Janet E. Cruse-Sawyer

Proinflammatory cytokine production by human keratinocytes stimulated with Propionibacterium acnes and P. acnes GroEL.

Background  Keratinocytes form the first line of defence in the skin and alert the host to danger by the production of a number of cytokines and chemokines. However, the interaction of commensal microorganisms with keratinocytes has not been well studied.

On the photodynamic therapy action spectrum of zinc phthalocyanine tetrasulphonic acid in vivo

The photodynamic therapy (PDT) activity of zinc phthalocyanine tetrasulphonic acid in a rodent tumour model was shown to be critically dependent on the wavelength of the excitation laser light over a relatively small wavelength range. Thus the sensitizer showed a doubling of the PDT activity with fibrosarcoma LSBD1 in BDIX rats when the wavelength of the illuminant was displaced from 680 to 692 nm. Under these conditions, the sensitizer is approximately three times more effective than polyhaematoporphyrin, whereas previously it has been considered to be of low PDT activity. This wavelength effect is attributed to a red shift of the absorption spectrum of the sensitizer in cells compared with that in solution. Fluorescence excitation studies with sensitizer absorbed in mouse 3T3 fibroblast cells are consistent with such a red shift.

Biodistribution of a methylene blue derivative in tumor and normal tissues of rats

By using a chemical extraction procedure and confocal laser scanning fluorescence microscopy we have investigated the kinetic patterns of uptake and biolocalization of a methylene blue derivative (MBD) in tumors and various normal tissues of Wistar rats bearing fibrosarcoma (Leeds ovarian tumor) after intravenous injection of MBD (10 mg kg-1 body weight). Similar kinetics of accumulation and elimination of MBD fluorescence were found in tumor tissue and surrounding normal skin and muscle tissues. However, the tumor:skin and tumor:muscle ratios of the MBD fluorescence intensity were found to be 9 and 4, respectively, 4 h after intravenous injection, indicating selective uptake of MBD by the tumor tissue. MBD was localized on the walls of all the vessels and extensively in the area of neoplastic cellular and tumorigenic fibrous components in the tumor tissue. Interestingly, no MBD fluorescence could be detected in the metastatic neoplastic cells in the remote lymph nodes. In the skin, MBD was mainly distributed in the keratinized epithelium of the epidermis, hair follicles and their accessories, while little was found both in the epidermis and dermis. In most other tissues, the maximal fluorescence intensity of MBD was found 1-4 h after injection, after which it decreased dramatically to almost undetectable levels 120 h postinjection. Strong fluorescence of MBD was seen in the tracheal mucosal epithelium, while little fluorescence was noted in the transitional epithelium of bladder. The kinetics of biolocalization of MBD in some other tissues (liver, spleen, kidney, brain, muscle, lung, heart) were also studied.

The influence of intracellular mTHPC concentration upon photobleaching dynamics

In this study, we report the effect of the local photosensitizer concentration upon the dynamics of the singlet oxygen-mediated photobleaching, within formalin-fixed keratinocytes. Although the cells were incubated at a single mTHPC dose, cell-to-cell variations in concentration were defined within the perinuclear region by differences in the initial amplitude of the laser-induced fluorescence emission, located around 652nm. At a fixed laser fluence-rate, it was found that the photobleaching, when plotted as a function of delivered light dose, proceeded more rapidly at higher drug concentration. The mTHPC spectral emission profile is shown to be approximately Lorentzian and remains unchanged as the photobleaching proceeds. This indicates that there is no perturbation of the detected signal due to the inner-filter effect.

Investigation of the Subcellular Localization of Zinc Phthalocyanines by Raman Mapping

Raman mapping has been used to determine the subcellular distribution of two substituted zinc phthalocyanines designed for use as photosensitizers in photodynamic therapy. Each compound was incubated over a range of time periods in two cultured cell lines, and the cells were subsequently formalin-fixed for analysis. Raman spectra were recorded at 1–2 μm steps across the cell, with the use of 782 nm laser excitation in order to minimize absorption by the photosensitizer chromophore and the consequent photodynamic activation of the system. Maps have been formed showing the distribution of the phthalocyanines within the cells. This distribution is seen to depend upon incubation time and the molecular structure of the phthalocyanine. Similar patterns of uptake are observed in both cell lines. Atomic force microscopy has been used to validate the technique used to correct the maps for variations in Raman sampling volume, resulting from inhomogenous cell thickness.

