The Validation of a New Vascular Damage Assay for Photodynamic Therapy Agents

Abstract

Abstract— The therapeutic effect of photodynamic therapy (PDT: photodynamic sensitizer + light) is partly due to vascular damage. This report describes a new vascular photodamage assay for PDT agents and a validation of the assay. The method described here quantitates changes in tissue blood perfusion based on the relative amount of injected fluorescein dye in treated and untreated tissues. A specially designed fluorometer uses chopped monochromatic light from an argon laser as a source for exciting fluorescein fluorescence. The fluorescent light emitted from the tissue is collected by a six element fiberoptic array, filtered and delivered to a photodiode detector coupled to a phase‐locked amplifier for conversion to a voltage signal for recording. This arrangement permits a rather simple, inexpensive construction and allows for the simultaneous use of the argon laser by other investigators. The routine assay for characterizing a specific photosensitizer at a standard dose consists of the sequential allocation of eight mice to a set of different light doses designed to span the dose‐response range of fluorescein fluorescence exclusion (measured 8–10 min after fluorescein injection). The assay validation experiment used an anionic photosensitizer, 2‐[l‐hexyloxyethyl]‐2‐devinyl pyropheophorbide‐a at a dose of 0.4 μmol/kg. The parameter estimates (n = 34 mice) from fitting the standard Hill dose‐response model to the data were: median fluorescence exclusion light dose FE50= 275 ± 8.3 J/cm2 and Hill sigmoidicity parameter m =−3.66 ± 0.28. Subsets of the full data set randomly selected to simulate a standard eight mice experiment yielded similar parameter estimates. The new assay provides reliable estimates of PDT vascular damage with a frugal sequential experimental design.

In Vivo Stability and Photodynamic Efficacy of Fluorinated Bacteriopurpurinimides Derived from Bacteriochlorophyll-a

“…Fluorescein-Exclusion Assay for Vascular Stasis. The fluorescein-exclusion assay was used to quantitatively determine PDT-induced vascular damage in normal mouse skin tissue. , As can be seen in Figure , immediately post-PDT, there was no exclusion of the fluorescein dye in the treatment site, noting that the blood vessels were initially compromised and leaky after PDT. At 4 h post-PDT, only partial fluorescein dye exclusion was observed in the treatment site compared to the hind leg (no light).…”

“…At 24 h postinjection, the mice (not inoculated with RIF) underwent PDT as described above. The mice were evaluated for PDT-induced vascular shutdown at various time points post-PDT (1, 4, and 24 h) . At each time point, 3 mice/group were injected iv with 0.20 cm 3 of fluorescein (λ ex = 490 nm, λ em = 520 nm), which has a very short plasma distribution half-life, and the vascular damage was evaluated 2−5 min postinjection.…”

“…The mice were evaluated for PDT-induced vascular shutdown at various time points post-PDT (1, 4, and 24 h). 24 At each time point, 3 mice/ group were injected iv with 0.20 cm 3 of fluorescein (λ ex ) 490 nm, λ em ) 520 nm), which has a very short plasma distribution half-life, and the vascular damage was evaluated 2-5 min postinjection. An in vivo fluorescence probe was utilized to quantitate fluorescein fluorescence in the treatment site (1 cm 2 area) as compared to the hind-leg non light treatment site.…”

The Role of the Peripheral Benzodiazepine Receptor in Photodynamic Activity of Certain Pyropheophorbide Ether Photosensitizers: Albumin Site II as a Surrogate Marker for Activity¶

“…With appropriate filtration, the 520 nm-emitted light was measured by a silicon photodiode and converted to voltage. Details of the method and instrumentation can be found in Bellnier et al (7). Vascular perfusion was expressed as the average fluorescence intensity (arbitrary units) within the treated areas compared with that in untreated areas for each animal.…”

Photodynamic Therapy: Critical PDT Theory†

“…PDT had been periodically used for cancer control since 1960 (12) but it was not until 1991 that Oleinick’s group identified apoptosis as a major death pathway initiated by photodamage (13). Vascular consequences have also been reported (14,15) as are immunologic effects (16). Autophagy has been identified as a cytoprotective response (17,18).…”