentally the performance was evaluated in optical phantoms with different optical properties employing a novel prototype clinical imaging system. The clinical feasibility of real-time, image-guided surgery was demonstrated in patients undergoing prostatectomy. Discussed are the reasons why the introduced method leads to an increased quantification performance followed by modifications so it can be applied to novel fluorescent molecular markers as phthalocyanine 4 and dual-fluorescent markers. These offer additional advantages as these can provide a linear response to marker concentration and further minimize the dependence on autofluorescence and optical properties, as demonstrated through modeling.

Assessing breast tissue density by transillumination breast spectroscopy (TIBS): an intermediate indicator of cancer risk.

Risk assessment by parenchymal density pattern, a strong physical indicator of future breast cancer risk, is available with the onset of mammographic screening programmes. However, due to the use of ionizing radiation, mammography is not recommended for use in younger women, thereby rendering risk assessment unattainable at an earlier age. Visible and near infrared light was used on 292 women with radiologically normal mammograms to determine whether transillumination breast spectroscopy (TIBS) can identify women with a high parenchymal density pattern as an intermediate indicator of breast cancer risk. Principal component analysis (PCA) was used to reduce the spectral data and generate density scores for each woman. To assess the accuracy of TIBS, logistic regression was used to calculate crude and adjusted odds ratios (OR) and 95% confidence intervals (CI) for each score. Receiver operator characteristic (ROC) curves and area under the curve (AUC) were also calculated for the crude and adjusted logistic models. Optical information relating to tissue chromophores, such as water, lipid and haemoglobin content, was sufficient to identify women with high parenchymal density. The resulting AUC for the final and most parsimonious multivariate logistic model was 0.922 (95% CI 0.878-0.967). TIBS provides information correlating to high parenchymal density and is a promising tool for risk assessment, particularly for younger women.

Optical transillumination spectroscopy to quantify parenchymal tissue density: an indicator for breast cancer risk.

Mammographic screening for early detection of breast cancer has proven valuable in improving breast cancer survival. However, breast cancer incidence is still increasing, and thus preventative oncology needs to receive more attention, with the goal of identifying women with increased risk of developing breast cancer in the future and offering them risk reduction interventions. Mammogram derived parenchymal density pattern has been shown by various authors to provide a high odds ratio for breast cancer. Near-infrared optical transillumination spectroscopy was employed to determine physiological properties of the breast tissue to quantify differences in women with low or high breast cancer risk. Specifically in this study, women who had a recent mammogram underwent examination of their breast tissue by optical transillumination spectroscopy. Areas of adipose and glandular tissues which give rise to mammographic density patterns also have characteristic optical transillumination spectra. Correlation between optical transillumination spectroscopy and mammographic density pattern was established using partial least squares analysis. Results show that predicted tissue density based on optical transillumination spectroscopy correlates with mammographic observed tissue density, with a Spearman Rank correlation coefficient of 0.72. This suggests that optical transillumination spectroscopy may be a promising tool to quantify and monitor changes in breast cancer risk.

Aspirin blocks binding of photosensitizer SnET2 into human serum albumin: implications for photodynamic therapy.

Tin etiopurpurin dichloride (SnET2) is one of the photosensitizers under investigation to be used in photodynamic therapy of prostate cancer. The drug is delivered intravenously, transported in vivo by liposomes and plasma proteins and localized within the prostate. SnET2 exists in two tautomeric forms (I - closed ring, II - open ring) with I converting spontaneously into the more energetically stable form II at physiological pH. Up to approximately 50% of the drug can be carried by serum albumin, although this association can increase photo-bleaching and diminish the drug efficiency. Molecular modeling and force field calculations indicate that Sudlow Site I in human serum albumin (HSA) is the most probable binding site for both forms of SnET2, with the porphyrin moiety nestling between domains IIA and IB, and the esterolytic side group oriented toward domain IIIA of HSA. Other drugs, including aspirin, bind to the same part of HSA. SnET2 does not bind to HSA when pre-incubated with aspirin, which confirms that its place of binding to this protein must be located near Lys199. This observation could be exploited to improve photo-efficiency of SnET2 by finding drugs that could compete with the photosensitizer for binding into Sudlow Site I of HSA.

