P-3

NMR (THF-d8) δ (ppm):

1H: 0.2 (-SiMe + -OSiMe3), 0.9–1.0 (-SiCH2- + -CH3), 1.7 (-CH2-CH3), 2.7 (-CH2S-), 3.2 (-SCH2- + -NHCH2-), 8.3 (-NH-)

13C: 0.8 (-SiMe + -OSiMe3), 10.6 (-CH2-CH3), 17.6 (-SiCH2-), 20.8 (-CH2-CH3), 26.5 (-SCH2-), 35.4 (-CH2S-), 41.1 (-NHCH2-), 170.8 (-C(O)NH-), 174.6 (-COOH) (residue)

29Si: −23.5, −21.2 (-CH2(Me)SiO2/2), 8.6 (-OSiMe3)

FTIR (cm−1): 3500–3000 (ν O-H), 3250 (ν N-H), 3000–2500 (ν C-H), 1700 (ν C=O) (carboxylic groups), 1650 amide I band (ν C=O), 1578 amide II band (δ NH in plane + ν C-N), 1379 (δ C-H), 1264 (δ Si-C), 1164 (δ Si-CH2), 1080 (ν Si-O-Si), 798 (ρ Si-CH3)

#### *3.2. Analytic Methods*

3.2.1. Nuclear Magnetic Resonance Spectroscopy (NMR)

Liquid state 1H-, 13C- and 29Si-NMR spectra for the functionalized polysiloxanes were recorded in THF-d8 as a solvent on a Bruker DRX-500 MHz spectrometer, with TMS as the reference (Billerica, MA, USA).

#### 3.2.2. Fourier Transform Infra-Red Spectroscopy (FT-IR)

The absorption spectra were recorded for thin films of samples cast on crystal KBr windows using a Nicolet 380 FTIR spectrometer (Thermo Scientific, Waltham, MA, USA). The spectra were obtained by adding 32 scans at a resolution of 1 cm<sup>−</sup>1. The position of characteristic vibration modes was correlated with the appropriate literature data [34,63].

#### 3.2.3. Thermogravimetric Analysis (TGA)

The thermal stability of the studied polymeric materials was analyzed with a Hi-Res TGA 2950 Thermogravimetric Analyzer (TA Instruments, New Castle, DE, USA) in nitrogen atmosphere (heating rate 10 ◦C/min, resolution 3, sensitivity 3).

#### 3.2.4. Differential Scanning Calorimetry (DSC)

The samples were studied using a DSC 2920 Modulated apparatus (TA Instruments, New Castle, DE, USA). Thermograms were taken for samples (sealed in aluminum pans) heated in N2 atmosphere at the rate of 10 ◦C/min from room temperature to 100 ◦C, ten cooled down at the same rate to −50 ◦C and heated again to 100 ◦C. The cooling/heating cycle was repeated and the temperatures of characteristic phase transitions were taken from the third measurement.

#### 3.2.5. Surface Energy Measurements

Thin polymer films were spread on commercially available clean glass slides (cleanready to use, Lab Glass) and 3-aminopropyltriethoxysilane pre-treated glass supports (APTES-glass, silane-prep slides, Sigma Aldrich) using a slit coating applicator (film thickness of 150 μm). Surface free energy was estimated by contact-angle measurements (sessile drop technique) at the film–air interface, as described earlier [22], using deionized water and glycerol (Chempur, pure p.a., anhydrous) as the reference liquids. Surface energies (including their polar and dispersive components) were estimated by the Owens–Wendt method [64].

