Willem M. Star

IN VIVO FLUORESCENCE SPECTROSCOPY AND IMAGING FOR ONCOLOGICAL APPLICATIONS

Keywords: Photomedicine group Reference LPAS-ARTICLE-1998-003View record in Web of Science Record created on 2007-07-20, modified on 2016-08-08

Fluorescence Photobleaching of ALA‐induced Protoporphyrin IX during Photodynamic Therapy of Normal Hairless Mouse Skin: The Effect of Light Dose and Irradiance and the Resulting Biological Effect

The photobleaching of 5‐aminolaevulinic acid (ALA)‐induced protoporphyrin IX (PpIX) was investigated during superficial photodynamic therapy (PDT) in normal skin of the SKH HRt hairless mouse. The effects of light dose and fluence rate on the dynamics and magnitude of photobleaching and on the corresponding PDT‐induced dam‐age were examined. The results show that the PDT damage cannot be predicted by the total light dose. Photo‐bleaching was monitored over a wide range of initial PpIX fluorescence intensities. The rate of PpIX photo‐bleaching is not a simple function of fluence rate but is dependent on the initial concentration of sensitizer. Also, at high fluence rates (50–150 mW/cm2, 514 nm) oxygen depletion is shown to have a significant effect. The rate of photobleaching with respect to light dose and the corresponding PDT damage both increase with decreasing fluence rate. We therefore suggest that the definition of a bleaching dose as the light dose that causes a 1/e reduction in fluorescence signal is insufficient to describe the dynamics of photobleaching and PDT‐induced dam‐age. We have detected the formation of PpIX photoproducts during the initial period of irradiation that were themselves subsequently photobleached. In the absence of oxygen, PpIX and its photoproducts are not photo‐bleached. We present a method of calculating a therapeutic dose delivered during superficial PDT that demonstrates a strong correlation with PDT damage.

Optical diffusion in layered media

In highly scattering media, light energy fluence rate distributions can be described by diffusion theory. Boundary conditions, appropriate to the diffusion approximation, are derived for surfaces where reflection of diffuse light occurs. Both outer surfaces and interfaces separating media with different indices of refraction can be treated. The diffusion equation together with its boundary conditions is solved using the finite element method. This numerical method allows much freedom of geometry.

In vivo fluorescence kinetics and photodynamic therapy using 5-aminolaevulinic acid-induced porphyrin: increased damage after multiple irradiations

The kinetics of fluorescence in tumour (TT) and subcutaneous tissue (ST) and the vascular effects of photodynamic therapy (PDT) were studied using protoporphyrin IX (PpIX), endogenously generated after i.v. administration of 100 and 200 mg kg-1 5-aminolaevulinic acid (ALA). The experimental model was a rat skinfold observation chamber containing a thin layer of ST in which a small syngeneic mammary tumour grows in a sheet-like fashion. Maximum TT and ST fluorescence following 200 mg kg-1 ALA was twice the value after 100 mg kg-1 ALA, but the initial increase with time was the same for the two doses in both TT and ST. The fluorescence increase in ST was slower and the maximum fluorescence was less and appeared later than in TT. Photodynamic therapy was applied using green argon laser light (514.5 nm, 100 J cm-2). Three groups received a single light treatment at different intervals after administration of 100 or 200 mg kg-1 ALA. In these groups no correlation was found between the fluorescence intensities and the vascular damage following PDT. A fourth group was treated twice and before the second light treatment some fluorescence had reappeared after photobleaching due to the first treatment. Only with the double light treatment was lasting TT necrosis achieved, and for the first time with any photosensitiser in this model this was accomplished without complete ST necrosis.

Topical Application of 5‐Aminolevulinic Acid Hexyl Ester and 5‐Aminolevulinic Acid to Normal Nude Mouse Skin: Differences in Protoporphyrin IX Fluorescence Kinetics and the Role of the Stratum Corneum¶

