KEYWORDS: Luminescence, Oxygen, Skin, Photodynamic therapy, In vivo imaging, In vitro testing, Spectroscopy, Tissue optics, Signal detection, Microscopes
We have applied a spectroscopic system capable of monitoring the fluorescence dynamics of photosensitiser at micron-scale locations within individual cells. This report shows that the accumulation of protoporphyrin IX (PpIX) within the nucleus of formalin-fixed keratinocytes, fibroblasts, and a metastatic squamous carcinoma cell line, following incubation with 5-aminolaevulinic acid (ALA), is dependent upon both incubation time and cell proliferation status. We
demonstrate that the process of photobleaching can be monitored via the depletion in PpIX fluorescence emission during exposure to 532 nm laser light. All spectra show a progressive reduction of the 634 nm PpIX peak - following a bi-exponential decay which is consistent with a singlet oxygen mediated process. The rate of photobleaching, when plotted as a function of light dose, increases with reduced incident laser power. The generation of the hydroxyaldehyde-chlorin photoproduct, as monitored by the increase in fluorescence emission centred on 672 nm, is also greatest when the lowest laser power is applied. When light is delivered in two fractions, there is evidence of PpIX fluorescence recovery during the dark period, and an increase in bleaching rate at the onset of the second exposure. These results are in qualitative agreement with measurements performed in vivo which demonstrate that the photodynamic dose is dependent upon fluence-rate and oxygen status.
Near-infrared Raman microscopy was used to study normal, adenomatous and carcinomatous colon tissue in vitro. Samples were fixed in formalin and it was shown that this procedure did not alter the observed Raman peaks of the tissue, and is therefore a suitable method of fixation. The samples were observed visually to be of varying shades ranging from cream to dark brown; this was independent of whether the samples were cancerous or normal. Differences were seen between the Raman spectra of the different colored tissue with the spectra of brown tissue displaying a greater number of resolved peaks. We believe these differences may be due to differing amounts of blood present in the tissue. This must be considered when using Raman spectra for diagnostic work to ensure that differences ascribed to cancerous changes are not in fact due to differing amounts of blood within the sample.
We have applied Raman spectroscopy to the study of the distribution within cells of photosensitizing compounds designed for use in photodynamic therapy. A human endothelial hybridoma cell-line was incubated in the presence of substituted zinc phthalocyanines, with the cells subsequently fixed in formalin. Microscopic Raman analysis was performed using 782 nm excitation in order to avoid photosensitizer absorption, thereby minimizing both the background fluorescence signal and the generation of cytotoxic species. A method of Raman mapping is described and we demonstrate that it is possible to identify the distribution of photosensitizer within the cell. The results indicate that sub-cellular photosensitizer localization is dependent upon both molecular structure and incubation time.
There is a widely recognized need for new approaches to effect endometrial ablation as an alternative to hysterectomy for treatment of menorrhagia. Photodynamic therapy (PDT) offers one such approach. We have investigated the use of 5-aminolaevulinic acid (ALA)-based PDT of the endometrium in model systems and in a series of patients. In all of this work, the ALA was administrated directly into the uterine cavity to reduce any possibility of systemic photosensitization. In a series of experiments in perfused ex vivo uteri, ALA was introduced into the cavity and protoporphyrin formation was measured in the endometrium, the underlying myometrium and the perfusate. ALA transfer into the perfusate was also measured. This work demonstrated that protoporphyrin formation in the endometrium was approximately ten fold that in the underlying myometrium and that systemic photosensitization would be unlikely to result form transfer of administered ALA from the uterus into the circulation. Similar results were found in studies carried out in vivo, where ALA was administered to patients scheduled for hysterectomy. Using a specially designed light source, the first patients have now been treated by giving intrauterine ALA followed by laser light. Two series of treatments - 10 in all - have been carried out. Eight patients have one year follow up. A reduction in measured menstrual blood loss was demonstrated in all but one patient. Complete symptomatic relief was obtained in tow women who did not require further treatment.
Tissue factor (TF) is not expressed by endothelial cells unless they have ben perturbed. Identifying the extrinsic pathway of coagulation as the process involved in clot formation demonstrated that PDT induces release of TF by endothelial cells. EAhy 926 cells were incubated with 10 (mu) g ml-1 of photosensitizer for 24 h prior to illumination at 40 J cm-2. A clotting assay was then performed. PDT caused a significant reduction in clotting time. This was repeated using factor VII- or X-deficient serum. In factor VII-deficient serum there was no difference between treated and untreated cells. There was no clot formation with any cells in the presence of factor x- deficient serum. The data indicate that PDT-induced procoagulation activity is via the extrinsic pathway of coagulation and results from direct initiation of endothelial cells to release tissue factor.