#### *3.3. Biological Studies*

#### 3.3.1. Biological Material

Reference strains *E. coli* ATCC 8738, and *S. aureus* ATCC 6538, as well as water-borne bacterial isolates *A. hydrophila* KC756842 and *A. tumefaciens* KJ719245 deposited at LOCK Culture Collection [65], were subcultured on Triptic Soy Agar slants (Merck Millipore, Burlington, MA, USA). In turn, mold *A. pullulans* LOCK0461 was cultivated on Malt Extract Agar slants (Merck Millipore). Tested microorganisms were cultivated at 28–30 ◦C for 48 h, and then maintained at 4 ◦C. For *C. vulgaris* incubation in liquid Bold Basal medium [66] at 25 ◦C for 7 days, the light was provided by a cool white LED (T5 15W 6400 K, 80 <sup>μ</sup>mol·m<sup>−</sup>2·s<sup>−</sup>1) with continuous illumination within the experimental period.

#### 3.3.2. Abiotic Surfaces

Bacterial adhesion was evaluated under laboratory conditions. Samples of the studied polymers (P-1, P-2, P-3 and LPSQ) were cast on clean glass slides (clean, ready to use, Lab Glass) using a slit coating applicator (film thickness of 150 μm). Experiments were conducted using white glass as the control carrier (Star Frost 76 mm × 26 mm, Waldemar Knittel Glasbearbeitungs GmbH, Bielefeld, Germany). Glass carriers were sterilized by autoclaving at 121 ◦C, and modified carriers were sterilized using UV light (265 nm, 2 h per each side).

#### 3.3.3. Assessment of Bacterial Adhesion

Sterile carriers (10 mm × 10 mm) were placed in sterile 25 mL Erlenmeyer flasks with 20 mL of culture medium (Table 4).

**Table 4.** Culture media.


The amount of cell inoculum (0.1 mL) was standardized densitometrically (1◦McF). The samples were incubated at 25 ◦C with agitation on a laboratory shaker (135 rpm) for 6 days. For *C. vulgaris* incubation, the cool white LED (T5 15 W 6400 K, 80 <sup>μ</sup>mol·m<sup>−</sup>2·s<sup>−</sup>1) with continuous illumination was used. Cell adhesion to the carriers was evaluated using both fluorescence microscopy and luminometry using ATP-free sampling pens (Merck KGaA, Darmstadt, Germany). Luminometric measurements were expressed in Relative Light Units (RLU) using a HY-LiTE2 luminometer (Merck KGaA, Darmstadt, Germany). The relative adhesion coefficient (A) was then calculated: the RLU result for adhered cells was divided by the RLU results for culture suspension in the given sample [67]. Adhered bacterial cells were observed after DAPI staining and using the fluorescence microscope NICON type BX41 fitted with a 50× lens and with top illumination of the tested surfaces by an external lamp. Images were captured with a digital camera.

3.3.4. Determination of Antimicrobial Activity of Working Solutions Used to Create Functional Polymers and Other Model Compounds

The routine antimicrobial susceptibility testing was based on the agar well diffusion method [68]. The agar plate surface was inoculated by spreading 200 μL of the microbial inoculum over the entire agar surface (TSA (Merck KGaA, Darmstadt, Germany) for bacteria, MEB (Merck KGaA, Darmstadt, Germany) for fungi, bold basal agar for algae). Then, a hole with a diameter of 5 mm was punched aseptically with a sterile cork borer, and a volume of 10 μL of the tested substance at the desired concentration was introduced into the well. Then, agar plates were incubated at 25 ◦C. The antimicrobial agen<sup>t</sup> diffused in the agar medium and inhibited the growth of the microbial strain tested. After incubation, the antimicrobial activity of the tested molecules was detected by the appearance of the inhibition zone (mm) around the well.

#### 3.3.5. Statistical Methods

The results of microbial adhesion were calculated as the means and standard deviations in the data from three independent tests. Analysis of variance (ANOVA) was used to examine the differences between group means, representing the adhesion results (OriginLab Corporation, Northampton, MA, USA). The results were compared to those for the control samples (glass carriers). Values with letters show statistically significant differences: a, *p* ≥ 0.05; b, 0.005 < *p* < 0.05; c, *p* < 0.005.
