2.1.

Chemicals

All chemicals were purchased from Sigma-Aldrich, Switzerland.

2.2.

CAM

Fertilized chicken eggs were purchased from Animalco AG, Switzerland. For 3 days, eggs were incubated at 37°C in a humidified and automatically rotating incubator with the blunt end up. At the embryo development day (EDD) 3, a small hole (diameter: 3 mm) was made at the pointed end of the shell and covered with tape. Afterward, eggs were placed back to the incubator in a stationary position until further use. On EDD 12, the hole was enlarged (diameter: 25 mm) enabling topical ALA and catechin administration followed by PDT.

2.3.

PpIX-PDT

PpIX-based PDT has been described in detail in our previous reports.¹⁸ Briefly, ALA was dissolved in an aqueous solution of NaCl (0.9% of NaCl and 20 mg of ALA per ml with a pH of 6.8). An amount of $20\mu \mathrm{l}$ of this solution was topically applied on the CAM. Then, 4 h after ALA administration, PDT was conducted with the light delivered by an epifluorescent microscope (Eclipse E 600 FN Nikon) utilizing a $4\times$ objective (Nikon, NA: 0.13, Plan Fluor, WD 17.1). PpIX produced endogenously was excited with light at 405 nm (filter cube BV-2A, Nikon, Japan). PpIX-PDT was performed with a light dose of $1.2\mathrm{J}/{\mathrm{cm}}^2$ (irradiance: $2.9\mathrm{mW}/{\mathrm{cm}}^2$). This light dose typically induces a grade 4 vascular damage according to the PDT effects scale defined by Lange et al.,¹⁹ i.e., vessels with a diameter $<70\mu \mathrm{m}$ are all closed by PDT.

2.4.

Catechin Administration

A stock solution of catechin was dissolved in EtOH before further dilution in NaCl (used for the topical administration on the CAM). Different concentrations of catechin (1, 5, 10, 20, 30, and $50\mu \mathrm{M}$) were applied topically (in $100\mu \mathrm{l}$ of NaCl) to the CAM at different time intervals ranging between 0 and 4 h prior to PpIX-PDT. In all experiments, the content of EtOH was $<1\%$. It should be noted that concentrations of catechin are relative to the weight of the embryo at given EDD, i.e., 5 g at EDD 12. For all control eggs, $100\mu \mathrm{l}$ of NaCl (without catechin) was topically applied on the CAM.

2.5.

Angiograms Acquisition and Analysis

Fluorescent angiograms of the CAM’s vascular network were recorded 24 h after PDT. Angiograms were obtained by an intravenous injection ($20\mu \mathrm{l}$) of fluorescein isothiocyanate–dextran (25 kD, 25 mg/ml) dissolved in NaCl. To further improve the contrast between the extravascular space and blood vessels of the CAM, India ink (Parker) was concurrently injected ($100\mu \mathrm{l}$) under the CAM. It has been proven that India ink, which is also used as a marker for tumor margination in the human skin, is not toxic to the CAMs in our conditions.²⁰ Angiograms were acquired with an epifluorescent microscope (Eclipse E 600 FN Nikon) utilizing a $4\times$ objective (Nikon, NA: 0.13, Plan Fluor, WD 17.1). PDT potency along with the assessment of the catechin photoprotection was quantified by the quantitative analysis of these fluorescent angiograms using an ImageJ Macro (NIH, Bethesda, Maryland) developed in our laboratory.²⁰

2.6.

PpIX Photobleaching Study

Fluorescent images for this substudy have been obtained with the same microscope setup as described above. A BV-2A filter cube (excitation: 405 nm; Nikon, Japan) combined with a longpass emission filter transmitting light above 610 nm (E610LP filter; Chroma, Irvine, California) was used for PpIX fluorescence visualization and relative quantification. Images were recorded at the beginning (first 5 s) and at the end (last 5 s) of the PDT irradiations. The ImageJ software was then used to quantify the PpIX fluorescence intensity changes (at the beginning and at the end of illumination) in the presence and absence of catechin. This study was carried out with a light dose of $10\mathrm{J}/{\mathrm{cm}}^2$ at 405 nm, and $10\mu \mathrm{M}$ catechin was administered 15 min before PDT.

3.1.