Apoptosis induced in vivo by photodynamic therapy in normal brain and intracranial tumour tissue.

The apoptotic response of normal brain and intracranial VX2 tumour following photodynamic therapy (PDT) mediated by 5 different photosensitizers (Photofrin, 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX), chloroaluminium phthalocyanine (AlCIPc), Tin Ethyl Etiopurpurin (SnET(2)), and meta -tetra(hydroxyphenyl)chlorin (m THPC)) was evaluated following a previous analysis which investigated the necrotic tissue response to PDT at 24 h post treatment. Free DNA ends, produced by internucleosomal DNA cleavage in apoptotic cells, were stained using a TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling) assay. Confocal laser scanning microscopy (CLSM) was used to quantify the local incidence of apoptosis and determine its spatial distribution throughout the brain. The incidence of apoptosis was confirmed by histopathology, which demonstrated cell shrinkage, pyknosis and karyorrhexis. At 24 h post PDT, AlClPc did not cause any detectable apoptosis, while the other photosensitizers produced varying numbers of apoptotic cells near the region of coagulative necrosis. The apoptotic response did not appear to be related to photosensitizer dose. These results suggest that at this time point, a minimal and fairly localized apoptotic effect is produced in brain tissues, the extent of which depends largely on the particular photosensitizer.

Lessons learned from laser tissue soldering and fibrin glue pyeloplasty in an in vivo porcine model.

PURPOSE: We compared sutured pyeloplasty to 2 newer techniques of tissue anastomosis, including laser soldered pyeloplasty using a diode laser with 50% albumin solder mixed with indocyanine green and fibrin glue. MATERIALS AND METHODS: We performed 53 pyeloplasties in 50 pigs using suture, laser or fibrin glue. In the immediate group anastomotic leak pressure was measured immediately postoperatively, and then animals were euthanized. At 1, 3 and 4 weeks postoperatively a pressure flow study at 10 cc per minute in cm. H2O was performed, and tissue was sent for histological and collagen content analysis. RESULTS: In the immediate studies laser soldering achieved a significantly higher mean anastomotic leak pressure (50.5 +/- 15.1 cm. H2O) than sutured (17.3 +/- 5.4) or fibrin glued (3.5 +/- 1.5) repairs. In the 1, 2 and 4-week studies animals in the sutured pyeloplasty group had no complications, and all pressure flow studies except 1 were normal. However, in the laser soldered groups we observed 8 urinomas in 19 animals, and most occurred during the first part of our study. This complication was prevented by stopping urine flow at the anastomotic site at laser irradiation and by improving application of the solder. Of the 11 animals in which pressure flow studies were performed only 2 were obstructed. Of the 7 chronic fibrin glue group 4 animals had urinomas and 2 had unobstructed pressure flow studies. Histological studies and immunohistochemical staining for collagen showed no differences in collagen distribution among the 3 procedures. CONCLUSIONS: Laser soldering and fibrin glue pyeloplasties are not superior in the long-term compared to sutured pyeloplasty. Fibrin glue in our animal model had the highest failure rate. Further improvements in the technical aspect of laser tissue welding need to be made to benefit from its theoretical advantages in minimally invasive surgery.

Changes in optical properties of ex vivo rat prostate due to heating.

