Abstract
A fluorometric analytical method was developed for quantification of protoporphyrin IX (PpIX) in skin samples and receptor phase solution after in vitro cutaneous penetration/permeation studies. Analytical conditions used were: excitation and emission wavelengths: 400 nm and 632 nm; bandwidth: 0.5 nm; excitation and emission slits: 10/10. PpIX was recovered from two different layers of skin, the stratum corneum (SC) and the epidermis plus dermis ([E+D]), by vortex homogenization, probe and bath sonication, using DMSO as an extraction solvent. The detection and quantification limits were 0.002 and 0.005 μg/mL, respectively. The assay was linear from 0.005 - 0.5 μg/mL. The within-day and between-day assay precision and accuracy in DMSO and receptor phase solution were each studied at the two concentration levels 0.04 and 0.2 μg/mL, and 0.01 and 0.08 μg/mL, respectively. The coefficients of variation and deviation from the theoretical values were lower than 5%. The skin recovery of PpIX from SC and [E+D] layers using two different concentrations (0.5 and 1.0 μg/mL) were all above 90.0%. The method described has potential application to in vitro penetration/permeation studies of PpIX using porcine skin as a biological membrane model.
Highlights
Fluorescence spectroscopy has gained increasing importance in recent years, especially in molecular and cellular biology, and in many associated areas of biochemical analysis, because it offers many advantages such as extreme sensitivity, a high degree of selectivity, and exceptional flexibility in measuring a range of solid and liquid sample formats (Miller, 2006).Fluorescence detection of a molecule capable of emitting fluorescence efficiently, called a photosensitizer (PS), is an analytical and sensitive method used to quantify the amount of PS in cells or tissues, and allows in vivo fluorescence imaging in living animals or patients to measure the pharmacokinetics and distribution of the PS (Castano, Demidova, Hamblin, 2004)
The study of the use of protoporphyrin IX (PpIX), an endogenous porphyrin, as a PS is promising because it has fast degradation in normal tissue compared to abnormal tissue, which leads to a short period of sensitization thereby reducing the collateral effects of the photodynamic therapy (Kennedy, Pottier, Pross, 1990)
We evaluated several parameters to demonstrate that the analytical procedure was suitable for analyzing PpIX in skin and receptor phase samples from in vitro skin penetration studies
Summary
Fluorescence detection of a molecule capable of emitting fluorescence efficiently, called a photosensitizer (PS), is an analytical and sensitive method used to quantify the amount of PS in cells or tissues, and allows in vivo fluorescence imaging in living animals or patients to measure the pharmacokinetics and distribution of the PS (Castano, Demidova, Hamblin, 2004). PSs are currently been used to treat different types of cancer, including non-melanoma skin cancers in a therapy called photodynamic therapy, that produces highly reactive lethal substances for tumorous cells through the combination of light, PS and oxygen (Brown, Brown, Walker, 2004; Zeitouni, Oseroff, Shieff, 2003). Its topical administration in the treatment of non-melanoma skin cancer in comparison to systemic application can be more efficient, since higher local concentrations in the cancerous tissue can be obtained (Rick et al, 1997). The main method to study the drug release performance into/through the skin is the in vitro test performed in a Franz Diffusion Cell using pig’s ear skin as a biological model for skin (Schmook, Meingassner, Billich, 2001)
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