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

In this work, the detailed crystallization process of 2′-deoxy-2′-fluoroguanosine (FGd) 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 FGd 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, FGdAg hydrogel exhibited low toxicity for normal oral keratinocyte cells (NOK-SI) and good antibacterial activities for Fusobacterium nucleatum in vitro.

However, to the best of our knowledge, the crystallization process of guanosine-based hydrogels has not been explored yet. Thus, determining their delicate structural balance between gelation and crystallization is critical, not only to gain more insight into the reasons for crystallization and further understand how to design new guanosine-based gelators but also to tune their lifetime stability and mechanical properties to develop multifunction supramolecular materials in the future. Inspired by this, the crystallization process of 2 0 -deoxy-2 0 -uoroguanosine ( F G d ) hydrogel has been studied in this work. F G d has the same base and similar sugar moiety as guanosine. It was rst synthesized by J. Imura in 1981 and thereaer, it was observed to exhibit signicant anti-virus activities for inuenza A and B, herpes simplex virus (HSV), and H7N1. [35][36][37][38][39] Furthermore, Christopher J. L. suggested that F G d is a powerful antifavoring tool to manipulate G-quadruplex polymorphism and folding topology. 40 Recently, the gel properties of F G d have been investigated by our group and the results indicated that F G d forms a transparent hydrogel only in the presence of K + . 41 In this work, the detailed crystallization process of F G d hydrogel was rst studied using single crystal X-ray diffraction, VT NMR, SEM, PXRD, and CD. Based on the above information, we observed that silver ions could not only block the crystallization of F G d but also induce F G d to form a long-term stable supramolecular hydrogel with favorable antimicrobial activities (Fig. 1).

Result and discussion
Recently, we reported the gel properties of F G d and the results indicated that F G d forms a transparent gel in the presence of K + and crystallizes in the presence of Li + , Na + , Rb + , and Cs + . 41 The possible self-assembling process of F G d hydrogel is shown in Fig. S1a † according to the previous guanosine-based hydrogel. To obtain an insight into the microstructure of the hydrogel formed by F G d , SEM was performed on xerogels containing KCl at a lower concentration to avoid the negative impact caused by the crystallization of salt. 3D networks with porous-like structures consisting of intensive slices were observed and the diameter of the hole was approximately 150 mm (see ESI, Fig. S1b and c †). Accordingly, atomic force microscope (AFM) measurements were carried out and a similar slice structure was obtained. Notably, some exible bers with a diameter of about 10-20 nm were observed on the surface of slices. These bers inter-tangled with each other to form highly ordered slice structures and thereaer formed 3D hydrogel networks. Nevertheless, F G d hydrogel was easily crystallized within hours similar to other guanosine-based hydrogels. Recent studies gave much evidence to explore the properties of gel to crystal transition for peptide, amino acid and sugar derivatives. [42][43][44][45] However, there is no literature to report the detail crystallization process of guanosine-based hydrogels. To nd out and understand the reason for the crystallization of F G d hydrogel, the visualize dynamic crystal process of F G d hydrogel has been investigated as shown in Fig. 2a. First, F G d formed a transparent gel at the concentration of 1.4 mg per 100 mL of 0.2 M KCl solution. Subsequently, the nucleoside F G d began to crystallize and the hydrogel broke down aer 24 h. Eventually, we got the single crystal of F G d in the above solution aer several months, which was, to the best of our knowledge, the rst free guanosine-based nucleoside crystal formed from hydrogel and would provide us more detailed information about the reason for crystallization at molecular level. Meanwhile, the crystal Fig. 3 (a) Molecular structure with systematic numbering and single crystal structure of F G d , which adopted an anti-conformation with an Ntype (3 0 -endo) sugar puckering and 5 0 -OH at ap position. (b) A detailed view of the mimic reverses Watson-Crick base pairs in the solid state of F G 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. process was also monitored by SEM at micro level (Fig. 2b). The results indicated that the micron-scale slice-like structures gradually grew to piece-like and even colorless millimeter-scale granule-like structures while the gel transformed to a single crystal.
