Thymol-Functionalized Hyaluronic Acid as Promising Preservative Biomaterial for the Inhibition of Candida albicans Biofilm Formation

Hyaluronic acid (HA) is a naturally occurring biopolymer that has been employed for a plethora of medicinal applications. Nevertheless, as HA is a natural polysaccharide, it can be a substrate able to promote microbial growth and proliferation. Biopolymer–drug conjugates have gained attention over the years to overcome drawbacks of each single component. Within this context, thymol (Thy), a phenolic compound occurring in essential oils (EOs) extracted from Thymus and Origanum, has been largely studied for its antimycotic applications. However, it is characterized by a low water solubility and moderate cytotoxicity. Herein, we report an innovative HA–thymol conjugate (HA-Thy) biomaterial to circumvent the drawbacks of free thymol use by providing the polymer conjugate with the beneficial properties of both components. Preliminary biological tests evidenced the decrease of thymol cytotoxicity for the HA-Thy conjugate, paired with a promising antibiofilm formation activity against Candida albicans, similar to pure thymol, highlighting its potential application as a preservative biomaterial in formulations.


Materials NMR spectroscopy
Infrared spectroscopy UV spectroscopy

Synthesis of Thy-ester (4)
Characterization of 4 S1: 1 H NMR spectrum for compound 4 and its structure.

S3:
Comparison of ATR-FTIR spectra of compound 4 and pure thymol.

S6
: 1 H NMR spectrum of HA-Thy-25 in D2O with peaks integrals and structure of compound 5a. S7: 1 H NMR spectrum of HA-Thy-50 in D2O with peaks integrals.

S8:
Comparison of 1 H NMR spectra in D2O of native HA, HA-Thy-25, HA-Thy-50 derivatives and Thy-ester. Table S2. Concentrations of thymol, HA and HA-Thy-25 used in the MTT assay experiments.

Candida albicans strain and Culture Conditions
Antifungal Susceptibility Testing (MIC)

Assays of Biofilm Inhibition (BMIC)
TableS4. Percentage of inhibition of C. albicans ATCC 10231 biofilm formation at 48 h for all the tested compounds.

NMR spectroscopy
Water soluble HA-Thy samples were dissolved in D 2 O at r.t. (6 mg in 0.6 ml of D 2 O). Thy-ester spectrum was recorded in CDCl 3 . NMR analyses were performed on a 400 MHz Bruker Avance III spectrometer and spectra were processed using MestReNova 6.0.2 (Mestrelab Research SL). Third order polynomial fit was used to correct base line. Residual internal solvent was used as standard.

Infrared spectroscopy
Fourier-transform infrared spectroscopy (FTIR) was used to characterized HA-Thy derivatives. Spectra, before and after chemical modification, were acquired in attenuated total reflection (ATR) using a Nicolet 6700 (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a Golden Gate single reflection diamond ATR accessory. All spectra were recorded in absorption mode with 200 scans/spectrum and at 4 cm -1 resolution in the region between 4000 -650 cm -1 . The software Omnic 8.0 (Thermo Fisher Scientific Inc.) was used for the elaboration of spectra.

UV spectroscopy
UV spectra were recorded on HP DIODE ARRAY instruments between 190 and 820 nm, with 2 nm resolution. Water soluble samples were dissolved in Milli-Q water and analyzed in quartz cuvettes.

Synthesis of HA-TBA
HA-TBA was synthetized slightly modifying reference N° 25. Sodium hyaluronate (1 g, 2.5 mmol) was mixed with TBAOH (TBAOH in a 40% wt/v in water, 87 mmol), after proper activation with Dowex®50WX-8-400 resin (58 mmol). The mixture was stirred for 5 h at r.t., filtered and HA-TBA solution was purified by extensive dialysis against distilled water. Finally, HA-TBA salt was freeze dried and it was collected as a cotton-like material. TBA + ion presence was confirmed by intense stretching peaks of methyl and methylene groups between 3000 and 2880 cm -1 in the infrared spectrum of HA-TBA salt. Furthermore, proton spectrum in D 2 O showed specific sets of signals assigned to the ammonium cation such as a triplet at 0.95 ppm for methyl groups and a sextuplet at 1.37, a quintuplet at 1.66 and a triplet at 3.20 ppm for the three methylene groups. Degree of substitution resulted to be close to 100% as indicated by 1 H NMR spectrum.

