## 2 Materials and Methods
### In Vitro Tests  
To perform in vitro tests, we collected biofilm materials growing on the Aurelian Walls in Rome, as a monument representative of biodeterioration patterns occurring in the Mediterranean area [13, 38, 39]. Specifically, we performed a survey on the vertical surfaces of the walls in the northern exposure where the biological growth is widely distributed, and not previously treated by restoration activities. Then, we selected and brushed superficially, employing a sterile lancet and a black humid patina. A part of the collected microorganisms was observed by the optical microscope with an immersion objective at 100-magnification (Olympus BX41,  
Figure 1: Structure of nanocontainers synthetized: (**a**) Silica Nanocapsule (NC): Scheme, SEM images of NC loaded with ZSciara sodium salt (ZS); (**b**) Mesoporous silica nanoparticles (MNP): Scheme, SEM images of NC loaded with ZSciara sodium salt (ZS). For the representation of Silica Nanocapsule and Mesoporous silica nanoparticles loaded with MBT, we refer to [PERSON] et al. [21]. Rome, Italy), following the procedures in UNI [40]. Thanks to the analytic keys of Guiry and Guiry [41], we identified the occurring species, which resulted in a mixed community of green algae, cyanobacteria, and meristematic fungi. Then, we cultured the detected species, according to the suggested nutritional and expositive requirements, in a BG-11 liquid media (Sigma-Aldrich) at 25 \({}^{\circ}\)C in conditions of natural sunlight, as previously reported [13]. Indeed, this type of media favored the proliferation of the algal component, and after two months, we obtained an almost pure culture of _Chlorococcus_ sp. that we used as target organisms to test the efficiency of our nanosystems. We tested eight different situations: the two free biocides (MBT and ZS); the two different nanosystems not loaded with biocides (NC, MNP); the two nanosystems loaded with zosteric sodium salt (NC_ZS; MNP_ZS) and with 2-mercaptoethanolphazothiazole (NC_MBT; MNP_MBT). The nanocontainers were loaded with a concentration of biocide related to the loading capability (respectively, NC_Zs = 2.1%; MNP_ZS = 7.8%; NC_MBT = 10%; MNP_MBT 8.2%). We tested, following the protocol fine-tuned in [PERSON] et al. [13], 3 mg of nanocontainers not loaded and loaded with the biocide, added to 1 mL of liquid culture. For the free biocide, we tested a minimum (1 mg) and maximum (3 mg) quantity, emphasizing the concentrations based on those loaded in the nanosystems (-0.01 mg/mL) and the weight put in 1 mL of culture (3 mg). Biocide efficiency was evaluated periodically, after the 1 st, 2 nd, 4 th, 6 th, and 8 th week, so we filled 40 test tubes for each product tested in each time control. We observed the control and the treated cultures, in triplicate form for each product, under an optical microscope (Zeiss Axioplan 2) equipped with a photo camera (LEICA DFC 450 C) at 20X magnification in visible and autofluorescencement light. Following the methodology of previous works [10; 19; 42; 43], we evaluated the loss of each cell's fluorescence as an indicator of biocidal efficiency. We observed the autofluorescenceence of cultured microorganisms with multi-channel detection, in blue (450-500 nm), purple (380-450 nm), and green (500-570 nm) wavelengths [44; 45]. The photosynthetic microorganisms, in our case green algae, contain chlorophylls, which show autofluorescence at 670-790 nm (excitation, 594 nm) [42; 43; 44; 45]. We used multiple channels to observe not only the disappearance of the chlorophyll but also of the other accessory pigments, evaluating the change in chlorophyll fluorescence from red to green until its disappearance. In the monitoring occurring over the weeks, we analyzed the pigment's autofluorescence to discriminate between active cells (the presence of chlorophyll autofluorescenceence) and inactive/dead cells (between the absence of chlorophyll autofluorescenceence and complete absence of fluorescence) [42].