Furthermore, to get more detail information about the crystal from the hydrogel in molecular level, the single crystal structure of F G d has been analyzed carefully from three levels: monomer molecular, base pair motif and three-dimension structure. Firstly, the monomer molecular structure with systematic numbering and the single crystal structure of F G d were shown in Fig. 3. The anti/syn conformation of the glycosyl bond was paramount to the canonical purine or pyrimidine nucleosides, which could be dened by the torsion angle c (O4 0 -C1 0 -N9-C4). The torsion angles c of F G d were measured to be À93.15 (33) , indicating that it adopted a normal anti conformation. Sugar puckering is another signicant conformational parameter dened by the pseudorotation phase angle (P) and maximum puckering amplitude (s m ). Two ranges of pseudorotation phase angle were initially observed in natural and synthetic nucleosides: C3 0 -endo with 0 # P # 36 (North) or C2 0endo with 144 # P # 180 (South). F G d adopted a typical N-type conformation with a twist of C3 0 -endo ( 3 T 2 , N, P ¼ 15.6(4) , s m ¼ 33.6(7) ). Moreover, it was also a crucial structural parameter for the orientation of the 5 0 -hydroxyl group dened by the torsion angle g (O5 0 -C5 0 -C4 0 -O4 0 ) relative to the sugar ring. For F G d , it was in the ap (gauche, trans) range with the O5B 0 -OH at the axial position and pointing outside the sugar ring (g ¼ 61.98 (33) ). Secondly, as F G d has the same base moiety as guanosine, which has three faces with hydrogen bonding donors and acceptors, it can self-assemble into complex and unique supramolecular structures, such as dimers, ribbons, and tetramers. In this case, the aforementioned structures were not obtained as the hydrogels broke down and it was only found that F G d could form mimic reverse Watson-Crick base pairs between two faces with an intermolecular hydrogen-bond (N10H-O11), which extended to form a linear ribbon structure with sugar residues located on both sides.
Finally, based on the base pair motif, a multilayered structure was formed at the 3D supramolecular level with complicated hydrogen bond networks including additional contributions from sugar residues (Fig. 4). To clearly display the hydrogen bond networks, the 3D supramolecular network was divided into three aspects: base-base, sugar-sugar, and basesugar interactions of different layers. The interaction between base moieties is shown in Fig. 4b. O11 held three molecules from three neighboring layers together as a bridge with two hydrogen bonds, and it was connected to C8H of one molecule and N10H of another molecule. The sugar-sugar interactions are displayed in Fig. 4c. The C2 0 -H of one molecule was directly connected to the O5 0 of another molecule. 5 0 -OH linked together three molecules from three adjacent layers with two hydrogen bonds (2 0 F-5 0 OH-3 0 O). The interactions of base-sugar are shown in Fig. 4d. There were four hydrogen bonds between four molecules at three neighboring layers (5 0 O-N1H, N7-3 0 OH, 3 0 O-N10H, O11-C2 0 H). In general, a complicated hydrogen bond network formed with nine hydrogen bonds was repeated innitely throughout the assembly process of the 3D multilayered supramolecular structure in the single-crystal state.
In order to explore their self-assembling properties in solution state, VT NMR spectrometry was used to investigate the formation of hydrogen bonds of F G d . As shown in Fig. S2, † the chemical shi of N10H atom of the amino group moved from d 6.60 (298 K) to d 6.46 ppm (338 K) with Dd ¼ 0.14 ppm, the chemical shi of 3 0 -OH atom of the hydroxyl group moved from    Both solid and solution results indicated that the K + -introduced G-quartet structure was not stable and easily broke down in hydrogel and subsequently formed a linear ribbon structure with mimic reverse Watson-Crick base pairs between the two faces with an intermolecular hydrogen-bond (N10H-O11), which may be the reason for the propensity of guanosine-based hydrogel to crystallize over time. To block the crystallization of F G d hydrogel getting a long lifetime stable supramolecular hydrogel, introducing stronger metal bonds would be a good choice. Previously, researchers determined that metal ions such as Pt 2+ , Hg 2+ , and Ag + tended to alter hydrogen bonding patterns between guanosine molecules by the coordination of the electron-rich nitrogen and oxygen groups around the purine ring to produce a range of metal ion-linked H-bonded architectures. 