## 3 Discussion
Hence, positively charged PSs generally lead to the more effective inactivation of Gram-negative bacteria than negatively charged and neutral PSs because of their ability to bind to the negatively-charged phosphate groups of the outer membrane and, when excited, contribute to damage to membrane-building lipids and proteins, including membrane-associated enzymes [28]. In our study, the MBC of CHL alone was 1000 \(\mu\)g mL\({}^{-1}\) with a long exposure time of 5 h against _P. aeruginosa_ (Figure 2). Taken together with the above-mentioned previous investigations, the possible explanation of the efficacy of CHL alone could be that the generation of the highly reactive \({}^{1}\)O\({}_{2}\) and other ROS that are capable of oxidizing proteins, lipids and nucleic acids requires a high concentration of CHL and a long illumination time and could occur without the necessity for direct contact between CHL and the cells. Moreover, the obtained results proved that the unilluminated CHL and the red light itself have no bactericidal effect on the _P. aeruginosa_ cells at the tested conditions, suggesting that _P. aeruginosa_ do not have to engage in adaptive survival mechanisms against CHL [19]. This result is consistent with [PERSON] and colleagues [43], who studied the susceptibility of _Staphylococcus aureus_, _Listeria monocytogenes_, _E. coli_ and _Salmonella_ spp. to CHL incorporated into gelatin film with no toxicity of the dark controls. The branched 25 kDa PEI Is a positively charged aliphatic polymer that contains amino nitrogen at every third atom of the polymer backbone as a mixture of primary, secondary, and tertiary amines, which provides its cationic nature upon protonation [44; 45]. This cationic characteristic can elicit apoptosis in eukaryotic cells [46]. However, branched 25 kDa PEI is widely used as a transfection agent and has various medical applications because it is a good compromise between toxicity and efficacy [47; 48]. We speculate that the electrostatic interaction between CHL and PEI or the ROS generated during the photodynamic process could open the door and be an advantage for the reduction in the cytotoxicity of PEI via the reduction in the cationic charges over the surface of the molecule, as it was previously proven that PEI/DNA complexes were less toxic by about 10% than PEI alone [49]. Moreover, oxidizing the amine groups of the branched 25 kDa PEI by hydrogen peroxide reduced its surface charge and its cytotoxicity [44]. Indeed, further investigations are needed to determine the cytotoxicity of the effective combinations of our study for a deeper understanding of the interaction between CHL and PEI. In the whole tested conditions in our study, PEI 25 kDa alone had no direct lethal effect on _P. aeruginosa_. Similarly, [PERSON] et al. [50] found that PEI 50 kDa enhanced the susceptibility of the Gram-negative bacteria _E. coli_, _P. aeruginosa_ and _Salmonella_ spp. to antibiotics without being directly lethal. On the other hand, 64 ug/mL of the branched PEI 1800 Da reduced the viability of _E. coli_ by 60% after 2 h, while the cell reduction was approximately 97% of the Gram-positive bacteria _Staphylococcus aureus_ in 10 min, suggesting that the cell structure played a role in the antibacterial activity of PEI [51]. _P. aeruginosa_ is recognized as a paradigm bacterium for biofilm formation because of its ability to adhere to a variety of surfaces, including human tissues, instruments and food; therefore, dislodging its biofilm is necessary [38]. Our study demonstrated that PEI (1600 ug mL\({}^{-1}\)) can detach approximately 72% of the established biofilm, and only 100 ug mL\({}^{-1}\) PEI in the presence of CHL at 125 ug mL\({}^{-1}\) under illumination conditions was required to detach approximately 76% of the biofilm. However, more than CHL/PEI 500/100 ug mL\({}^{-1}\) was required to eradicate the bacteria in the colony biofilm. Similarly, [PERSON] and colleagues [52] proved that _P. aeruginosa_ biofilm can be disrupted via PEI linked with polyethylene glycol.