A study of the vascular effect of photodynamic therapy with oxygen variation
on chorioallantoic membrane model
Authors:   , 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.