34,46,47 For example, Kraatz et al. reported that an Ag + induced G gel was exploited for the light triggered in situ fabrication of uniform AgNPs within a gel to make a nano-bio hybrid material; Mann et al. found that supramolecular hydrogels produced by spontaneous self association of disodium guanosine 5 0 -monophosphate (Na 2 5 0 GMP) in the presence of Ag + ions. Inspired by this, Ag + was introduced in this work and the results showed that F G d can not only form hydrogel in the presence of silver ions but also demonstrate long lifetime stability (>6 months). Furthermore, F G d hydrogelation experiments were carried out at different concentrations of AgNO 3 and F G d (0.0125 M to 0.2 M AgNO 3 and 0.175 mg to 2.8 mg of F G d per 100 mL of solution). The phase diagram in Fig. 5 revealed that the formative qualication of the hydrogel. The concentration control between AgNO 3 and F G d was observed to play a critical role in the formation process of F G d hydrogel. Interestingly, F G d tended to form a hydrogel at low silver ion concentration, which suggested that the hydrogel had signicant potential for biological or medical applications. Furthermore, the mechanical properties of the hydrogel were analyzed in detail using rheological measurements. The storage modulus (measurement of elastic property) and loss modulus (measurement of uidity) were expressed as G 0 and G 00 , respectively. As shown in Fig. 6, the typical elastic nature of F G d + Ag + ( F G d Ag) hydrogel was evident from the fact that G 0 > G 00 (solidlike behavior) via strain amplitudes ranging from 0.001 to 0.1% at 6.28 rad s À1 . Moreover, the frequency sweep experiments of F G d Ag hydrogel were performed in the angular frequency range of 0.1-100 rad s À1 under an initial strain of 0.1% and the results showed that F G d Ag hydrogel had a higher storage modulus G 0 than loss modulus G 00 over the entire applied frequency range, indicating that it exhibited a solid-like behavior.
To obtain microstructures of the F G d Ag hydrogel, we prepared xerogels for SEM observations (Fig. 7a-f). The hydrogels were prepared in a sample tube (0.7 w/v%) containing 0.025 M AgNO 3 and they were subsequently frozen. The frozen samples were lyophilized using a vacuum pump. Prior to examination, the xerogel was attached to the silica wafer and coated with a thin layer of gold. The SEM images revealed the three-dimensional uniform porous-like structures with a diameter of approximately 500 nm. Careful inspection of the images revealed that exible bers with a diameter of approximately 20-30 nm and several micrometers in length were formed spontaneously during the formation of hydrogel. The bers intertangled with each other to form highly ordered lm-like structures. Energy-dispersive X-ray spectroscopy analysis conrmed the presence of Ag in the bers. AFM was carried out to further analyze and evaluate the structures of the hydrogel. Fig. 7g-i reveals the existence of an interconnected brous network with a diameter of approximately 20-30 nm, height of 6-8 nm, and length of several micrometers, which are consistent with the above results.
To explore more molecular-level evidences of the hydrogel, CD spectroscopy, PXRD, and NMR were carried out. Previous reports indicated that a planar quartet system show peaks at 240 nm and 260 nm in CD spectrum. 28 Here, the F G d Ag hydrogel showed two negative peaks at 220 and 290 nm (see ESI, Fig. S4 †), which indicated that the G-quartet structures were not observed in this hydrogel. As shown in Fig. S5, † the freeze-dried sample of F G d Ag hydrogel was studied using PXRD and it exhibited similar patents for F G d Ag hydrogel and the crystal of F G d hydrogel in the presence of K + . All of them exhibited a signicant peak at 2q z 6.0 and 26.8 (d ¼ 14.5Å and 3.4Å, respectively) consistent with the monomeric length and Pi-Pi stacking distance between two layers of G rings. 31 Subsequently, F G d and F G d Ag were characterized using 1 H NMR spectra at 298 K (see ESI, Fig. S6 †). The results showed that N10H peak almost disappeared when Ag + was introduced to F G d , indicating that the amino group may participate in the silver base pair. VT NMR experiments were carried out to further investigate the formation of hydrogen bonds of F G d Ag. As shown in Fig. 8 and Stoichiometric titration experiments were performed to verify the formation of silver-mediated pairs and determine the amount of silver ions bound to the nucleoside F G d . As shown in Fig. 9, the changes in UV wavelengths were plotted versus the ratio of silver equivalents/ F G d , suggesting that two silver ions were captured by one F G d ( Fig. 9a and b). Accordingly, positive mode electrospray ionization-mass spectroscopy analysis was carried out and the corresponding silver complexes of 1 : 1 and 1 : 2 peaks at 475.1 and 624.1, respectively, were observed (see ESI, Fig. S7 †), which provided powerful evidence further indicating that the two silver ions were captured by one F G d . Based on the above results, the possible silver-mediated base pair motifs are shown in Fig. 9c. To validate the assumption, 1 H-1 H NOE experiments were carried out and the results suggested that C8-H produced a strong NOE at NH2a, NH2b, and NH simultaneously (Fig. 9c), which provided an unambiguous interpretation of the G-ribbon structures.