Study of the Photoprotective Effects of Catechin Applied at Different Concentrations

As mentioned above, PpIX is one of the most frequently used PSs for PDT. Its phototoxic effects are well documented, in particular, those induced in the vascularization.^18,21–23 Hence, in this study, it has been used as PS for the induction of vascular damages in the CAM. On the basis of a preliminary study, we established the spectral and radiometric conditions inducing grade 4 vascular damages according to Lange et al.¹⁹ (data not shown). We concluded that light at 405 nm applied with a dose of $1.2\mathrm{J}/{\mathrm{cm}}^2$ and an irradiance of $2.9\mathrm{mW}/{\mathrm{cm}}^2$ was appropriate. These conditions were used in all experiments presented in Secs. 3.1 and 3.2.

In the first step, we studied the photoprotective effects of catechin applied topically at different concentrations 15 min before PDT (1 to $50\mu \mathrm{M}$). Figure 2(a) shows illustrative fluorescence angiograms acquired 24 h after PDT. A quantitative analysis of fluorescence angiograms [Fig. 2(b)] to assess the efficacy of PpIX-PDT, i.e., to determine the diameter of the smallest vessels that did not close after PpIX-PDT, showed that, in the control samples, this diameter was in the order of $100\mu \mathrm{m}$. On the other hand, PpIX-PDT was significantly less potent at all concentrations of catechin. More precisely, the small concentrations (1 and $5\mu \mathrm{M}$) reduced the PpIX-PDT effects in such a way that the smallest vessels that did not close had an average diameter of about $45\mu \mathrm{m}$. Similarly, the administration of catechin at higher concentrations (20, 30, and $50\mu \mathrm{M}$) led to a reduction of the smallest not closed vessel down to 39, 47, and $64\mu \mathrm{m}$, respectively. An amount of $10\mu \mathrm{M}$ of catechin showed the most significant photoprotective effect since the average diameter of the smallest unclosed vessels was about $23\mu \mathrm{m}$. It should be noted that “no light” controls (in the presence of catechin only or a combination of catechin with ALA) were conducted, resulting in an absence of vascular changes (data not shown).

Fig. 2

Assessment of PpIX-PDT on the CAM in the presence of different concentrations of catechin. (a) Representative FITC-dextran fluorescent angiographies of the CAM in control samples (no catechin) and in the presence of different concentrations of catechin. Images were acquired 24 h after PDT. All images have the same magnification. A typical unclosed vessel as well as the illuminated circular area is presented in the control image only for the sake of clarity. Red asterisks in the images represent the centers of the illumination with a diameter of 2 mm. (b) Quantitative analysis of the fluorescent images. Number of eggs: $n=35$. The error bars represent the standard error of the mean. Significant $p$ values are represented as **$p\le 0.01$, ***$p<0.001$.

Although the family of catechins is well recognized for its antioxidant properties, it should be noted that they are also able to act as pro-oxidants generating ROS.^24,25 Indeed, although their antioxidant properties are due to their ability to act as free radical/ROS scavengers, their presence can stimulate hemoglobin-induced protein oxidation. This could be caused by their antioxidant capability, which promptly induces an oxidative degradation of hemoglobin,²² an effect that may explain the concentration-dependent photoprotective properties of catechin.

3.2.

Determination of the Optimal Catechin Administration Time Before PpIX-PDT

Following the catechin concentration study reported in Sec. 3.1, we investigated the influence of the role played by the time separating the catechin administration and PpIX-PDT. Since $10\mu \mathrm{M}$ of catechin was found to be optimal, this concentration was used for the second substudy. Figure 3 shows the vascular effects of PpIX-PDT when catechin was applied topically at different times before illumination on the CAM.

Fig. 3

Effects of PpIX-PDT on the CAM when catechin is applied at different times before illumination. (a) Representative FITC-dextran fluorescent angiographies of the CAM in control samples (no catechin) and in the presence of $10\mu \mathrm{M}$ of catechin administered at different times before PpIX-PDT. Images were acquired 24 h after PDT. Red asterisks in the images represent the center of the illumination spots with a diameter of 2 mm. (b) Quantitative analysis of the fluorescent images for different times between catechin administration and PpIX-PDT. Number of eggs: $n=35$. The error bars represent the standard error of the mean. Significant $p$ values are represented as *$p\le 0.05$, **$p\le 0.01$, ***$p<0.001$.

