Although the benefits of topical sensitizer administration have been confirmed for photodynamic therapy (PDT), ALA-induced protoporphyrin IX is the only sensitizer clinically used with this administration route. Unfortunately, ALA-PDT results in poor treatment response for thicker lesions. Here, selectivity and depth distribution of the highly potent sensitizer meso-tetra(hydroxyphenyl)chlorin (mTHPC), supplied in a novel liposome formulation was investigated following topical administration for 4 and 6 h in a murine skin tumor model. Extraction data indicated an average [± standard deviation (SD)] mTHPC concentration within lesions of 6.0(±3.1) ng/mg tissue with no significant difference (p<0.05) between 4- and 6-h application times and undetectable levels of generalized photosensitivity. Absorption spectroscopy and chemical extraction both indicated a significant selectivity between lesion and normal surrounding skin at 4 and 6 h, whereas the more sensitive fluorescence imaging setup revealed significant selectivity only for the 4-h application time. Absorption data showed a significant correlation with extraction, whereas the results from the fluorescence imaging setup did not correlate with the other methods. Our results indicate that this sensitizer formulation and administration path could be interesting for topical mTHPC-PDT, decreasing the effects of extended skin photosensitivity associated with systemic mTHPC administration.
Meso-tetra(hydroxyphenyl)chlorin (mTHPC)(international generic name Temoporfin) is a potent photosensitizer used
for photodynamic therapy (PDT). In this study the pharmacokinetics of a systemically administered novel lipid
formulation of Temoporfin in a murine tumor model has been investigated. Fluorescence spectroscopy measurements
were performed at several time intervals following drug administration, yielding information on the Temoporfin
concentration within excised internal organs as a function of time after injection. Both point-monitoring and imaging
setups were used. The acquired fluorescence data were correlated to the concentration of Temoporfin obtained with High
Performance Liquid Chromatography (HPLC). There was a significant correlation between the fluorescence methods and
HPLC for most organs investigated. The pharmacokinetics of this new liposomal formulation of Temoporfin exhibited a
rather flat temporal profile in the time interval 2-8 hours in this study.
Fluorescence molecular tomography (FMT) suffers from inherent ill-posedness due to the vast number of
possible solutions to the reconstruction problem. To increase the robustness of such a problem one need
prior information. We present here a method for rendering a priori information of the position of a
fluorescent inclusion inside turbid media. The method utilizes solely two spectral bands within the
fluorescence spectrum emitted from the fluorophore. The method is presented and verified using
experimental data from a tissue phantom. The confinement is also used to impose weights onto the voxels
before the inversion of the linear set of equations describing the FMT problem.
m-THPC photodynamic therapy has been successfully studied in skin cancer, but no research effort concerning its topical application has been performed until now. Determination of the biodistribution of a special m-THPC thermogel formulation and its tumour selectivity was studied after topical application on hairless SKH-1 mice bearing non-melanoma skin carcinomas. 20 μl/cm2 of m-THPC thermogel (0.5 mg m-THPC/ml) were applied on normal and tumour area and the concentration or demarcation of tumor by mTHPC fluorescence was measured at 2, 4 and 6 hours after drug application by three methods: 1. A fluorescence imaging system capturing images at two emission wavelengths (500 and 654 nm) following 405 nm excitation. Signals from different regions of interest were averaged and the intensity ratio at 654 to 500 was calculated. 2. A fluorescence spectrometer with a fiber bundle for in vivo spectra recording after 420 nm excitation. 3 Each animal was euthanized and the photosensitizer was chemically extracted from liver, spleen, muscle, normal skin and tumour. The photosensitizer concentrations in the extracts and in plasma were determined by fluorescence spectroscopy. The in vivo methods showed a remarkable difference in the concentration of photosensitizer in normal skin and tumour. The highest concentration in tumour was observed 6h after drug application and the highest fluorescence intensity ratio of m-THPC in tumour to normal tissue was observed at 4 hours. Furthermore, no m-THPC was detected in normal tissues or plasma after drug topical application. In vivo and ex vivo results were consistent.
