Monte Carlo Model of Stricture Formation in Photodynamic Therapy of Normal Pig Esophagus

Photodynamic therapy (PDT) is FDA-approved for use in patients with Barrett's esophagus using porfimer sodium (2 mg per kg) and a recommended light dose of 130 J cm⁻¹ for high grade dysplasia. Despite uniform drug and light doses, the clinical outcome of PDT is variable. A significant number of PDT cases result in esophageal strictures, a side effect related to excessive energy absorption. The purpose of this project was to model esophageal stricture formation with a Monte Carlo simulation. An original multilayer Monte Carlo computer simulation was developed for esophageal PDT. Optical absorption and scattering coefficients were derived for mucosal and muscle layers of normal porcine esophagus. Porfimer sodium was added to each layer by increasing the absorption coefficient by the appropriate amount. A threshold-absorbed light dose was assumed to be required for stricture formation and ablation. The simulation predicted irreversible damage to the mucosa with a 160 J cm⁻¹ light dose and damage to the muscle layer with an additional 160 J cm⁻¹ light dose for a tissue porfimer sodium content of 3.5 mg kg⁻¹. The simulation accurately modeled photodynamic stricture formation in normal pig in vivo esophageal tissue. This preliminary work suggests that the absorbed light threshold for stricture formation may be between 2 and 4 J per gram of tissue.     

Measurement of Tumor Vascular Damage in Mice with 99m Tc-MIBI Following Photodynamic Therapy

Abstract— The clinical perfusion agent ^99mTc-MIBI was used to monitor changes in tumor vascular perfusion (TVP) induced by Photofrin® (Pll)-mediated photodynamic therapy (PDT). BALB/c mice bearing an EMT-6 tumor on each hind thigh were given an intravenous injection of 1, 2 or 5 mg kg⁻¹ PII. Twenty-four hours later, one tumor was illuminated (600–650 run, 200 mW cm⁻² 400 J cm⁻²) while the other served as a control. At various time intervals after PDT (0, 2 and 24 h) mice received an intravenous injection of ^99mTc-hexakismethoxy(sobutyusonitri-le (MIBI) (0.18 MBq g⁻¹) and were sacrificed 2 min later. The light-treated and the untreated tumors were then dissected, the radioactivity was counted and the percentage of the injected dose per gram of tumor (%ID g⁻¹) was calculated as a measure of TVP. We observed that TVP is drug dose dependent, develops progressively with time post-PDT and is inversely related to PDT efficacy. Our data show that early tumor retention of ^99mMIBI is a simple method to assess TVP and vascular damage induced by PDT.     

Combination Therapy Using Verteporfin and Ranibizumab; Optimizing the Timing in the CAM Model

Combining photodynamic therapy (PDT) using verteporfin (Visudyne^®) with ranibizumab (Lucentis^®) can optimize the overall treatment outcome by providing more efficacy in vessel closure, and thus reduce the need for retreatment in patients with wet age-related macular degeneration. In this preclinical study in the chorioallantoic membrane (CAM) of the chicken embryo, we compare the vascular occlusion effects of verteporfin and ranibizumab as monotherapies with those observed in the combined therapy. In order to optimize the combination therapy, we varied the timing and sequence of the PDT and antivascular endothelial growth factor modalities. We observed that 1 day after PDT, the smaller blood vessels (∅ < 70 μm) of the CAM were selectively occluded, but as early as 2 days after PDT, both significant reperfusion and regrowth of new vessels were observed. Both these phenomena could be significantly delayed by application of ranibizumab. Ranibizumab itself did not induce any vascular occlusion. Under the applied conditions of combination therapy, the occlusion of the targeted blood vessels could be significantly extended to 3 days in this model compared with 1 day in the case of verteporfin monotherapy. Thus, in the present preclinical study, we demonstrate that for the applied conditions, the optimal time to administer ranibizumab is 24 h after PDT.     