Abstract An important limitation of topical 5-aminolevulinic acid (ALA)-based photodetection and photodynamic therapy is that the amount of the fluorescing and photosensitizing product protoporphyrin IX (PpIX) formed is limited. The reason for this is probably the limited diffusion of ALA through the stratum corneum. A solution to this problem might be found in the use of ALA derivatives, as these compounds are more lipophilic and therefore might have better penetration properties than ALA itself. Previous studies have shown that ALA hexyl ester (ALAHE) is more successful than ALA for photodetection of early (pre)malignant lesions in the bladder. However, ALA pentyl ester slightly increased the in vivo PpIX fluorescence in early (pre)malignant lesions in hairless mouse skin compared to ALA. The increased PpIX fluorescence is located in the stratum corneum and not in the dysplastic epidermal layer. In the present study, ALA- and ALAHE-induced PpIX fluorescence kinetics are compared in the normal nude mouse skin, of which the permeability properties differ from the bladder. Application times and ALA(HE) concentrations were varied, the effect of a penetration enhancer and the effect of tape stripping the skin before or after application were investigated. Only during application for 24 h, did ALAHE induce slightly more PpIX fluorescence than ALA. After application times ranging from 1 to 60 min, ALA-induced PpIX fluorescence was higher than ALAHE-induced PpIX fluorescence. ALA also induced higher PpIX production than ALAHE after 10 min of application with concentrations ranging from 0.5 to 40%. The results of experiments with the penetration enhancer and tape stripping indicated that the stratum corneum acts a barrier against ALA and ALAHE. Use of penetration enhancer or tape stripping enhanced the PpIX production more in the case of ALAHE application than in the case of ALA application. This, together with the results from the different application times and concentrations indicates that ALAHE diffuses more slowly across the stratum corneum than ALA.

What does photodynamic therapy have to offer radiation oncologists (or their cancer patients)

Major advances have recently been made in photodynamic therapy (PDT) for clinical application, including the development of more powerful photosensitizers and light sources and suitable light applicators. PDT is emerging as an attractive new form of cancer therapy, suitable for treating superficial lesions (less than 1 cm in depth) and carcinoma in situ, or as an adjuvant to surgery for more bulky disease. PDT is therefore complementary to radiotherapy which is better suited to treating larger tumours. There are some qualitative similarities between light distribution in tissue during superficial illumination and ionizing radiation dose distributions during external beam irradiation, or between interstitial PDT and brachytherapy, although the geometric scale is very different (visible light penetrates a maximum of 5-10 mm in tissue). The contribution of scattered light to tissue irradiance is much greater than for ionizing radiation and in situ light dosimetry is very important (although rather complicated) to ensure adequate illumination without over-treating. Dosimetry and treatment planning are highly advanced for ionizing radiation and are routine in all radiotherapy departments. Proper in situ light dosimetry and dose distribution calculation for PDT is in its infancy. Physicists have an important role to play in the further optimization of clinical PDT and much of the infrastructure and expertise present in the radiotherapy department is ideally suited to accommodate PDT. In this review, parallels and contrasts are made between PDT and ionizing radiation for both mechanistic and dosimetric aspects of the therapies. A summary of the most interesting clinical applications is also given.

Protoporphyrin IX Fluorescence Photobleaching during ALA-Mediated Photodynamic Therapy of UVB-Induced Tumors in Hairless Mouse Skin

Abstract— Fluorescence photobleaching of protoporphyrin IX (PpIX) during superficial photodynamic therapy (PDT), using 514 nm excitation, was studied in UVB‐induced tumor tissue in the SKH‐HR1 hairless mouse. The effects of different irradiance and light fractionation regimes upon the kinetics of photobleaching and the PDT‐induced damage were examined. Results show that the rate of PpIX photobleaching (i.e. fluorescence intensity vs fluence) and the PDT damage both increase with decreasing irradiance. We have also detected the formation of fluorescent PpIX photoproducts in the tumor during PDT, although the quantity recorded is not significantly greater than generated in normal mouse skin, using the same light regime. The subsequent photobleaching of the photoproducts also occurs at a rate (vs fluence) that increases with decreasing irradiance. In the case of light fractionation, the rate of photobleaching increases upon renewed exposure after the dark period, and there is a corresponding increase in PDT damage although this increase is smaller than that observed with decreasing irradiance. The effect of fractionation is greater in UVB‐induced tumor tissue than in normal tissue and the damage is enhanced when fractionation occurs at earlier time points. We observed a variation in the distribution of PDT damage over the irradiated area of the tumor: at high irradiance a ring of damage was observed around the periphery. The distribution of PDT damage became more homogeneous with both lower irradiance and the use of light fractionation. The therapeutic dose delivered during PDT, calculated from an analysis of the fluorescence photobleaching rate, shows a strong correlation with the damage induced in normal skin, with and without fractionation. The same correlation could be made with the data obtained from UVB‐induced tumor tissue using a single light exposure. However, there was no such correlation when fractionation schemes were employed upon the tumor tissue.