Synthesis of HA-Thy (5)
To a solution of HA-TBA (50 mg, 0.07 mmol) in DMSO (2.5 ml), Thy-ester (4) was added using two different reactants ratio (HA-TBA:Thy-ester=1:0.25 and HA-TBA:Thy-ester=1:0.50). TBAI salt was added as catalyst (0.02 mmol). Solutions were kept under stirring until TLC (Hex:Et=8:2) revealed consumption of 4. Generally, 5 days of reaction were needed for its the complete consumption. After reaction time, the polymeric product was precipitated adding brine (1 ml) and ethanol (20 ml) to the mixture and stirred for additional 3 h. The HA precipitate was separated by centrifugation and purified by dialysis (cut-off 14 kDa) against Milli-Q water for 5 days. Extensive dialysis was necessary for the complete purification of HA derivatives from bromide anion, excess of NaCl or solvents. Finally, HA-Thy bioconjugate was frozen in liquid nitrogen, freeze dried and collected as a white cotton-like material.
Degree of thymol functionalization (TF%) was calculated from the ratio between aromatic signals of thymol between 7.5 and 6.7 ppm and methyl peak of HA unit at 2.0 ppm.  Figure S4. ATR-FTIR spectra of HA-Thy-25 (orange) and HA-Thy-50 (red) and Thy-ester (black) compared to that of native HA (black). The formation of a new ester bond by the linkage of thymol causes the increase of ester absorption band at 1745 cm -1 at the expense of COONa band at 1605cm -1 . Figure S5. UV absorption spectra of pristine HA (blue), thymol (green), HA-Thy-25 (orange) and HA-Thy-50 (red) dissolved in water. The functionalization of HA with thymol causes the appearance of an absorption peak situated at 264 nm in the two HA derivatives. Free-phenolic thymol structure absorbs at 276 nm.

Cell cultures and cellular viability MTT assay
COS-7 fibroblast cells (ECACC 87021302) were seeded at a density of 100,000 cells per well in a 24-well plate containing complete high glucose DMEM medium supplemented with 10% glutamine and 10% FBS. The compounds HA, HA-Thy-25, and free thymol were weighed under semi-sterile conditions and mechanically dissolved in Optimem Medium for 24 h at r.t. After 24 h, these solutions were added in the 24 well plate starting from the initial concentration of 12.8 mM for HA and HA-Thy-25, and 3.2 mM for free thymol, and then diluted 1:2 in Optimem Medium directly in well. Following a 48-hour incubation, cell viability was assessed using the MTT colorimetric assay (Abcam, ab211091), following the manufacturer's protocol. Briefly, the assay involved an incubation of 1.5 h at 37 °C, followed by measurement of the absorbance of the resulting solutions at 590 nm using a spectrophotometer (ThermoFisher). Five colonies of C. albicans were then collected with phosphate-buffered saline (PBS) and the inoculum was determined by spectrophotometric reading (Ultrospec™ 2100 pro) and was confirmed with the reading at the Bürker chamber.

Effect of Thymol, HA and HA-Thy-25 on Candida albicans planktonic cells.
The minimum inhibitory concentration (MIC) of thymol, HA-Thy and HA against C. albicans (ATCC 10231) was determined according to the standardized method (CLSI M27-A3 document; CLSI M27-S4). The final concentration of the inoculum was 1 × 10 3 -5 × 10 3 cells/mL. The compounds in RPMI were added to the wells. The concentrations of thymol ranged from 500 µg/mL to 0.977 µg/mL. The plates, incubated at 37 °C for 24 h and 48 h, were observed for growth inhibition compared to untreated growth controls. The minimum inhibitory concentration that caused growth inhibitions ≥ 50% (MIC) was determined. The antifungal activities are the result of three independent experiments performed in triplicate and reported as median. All experiments were carried out, in triplicate, at least three times on separate dates and on different HA-Thy-25 batches.