Development of noncytotoxic silver–chitosan nanocomposites for efficient control of biofilm forming microbes

We describe the synthetic pathway to produce efficient bactericidal, fungicidal and non-cytotoxic chitosan–ascorbic acid–silver composites as solid films.


Fourier transformed infrared spectroscopy (FTIR)
Fourier transformed infrared spectroscopy (FTIR) was used to study the interactions between the polymer functional groups and the metal particles surface. The spectra were collected in the range of 4000-400 cm -1 with a 1 cm -1 step (Perkin Elmer Spectrum Two ATR).

Contact angle measurements
The wettability of nanocomposites was determined through contact angle measurements via the sitting drop technique on a goniometer Surftens Universal (OEG GmbH). Static contact angles of water were calculated using Surftens 4.3-windows image processing software for digital images. High purity water was applied in the measurements with a constant drop volume of 2.5 µL. All measurements were performed at room temperature and the results were presented as mean values (n=10) ± SD.

Cell viability test protocol
Cells after the experiment were washed with PBS, treated with MTT solution (0.5 mg/mL) and incubated for 3 h at 37°C. The formazan crystals were dissolved in a DMSO:CH 3 OH mixture (1:1) and the absorbance at 570 nm was measured (Infinite 200 microplate reader, Tecan). A reference wavelength 670 nm was recorded to exclude potential scattering. In the Alamar assay, cells were washed with PBS and incubated with resazurin sodium salt solution (25 µM in PBS) for 3 h at 37°C in the dark. The fluorescence caused by the cellular metabolic activity was measured at 605 nm (excitation wavelength 560 nm).

Results
The mean values of silver concentration were listed in Table S1. In all cases, the obtained concentrations were close but lower than the theoretical values, which might be caused by the dilution error or some losses upon synthesis. The theoretical concentrations were calculated for silver solutions in the polymer solution. Colloids stability: Zeta-potential (ζ) of chitosan-silver nanoparticles dispersions The surface charge of chitosan-silver nanoparticles after synthesis was determined by measurement of the zeta potential at pH = 4.5 (Zetasizer Nano ZS, Malvern Instruments, Malvern, UK). The value of electrokinetic potential determines the colloidal system stability and potential aggregation. The long-term stability was checked two months after synthesis.
Samples were stored for this purpose in the dark. Measurements performed in fourfold repetition were presented as mean values ± standard deviation (SD) in Table S2. With the increase in the absolute value of the potential, the degree of electrostatic repulsion between nanoparticles increases and thus the more stable the colloidal system is. In such formulations, the aggregation and flocculation are less probable. Chitosan-based silver nanoparticles exhibited positive ζ-potential values, indicating a positively-charged polymeric layer on the AgNPs surface. Values above +45 mV were obtained for all colloids at each silver concentration, which corresponds to a colloidal system with good stability. Depending on the synthetic protocol and applied reducing and stabilizing agent, AgNPs possess positive or negative values of zeta potential e.g. negative for sodium borohydride reduced silver nanoparticles or positive for chitosan-based colloids/ 2, 3 Since chitosan solution provides sufficient positive charge coming from the amino groups and also steric stabilization, the obtained silver nanoparticles were electrostatically stable. Moreover, the long-term stability of the colloids was confirmed by almost constant values of zeta potential which is consistent with the literature, where sterically stabilized nanoparticles are considered as more stable than stabilized only by surface charge. 4,5 Energy dispersive spectroscopy analysis The STEM-HAADF micrographs with EDS analysis confirmed the AgNPs chemical identity and presence in the nanocomposites ( Figure S1). In all MAg/VC composites with different silver loadings, silver presence was proven; the M26/VC analysis is presented as an example. Figure S1. Confirmation of the presence of silver nanoparticles by using Energy Dispersive Elemental Analysis (EDS). The analysis was performed during STEM-HADF mode visualization.  Chitosan functional groups -silver nanoparticles surface interactions: IR-ATR measurements