In vitro survival of nonsmall cell lung cancer cells following combined treatment with ionizing radiation and photofrin-mediated photodynamic therapy

It has been suggested that combination high dose rate (HDR) intraluminal brachytherapy and photodynamic therapy (PDT) in nonsmall cell lung cancer (NSCLC) may improve efficacy of treatment, reduce toxicity and enhance quality of life for patients. To provide a cellular basis for this we examined the in vitro sensitivity of MRC5 normal lung fibroblasts and four NSCLC cell lines following HDR radiation, PDT and combined HDR radiation and PDT. HDR radiation was cobalt-60 gamma rays (1.5–1.9 Gy min⁻¹). For PDT treatment, cells were exposed to 2.5 μg mL⁻¹ Photofrin for 18–24 h followed by light exposure (20 mW cm⁻²). For combined treatment cells were exposed to Photofrin and then either exposed to light and 15–30 min later exposed to HDR radiation or exposed to HDR radiation and 15–30 min later exposed to light. D₃₇ values calculated from clonogenic survival curves indicated a six-fold difference in HDR radiation sensitivity and an eight-fold difference in PDT sensitivity. The effect of combined treatment was not significantly different from an additive effect of the individual treatment modalities for the NSCLC cells, but was significantly less than additive for the MRC5 cells. These results suggest an equivalent tumor cell kill may be possible at reduced systemic effects to patients.     

Photochemical Internalization of Bleomycin is Superior to Photodynamic Therapy Due to the Therapeutic Effect in the Tumor Periphery

Photochemical internalization (PCI) is under development for clinical use in treatment of soft tissue sarcomas and other solid tumors. PCI may release endocytosed bleomycin (BLM) into the cytosol by photochemical rupture of the endocytic vesicles. In this study, the human fibrosarcoma xenograft HT1080 was transplanted into the leg muscle of athymic mice. The photosensitizer disulfonated aluminum phthalocyanine (AlPcS_2a) and BLM were systemically administrated 48 h and 30 min, respectively, prior to light exposure at 670 nm (30 J cm⁻²). The purposes of this study were to evaluate the treatment response to AlPcS_2a-photodynamic therapy (PDT) and AlPcS_2a-PDT in combination with BLM ( i.e. PCI of BLM) in an orthotopic, invasive and clinically relevant tumor model and to explore the underlying response mechanisms caused by PDT and PCI of BLM. The treatment response was evaluated by measuring tumor growth, contrast-enhanced magnetic resonance imaging (CE-MRI), histology and fluorescence microscopy. The results show that PCI of BLM is superior to PDT in inducing tumor growth retardation and acts synergistically as compared to the individual treatment modalities. The CE-MRI analyses 2 h after AlPcS_2a-PDT and PCI of BLM identified a treatment-induced nonperfused central zone of the tumor and a well-perfused peripheral zone. While there were no differences in the vascular response between PDT and PCI, the histological analyses showed that PDT caused necrosis in the tumor center and viable tumor cells were found in the tumor periphery. PCI caused larger necrotic areas and the regrowth in the peripheral zone was almost completely inhibited after PCI. The results indicate that PDT is less efficient in the tumor periphery than in the tumor center and that the treatment effect of PCI is superior to PDT in the tumor periphery.     

Amelioration of Airway Stenosis in Rabbit Models by Photodynamic Therapy with Talaporfin Sodium (NPe6)

It is difficult to treat patients with acquired airway stenosis, and the quality of life of such patients is therefore lowered. We have suggested the application of photodynamic therapy (PDT) as a new treatment for airway stenosis and have determined the efficacy of PDT in animal disease models using a second-generation photosensitizer with reduced photosensitivity. An airway stenosis rabbit model induced by scraping of the tracheal mucosa was administered NPe6 (5 mg kg⁻¹), and the stenotic lesion was irradiated with 670 nm light emitted from a cylindrical diffuser tip at 60 J cm⁻² under bronchoscopic monitoring. PDT using NPe6 improved airway stenosis ( P  = 0.043) and respiratory stridor. A significant prolongation of survival time was seen in the PDT-treated animals compared to that in the untreated animals ( P  = 0.025) and 44% of the treated animals achieved long-term survival (>60 days). In conclusion, PDT using NPe6 is effective for improvement in airway stenosis.     