This study examines the effectiveness of a single, first-order Arrhenius process in accurately modelling the thermally induced changes in the optical properties, particularly the reduced scattering coefficient, mu(s)', and the absorption coefficient, mu(a), of ex vivo rat prostate. Recent work has shown that mu(s)' can increase as much as five-fold due to thermal coagulation, and the observed change in mu(s)' has been modelled well according to a first-order rate process in albumen. Conversely, optical property measurements conducted using pig liver suggest that this change in mu(s)' cannot suitably be described using a single rate parameter. In canine prostate, measurements have indicated that while the absorption coefficient varies with temperature, it does not do so according to first-order kinetics. A double integrating sphere system was used to measure the reflectance and transmittance of light at 810 nm through a thin sample of prostate. Using prostate samples collected from Sprague Dawley rats, optical properties were measured at a constant elevated temperature. Tissue samples were measured over the range 54-83 degrees C. The optical properties of the sample were determined through comparison with reflectance and transmittance values predicted by a Monte Carlo simulation of light propagation in turbid media. A first order Arrhenius model was applied to the observed change in mu(s)' and mu(a) to determine the rate process parameters for thermal coagulation. The measured rate coefficients were Ea = (7.18 +/- 1.74) x 10(4) J mol(-1) and Afreq = 3.14 x 10(8) s(-1) for mu(s)'. It was determined that the change in mu(s)' is well described by a single first-order rate process. Similar analysis performed on the changes in mu(a) due to increased temperatures yielded Ea = (1.01 +/- 0.35) x 10(5) J mol(-1) and Afreq = 8.92 x 10(12) s(-1). The results for mu(a) suggest that the Arrhenius model may be applicable to the changes in absorption.

Localized drug delivery using crosslinked gelatin gels containing liposomes: factors influencing liposome stability and drug release.

We describe a drug-delivery vehicle that combines the sustained release properties of liposomes with the structural advantages of crosslinked gelatin gels that can be implanted directly or coated onto medical devices. Liposome inclusion in gelatin gels does not compromise thermal stability nor does it interfere with the resiliency of gels to tensile force. However, electron spin resonance analysis of sequestered DPPC liposomes revealed a slight depression (ca. 1.0 degrees C) of the gel-to-fluid phase transition relative to liposomes in suspension. The level of liposome release from gels was determined by liposome concentration, liposome size, and the presence of poly(ethylene oxide) chains in the gel matrix or in the liposome membrane. Both neutral and charged liposomes displayed relatively high affinities for poly(ethylene glycol)gelatin gels, with only 10-15% release of initially sequestered liposomes while liposomes in which poly(ethylene glycol) was included within the membrane were not as well retained (approximately 65% release). The in vitro efflux of ciprofloxacin from liposomal gels immersed in serum was nearly complete after 24 h compared to 38% release of liposomal chlorhexidine after 6 days. The serum-induced destabilization of liposomal ciprofloxacin depended on the accessibility of serum components to gels as partly immersed gels retained approximately 50% of their load of drug after 24 h. In vivo experiments using a catheterized rabbit model of urinary tract infection revealed the absence of viable Escherichia coli on coated catheter surfaces in seven out of nine cases while all untreated catheter surfaces examined (n = 7) were contaminated.

Low-level laser therapy for wound healing: feasibility of wound dressing transillumination.

OBJECTIVE: The purpose of this study was to assess the feasibility of exposing wounds during low-level laser therapy (LLLT) by transillumination of the wound dressings. BACKGROUND DATA: LLLT has been associated with accelerated wound healing in chronic ulcers. The usual approach is to remove wound dressings prior to exposure and to treat three to five times weekly. Frequent change of wound dressings is time consuming and costly; it disrupts the healing process, increases the risk of wound infection, and may be traumatic for the patient. METHODS: A double integrating sphere setup was employed to quantify the diffuse transmittance and reflectance of various wound dressings. Differences in transmittance for large area sources and point sources were demonstrated through the use of a diode laser and an incoherent light source. RESULTS: There were a number of gels and membrane style wound dressings with diffuse transmittance of more than 50%. Hence, for these dressings the prescribed radiant exposure to the wound surface could be achieved by increasing the exposure duration, while maintaining reasonable overall treatment times. CONCLUSIONS: Although LLLT by transillumination of wound dressings is feasible for a variety of wound dressings without significant commitments in additional treatment time, the specific transmission of products not included in this study needs to be determined at the intended treatment wavelength. A transillumination approach may facilitate a faster rate of wound healing than LLLT applied to exposed wounds by reducing trauma and the risk of infection.

