A study of the vascular effect of photodynamic therapy with oxygen variation on chorioallantoic membrane model
Authors:   ,   Pontificia Universidad Católica de Chile. Facultad de Fí­sica, Aizpuru Vargas Luis Lauro 
Published: November 2024

Chicken chorioallantoic membrane (CAM) assays have been proven to be very effective models to study the impact of chemical and biological agents on its vascular network. The study of pharmaceutical drugs, xenografts and photosensitizers on these assays are current areas of research. In particular, the use of photodynamic therapy (PDT) has been shown to induce a significant decrease in both the diameter and number of blood vessel ramifications of the CAM. Given that molecular oxygen is so important for the effectiveness of this therapy, it opens the possibility of testing bio-compatible photosynthetic microalgae capable of locally producing oxygen. Even though there is ample evidence of the use of bio-compatible microalgae in scaffolds for wound recovery, there is little to no research done on the combination of microalgae and PDT in CAM essays. In this thesis work, we present a study that explores how microalgae, PDT and the combination of these two change the vascularization of the CAM. Throughout this experiment, the photosensitizer that was used was methylene blue whilst the microalgae that was used was Chlamydomonas reinhardtii. In order to perform the quantitative measurements, an image analysis software allowed us to count the number of bifurcation points. Firstly, four groups were defined to study how the PDT at different days of embryonic development (EDD) might change blood vessel ramifications. These groups were defined as control, only light, only methylene blue and the PDT groups. It was found that after 24 hours, for EDD8, the ramifications for the PDT case dropped by 40% whilst for EDD10 they dropped by 60% with light at 630 nm with 30 mW/cm2 for 5 minutes. Second, it was explored how illuminated microalgae might possibly increase the vascular networks with the same light parameters. It was found that after 24 hours for EDD8 the network grew by 30% and for EDD10 it grew by 20%. Moreover, the combination of a photosensitizer and microalgae was studied to analyze if it might change the effectiveness of the PDT. It was found after 24 hours that for all the explored permutations for both EDD8 and EDD10 all the eggs were dead, showcasing the effect of an oxygen-enriched PDT. Given that the effect was way to strong, the methylene blue solution was diluted by a factor of x10 (0.03 mg of MB/mL) to see if this resulted in a measurable effect on the eggs for the combination case. Indeed, it was found that the decrease from the baseline ramification levels was 30% for the PDT and 60% for the oxygen enhanced PDT. The novel element of this work was that it was one of the first to explore the possibility of creating an oxygen-rich PDT in a CAM assay. This showed that it is possible to combine microalgae and a photosensitizer in a way that enhances the effectiveness of the PDT. This might translate into the possibility of employing this technique in an in-vivo trail with mice or other mammals to guarantee its safety. In conclusion, we found that illuminated photosynthetic microalgae raises the effectiveness of PDT in the vascular network of CAM assays.