Antibacterial, Antibiofilm, and Antiadhesive Properties of Different Quaternized Chitosan Derivatives

In the era of antimicrobial resistance, the identification of new antimicrobials is a research priority at the global level. In this regard, the attention towards functional antimicrobial polymers, with biomedical/pharmaceutical grade, and exerting anti-infective properties has recently grown. The aim of this study was to evaluate the antibacterial, antibiofilm, and antiadhesive properties of a number of quaternized chitosan derivatives that have displayed significant muco-adhesive properties and wound healing promotion features in previous studies. Low (QAL) and high (QAH) molecular weight quaternized chitosan derivatives were synthetized and further modified with thiol moieties or pendant cyclodextrin, and their antibacterial activity evaluated as minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC). The ability of the derivatives to prevent biofilm formation was assessed by crystal violet staining. Both QAL and QAH derivatives exerted a bactericidal and/or inhibitory activity on the growth of P. aeruginosa and S. epidermidis. The same compounds also showed marked dose-dependent anti-biofilm activity. Furthermore, the high molecular weight derivative (QAH) was used to functionalize titanium plates. The successful functionalization, demonstrated by electron microscopy, was able to partially inhibit the adhesion of S. epidermidis at 6 h of incubation. The shown ability of the chitosan derivatives tested to both inhibit bacterial growth and/or biofilm formation of clinically relevant bacterial species reveals their potential as multifunctional molecules against bacterial infections.


ATTENUATED TOTAL REFLECTANCE FOURIER TRANSFORM INFRARED SPECTROSCOPY
The titanium surfaces were also characterized by microscopy coupled to infrared (Cary 660 series FT-IR, Agilent Technologies). Spectra were acquired in attenuated total reflectance (ATR), in percent transmittance, in a range between 500 cm -1 and 4000 cm -1 with a resolution interval of 4 cm -1 and a scanning number of 1000. The spectrum of titanium subjected to etching was subtracted from the acquired spectra of titanium samples.

Preliminary screening of silanization by using APTES
Step 3 of the titanium surface functionalization involved the reaction with 3-aminopropyltrietoxysilane (APTES, Sigma-Aldrich). For the setup of functionalization conditions, ethanol, ethanol/HCl 0.05 mM 33:1, or ethanol/H2O (1:1), or toluene were arrayed. Each titanium surface previously etched was immersed in 5 mL of a 5% (v/v) APTES solution (in either ethanol, ethanol/HCl, ethanol/H2O, or toluene) and the reaction was proceeded for 24 h in a shaker water bath (25 °C, 80 rpm). In case of ethanol and toluene, the reaction proceeded under N2 atmosphere. Next, the samples were washed with 20 mL each of reaction solvents, followed by ethanol and deionized water; dried by wiping with filter paper, then under vacuum. Samples thermal curing was performed by heating to 110 °C in an oil bath for 20 min under an N2 stream. Table S1 reports the quantification of amine moieties on titanium surfaces resulting from the use of different solvents; as determined by picric acid method. Etahanol + HCl 0.5 mM 5.8 • 10 -2 ± 2 • 10 -3 Toluene 59.2 • 10 -2 ± 3 • 10 -2

Method of multilayering conjugation of QAH-Pro on titanium surface -Ti-QAH-Pro-ML
For multilayering grafting of QAH-Pro on titanium surface, stages 4 and 5 of the functionalization protocol were repeated 4 times. In details, silanized titanium samples were immersed in 5% glutaraldehyde solutions in 50 mM pH 7.5 phosphate buffer for 4 h at 25 °C, washed three times by dipping in 20 mL of deionized water in sequence and wiped dry by capillarity with filter paper, subsequently transferred to aqueous solutions (pH 7) of polymers (0.5mg/mL) and incubated 24 h at 25 °C. This procedure was repeated 4 times, finally the samples were washed twice with 20 mL of deionized water, wiped dry with filter paper and vacuum dried at 37 °C.

Method of Grafting on Titanium surfaces by using HMDI as spacer (Ti-QAH-Cyan and Ti-QAL-Cyan)
As an alternative to glutaraldehyde, hexamethylene diisocyanate (HMDI) was used as a longer crosslinker. Silanized titanium surfaces were dried under vacuum, 700 µL of DMSO, 100 µL of HMDI, and 10 µL of triethylamine (TEA) were added under N2 atmosphere and kept for 24 h. Freshly distilled HMDI, DMSO and TEA were used. Following incubation, the samples were washed with DMSO then transferred to 1mL of polymer solution (0.5 mg/mL in DMSO, 1% TEA) and left for 24 h at 25°C. Subsequently, they were abundantly washed with DMSO, deionized water, ethanol and ether.

CHARACTERIZATION OF POLYMERS
1 H-NMR NMR measurements of quaternized and cyclodextrin grafted polymers were carried out with a Bruker Avance II operating at 250.13 MHz. During data acquisition the temperature was kept at 25 ± 0.1 °C. For analysis the samples were dissolved in D2O (1-2 %). The acquisition of 1 H-NMR spectra, shown in Figures S7-S10, allowed for the determination of quaternarization degree and cyclodextrin conjugation fractions. The proton unit of the quaternized chitosan was determined from the area under the signals at 4.2-2.2 ppm, corresponding to the protons of the chitosan repeating unit, but for the anomeric one, and the methylene of the pendant chain. In addition, the following were calculated: deacetylation degree, from the diagnostic acetyl groups, at 2.0 ppm; quaternization degree, in terms of quaternary ammonium moieties per repeating unit; and mean length of pendant chains, from the signals of methyl end groups at 1.6-0.7 ppm. Concerning the functionalization with cyclodextrin, the signal at 2.5 ppm of the terminal methylene groups of the quaternized pendant was used as reference and the signals of the cyclodextrin anomeric protons at 5.2 and 5.0 ppm as diagnostic.

Method of thiol determination and protection
The quantification of free thiol moieties was performed for both QAH-Pro and QAL-Pro. Polymers were dissolved in 10 mL of water, 1% starch aqueous solution (1 mL) was added, the pH was adjusted to 3with 1 M HCl and the solution was titrated with 1 mM aqueous iodine until a permanent light blue discoloration was observed. The quantification of degree of substitution by thiol-bearing groups in both polymers was performed after borohydride red-ox reaction. In details, 8 mL of 10% aqueous sodium borohydride was added to a solution of 15 mg polymer in 2 mL of water, and stirred for 1 h. Then the excess of sodium borohydride was destroyed by adjusting to pH 3 with1 M HCl and the thiol content was determined by iodometric titration. The quantification of the protected thiols was performed on samples of 0.1% polymer solution (5 mL), reduced with glutathione (0.1%; 2 h), then analyzed spectrophotometrically at 307 nm. A calibration curve was obtained with 6-MNA (6-mercaptonicotinamide) standard solutions. The content of conjugated aromatic ligand was expressed as the percent of degree of substitution on polymer repeating units. analyzes were carried out in quartz cuvettes, setting the analysis temperature at 25 °C, with toluene used as a reference. The differential increase value of the refractive index as a function of concentration (dn/dc) was set at 0.192 mL/g for plain chitosans [1] and calculated on the basis of size exclusion chromatography linear regressions for chitosan derivatives, according to the procedure adopted for polymer grafting quantification.