Interpretation of intrinsic optical signals and calcein fluorescence during acute excitotoxic insult in the hippocampal slice.

Immediate (acute) neuronal damage in response to overstimulation of glutamate receptors results from toxic exposure to food poisons acting as glutamate analogues. Glutamate agonist application evokes dramatic intrinsic optical signals (IOSs) in the rat hippocampal slice preparation, particularly in the CA1 region. Theoretically IOSs are generated by alterations to neuronal and glial structure that change light transmittance (LT) in live brain tissue. To better understand such signals, IOSs evoked by the glutamate agonist N-methyl-D-aspartate were imaged in the rat hippocampal slice. We correlated these excitotoxic signals with: (1) biophysical principles governing light transport, (2) tissue volume changes as measured using a free intracellular fluorophore (calcein), (3) dendritic morphology visualized by dye injection, and (4) standard histopathology. In theory LT elevation evoked during acute excitotoxic swelling is generated by change to subcellular structure that reduces light scattering during cell swelling. However, in responsive dendritic regions, initial LT elevation caused by cell swelling was overridden by the formation of dendritic beads, a conformation that increased light scattering (thereby reducing LT) even as the calcein signal demonstrated that the tissue continued to swell. Thus IOS imaging reveals acute somatic and dendritic damage during excitotoxic stress that can be monitored across slices of brain tissue in real time.

Photodynamic therapy using Photofrin in combination with buthionine sulfoximine (BSO) to treat 9L gliosarcoma in rat brain.

BACKGROUND AND OBJECTIVE: The reactive oxygen mechanisms associated with cell damage after photodynamic therapy (PDT) may be exploited to enhance tumor destruction. Pharmacological reduction of glutathione (GSH), an inhibitor of reactive oxygen species, can be induced by administration of buthionine sulfoximine (BSO). STUDY DESIGN/MATERIALS AND METHODS: BSO was administered in combination with Photofrin as the photosensitizer in order to promote PDT induced cell damage. Photofrin (12.5 mg/kg) or Photofrin with BSO (440 mg/kg) were administered to male Fischer rats (n = 27) containing an intracerebral 9L gliosarcoma or to non tumored rats. Brain tumor or non tumored brain was treated with an optical (632 nm) irradiance of 140 J/cm2. Animals were sacrificed 24 h after PDT and the volume of tissue necrosis was measured. Brain Photofrin concentration was measured in tumor and in non tumor bearing animals administered either Photofrin or Photofrin with BSO. GSH was measured by high pressure liquid chromatography in tumor and homologous non tumor tissue in animals administered BSO or control solution. RESULTS: The volume of tumor necrosis was significantly greater in animals administered Photofrin and BSO than in animals administered only Photofrin. No differences were detected in non tumored tissue damage between groups. No differences in Photofrin concentration were detected in tumored or nontumored animals between animals administered Photofrin and animals administered Photofrin and BSO. BSO administration preferentially and significantly reduced GSH in tumor compared to non tumor tissue. CONCLUSIONS: Our data suggest that BSO administration preferentially augments tumor destruction without compromising non tumored tissue.

Light dosimetry for intraperitoneal photodynamic therapy in a murine xenograft model of human epithelial ovarian carcinoma.

