Priscila Fernanda Campos de Menezes

Can efficiency of the photosensitizer be predicted by its photostability in solution

We have investigated a possible correlation between the photostability and photodynamic efficacy for different photosensitizers; hematoporphyrin derivatives and chlorines. To perform such analysis, we combined the depth of necrosis (dnec) measurement, expressed by the light threshold dose and a photodegradation parameter, measured from investigation of photosensitizer degradation in solution. The dnec analysis allows us to determine the light threshold dose and compare its value with the existent results in the literature. The use of simple models to understand basic features of Photodynamic Therapy (PDT) may contribute to the solid establishment of dosimetry in PDT, enhancing its use in the clinical management of cancers and others lesions. Using hematoporphyrin derivatives and chlorines photosensitizers we investigated their properties related to the photodegradation in solution and the light threshold dose (Dth) in rat livers.

Correlation of cytotoxicity and depth of necrosis of the photoproducts of photogem

Photodynamic therapy (PDT) is an approved modality for cancer treatment, which involves the administration of a photosensitive drug (PS) that is selectively accumulated in neoplastic tissues and their vasculature and subsequently can be activated with light at the appropriate wavelength to generate reactive molecular species that are toxic to tissues. In PDT, a great part of the used PS suffers degradation by light (photobleaching) that involves a decrease in the absorption and intensity of fluorescence of the photosensitizer as well as photoproduct formation evidenced by the appearance of a new absorption band. In this study, we investigated the correlation of cytotoxicity and depth of necrosis of Photogem and its photoproducts obtained previously by irradiation at 514 and 630 nm. The cytotoxicity for degraded Photogem decreases with the previous irradiation time of Photogem solution suggesting that the photoproducts of Photogem are less cytotoxics than the original formulation. A transition between the necrosed epithelium and healthy epithelium of normal liver of rats after irradiation at 630 nm was observed with irradiated and nonirradiated PS. It is observed that the depth of necrosis only at irradiation dose of 150 J/cm2 in both concentrations is greater for Photogem followed by Photogem degradated previously at 514 and then at 630 nm. The results obtained suggest that the threshold of necrosis values is lower for Photogem followed by its photoproducts formed, suggesting that the photoproducts present a low photodynamic activity. If the photosensitizer degradation happens at the same time as tumor destruction, the drug degradation can be complete before reaching the threshold of necrosis; then it is very important to control the drug concentration and light intensity of irradiation during PDT.

Photodynamic activity of different dyes

Photodynamic therapy (PDT) is a technique for inducing tissue damage with light irradiation of a drug selectively retained in malignant tissue. Many kinds of compounds are known with photosensitizing properties including dyes, drugs, cosmetics, chemicals, and many natural substances. There are different classes of sensitizers used for medical purposes such as tetrapyrroles (porphyrins and derivatives, chlorophyll, phylloerythrin, phthalocyanines), tricyclic dyes with different meso-atoms (acridine orange, proflavine, riboflavine, methylene blue, fluorescein, eosine, erythrosine, rose bengal), and furocoumarins (psoralen and its methoxyderivatives xanthotoxin, bergaptene). In this work, we performed one comparative cytotoxic study of the photodynamic activity presented by tricyclic dyes (methylene blue, fluorescein and erythrosine) and the commercial Russian photosensitizer Photogem® (hematoporphyrin derivative). For this purpose, three cell lines were used: HEp-2 (tumor cells), VERO and McCoy (nontumor cells), and a yeast strain. The wavelength used for irradiation was 630 nm, the same as used in PDT for medical purposes, since it is in the therapeutic window, i.e., where light can penetrate more into the tissues. The results suggest that Photogem® is more cytotoxic and more photocytotoxic than the studied tricyclic dyes in nontumor and tumor cells. These dyes present less cytotoxicity (around half) in normal cells (dark and light) than in tumor cells. In the experiments with microorganisms, methylene blue presented a better photodynamic effect than Photogem®. These results can be explained by the fact that it is more difficult for Photogem® to penetrate in microorganism membranes than mammalian cell membranes. As for Photogem®, these tricycle dyes present a higher cytotoxicity in tumor cells. These data suggest that methylene blue can be an option in photodynamic inactivation as well as in photodynamic therapy, mainly for superficial lesions.