THE INFLUENCE OF SODIUM PENTOBARBITAL ANESTHESIA ON in vivo PHOTODYNAMIC THERAPY

Abstract The use of sodium pentobarbital anesthesia 50 jig gm⁻¹ during localized photodynamic therapy (PDT) was examined in C57BL/6 mice transplanted with the pigmented B-16 melanoma. A 10 mg kg⁻¹ i.p. injection of Photofrin II was administered 24 h prior to light exposure (630 nm, 150 mW, cm⁻², 300-500 J cm⁻²). Separate groups of mice were utilized to monitor tumour temperature and PDT tumor response. Core tumor temperatures decreased by approx. 10^oC following sodium pentobarbital administration. Tumor responses were determined by documenting the percentage of treated animals without tumor recurrences for a period of 50 days following PDT. Superior PDT induced tumor responses were obtained in control (non-anesthetized) mice following light doses of 400 and 500 J cm⁻². The results of this study indicate that sodium pentobarbital can induce a protective effect on B-16 melanomas treated with PDT.     

TUMOR DESTRUCTION IN PHOTODYNAMIC THERAPY

Abstract The effects of photodynamic therapy (PDT) on the tumor microvasculature in the first few hours after treatment was studied at the light microscope (LM) and electron microscope (EM) levels in DBA/2Ha mice bearing SMT-F tumors. Animals received intraperitoneal injections of 10 mg kg⁻ of Photofrin II and 24 h later tumors were treated with 100 J cm⁻² of light (630 nm). Animals were sacrificed and their tumors removed at time 0, 30 min, 1, 2, 4, 8, 16 and 24 h after treatment. The results indicate that the effects of PDT are initially direct destruction of the microfibrils in the subendothelial zone of the tumor capillaries with subsequent tumor cell death secondary to hemorrhage and vascular collapse.     

The Relationship of Phthalocyanine 4 (Pc 4) Concentrations Measured Noninvasively to Outcome of Pc 4 Photodynamic Therapy in Mice

The ability to noninvasively measure photosensitizer concentration at target tissues will allow optimization of photodynamic therapy (PDT) and could improve outcome. In this study, we evaluated whether preirradiation tumor phthalocyanine 4 (Pc 4) concentrations, measured noninvasively by the optical pharmacokinetic system (OPS), correlated with tumor response to PDT. Mice bearing human breast cancer xenografts were treated with 2 mg kg⁻¹ Pc 4 iv only, laser irradiation (150 J cm⁻²) only, Pc 4 followed by fractionated irradiation or Pc 4 followed by continuous irradiation. Laser irradiation treatment was initiated when the tumor to skin ratio of Pc 4 concentration reached a maximum of 2.1 at 48 h after administration. Pc 4 concentrations in tumor, as well as in Intralipid in vitro , decreased monoexponentially with laser fluence. Pc 4-PDT resulted in significant tumor regression, and tumor response was similar in the groups receiving either fractionated or continuous irradiation treatment after Pc 4. Tumor growth delay following Pc 4-PDT correlated with OPS-measured tumor Pc 4 concentrations at 24 h prior to PDT ( R ² = 0.86). In excised tumors, OPS-measured Pc 4 concentrations were similar to the HPLC-measured concentrations. Thus, OPS measurements of photosensitizer concentrations can be used to assist in the scheduling of Pc 4-PDT.     