Few studies have been published to date measuring spatially resolved fluence rates in complex tissue geometries. Here the light distributions of three different intraperitoneal light delivery geometries in a murine ovarian cancer model were investigated to assess their influence on the tumorcidal efficacy of photodynamic therapy (PDT). In vivo fluence rate measurements in the peritoneal cavities of mice, with the light intensity being mapped in three transverse planes, were performed using fiber-optic detectors. Three different source fiber designs and placements were tested for their ability to provide uniform irradiation of the peritoneal cavity. The biological response to a PDT protocol comprising three separate treatments administered at 72 h intervals, each consisting of a 0.25 mg kg-1 intraperitoneal injection of benzoporphyrin derivative-mono acid ring A followed 90 min later by delivery of 15 J of 690 nm light, was measured. The tissue response was evaluated by measuring the number of remaining visible lesions and the total residual tumor mass. Fluence rate measurements showed large variations in the fluence rate distribution for similar intended treatments. The most uniform and reproducible illumination was achieved using two 18 mm long cylindrical emitting optical fibers. The biological response was comparable to that produced when a flat-cleaved end optical fiber is used to illuminate the four quadrants of the abdomen sequentially. While a good reproducibility in tumor induction in this animal model exists, no correlation was found between the fluence rate distribution measured in one group of animals and the biological response in a separate group of similarly treated animals. Due to the large intra-animal variability in fluence rate distribution, representative fluence rate mapping in complex tissue geometries is of limited value when applied to an individual PDT treatment. Thus, surveillance of the fluence rate during individual treatments will be required for acceptable PDT dosimetry. To improve the versatility of this particular animal model for PDT research, a large number of extended sources are required to increase uniformity of the illumination in order to reduce unwanted cytotoxic side effects resulting from foci of high fluence rates. In this way, subsequent increase of the total energy delivered to the tumor may be possible.

Photodynamic therapy of intracranial tissues: a preclinical comparative study of four different photosensitizers.

OBJECTIVE: The effectiveness of four different photosensitizers for intracranial photodynamic therapy (PDT) of normal brain tissues and an intracranial tumor was investigated in rabbits, using the photodynamic threshold model. SUMMARY: PDT is currently being investigated as an adjuvant treatment to surgical resection and/or radio chemotherapy of intracranial neoplasms. While possible neurotoxic side effects of the treatment have been noted, only limited preclinical data quantifying the response of intracranial normal and tumor tissues following PDT are available. MATERIALS AND METHODS: The photodynamic threshold dose values for the four photosensitizers, Photofrin, 5-aminolevulinic acid (ALA)-induced Protoporphyrin IX (PpIX), Tin Ethyl Etiopurpurin (SnET2), and chloroaluminum phthalocyanine (AlClPc), were determined using measured light fluence distributions, photosensitizer concentration in tissue, and histologically-determined extent of necrosis following PDT. These measurements were made in normal rabbit brain and in an intracranially-implanted carcinoma (VX2). RESULTS: For Photofrin, AlClPc, and SnET2 (in an emulsion delivery vehicle) normal grey and white matter were very sensitive to PDT, showing a significantly lower threshold dose value than VX2-tumor. For ALA-induced PpIX and SnET2 (in liposome) very little or no white matter damage was observed. Additionally, ALA-PpIX showed significantly lower concentration in white matter than in cortex and tumor. Normal brain structures lacking a blood-brain barrier showed high uptake of all photosensitizers and, hence, are at risk of collateral damage during PDT. CONCLUSIONS: For clinical PDT of most adult intracranial neoplasms ALA-induced PpIX appears to be promising, and SnET2 (liposomal) has potential for selective tumor destruction with relative sparing of white matter. Other normal brain structures and, for the other photosensitizers, also white matter are at risk of collateral damage, if exposed to light during PDT.

Photodynamic therapy of U87 human glioma in nude rat using liposome-delivered photofrin.

BACKGROUND AND OBJECTIVE: Liposomes as photosensitizer carriers may enhance the photodynamic effect on tumors. STUDY DESIGN/MATERIALS AND METHODS: To test this hypothesis, we treated U87 human glioma in rat brain with photodynamic therapy (PDT) using Photofrin encapsulated in a liposome carrier or Photofrin in dextrose. Nontumored brain was also treated and Photofrin content ratios were measured in tumor and nontumored brain. RESULTS: PDT using the liposome encapsulated photosensitizer significantly increased tumor destruction compared to PDT with Photofrin in dextrose (P = 0.007), whereas no difference in tissue damage was detected in nontumored brain with or without liposome carrier. Photofrin uptake was also significantly elevated in the liposome vehicle group compared to the dextrose (P < 0.05) group. CONCLUSIONS: Our data suggest that Photofrin encapsulated in a liposome may enhance the PDT treatment of human brain tumors.