Objective:
To assess the efficacy of PDT to:
Palliate symptoms, control disease and extend survival in patients
with advanced inoperable cancer.
Subject and Method:
55 Males and 23 females aged between 45-81 years (mean 66 years)
with inoperable and advanced lung cancer with > 5O. obstructive
lesions of the main, lobar or segmental bronchi. Patients had
pre-treatment routine clinical radiological, functional and
endoscopic assessment with proven histological diagnosis.
Protocol of PDT was; Intravenous injection of 2 mg/Kg bodyweight
Polyhaematoporphyrin (equivalent to Photofrin) or Photofrin
followed 24-72 hours later by illumination of tumour using 630 nm
light (Oxford Laser) delivered via an optical fibre with end
diffuser. Treatments were carried out under general anaesthesia
as a day case procedure. Patients were rebronchoscoped for
debridement/retreatment 4-7 days later.
Results:
There was no treatment related mortality. Two patients developed
mild photosensitivity reaction. All patients showed symptomatic
improvement with good initial functional and radiological
amelioration. Every patient responded to treatment. Seven
patients had complete response and negative histology for 3-12
months. After the first treatment average Forced Vital Capacity
(FVC) and Forced Expiratory Volume in one second (FEV1)
improvement was 0.5 litres and 0.4 litres respectively. Twenty
five percent of patients (nr 19) survived more than 2 years, 10'.
(nr=8) between 1-2 years and the remaining 51 patients less than a
year.
Conclusion:
PDT should be considered as a therapeutic modality for all stages
of lung cancer and is an excellent treatment modality for
palliation in advanced bronchial malignancies.
The EAhy 926 cell is a hybridoma line derived from human endothelium and A549/8 cells. They display stable endothelial characteristics and may provide an indication of how endothelial cells respond to photodynamic therapy. Cells were grown as monolayers, seeded at a density of 104 cells/35 mm dish, and then incubated with zinc (II) tetrasubstituted phthalocyanines (carboxylated, sulphonated, pyridinium or diethanolamine sulphonamide). After 24 hours, the cells were illuminated with laser light at 680 nm or 692 nm as appropriate. The response to each photosensitizer was evaluated using cell proliferation, clonogenicity, and release of tissue factor.
We have used ultra-low light level fluorescence microscopy to examine the suggestion that the relatively poor response of human basal cell carcinomas (BCC) to topical 5-aminolaevulinic acid (ALA)-based photodynamic therapy (PDT) arises from limited drug penetration into the lesion. The distribution of ALA-induced protoporphyrin IX (PpIX) in human BCC and Bowen's disease was examined and, in almost all cases, was found to be most intense in those regions of tumor immediately adjacent to the dermis. This distribution was independent of tumor type, and did not appear to be affected by tumor depth in the skin. It is suggested that ALA penetration may not limit the efficacy of ALA-PDT in the treatment of BCC. Failure of superficial ALA-based PDT in basal cell carcinoma may, instead, be related to the histological structure of this type of lesion.
Aminolaevulinic acid (ALA) induced porphyrin biosynthesis and the resulting in vitro phototoxicity have been determined in both SV40 transformed Swiss mouse 3T3 fibroblasts and PtK2 epithelial cells. Both cell lines respond to the addition of exogenous ALA, producing porphyrin linearly with ALA concentrations up to 0.3 mM. Notably the only accumulating porphyrin detected by HPLC was PpIX. Although the levels of PpIX are both dependent on the time and concentration used, the final intracellular porphyrin concentration is dictated by the presence of serum. When ALA is added in medium containing 10% new born calf serum, 90 - 95% of the induced porphyrin appears in the incubation medium. In the absence of serum, the intracellular PpIX levels are maintained and only under these conditions can successful in vitro PDT be performed. Gel permeation chromatography has indicated that the afflux of PpIX is promoted by the low density and high density lipoproteins, with an unknown protein (mw < 66000) contributing significantly to the effect seen. It appears that this protein is present at very low concentrations in both foetal and new born calf serum.
At present lasers are chosen in photodynamic therapy because they produce light of superior beam and spectral qualities and of more penetrating longer wavelengths. However there is stronger absorption by porphyrins at less penetrating shorter wavelengths. The response of a rat intradermal fibrosarcoma to topical 5-aminolaevulinic acid photodynamic therapy was compared using 630 nm light from a copper vapor/due laser or broad spectrum red or white light from a modified slide projector. When the tumors were between 2 - 3 mm thick they were randomized to treatment with one light source at a range of fluences all at the same sub- hyperthermic fluence rate. Laser light and white light produced a greater tumor growth delay than broad spectrum red light, and at higher fluences white light was comparable to laser light. For the treatment of thin, superficial tumors using topical aminolaevulinic acid photodynamic therapy an inexpensive, portable, white light source may be an effective alternative to a laser.
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