Measurements of concentration of sensitizers for photodynamic therapy can provide important information in the dosimetry planning and can also give input to the optimal time for treatment. There has been skepticism towards fluorescence techniques for this purpose, as the signal depends on the fluorescence yield and optical properties of the
tissue. Absorption based techniques, lack on the other hand, often the sensitivity required for many sensitizers with relative weak absorption in a wavelength region where hemoglobin absorption is dominant. A direct comparison between absorption and fluorescence techniques for measuring mTHPC concentration after topical application on hairless SKH-1 mice bearing skin carcinomas has been performed. 20 μl/cm2 of m-THPC thermogel (0.5 mg m-THPC/ml) were applied on normal and tumor area and the concentration of mTHPC was measured at 4 and 6 hours after drug application by two methods: 1. A fluorescence imaging system capturing images at two wavelengths (500 and 650 nm) following 405 nm excitation. Signals from different regions of interest were averaged and the intensity ratio at 650 to 500 was calculated. 2. A diffuse reflectance spectroscopy system with a fiber separation of 2 mm, providing the absorbance at 652 nm. Both
systems provided consistent results related to the photosensitizer concentration. The methods show a remarkable difference in the concentration of photosensitizer in normal skin and tumor. No significant difference in mTHPC concentration in tumor could be observed between the 4 and 6h groups after drug application.
A way to determine the depth of an embedded fluorescent object, for example deep-lying tumors marked with a fluorescent probe, is to detect fluorescent light that has propagated through the medium at two different wavelength bands. A ratio can then be calculated between the corresponding intensities. The wavelength regions should be chosen such that there is a difference in the absorption in the medium. This spectral information could be used as a complement in other methods, for example in tomography, due to its straightforward implementation. In this study we have performed phantom measurements to determine the depth of a fluorescent object, filled with fluorophores. The transmission of yellow and red fluorescence was measured and a ratio of yellow to red fluorescence was calculated for several depths in tissue with a thickness of 2 cm. The ratio showed a clear dependence on the depth of the object.
We present a novel method for estimating the depth of a fluorescent lesion in tissue based on measurements of the fluorescence signal in different wavelength bands. The measured fluorescence spectrum following irradiation by excitation light at the surface is a function of several parameters, because the fluorescence light has to pass through tissue with characteristic scattering and absorption properties. Thus, the intrinsic fluorescence spectrum will be altered, in a way determined by the tissue optical properties, the depth of the fluorophore, and also by the geometry of the light irradiation and the detection system. By analyzing the ratio between the signals at two wavelengths we show that it is possible to estimate the depth of the lesion. We have performed Monte Carlo simulations and measurements on an Intralipid phantom in the wavelength range 850 - 1000 nm. By taking the ratio between the signals at the wavelengths 875 and 930 nm the depth of a fluorescing layer could be determined with 0.8 mm accuracy down at least a depth of 10 mm. Monte Carlo simulations were also performed for different tissue types with various composition. The results indicate that depth estimation of a lesion is possible with no assumptions made about the optical properties for a wide range of tissues.
During δ-aminolevulinic acid (ALA) based Interstitial Photodynamic Therapy (IPDT) a high light fluence rate is present close to the source fibers. This might induce an unintentional tissue temperature increase of importance for the treatment outcome. In a previous study, we have observed, that the absorption in the tissue increases during the treatment. A system to measure the local tissue temperature at the source fibers during IPDT on tissue phantoms is presented. The temperature was measured by acquiring the fluorescence from small Cr3+-doped crystals attached to the tip of the illumination fiber used in an IPDT-system. The fluorescence of
the Alexandrite crystal used is temperature dependent. A ratio of the intensity of the fluorescence was formed between two different wavelength bands in the red region. The system was calibrated by immersing the fibers in an Intralipid solution placed in a temperature controlled oven. Measurements were then performed by placing the fibers interstitially in a pork chop as a tissue phantom. Measurements were also performed superficially on skin on a volunteer. A treatment was conducted for 10 minutes, and the fluorescence was measured each minute during the illumination. The fluorescence yielded the temperature at the fiber tip through the calibration curve. The measurements indicate a temperature increase of a few degrees during the simulated treatment.
In this project a technique to optically measure the temperature is evaluated. The measurement is to be performed through optical fibres during photodynamic laser treatments or laser thermo therapy of malignant tumours. For this technique Cr3+-doped crystals were used. The lifetime of the ions’ fluorescence were measured, since the fluorescence is strongly temperature dependent. A piece of a crystal was attached to the tip of an optical fibre. The crystal was excited at 635 nm, which is the wavelength most frequently used for photodynamic treatment.
An accuracy in the temperature measurement of ± 0.3 °C was obtained for Cr:LiSAF in the region 20 - 70 °C. This is well within the requirements for this application. Alexandrite and Cr:YAG were also evaluated in this study, also yielding a very good accuracy. A laser treatment was simulated using pork chop as tissue phantom and the temperature was measured.
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