ULTRASTRUCTURAL STUDIES ON THE MECHANISM OF THE PHOTODYNAMIC THERAPY OF TUMORS

Abstract Balb/c mice bearing a transplanted MS-2 fibrosarcoma were injected with 2.5 mg kg ¹ of either tetra(4-sulfonatophenyl/porphine (TPPS) in phosphate-buffered saline or 0.5 mg kg⁻¹ of Zn²⁺-phthalocyanine (Zn-Pc) incorporated into unilamellar liposomes of dipalmitoyl-phosphatidylcholine. Chromatographic studies showed that TPPS is mainly transported in the serum by globulins and albumin, while Zn-Pc is specifically bound by lipoproteins. Exposure of the injected mice to red light (300 J cm⁻²) caused extensive tumor necrosis. The ultrastructural analysis of tumor specimens taken from mice at 15 h after PDT showed that TPPS photoinduces a preferential necrosis of the neoplastic cells, while Zn-Pc causes severe photodamage to both the vascular system and the neoplastic cells. The different modes of tumor photosensitization by TPPS and Zn-Pc are discussed on the basis of the transport mechanism of the two dyes.     

PARTIAL PROTECTION OF PHOTODYNAMIC-INDUCED SKIN REACTIONS IN MICE BY N-ACETYLCYSTEINE: A PRECLINICAL STUDY

Abstract The major side effect of photodynamic therapy (PDT) using Photofrin® is enhanced skin sensitivity for sunlight, which persists for 3-8 weeks after injection. Formation of singlet oxygen and radicals is believed to be involved in the basic mechanism of inducing skin damage. Reducing this side effect would make PDT more widely acceptable, particularly for palliative use. Hairless dorsal skin patches of mice, injected with 10 mg kg⁻¹ photofrin intraperitoneally (i.p.) 24 h before illumination, were used to evaluate the effect of increasing light doses. The light was obtained from a halogen lamp and transmitted via a fiber optic to illuminate a field of 2.5 cm². After establishing a dose-response relationship for single or fractionated light dose illumination of the skin, drugs known to scavenge radicals, quench singlet oxygen or interfere with histamine release were tested for their protective effect. N -acetylcysteine (NAC), a radical scavenger, administered i.p. (1000 and 2000 mg kg⁻¹) 1 h before illumination produced a significant decrease in skin damage at light doses >50 J cm⁻² (protection factor of 1.3-1.8). When NAC was administered in a dose of 500 mg kg⁻¹, no protection was observed. Fractionated illumination experiments in combination with multiple injections of NAC (1000 mg kg⁻¹) also failed to show any protection. The addition of Ranitidine®, a histamine blocking agent (25-100 mg kg⁻¹, given prior to illumination, resulted in a limited protection at higher light doses. From this study we conclude that NAC could be of value in amelioration of the photosensitivity in patients treated with PDT.     

Alterations in Skin Immune Response Throughout Chronic UVB Irradiation—Skin Cancer Development and Prevention by Naproxen

Skin exposure to UVB radiation has been reported to produce both a significant inflammatory response and marked immunosuppression. This work was aimed to evaluate whether the response of murine skin to an acute UVB dose was modified by pre-exposure to chronic UVB irradiation and by topical treatment with naproxen, a nonsteroidal anti-inflammatory drug. Moreover, the effect of naproxen on the incidence of UV-induced skin tumors was studied. Prostaglandin E₂ (PGE₂) and tumor necrosis factor alpha (TNF-α) levels were increased 96 h post-UVB in acutely irradiated animals and both mediators were modified by topical naproxen application—PGE₂ was decreased while TNF-α was increased. Such inflammatory response was suppressed when mice were chronically irradiated. Naproxen application on chronically irradiated mice reduced the incidence of tumor lesions. Taken together, our data suggest that chronic UVB irradiation generates an immunosuppressive state that prevents skin cells from responding normally to an acute irradiation challenge, thus impairing the protective effect of TNF-α against skin tumor development. Furthermore, reduction in the incidence of tumor lesions by naproxen may be due to its ability to increase TNF-α levels as well as to decrease PGE₂.     