A solubilization technique for photosensitizer quantification in ex vivo tissue samples.

The determination of the photosensitizer concentration in ex vivo tissue samples is commonly used for pharmacokinetic and dosimetric studies of photodynamic therapy, both clinically and pre-clinically. In this report, a new method is presented based on tissue solubilization and subsequent fluorometry. This method has the advantages of good sensitivity, accuracy and reproducibility, as well as low cost and ease of handling of the tissue samples. The method was tested for six different photosensitizers in a variety of tissues. The accuracy and concentration detection limits are compared with those of other published extraction methods.

Photodynamic therapy of 9L gliosarcoma with liposome-delivered photofrin.

The effect of Photofrin encapsulated in a liposome delivery vehicle for photodynamic therapy (PDT) of the 9L gliosarcoma and normal rat brain was tested. We hypothesized that the liposome vehicle enhances therapeutic efficacy, possibly by increasing tumor tissue concentration of Photofrin. Male Fisher rats bearing a 9L gliosarcoma were treated 16 days after intracerebral tumor implantation with either Photofrin in dextrose (n = 5) or Photofrin in liposome (n = 6). Nontumor-bearing animals were treated with Photofrin delivered either in dextrose (n = 4) or liposome (n = 4) vehicle. Tissue concentrations of Photofrin delivered either in dextrose (n = 4) or liposome (n = 4) vehicle were measured in tumor, brain adjacent to tumor and in normal brain tissue. Photofrin was administered (intraperitoneally) at a dose of 12.5 mg/kg and PDT (17 J/cm2 of 632 nm light at 100 mW/cm2) was performed 24 h after Photofrin administration. Brains were removed 24 h after PDT and stained with hematoxylin and eosin for analysis of cellular damage. The PDT using Photofrin in the liposome vehicle caused significantly more damage to the tumor (P < 0.001) than did PDT with Photofrin in dextrose. The PDT of tumor with Photofrin delivered in liposomes caused a 22% volume of cellular necrosis, while PDT of tumor with Photofrin delivered in dextrose caused only scattered cellular damage. Photofrin concentration in tumors was significantly higher (P = 0.021) using liposome (33.8 +/- 18.9 micrograms/g) compared to dextrose delivery (5.5 +/- 1.5 micrograms/g). Normal brain was affected similarly in both groups, with only scattered cellular necrosis. Our data suggest that the liposome vehicle enhances the therapeutic efficacy of PDT treatment of 9L tumors.

Absorbed photodynamic dose from pulsed versus continuous wave light examined with tissue-simulating dosimeters.

A dosimetric system has been developed to measure the spatially resolved light dose absorbed by a photosensitizer in a tissue-simulating medium. These gelatin-based dosimeters had macroscopic optical scattering and absorption properties that are typical for homogeneous tissue and contained the photosensitizer benzoporphyrin derivative monoacid (BPD-MA). A reporter molecule, 2?7?-dichlorofluorescin diacetate (DCF-DA), served as an actinometer, which could be photosensitized by BPD-MA to generate a highly fluorescent photoproduct. The relative photosensitizing efficiencies of high-intensity pulsed and cw laser light were compared in these tissue-simulating dosimeters. These measurements demonstrate an increase in penetration for pulsed light as compared with cw light in the dosimeters. A numerical simulation of the light propagation based on optical diffusion theory was used along with the energy levels of the photosensitizer molecule to examine the mechanisms involved in the absorbed dose. The increased penetration of high-intensity pulsed light was due to a transient decrease in the absorption of the photosensitizer, resulting from saturation of the photosensitizer optical transitions. This study provides the first direct comparison of the photodynamic dose absorbed by a photosensitizer using both high-intensity pulsed and cw laser light in a tissue-simulating medium. These measurements demonstrate that a small increase in depth of treatment is possible with pulsed laser light as compared with cw laser light simply on the basis of the unique photochemistry of the photosensitizer. However, this effect still needs to be examined carefully in tumor tissue, where other biological or chemical effects may become significant.