Fluence-rate effects upon m-THPC photobleaching in a formalin-fixed cell system

We have applied a micro-spectroscopic technique in order to record the laser-induced fluorescence emission of the PDT photosensitiser m-THPC (Foscan) from micron-scale locations within individual formalin-fixed keratinocytes. We demonstrate that m-THPC is highly photolabile in this cellular environment, and that the process of photobleaching can be monitored via the depletion in fluorescence emission during continuous irradiation with 410nm laser light. The progressive reduction of the characteristic 652nm m-THPC fluorescence peak can be described with bi-exponential decay kinetics, consistent with a singlet oxygen-mediated process. The rate of photobleaching, when plotted as a function of light dose, shows inverse fluence-rate dependence. Specifically, the rate of photobleaching induced by the higher laser powers appears to be limited by oxygen availability, as demonstrated by an increase in the (1/e) bleaching dose. Fractionated irradiation provides evidence of intracellular re-oxygenation. These results are in qualitative agreement with previous in vitro and in vivo studies, which indicate that the photodynamic dose delivered during light irradiation is critically dependent upon local fluence rate and oxygen partial pressure.

The photodynamic response of two rodent tumour models to four zinc (II)-substituted phthalocyanines

Four novel zinc (II)-substituted phthalocyanines, varying in charge and hydrophobicity, were evaluated in vivo as new photosensitizers for photodynamic therapy. Two rat tumours with differing vascularity were used: a mammary carcinoma (LMC1) and a fibrosarcoma (LSBD1), with vascular components six times higher in the latter (10.8%+/-1.5) than in the former (1.8%+/-1.4). Each sensitizer was assessed for tumour response relative to normal tissue damage, and optimum doses were selected for further study, ranging from 0.5 to 20 mg kg(-1). Interstitial illumination of the tumours was carried out using a 200-microm-core optical fibre with a 0.5 cm length of diffusing tip, at either 680 or 692 nm, depending on the sensitizer. Light doses of between 200 and 600 J were delivered at a rate of 100 mW from the 0.5-cm diffusing section of the fibre. Maximum mean growth delays ranged from 9 to 13.5 days depending on sensitizer and type of tumour, with the most potent photosensitizer appearing to be the cationic compound. Histopathological changes were investigated after treatment to determine the mechanism by which tumour necrosis was effected. The tumours had the appearance of an infarct and, under the conditions used, the observed damage was shown to be mainly due to ischaemic processes, although some direct tumour cell damage could not be ruled out.

Fluorescence microspectroscopy technique for the study of intracellular protoporphyrin IX dynamics.

We describe a technique designed to monitor the fluorescence dynamics of photosensitizers used in photodynamic therapy (PDT) at micrometer-scale locations within individual formalin-fixed cells. The accumulation of protoporphyrin IX (PpIX) within keratinocytes and fibroblasts, following incubation with 5-aminolaevulinic acid (ALA), is shown to be dependent upon both incubation time and cell proliferation status. Also, the process of photobleaching within these cells is demonstrated via the depletion in PpIX fluorescence emission during exposure to 532 nm light. All spectra show a progressive reduction of the 634 nm PpIX peak, following a biexponential decay that 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 (photoprotoporphyrin), as monitored by the increase in fluorescence emission centered on 672 nm, is also greatest when the lowest laser power is applied. When light is delivered in two fractions, PpIX fluorescence recovers during the dark period and there is an increase in bleaching rate at the onset of the second exposure. These results are qualitatively consistent with measurements performed in vivo, which demonstrate that the photodynamic dose is dependent upon fluence rate and oxygen status.

Mapping the distribution of zinc phthalocyanine derivatives in EAhy 926 cells using Raman microscopy

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.

Tunable laser-based illumination system for cells in monolayer

A system of testing of potential photosensitizing agents for PDT by the illumination of cells in monolayer is described. The system allows tunability of the excitation wavelength, uniform illumination over a 35 mm cell dish, variation of the irradiance and monitoring of the power delivered throughout the duration of illumination. Knowledge of these parameters allows rapid processing, precise light dosimetry, limitation of possible heating effects as well as optimization of the excitation efficiency. The system is applied to the illumination of an endothelial hybridoma cell line using 680 nm and 692 nm light in the presence of phthalocyanine sensitizers.