Phototransformation of hematoporphyrin in aqueous solution: Anomalous behavior at low oxygen concentration

The photoactivation of a photosensitizer is the initial step in photodynamic therapy (PDT) where photochemical reactions result in the production of reactive oxygen species and eventually cell death. In addition to oxidizing biomolecules, some of these photochemical reactions lead to photosensitizer degradation at a rate dependent on the oxygen concentration among other factors. We investigated photodegradation of Photogem ® (28 μM), a hematoporphyrin derivative, at different oxygen concentrations (9.4 to 625.0 μM) in aqueous solution. The degradation was monitored by fluorescence spectroscopy. The degradation rate (M/s) increases as the oxygen concentration increases when the molar ratio of oxygen to Photogem® is greater than 1. At lower oxygen concentrations (<25 μM) an inversion of this behavior was observed. The data do not fit a simple kinetic model of first-order dependence on oxygen concentration. This inversion of the degradation rate at low oxygen concentration has not previously been demonstrated and highlights the relationship between photosensitizer and oxygen concentrations in determining the photobleaching mechanism(s). The findings demonstrate that current models for photobleaching are insufficient to explain completely the effects at low oxygen concentration.

Determination of threshold dose of photodynamic therapy to measure superficial necrosis.

BACKGROUND DATA Photodynamic therapy (PDT) involves the photoinduction of cytotoxicity using a photosensitizer agent, a light source of the proper wavelength, and the presence of molecular oxygen. A model for tissue response to PDT based on the photodynamic threshold dose (D(th)) has been widely used. In this model cells exposed to doses below D(th) survive while at doses above the D(th) necrosis takes place. OBJECTIVE This study evaluated the light D(th) values by using two different methods of determination. One model concerns the depth of necrosis and the other the width of superficial necrosis. MATERIALS AND METHODS Using normal rat liver we investigated the depth and width of necrosis induced by PDT when a laser with a gaussian intensity profile is used. Different light doses, photosensitizers (Photogem, Photofrin, Photosan, Foscan, Photodithazine, and Radachlorin), and concentrations were employed. Each experiment was performed on five animals and the average and standard deviations were calculated. RESULTS A simple depth and width of necrosis model analysis allows us to determine the threshold dose by measuring both depth and surface data. Comparison shows that both measurements provide the same value within the degree of experimental error. CONCLUSION This work demonstrates that by knowing the extent of the superficial necrotic area of a target tissue irradiated by a gaussian light beam, it is possible to estimate the threshold dose. This technique may find application where the determination of D(th) must be done without cutting the tissue.

Influence of pH on the phototransformation process of Photogem

Photogem® is a hematoporphyrin derivative that has been used as a photosensitizer in experimental and clinical Photodynamic Therapy (PDT) in Brazil. Photosensitizers are degraded under illumination. This process, usually called photobleaching, can be monitored by decreasing in fluorescence intensities and includes the following photoprocesses: photodegradation, phototransformation, and photorelocalization. Photobleaching of hematoporphyrin-type sensitizers during illumination in aqueous solution is related not only to photodegradation but is also followed by the formation of photoproducts with a new fluorescence band at around 640–650 nm and with increased light absorption in the red spectral region at 640 nm. In this study, the influence of pH on the phototransformation process was investigated. Photogem® solutions, 40 μg/ml, were irradiated at 514 nm with intensity of 100 mW/cm2 for 20 min with different pH environments. The controls were performed with the samples in the absence of light. The Photogem® photodegradation is dependent on the pH. The behavior of photodegradation and photoproducts formation (monitored at 640 nm) is distinct and depends on the photosensitizer concentration. The processes of degradation and photoproducts formation were monitored with Photogemin the concentration of 40 μg/mL since that demonstrated the best visualization of both processes. While below pH 5 the photodegradation occurred, there was no detectable presence of photoproducts. The increase of pH led to increase of photoproducts formation rate with photodegradation reaching the highest value at pH 10. The increase of photoproducts formation and instability of Photogem® from pH 6 to pH 10 are in agreement with the desired properties of an ideal photosensitizer since there are significant differences in pH between normal (7.0 < pH < 8.6) and tumor (5.8 < pH < 7.9) tissues. It is important to know the effect of pH in the process of phototransformation (degradation and photoproduct formation) of the molecule since low pH values promotes increase in the proportion of aggregates species in solution and high pH values promotes increase in the proportion of monomeric species. There must be an ideal pH interval which favors the phototransformation process that is correlated with the singlet oxygen formation responsible by the photodynamic effect. These differences in pH between normal and tumor cells can explain the presence of photosensitizers in target tumor cells, making PDT a selective therapy.