We observed that the presence of $10\text{-}\mu \mathrm{M}$ catechin reduces the PpIX-PDT effect at all times. When administered just (i.e., $<30\mathrm{s}$) before PpIX-PDT illumination, the smallest vessel that did not close was in the order of $51\mu \mathrm{m}$. Increasing the time between catechin administration and illumination up to about 15 min led to more potent inhibitions of the vascular effects of PpIX-PDT. Indeed, when catechin was applied 7 min before PDT, the average diameter of the smallest unclosed vessel was $48\mu \mathrm{m}$, whereas this diameter went down to $23\mu \mathrm{m}$ at the optimal time of 15 min. With longer times (30 min and more), catechin became less and less potent to generate a photoprotection.

According to our knowledge, there is no literature on the catechin pharmacokinetics in the CAM. However, studies conducted on rodents, dogs, rabbits, and humans showed that, after oral administration, catechin is mainly metabolized by phase-2 conjugation process in the liver and intestine.²⁶ In our study conducted on the CAM, the reduced photoprotection observed at times $>30\min$ could be explained by the catechin internalization by the CAM and chicken embryo itself.

3.3.

Assessment of the ROS Production During PDT Based on the Measurement of the PpIX Fluorescence Photobleaching

Measurements conducted in this part of the study were based on the observation that PpIX photobleaches during PDT, an effect that is directly related to the ROS (mostly singlet oxygen) production.²⁷ Indeed, Moan and Berg²⁸ demonstrated that the more ROS are created, the more PpIX is photobleached. Therefore, it is of interest to determine if a correlation exists between the photoprotective effects of catechin and the PpIX photobleaching. Thus, this estimation of the protective effect of catechin was compared with its ability to prevent photobleaching of PpIX. Figure 4 shows the ratio of the fluorescence intensity of PpIX measured at the beginning (first 5 s) divided by this intensity measured at the end of PDT (last 5 s), both for a control group (no catechin) and in the presence of $10\text{-}\mu \mathrm{M}$ catechin applied 15 min before PpIX-PDT. If no photobleaching would occur, the ratio of the PpIX fluorescence at the beginning and at the end of illumination would be equal to 1.

Fig. 4

Reduction of the PpIX photobleaching with catechin. PpIX fluorescence intensity was recorded in the first 5 s and last 5 s of the PDT illumination. These values were divided and compared for the control samples and when $10\text{-}\mu \mathrm{M}$ catechin was administered 15 min before PDT. Number of studied eggs: $n=12$. The error bars represent the standard error of the mean. Significant $p$ value is represented as ***$p<0.001$.

For the purpose of acquiring a good signal-to-noise ratio, the light dose used for this part of the study was increased to $10\mathrm{J}/{\mathrm{cm}}^2$ for all conditions (controls and $10\text{-}\mu \mathrm{M}$ catechin). It should be noted that increasing the light dose did not preclude the photoprotective effect of catechin. At the same time, it is interesting to point out that when photobleaching experiments of PpIX were conducted with $1.2\mathrm{J}/{\mathrm{cm}}^2$ (see Secs. 3.1 and 3.2), the PpIX photobleaching was almost not measurable (data not showed), although the tissue damages were significant. On the whole, these observations suggest that only a small fraction of the PpIX present on the CAM membrane 4 h after ALA application is actually localized in compartment(s), probably the mitochondria, playing a crucial role in the photosensitization process. This suggests that the majority of the PpIX fluorescing in the CAM in our conditions plays a reduced role in the photosensitization process, if any, probably because it is outside these critical compartments.

Nevertheless, as shown in Fig. 4, $10\text{-}\mu \mathrm{M}$ catechin not only reduces the PpIX photobleaching, but leads to an increase of the PpIX fluorescence in the CAM. This effect is probably due, at least in part, to the ability of catechin to reduce the ROS concentration. It could also be that the increase of PpIX fluorescence observed during PDT in the presence of catechin is due to a chelation of iron by this flavanol, as postulated by Samman et al.²⁹ This mechanism may impair the availability of iron to convert PpIX in heme, thus leading to an increase of the PpIX fluorescence detected in the CAM.