LIGHT DOSIMETRY FOR PHOTODYNAMIC THERAPY BY WHOLE BLADDER WALL IRRADIATION

Abstract In Photodynamic Therapy (PDT) there is a need for accurate quantitative light dosimetry. This has become particularly apparent in the treatment of superficial bladder cancer, either by focal or by whole bladder wall irradiation. We have studied this problem using a spherical model of the bladder, consisting of two concentric thin‐walled plastic spheres, 8 and 10 cm in diameter. The inner sphere was filled with water or with a light‐scattering medium. The space between the spheres was filled with an optically tissue equivalent liquid. An isotropic light source was placed at the center of the spheres. Light energy fluence rates (light “dose rates”) during PDT of the bladder simulated in this manner, were measured using a specially developed miniature light detector and were also calculated using a mathematical model. These data were confirmed by measurements in vivo (dog bladder). In the case of focal irradiation at a wavelength of 630 nm, the ratio (R) between the true light fluence rate at the bladder surface and the fluence rate due to the primary light beam is somewhat larger than 1, depending on the diameter of the primary beam. The maximum ratio is 2, for a beam diameter of several centimeters. In the case of whole bladder wall PDT, R is larger than 5. This is due to the strong scattering of (red) light by tissue and indicates that the intensity of the primary beam, which is usually reported, is not a good measure of the true fluence rate for whole bladder wall PDT. When the light source is moved away from the center of the spheres, the rate of change of the fluence rate at the bladder wall is more than a factor of 2 larger when the bladder cavity is filled with a light‐scattering suspension, as compared with water. The use of a light‐scattering medium may therefore not be advantageous to achieve a uniform light distribution across the bladder wall.

Diffusion Theory of Light Transport

In this chapter light is principally described as particles with energy hν and velocity c. These particles are scattered or absorbed by structures in turbid media such as biological tissues and are reflected at boundaries between media with different refractive index, according to the laws of Fresnel. We will only consider monochromatic light, which covers most practical situations in medical and biological laser applications. The formulations are easily extended to polychromatic light as long as scattering effects are elastic. The theory becomes more complicated for inelastic scattering, like in fluorescence. However, even then the diffusion theory is a straightforward extension of the discussions presented in the following sections. Finally, we neglect polarization and interference. To include polarization one would need four diffusion equations instead of one [1].

Topical 5-Aminolevulinic Acid-photodynamic Therapy of Hairless Mouse Skin Using Two-fold Illumination Schemes: PpIX Fluorescence Kinetics, Photobleaching and Biological Effect†¶

Abstract Light fractionation with dark periods of the order of hours has been shown to considerably increase the efficacy of 5-aminolevulinic acid-photodynamic therapy (ALA-PDT). Recent investigations have suggested that this increase may be due to the resynthesis of protoporphyrin IX (PpIX) during the dark period following the first illumination that is then utilized in the second light fraction. We have investigated the kinetics of PpIX fluorescence and PDT-induced damage during PDT in the normal skin of the SKH1 HR hairless mouse. A single illumination (514 nm), with light fluences of 5, 10 and 50 J cm−2 was performed 4 h after the application of 20% ALA, to determine the effect of PDT on the synthesis of PpIX. Results show that the kinetics of PpIX fluorescence after illumination are dependent on the fluence delivered; the resynthesis of PpIX is progressively inhibited following fluences above 10 J cm−2. In order to determine the influence of the PpIX fluorescence intensity at the time of the second illumination on the visual skin damage, 5 + 95 and 50 + 50 J cm−2 (when significantly less PpIX fluorescence is present before the second illumination), were delivered with a dark interval of 2 h between light fractions. Each scheme was compared to illumination with 100 J cm−2 in a single fraction delivered 4 or 6 h after the application of ALA. As we have shown previously greater skin damage results when an equal light fluence is delivered in two fractions. However, significantly more damage results when 5 J cm−2 is delivered in the first light fraction. Also, delivering 5 J cm−2 at 5 mW cm−2 + 95 J cm−2 at 50 mW cm−2 results in a reduction in visual skin damage from that obtained with 5 + 95 J cm−2 at 50 mW cm−2. A similar reduction in damage is observed if 5 + 45 J cm−2 are delivered at 50 mW cm−2. PpIX photoproducts are formed during illumination and subsequently photobleached. PpIX photoproducts do not dissipate in the 2 h dark interval between illuminations.