Fusobacterium nucleatum and Porphyromonas gingivalis, two Gram-negative anaerobes, are the most abundant microorganisms present in the oral cavity during periodontal disease. 48,49 In this study, the antibacterial activities of F G d and F G d Ag hydrogels in vitro were preliminarily evaluated. Initially, we assessed the biocompatibility of F G d ( F G d dissolved in PBS) and F G d Ag hydrogels in vitro, the cell viability of the immortalized normal oral keratinocyte cells (NOK-SI) was assessed using CCK8 assay. As shown in Fig. 10, when the concentration of F G d and F G d Ag increased to 2.0 mg mL À1 , the viability of the NOK-SI cell line was higher than 97%. As the concentration of F G d and F G d Ag increased to 8.0 mg mL À1 , the viability of NOK-SI cell line was higher than 86%. As expected, these results indicated that the F G d and F G d Ag hydrogels had only slight toxicity in vitro to be used as a safe biomaterial. Furthermore, the preliminary in vitro antimicrobial activities of F G d and F G d Ag hydrogels on Fusobacterium nucleatum and Porphyromonas gingivalis were evaluated by measuring the inhibition growth diameter and minimal bactericidal concentrations (MBCs). The results presented F G d and F G d Ag hydrogels had no obvious inhibition effect on Porphyromonas gingivalis. However, F G d Ag hydrogel exhibited excellent antibacterial activities for Fusobacterium nucleatum (MBC: 31.25 mg mL À1 ), compared to the control group, F G d hydrogel, which exhibited no antibacterial activities, silver ions as positive control (see ESI, Fig. S8 †). In Fig. 10c and d, the diameters of Fusobacterium nucleatum inhibiting loops were approximately 0.57, 1.3, 1.9, 2.67 mm for F G d Ag hydrogel 31.5, 62.5, 125, and 250 mg mL À1 , respectively. Therefore, these ndings suggested that F G d Ag might be a possible biomaterial for antimicrobial medical applications.

Conclusion
In summary, the detailed crystallization process of F G d hydrogel was studied using SEM. The results indicated that the micronscale slice-like structure gradually grew to piece-like and even colorless millimeter-scale granule-like structure while the gel transformed to single crystal, which is, to the best of our knowledge, the rst free guanosine-based nucleoside crystal formed from a hydrogel. The single crystal X-ray diffraction analysis indicated that F G d adopted an anti-conformation with an N-type (3 0 -endo) sugar puckering and 5 0 -OH at ap position, formed mimic reverses Watson-Crick base pairs with an intermolecular hydrogen-bond (N10H-O11), and had nine hydrogen bonds innitely repeated in the entire assembly to form the 3D multilayered supramolecular structure in the solid state. These hydrogen bonds were also further investigated using VT NMR in solution state, and the results were consistent with the solid state. Then, Ag + was introduced to block its crystallization and form hydrogel with long lifetime stability (>6 months). Rheological measurements revealed that F G d Ag gel has a higher storage modulus G 0 than loss modulus G 00 over the entire applied frequency range (solid-like behavior). SEM and AFM studies demonstrated that exible bers with a diameter of approximately 20-30 nm, height measurements of approximately 7 nm, and length of several micrometers were formed spontaneously during the formation of F G d Ag gel whereas F G d formed a slice structure. Possible silver-mediated base pair motifs were suggested using PXRD, CD NMR, UV, and MS. Finally, F G d Ag hydrogel exhibited low toxicity for NOK-SI cell and good antibacterial activities for Fusobacterium nucleatum in vitro, whereas F G d exhibited no antibacterial activity. The above results suggested that F G d Ag might be useful for future applications in the eld of antimicrobial medicines or drug delivery.

Conflicts of interest
The authors declare no conict of interest.