PHOTODYNAMIC THERAPY OF B16 PIGMENTED MELANOMA WITH LIPOSOME-DELIVERED Si(IV)-NAPHTHALOCYANINE

The possibility of extending photodynamic therapy to the treatment of highly pigmented neoplastic lesions was tested by using Si(IV)-naphthalocyanine (SiNc) as a tumor-localizing agent. Si(IV)-naphthalocyanine displays intense absorbance at 776 nm (ɛ= 5 × 10⁵ M⁻¹ cm⁻¹), where melanin absorption becomes weaker. As an experimental model we selected B16 pigmented melanoma subcutaneously transplanted to C57BL mice. Upon injection of 0.5 or 1 mg kg⁻¹ of liposome-incorporated SiNc, maximal accumulation of the photosensitizer in the tumor was observed at 24 h with recoveries of 0.35 and 0.57 μg g⁻¹, respectively. However, the tumor targeting by SiNc shows essentially no selectivity, since the photosensitizer concentrations in the skin (peritumoral tissue) were very similar to those found in the tumor at all postinjection times examined by us. Irradiation of SiNc-loaded melanoma with 776 nm light from a diode laser at 24 h postinjection induces tumor necrosis and delay of tumor growth. The effect appears to be of purely photochemical nature at dose rates up to 260 mW cm⁻²; at higher dose rates, thermal effects are likely to become important.     

LIGHT INDUCED FLUORESCENCE SPECTRAL CHANGES IN NATIVE PHYTOCHROME FROM Secale cereale L. AT LIQUID NITROGEN TEMPERATURE

Abstract— Fluorescence spectra of native rye phytochrome were determined under different light conditions at liquid nitrogen temperature. Fluorescence spectrum of the red-light-absorbing form (Pr) had a major peak at about 685 nm (14 600 cm⁻¹) and a broad sub-peak at about 515 nm (19 400 cm⁻¹). The peak height at 685 nm was reduced by irradiation with monochromatic light of 640 nm, and a new peak became obvious at about 702 nm (14250 cm⁻¹). This spectral change was almost completely reversed by subsequent irradiation with 700-nm light. Fluorescence spectrum of the photoequilibrium mixture of Pr and far-red-light absorbing form under continuous red light showed a sharp peak at about 685 nm having a peak height ca. 12% of Pr, and a broad sub-peak at about 508 nm (19 700 cm⁻¹). Light of 730 nm did not reduce the peak height at about 685 nm but induced a new shoulder at about 699 nm (14300 cm⁻¹). Monochromatic light of 640 and 700 nm given following the light of 730 nm could not reverse the spectral change at 699 nm induced by the irradiation with 730-nm light. Fluorescence spectrum of Pr in partially degraded phytochrome was similar to that in native phytochrome but the peak position in the red region was shifted by about 5 nm (100 cm⁻¹) to the blue.     

LIGHT-INDUCED FOURIER TRANSFORM INFRARED SPECTROSCOPIC INVESTIGATIONS OF THE INTERMEDIARY ELECTRON ACCEPTOR REDUTION IN BACTERIAL PHOTOSYNTHESIS

Abstract— Molecular changes associated with the light-induced reduction of the intermediary electron acceptor I (bacteriopheophytin, BPh) in bacterial photosynthesis were studied by means of Fourier transform infrared (FTIR) difference spectroscopy. Chromatophore membranes and reconstituted reaction centers (RCs) of Rhodopseudomonas viridis were prereduced with sodium dithionite and illuminated in order to trap photochemically the state I⁻. Fourier transform infrared spectra of these samples were recorded before, during and after illumination, with an accuracy better than 10⁻³ absorbance units. Difference spectra of I⁻ in chromatophores and in RCs closely correspond to each other. In the carbonyl stretching frequency region between 1640 and 1750 cm⁻¹, bands are tentatively attributed to a shift (from 1713 to 1683 cm⁻¹) of a keto carbonyl group, a change of an acetyl carbonyl grou at 1656 cm⁻¹ and a decrease in absorbance strength of ester carbonyl groups (at 1746 and 1732 cm⁻P) after reduction of I. These groups likely belong to the BPh molecule, although at least one of the ester carbonyls could be assigned to an amino acid side chain. The absence of strong bands in the amide I and amide II region excludes large protein conformational changes associated with I reduction.     