X-Ray photoelectron spectroscopy analysis of MAg/VC composites (XPS)
The FTIR spectroscopy was used to confirm the specific interaction of chitosan functional groups with the silver nanoparticles. Spectra of pure chitosan films with medium average molecular weight and MAg/VC composites were collected. A typical chitosan spectrum ( Figure S2) presents the characteristic vibrational bands of chitosan at: 1650 cm -1 and 1590 cm -1 corresponding to amide I groups, C-O stretching along with N-H deformation mode (acetylated amine, and to free amine groups, respectively). Bending vibrations of -CH 2 and -CH 3 may be assigned to the absorption bands at 1376 and 1409 cm -1 , respectively. 6 Also, 1320 cm -1 and 1259 cm -1 bands are detected, corresponding to CH 2 wagging vibration in primary alcohol and the amide III vibration coming from a combination of N-H deformation and C-N stretching. Due to electrostatic interactions between the polymer and the AgNPs, a significant shift to lower wavenumbers for the amino group band (1590 cm -1 for pure polymer) is observed.
The second representative shift to lower wavenumbers for chitosan-silver nanocomposites occurs at 1409 cm -1 (for pure polymer). The spectra clearly demonstrate the interactions between the primary amino groups of the polymer with the metal nanoparticles surface. 7,8 Results stay in agreement with our previous report 9 and results obtained for chitosan-silver materials by Potara et al. and Wei et al.. 10,11 Interactions between the polymer and the surface of silver nanoparticles are crucial for the stability of the composites preventing from an excessive release of NPs to the environment.
FTIR analysis was also used to evaluate the potential polymer thermal degradation in the acidic solution used during the synthesis. The spectrum of pure chitosan films with and without applying the temperature conditions, used in the silver nanoparticles synthesis, are presented in Figure S3. The influence of the synthesis protocol on the polymer oxygencontaining groups and potential chitosan degradation were evaluated. In this regard, chitosan medium solution was heated up to 95°C for ~15 h. Afterward, films of chitosan before and after heating were casted and neutralized with1% sodium hydroxide and water.
As described in the main manuscript, the reduction of silver ions in the chitosan solution is coupled with the oxidation and/or hydrolysis of the polymer hydroxyl groups. No significant changes in the shape and position of the band assigned to the hydroxyl groups (~3500cm -1 ) were observed. The slight intensity increase might be a consequence of the different water content in the films. Based on this finding, it may be assumed that after the temperature treatment, in the absence of silver ions, no oxidation and/or hydrolysis of the polymer hydroxyl groups occur. thermo-oxidative process, was observed above 500°C 15 . Therefore, 95° is far below the temperature of the polymer degradation.
Next to the thermal, also acidic decomposition of chitosan is possible. Chitosan depolymerization, hydrolysis, fragmentation or even decomposition in hydrochloric acid is well described in the literature. In most of the reports, studies were focused on depolymerization of chitosan using a high concentration of HCl [16][17][18][19][20] . Enzymatic hydrolysis of chitosan is another well-known approach 21 . Acetic acid used for chitosan dissolution is not described as a degradative agent, even at high temperatures.
To conclude, no signs of chitosan depolymerization or degradation was observed on the FTIR spectra after the temperature treatment used.

Contact angle measurements
The most popular approach in the development of antibacterial surfaces is creating hydrophilic or superhydrophobic assemblies since hydrophobic ones with contact angles between 90° and 150° are known to be more favorable for promoting bacterial cells adhesion.
However, this phenomenon also strongly depends on cell structure. 22,23 Another approach is a typical example of antibacterial surface acting by either being toxic when coming into contact with bacteria or by releasing antibacterial agent from the surface. 24 Nevertheless, the determination of the composites surfaces properties remains as an important aspect of their optimization process and enables for appropriate selection of procedures for nanocomposites in vitro biological activity assessment.
The surface properties of CS and CS-Ag films were investigated via contact angle measurements. The mean values of contact angles for both, pure chitosan films and nanocomposites containing silver nanoparticles, remain above 90° defining the hydrophilicity/hydrophobicity transition (Table S5).  31 Even though a hydrophobic character of MAg/VC composites was proven, the bactericidal and fungicidal effect of silver species together with the bacteriostatic activity of chitosan presumably did not enable for microorganisms attachment and biofilm formation after incubation with the most effective composites as we will demonstrate later on.

Cytotoxicity tests results
The cytotoxicity tests results of chitosan L/M/H based composites containing silver towards three selected cell lines: A549, CT26, and HaCaT are shown in Table S6.