Silver ions blocking crystallization of guanosine-based hydrogel for potential antimicrobial applications

Abstract

In this work, the detailed crystallization process of 2'-deoxy-2'-fluoroguanosine (^FG_d) hydrogel has been studied using single crystal X-ray diffraction, variable-temperature nuclear magnetic resonance (VT-NMR), and scanning electron microscopy (SEM). Both solid and solution results indicated that the K⁺-mediated G-quartet structures were unstable and easily resulted in the breakdown of the hydrogel to form linear ribbon structures by forming mimic reverse Watson-Crick base pairs between the two faces with an intermolecular hydrogen-bond (N10H-O11). Accordingly, Ag⁺ was introduced to block the crystallization of ^FG_d to form long lifetime stable supramolecular hydrogel (>6 months) and possible silver-ions-mediated base pair motifs were suggested via NMR, UV, and mass spectroscopy (MS) in combination with powder X-ray diffraction (PXRD) and circular dichroism spectroscopy (CD). Furthermore, ^FG_dAg hydrogel exhibited low toxicity for normal oral keratinocyte cells (NOK-SI) and good antibacterial activities for Fusobacterium nucleatum in vitro.

Fig. 1. Schematic illustration of a long lifetime stable supramolecular hydrogel formed when silver ions were used to block the crystallization of ^FG_d. The hydrogel further showed highly antimicrobial activities.

Fig. 2. (a) Images of the crystal process of ^FG_d hydrogel at a concentration of 1.4 mg per 100 μL of 0.2 M KCl solutions. (b–d) SEM images of the xerogels, crystal, and single crystal prepared from ^FG_d. Scale bars: 10 μm (b), 25 μm (c), and 0.5 mm (d).

Fig. 3. (a) Molecular structure with systematic numbering and single crystal structure of ^FG_d, which adopted an anti-conformation with an N-type (3′-endo) sugar puckering and 5′-OH at ap position. (b) A detailed view of the mimic reverses Watson–Crick base pairs in the solid state of ^FG_d and the repeated hydrogen bonds unit in the entire assembly. Atoms were coded as follows: red, oxygen; blue, nitrogen; gray, carbon; green, fluorine; black, hydrogen.

Fig. 4. Complicated hydrogen bond networks of ^FG_d. (a) The overall multilayered supramolecular structure of ^FG_d. (b) The interactions between bases. (c) The interactions between sugars. (d) The interactions between base and sugar. Nine hydrogen bond units repeated across the different layers in the entire assembly were highlighted in green color. Atoms were coded as follows: red, oxygen; blue, nitrogen; gray, carbon; green, fluorine; black, hydrogen.

Fig. 5. Preparation and characterization of ^FG_dAg hydrogel in water. (a) The formation of a long-lasting stable supramolecular hydrogel via Ag-ions-mediated ^FG_d in water at room temperature. (b) Phase diagram of gelation of ^FG_d at different concentrations of aqueous AgNO₃. G: gel, S: solution, P: precipitate.

Fig. 6. Evaluation of G′ and G′′ as a function of the strain (a) and frequency sweep (b). The compound was measured at the concentration of 0.7 mg per 100 μL in 0.025 M AgNO₃ solutions at 25 °C.

Fig. 7. Microstructures of ^FG_d hydrogel at the concentration of 0.7% in 0.025 M AgNO₃ solution. (a–f) SEM images of ^FG_d hydrogel. (g–i) AFM images of ^FG_d hydrogel. Scale bars: 5 μm (a and d), 1.5 μm (b and e), 500 nm (c and f), 1.5 μm (g), 500 nm (h), and 50 nm (i).

Fig. 8. VT ¹H NMR spectra of ^FG_dAg recorded from 298 K to 338 K.

Fig. 9. Analysis and evaluation of possible silver-ions-mediated base pair in ^FG_dAg hydrogel. (a) UV spectrophotometric titration of 0.25 μM ^FG_d with the increase in Ag⁺ concentration measured in H₂O. (b) Graphs of the ratio of silver ion/duplex versus absorbance changes measured at different wavelengths. (c) The proposed silver-mediated self-assembling structure of ^FG_d and its ¹H–¹H NOE spectra.

Fig. 10. Demonstration of low cytotoxicities and excellent antimicrobial efficiencies of ^FG_d and ^FG_dAg in vitro. (a and b) CCK8 assay of the cytotoxicities of ^FG_d and ^FG_dAg on immortalized normal oral keratinocyte cells (NOK-SI). The results represent mean ± SD. (c) The antibacterial activities in vitro via evaluating the inhibition growth diameter of ^FG_d and ^FG_dAg hydrogels against Fusobacterium nucleatum. In the sterilization experiment, (d) the concentrations of ^FG_d and ^FG_dAg hydrogels were 0 μg ml⁻¹ (1); 15.5 μg ml⁻¹ (2); 31.5 μg ml⁻¹ (3); 62.5 μg ml⁻¹ (4); 125 μg ml⁻¹ (5) and 250 μg ml⁻¹(6), respectively. The dates represent the growth inhibition diameter (in mm) of each sample in three independent experiments.