## 4 Discussion  
In this pre-clinical work on healthy mice, we aimed to answer the question of what kind of photosensitizer is more appropriate for PDT-opening of the BBB. To answer this question, we studied PDT-opening of the BBB using commercial PS such as 5-ALA and AlPcS as well as new synthetized PS such as ZnPc and functionalized its form GalZnPc. Our results reveal a dose-depended PDT opening of the BBB using all types of PS. The PDT-opening of the BBB using 5-ALA and AlPcS also have been shown by [PERSON] et al. [9], [PERSON] [10], and in our previous experiments [11; 12; 13; 14]. Using spectrofluorimetric assay for quantitative analysis of the BBB permeability to EBAC and confocal imaging of FITCD extravasation, our results demonstrate that PCs induce stronger ECAB leakage compared with 5-ALA. Among PCs, GalZnPc causes the more pronounced increase in the BBB permeability to EBAC vs. AlPcS, ZnPc. Phthalocyanines generate singlet oxygen directly under light irradiation [35], while 5-ALA does not produce single oxygen itself but only via metabolization in the  
Figure 2: (**a**) Fluorescence spectra of murine brain in control (untreated with photosensitizer) and experimental group treated with 5-ALA; (**b**) Fluorescence intensities comparison before and after irradiation with 635 nm for 5-ALA/PplX signal obtained from 5-ALA treated mice; (**c**) Fluorescence spectra of murine brain in control (untreated with photosensitizer) and experimental group treated with GalZnPc; (**d**) Fluorescence intensities comparison before and after irradiation with 670 nm for GalZnPc signal obtained from phthalocyanine-treated mice; (**e**) excitation and emission spectra of AlPc; (**f**) excitation and emission spectra of ZnPc; (**g**) excitation and emission spectra of GalZnPc; Spectra in e, f and g are normalized with respect to maximum of excitation and emission signals for corresponding phthalocyanines.  
cells to protoporphyrin IX [36]. These facts can explain the lesser PDT-5-ALA opening of the BBB to EBAC compared with different forms of used phthalocyanines (AlPc\(\backslash\)S, ZnPc, GalZnPc). The stronger effect on the BBB permeability by GalZnPc can be due to enhancing the transendothelial permeability by galactose via activation of protein kinase C (PKC), a family of kinases vital to intracellular signaling that is a common effect of monosaccharides on vascular endothelium [22; 23].  
To better understand mechanisms responsible for PDT-induced opening of the BBB to high molecular weight molecules (EBAC and FITCD), we explored PDT-related changes in the fluorescent signal of TJ trans-membrane proteins such as CLND, OCC, and JAM. The fluorescence of these molecular factors was analyzed before PDT (the control groups), 1 h after PDT (when BBB was opened), and 4 h after PDT (when the BBB was closed and completely recovered). Our results uncovered a decrease in the fluorescence of TJ proteins in the time of PDT-opening of the BBB with a rapid normalization of the fluorescence of these proteins till the moment of BBB closing.  
These findings are consistent with data of others showing that PDT has a direct effect on increasing the gaps of the TJs between endothelial cells via changes of cytoskeleton and vascular tone loss due to microtubule depolymerization [37; 38; 39]. One of the mechanisms by which PDT opens the BBB can be an increase in the singlet oxygen/ROS production and development of oxidative stress that we observed in our previous work using 5-ALA [14] and that was also described for phthalocyanines [35]. We assume that a PDT-induced increase in oxidative stress and associated with these changes in TJ proteins might be one mechanism underlying PDT-related BBB opening.