PHOTOCHEMICAL REACTION OF 13-CIS-BACTERIORHODOPSIN STUDIED BY LOW TEMPERATURE SPECTROPHOTOMETRY

Abstract— The photoreaction cycle of 13- cis -bacteriorhodopsin (13- cis -bR) was investigated by low temperature spectrophotometry using two different preparations; 13- cis -bR constituted from bacterioopsin and 13- cis -retinal, and dark-adapted bacteriorhodopsin (bR^D), which is an equi-molar mixture of 13- cis -bR and trans -bR.
By irradiation with 500 nm light at — 190°C, 13- cis -bR was converted to its batho-product, batho-13- cis -bR (batho-bR¹³), which is different from batho-product from trans -bR, batho-bR^t. On warming batho-bR¹³ to -5°C in the dark, it completely changed to trans -bR. We estimated the composition of 13- cis -bR and trans -bR in the warmed sample spectrophotometrically and then the absorption spectrum of batho-bR¹³ was calculated. The absorption maximum lies at 608 nm, 1250 cm⁻¹ longer than that of 13- cis -bR; the molar extinction coefficient (ε) is about 74000 M ⁻¹ cm⁻¹, larger than that of 13- cis -bR (52000 M ⁻¹ cm⁻¹).
On the warming the sample containing batho-bR¹³ formed by irradiating 13- cis -bR or bR^D at — 190°C, we could not detect other intermediates such as the lumi- or meta-intermediates seen in trans-bR system.     

Preclinical Studies with 5,10,15-Tris(4-Methylpyridinium)-20-Phenyl-[21H,23H]-Porphine Trichloride for the Photodynamic Treatment of Superficial Mycoses Caused by Trichophyton rubrum

Dermatophytes are fungi that cause infections of keratinized tissues. We have recently demonstrated the susceptibility of the dermatophyte Trichophyton rubrum to photodynamic treatment (PDT) with 5,10,15-Tris(4-methylpyridinium)-20-phenyl-[21 H ,23 H ]-porphine trichloride (Sylsens B) in 5 m m citric acid/sodium citrate buffer (pH 5.2, formulation I). In this work, we examined the penetration of Sylsens B in healthy and with T. rubrum infected skin and we investigated the susceptibility of T. rubrum to PDT using formulation I and UVA-1 radiation (340–550 nm). Skin penetration studies were performed with formulations I and II (Sylsens B in PBS, pH 7.4) applied on dermatomed skin, human stratum corneum (SC), disrupted SC by T. rubrum growth and SC pretreated with a detergent. No penetration was observed in healthy skin. Disruption of SC by preceding fungal growth caused Sylsens B penetration at pH 7.4, but not at pH 5.2. However, chemically damaged SC allowed Sylsens B to penetrate also at pH 5.2. UVA-1 PDT was applied ex vivo during two fungal growth stages of two T. rubrum strains (CBS 304.60 and a clinical isolate). Both strains could be killed by UVA-1 alone (40 J/cm²). Combined with formulation I (1 and 10 μ m Sylsens B for, respectively, CBS 304.60 and the clinical isolate), only 18 J/cm² UVA-1 was required for fungal kill. Therefore, PDT with 10 μ m Sylsens B (formulation I) and 18 J/cm² UVA-1 could be considered as effective and safe. This offers the possibility to perform clinical studies in future.     