Determination of post-mortem interval using in situ tissue optical fluorescence

In this study we have used fluorescence spectroscopy to determine the post-mortem interval. Conventional methods in forensic medicine involve tissue or body fluids sampling and laboratory tests, which are often time demanding and may depend on expensive analysis. The presented method consists in using time-dependent variations on the fluorescence spectrum and its correlation with the time elapsed after regular metabolic activity cessation. This new approach addresses unmet needs for post-mortem interval determination in forensic medicine, by providing rapid and in situ measurements that shows improved time resolution relative to existing methods.

Fluorescence guided PDT for optimization of the outcome of skin cancer treatment

The photodynamic therapy (PDT) is an alternative technique that can be indicated for superficial basal cell carcinoma (sBCC), Bowen’s disease and actinic keratosis with high efficiency. The objective of this study is to present the importance of fluorescence imaging for PDT guidance and monitoring in real time. Confirming that the lesion is well prepared and the photosensitizer shows a homogenous distribution, the outcome after few PDT sessions will be positive and the recurrence should be lower. Our proposition in this study is use the widefield fluorescence imaging to evaluate the PDT protocol in situ and in real time for each lesion. This evaluation procedure is performed in two steps: first with the monitoring of the production of protoporphyrin IX (PpIX) induced by methyl aminolevulinate (MAL), an derivative of 5-aminolevulinic acid (ALA) and second with the detection of PpIX photobleaching after illumination. The fluorescence images provide information correlated with distinct clinical features and with the treatment outcome. Eight BCC lesions are presented and discussed in this study. Different fluorescence patterns of PpIX production and photobleaching could be correlated with the treatment response. The presented results show the potential of using widefield fluorescence imaging as a guidance tool to customized PDT.

Optimization of Photodynamic Therapy Using Negative Pressure

OBJECTIVE The goal of this study is to demonstrate an alternative procedure to perform topical photodynamic therapy (PDT). Here, we propose the combined use of negative pressure and a 5-Aminolevulinic acid (5-ALA) cream occlusion to increase protoporphyrin IX (PPIX) formation. BACKGROUND DATA PDT using topical 5-ALA as a prodrug and precursor of PPIX has been used in the treatment and diagnosis of different types of cancer and skin diseases. The use of 5-ALA offers many advantages as a localized and non-systemic application, but it shows limitations in relation to skin penetration. Many authors have discussed the limitations of 5-ALA penetration through the skin. The skin penetration of 5-ALA can be optimized using mechanical devices associated with typical PDT procedure. METHODS For this study, 20% 5-ALA cream was applied to a 9 cm(2) area of skin, and an occlusive dressing was placed. The PPIX production was collected at the skin surface, using fluorescence spectroscopy and widefield fluorescence imaging, for 7 h, and after 24 h. RESULTS We observed that in the presence of negative pressure therapy, the PPIX production, distribution, and elimination are greater and faster than in the control group. The PPIX formation was ∼30% in deeper skin layers, quantified by fluorescence spectroscopy analysis, and ∼20% in surface skin layers, quantified by widefield fluorescence imaging analysis. CONCLUSIONS Negative pressure induction can also help PDT application in the case of inefficient PPIX production. These results can be useful for optimizing the PDT.

Fluorescence evaluations for porphyrin formation during topical PDT using ALA and methyl-ALA mixtures in pig skin models

BACKGROUND Photodynamic Therapy (PDT) using Aminolevulinic acid (ALA) and derivative molecules as topical medication and as a precursor of protoporphyrin (PPIX), is limited due to low permeation through skin or efficiency in porphyrin production. This behavior affects the production and homogeneity of PPIX distribution on superficial skin and in the deeper skin layers. Many authors propose alternatives to solve this such as, modification in the ALA and derivativemolecules, modifying the chemical properties of emulsion external phase or incorporating a delivery system to the emulsion. The goal of this study is to discuss what proportion of ALA and Methyl aminolevulinate (MAL) on mixtures increase the amount and uniformity of PPIX formation at superficial skin by fluorescence evaluations. METHODS The study was conducted in vivo using a pig skin model. PPIX production was monitored using fluorescence spectroscopy and widefield fluorescence imaging on skin surface. 20% of ALA and MAL cream were done mixing the following proportions: ALA, M2 (80% ALA-20% MAL), M3 (60% ALA-40% MAL), M4 (50% ALA-MAL), M5 (40% ALA-60% MAL), M6 (20% ALA-80% MAL) and MAL. RESULTS Mixtures M3, M4, and M5 showed the most PPIX production on skin by widefield fluorescence imaging and fluorescence spectroscopy in 3h of incubation. These results suggest that 50% of ALA and MAL in the same mixture increase the PPIX production in amount, homogeneity and time production when compared to ALA and MAL. This has a positive impact on photodynamic damage optimizing the PDT treatment.