Hetero-phase bactericidal agents seem to be very attractive due to their wide range of potential medical and environmental applications. Immobilized PSs in a solid phase are a promising solution for development of long-term disinfection systems suitable for a continuous modus operandi. The photodynamic effectiveness of immobilized PSs was shown earlier by several groups. PSs immobilized on silicon polymer complexes of Ru(II) with poly-azo-heterocyclic compounds were shown to generate reactive oxygen species (ROS) under sunlight illumination . Zn(II) phthalocyanine tetrasulfonic acid, covalently connected to a membrane polymer, was shown to have a high photodynamic efficacy in wastewater disinfection . Several PSs incorporated under pressure and heat into poly(vinylidene fluoride) demonstrated high antimicrobial properties against Gram-positive and Gram-negative bacteria, decreasing their concentration by 4 log10 . However, in some cases the rate of ROS formation by immobilized PSs can be significantly lower than with the free PSs .
The PSs studied in this work dissolve well in water, which allowed using them in a free form in aqueous solutions on the one hand, and demonstrate good solubility in organic solvents on the other hand, thus enabling dissolving them along with various polymers. This PS-polymer solution can be applied on chosen surfaces, and after solvent evaporation, the surfaces become coated with the active polymer. In this study, we chose polystyrene as the support polymer, which was able to mix with the tested PSs in a wide range of ratios due to the presence of aromatic rings in both PSs (Table 1) as well as in the polystyrene structure. Another advantage of the applied method was a high efficiency of immobilization (more than 99%), suggesting that only a negligible part of the PS may be released from the polymer support into the treated aqueous phase. The polymer was washed after the immobilization to remove traces of free PS, to ensure an antimicrobial effect of the immobilized PS without hindrance of free PS. However, in future practical applications of this system, it will be possible to skip this step because of low release of the free PS. The observed leakage of PS from the polymer can be presumably assigned to a minor destruction of the polystyrene under illumination. The data obtained in our study indicate that immobilized PSs caused a significant decrease in the concentration of the Gram-positive S. aureus and Gram-negative E.coli bacteria, up to their total eradication. The results of the experiments with the MB leaked from the polymer, show that the antibacterial activity of the MB-polystyrene was due only to the immobilized MB. In the case of RB-polystyrene, the total PACT effect was composed of the effect of the immobilized RB and of the effect of the RB leaked to the solution. It is important to notice that the observed antibacterial activity of the RB-polystyrene could not be solely explained by the presence of the leaked RB. We propose an explanation for the process of bacterial eradication by PSs immobilized in a solid phase. As shown in Fig. 2, round pores were formed on the surface of the polystyrene, which were big enough for enabling bacteria cells to enter into them, so that the effective surface area was enlarged compared to even and smooth polymer surfaces. Furthermore, according to the results obtained after a continual incubation of both S. aureus and E. coli on polystyrene films that did not contain a PS, the cells readily attached to the polymer surface, probably owing to its high hydrophobic properties. These factors led to increased contact between the polymer and the bacterial cells and to a possibility of high cell sorption by the polymer. Since a photodynamic effect can occur only in close proximity between the PS and the cells, the high polymer contact area was an important factor for exhibiting bactericidal activity by the examined system. Polystyrene by itself was not toxic to the cells at all, and both types of cells not only grew and multiplied on the polymer surface, but even started to form biofilms. In contradistinction, polymer films with the incorporated PSs did not contain cell aggregates and moreover, the single bacterial cells which were detected on the polystyrene surface had actually already been destroyed. These facts indicate that the hydrophobic attachment of the cells to the polymer, amplified by the porous polystyrene structure, facilitated the photodynamic activity of the immobilized PSs. PS molecules excited by light transferred energy to dissolved oxygen in the nearby polymer layer, promoting ROS formation, which in turn eradicated the bacterial cells attached to the polymer surface. Interestingly, the immobilized PS exhibited equal killing effects on E. coli and S. aureus. Apparently, for exhibiting the PACT effect, PS must not necessarily bind to the cell envelopes or enter into the cells. The obtained data open wide prospects for further applications of immobilized PS, for instance for inner coating of baths for aseptic holding of medical devices and for wastewater disinfection in a continuous regime. The latter application is now being studied by our group. In addition, using for immobilization of PSs polymer supports labile under illumination, systems of controlled release of PSs can be constructed.