Comparison of the Disinfection Effects of Vacuum-UV (VUV) and UV Light on Bacillus subtilis Spores in Aqueous Suspensions at 172, 222 and 254 nm

The efficacy of UV and vacuum-UV (VUV) disinfection of Bacillus subtilis spores in aqueous suspensions at wavelengths of 172, 222 and 254 nm was evaluated. A Xe₂* excilamp, a KrCl* excilamp and a low-pressure mercury lamp were used as almost monochromatic light sources at these three wavelengths. The first-order inactivation rate constants at 172, 222 and 254 nm were 0.0023, 0.122 and 0.069 cm² mJ⁻¹, respectively. Therefore, a 2 log reduction of B .  subtilis spores was reached with fluences (UV doses) of 870, 21.6 and 40.4 mJ cm⁻² at these individual wavelengths. Consequently, for the inactivation of B .  subtilis spores, VUV exposure at 172 nm is much less efficient than exposure at the other two wavelengths, while exposure at 222 nm is more efficient than that at 254 nm, which is probably because triplet energy transfer from DPA to thymine bases at 222 nm is higher than that at 254 nm. This research indicated quantitatively that VUV light is not practicable for microorganism disinfection in water and wastewater treatment. However, in comparison with other advanced oxidation processes ( e.g. UV/TiO₂, UV/H₂O₂ or O₃/H₂O₂) the VUV-initiated photolysis of water is likely more efficient in generating hydroxyl radicals and more effective for the inactivation of microorganisms.     

PHOTODYNAMIC THERAPY IN THE TREATMENT OF PANCREATIC CARCINOMA: DIHEMATOPORPHYRIN ETHER UPTAKE and PHOTOBLEACHING KINETICS

Abstract Results of dihematoporphryin ether (DHE) uptake and fluorescence kinetics show that the concentration in the pancreas is on the order of 40-60 μg DHE g⁻¹ of tissue at an injected dose of 40 mg kg⁻¹. Previously concentrations on this order have primarily been found in organs of the reticuloendothelial system. Two intra-pancreatic carcinoma models, one of acinar origin (rat) and one of ductal origin (hamster), were studied. Both showed equal or higher concentrations of DHE as compared with normal pancreas when fluorescence measurements and chemical extraction procedures were performed. Photodynamic therapy (PDT) treatment of the normal pancreas and pancreatic tumors yielded atypical results. When the normal pancreas with DHE present is exposed to 630-nm light from a dye laser (75 mW cm⁻², 30 min), the normal photobleaching measurable by fluorescence decay does not occur. Yet the pancreatic tumor responds with a relatively normal fluorescence decay pattern, with hemorrhaging and a resultant loss of measurable DHE concentration.     

In vitro photodynamic eradication of Pseudomonas aeruginosa in planktonic and biofilm culture

Photodynamic disinfection (PDD) is a nonantibiotic approach to treating drug-resistant bacterial infections. Pseudomonas aeruginosa , an opportunistic pathogen, is problematic because of its propensity to develop antibiotic resistance and its ability to secrete a protective biofilm matrix. This study examined the ability of PDD to eradicate planktonic and biofilm cultures of P. aeruginosa in vitro . Planktonic P. aeruginosa cultures were briefly exposed to a methylene blue-based photosensitizer formulation and subjected to energy doses ranging from 1.7 to 20.6 J cm⁻² using a 670 nm nonthermal diode laser. Biofilms were grown for 24 and 48 h and exposed to photosensitizer for 30 s before illumination with 13.2 or 26.4 J of energy. A single exposure of planktonic P. aeruginosa to photosensitizer at >15.5 J cm⁻² resulted in 100% eradication (>7 log₁₀ reduction from control), an effect that could be decreased significantly in the presence of the singlet oxygen quenchers l -tryptophan and sodium azide. Decreasing the energy dose below this threshold by varying both power density and illumination duration resulted in a dose-dependent decrease in bacterial kill. In addition, 24 h biofilm viability was reduced by 99% with single exposure and 99.9% with double exposure, while 48 h biofilm viability was reduced by >99.999% with both single and double exposures. This study shows that PDD is effective in eradicating planktonic and biofilm cultures of P. aeruginosa, supporting the concept that translation into clinical practice for indications such as otitis externa and wound disinfection is a viable option.